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Heidi Vierimaa

SALMON CARDIACPEPTIDE AS A MODELFOR NATRIURETICPEPTIDE SECRETIONTHE ROLE OF MECHANICAL LOAD, TEMPERATURE AND ENDOTHELIN-1

FACULTY OF MEDICINE, DEPARTMENT OF PHYSIOLOGY, BIOCENTER OULU, UNIVERSITY OF OULU

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eidi Vierimaa

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A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 8 9 0

HEIDI VIERIMAA

SALMON CARDIAC PEPTIDEAS A MODEL FOR NATRIURETIC PEPTIDE SECRETIONThe role of mechanical load, temperature and endothelin-1

Academic dissertation to be presented, with the assent ofthe Faculty of Medicine of the University of Oulu, forpublic defence in Auditorium F101 of the Department ofPhysiology (Aapistie 7), on September 29th, 2006,at 12 noon

OULUN YLIOPISTO, OULU 2006

Copyright © 2006Acta Univ. Oul. D 890, 2006

Supervised byProfessor Olli Vuolteenaho

Reviewed byDocent Mika LaineProfessor Mikko Nikinmaa

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Cover designRaimo Ahonen

OULU UNIVERSITY PRESSOULU 2006

Vierimaa, Heidi, Salmon cardiac peptide as a model for natriuretic peptide secretion.The role of mechanical load, temperature and endothelin-1Faculty of Medicine, Department of Physiology, Biocenter Oulu, University of Oulu, P.O. Box5000, FI-90014 University of Oulu, Finland Acta Univ. Oul. D 890, 2006Oulu, Finland

AbstractThe natriuretic peptides are a family of hormones secreted by the heart. They play a fundamental rolein salt and water balance and blood pressure regulation. Atrial natriuretic peptide (ANP), brainnatriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are the known members of themammalian natriuretic peptide family. A major stimulus for the secretion of cardiac natriureticpeptides is myocyte stretch. Therefore, the secretion of natriuretic peptides is increased in responseto elevated blood volume. Natriuretic peptide production and release is also affected by several otherfactors, such as endothelin-1 (ET-1), acting in paracrine fashion.

The aim of this study was to elucidate factors regulating the novel cardiac peptide hormone,salmon cardiac peptide (sCP), belonging to the family of natriuretic peptides. The role of mechanicalload, temperature and ET-1 in sCP secretion and production was studied using in vitro (isolatedperfused ventricle preparation) and in vivo methods. Comparisons between the natriuretic peptidesystems in fish and mammals were done to clarify functional evolution of this hormone family.Salmon (Salmo salar) was selected as a model, since it has an outstanding adaptability to widevariations in environmental salinity and has developed defence mechanisms against volume or saltload.

The results showed that salmon ventricle stores large amounts of the prohormone of sCP, whereasthe secreted form is the mature 29-amino acid form. The N-terminal fragment of pro-sCP is co-secreted with sCP in equimolar amounts. sCP is released rapidly in response to appropriate stimulus,whereas induction of its gene expression is slower. Mechanical load is an important regulator of sCPsecretion. Temperature also plays a major role in regulating sCP plasma concentration by affectingits elimination from circulation. Additionally, ET-1 is a potent secretagogue of the sCP system andan inotropic agent in salmon heart. Furthermore, the present results reveal remarkable synergismbetween the cardiac effects of ET-1 and β-adrenergic stimulation.

In conclusion, the sCP system in salmon ventricle largely resembles the ANP system inmammalian atrium, while also having specific characteristics, such as a regulated ventricularnatriuretic peptide secretion pathway. Therefore, the sCP system offers a unique model for studyingmechanisms of natriuretic peptide biology.

Keywords: adrenergic, endothelin-1, heart, mechanical load, natriuretic peptides,perfusion, salmon

To my family

Acknowledgements

This work was carried out at the Department of Physiology, University of Oulu, during the years 1998-2006. I want to express my sincere gratitude to my supervisor, Professor Olli Vuolteenaho, for giving me an opportunity to prepare this thesis as a member of his research group. Olli has always given constructive comments and invaluable support when planning and implementing the different studies and preparing the manuscripts. I also regard him as a thorough and responsible scientist and a good example for a young researcher. I wish to thank Professor Heikki Ruskoaho and Professor Emeritus Juhani Leppäluoto for their support and for the excellent research facilities provided by the Department of Physiology and Biocenter Oulu.

I would like to acknowledge all the past and present workmates from the Department of Physiology. I am especially thankful to my closest colleagues Minna Ala-Kopsala, Mika Ilves and Theresa Majalahti for their friendship and, generally, not scientific but non-scientific conversations. My past workmates and co-authors Docent Olli Arjamaa and Dr. Virpi Tervonen deserve my grateful thanks for introducing me to the methods and for all their help with this work. I also wish to thank all the other co-authors, especially Kati Kokkonen and Jarkko Ronkainen, for collaboration. Docent Pasi Tavi deserves thanks for knowledgeable feedback. I owe my thanks for superior laboratory assistance to all the past and present laboratory staff at the Department of Physiology, especially Helka Koisti, Seija Linnaluoto, Tuula Lumijärvi, Anneli Rautio, Tuula Taskinen and Ulla Weckström. In addition, I would like to thank Helka Koisti and Anneli Rautio for their friendship and for being an emotional anchor for me all the while. The expert help of Alpo Vanhala, who built a lot of new equipment and experimental set-ups I used during this work, was of great importance. I am also very grateful to our laboratory manager Eero Kouvalainen for his eager and expert help with computer problems and statistical analysis. I also thank Marika Hämeenaho for her skilful secretarial assistance and kind help with everything.

The staff of the Game and Fisheries Institute in Taivalkoski, Muonio and Laukaa and the hatcheries of Montan lohi and Imatran Voima power plant in Muhos and Ii are gratefully acknowledged for providing the fish used in this study. I am grateful to the official examiners of the thesis, Docent Mika Laine and Professor Mikko Nikinmaa, for

their critical evaluation and valuable comments on the thesis. Anna Vuolteenaho is acknowledged for careful revision of the English language of the thesis.

My parents Kirsti and Hannu and my brother Juha merit my warmest thanks for all the support and care they have shown me throughout my life. All my friends are very important for me, giving me numerous relaxing, fun and even comical moments besides work. Especially, I wish to thank "true friends" Sini and Keksi, for always being there for me. Last, but not least, my dearest Tom Sirviö deserves my deepest gratitude for all his loving support, understanding and patience during these years.

This study was supported by the Academy of Finland, Biocenter Oulu, Kemira Foundation, Olvi Foundation, Oulu University Scholarship Foundation, Sigrid Juselius Foundation and Tyyni Tani Foundation.

Oulu, September 2006 Heidi Vierimaa

Abbreviations

ANOVA analysis of variance ANP A-type or atrial natriuretic peptide AT1 angiotensin type 1 receptor BNP B-type or brain natriuretic peptide BSA bovine serum albumin cAMP cyclic adenosine monophosphate cDNA complementary deoxyribonucleic acid cGMP cyclic guanosine monophosphate CNP C-type natriuretic peptide C-terminal carboxy-terminal DAG diacylglycerol ECE endothelin-converting enzyme ET-1 endothelin-1 ETA endothelin type A receptor ETB endothelin type B receptor GMP guanosine monophosphate GST gluthathione S-transferase Gx G protein subtype HPLC high performance liquid chromatography IP3 inositol triphosphate mRNA messenger ribonucleic acid NEP neutral endopeptidase NHE-1 Na+/H+ exchanger NO nitric oxide NPR natriuretic peptide receptor NPR-A type A natriuretic peptide receptor NPR-B type B natriuretic peptide receptor NPR-C type C natriuretic peptide receptor N-terminal amino-terminal NT-proANP amino-terminal fragment of proANP NT-proBNP amino-terminal fragment of proBNP

NT-pro-sCP amino-terminal fragment of pro-sCP PCR polymerase chain reaction PIP2 phosphatidylinositol 4,5-diphosphate PKC protein kinase C PLC phospholipase C RIA radioimmunoassay RNA ribonucleic acid SA channel stretch-activated ion channel sCP salmon cardiac peptide SE standard error of mean TFA trifluoroacetic acid VNP ventricular natriuretic peptide

List of original articles

This thesis is based on the following articles, which are referred to in the text by their Roman numerals:

I Kokkonen K*, Vierimaa H*, Bergström S, Tervonen V, Arjamaa O, Ruskoaho H, Järvilehto M & Vuolteenaho O (2000) Novel salmon cardiac peptide hormone is released from the ventricle by regulated secretory pathway. American Journal of Physiology Endocrinology and Metabolism 278: E285-E292.

II Tervonen V, Kokkonen K*, Vierimaa H*, Ruskoaho H & Vuolteenaho O (2001) Temperature has a major influence on cardiac natriuretic peptide in salmon. Journal of Physiology 536: 199-209.

III Vierimaa H, Hirvinen M, Tervonen V, Arjamaa O, Ruskoaho H & Vuolteenaho O (2002) Pronatriuretic peptide is a sensitive marker of the endocrine function of teleost heart. American Journal of Physiology Endocrinology and Metabolism 282: E843-E850.

IV Vierimaa H, Ronkainen J, Ruskoaho H & Vuolteenaho O (2006) Synergistic activation of salmon cardiac function by endothelin and beta-adrenergic stimulation. American Journal of Physiology Heart and Circulatory Physiology 291: H1360-H1370.

* equal contribution

Contents

Abstract Acknowledgements Abbreviations List of original articles Contents 1 Introduction ................................................................................................................... 17 2 Review of the literature ................................................................................................. 19

2.1 Natriuretic peptide system in mammals..................................................................19 2.2 Structure and biosynthesis of mammalian natriuretic peptides...............................20

2.2.1 Biosynthesis of ANP........................................................................................21 2.2.2 Biosynthesis of BNP........................................................................................23 2.2.3 Biosynthesis of CNP........................................................................................23

2.3 Regulation of mammalian natriuretic peptides .......................................................24 2.3.1 Regulation of ANP ..........................................................................................24

2.3.1.1 Hemodynamic factors...............................................................................24 2.3.1.2 Vasoactive agents......................................................................................27 2.3.1.3 Adrenergic stimulation..............................................................................28 2.3.1.4 Hormones and other factors......................................................................29 2.3.1.5 Hypoxia ....................................................................................................30

2.3.2 Regulation of BNP ..........................................................................................30 2.3.3 Regulation of CNP ..........................................................................................33

2.4 Natriuretic peptide receptors in mammals ..............................................................33 2.5 Elimination of natriuretic peptides in mammals.....................................................35 2.6 Physiological effects of natriuretic peptides in mammals.......................................35

2.6.1 Effects of ANP and BNP .................................................................................36 2.6.1.1 Hypotensive nature of ANP and BNP.......................................................36 2.6.1.2 Renal effects .............................................................................................36 2.6.1.3 Inhibition of renin-angiotensin-aldosterone system..................................37 2.6.1.4 Vascular effects and effects on intravascular volume ...............................37 2.6.1.5 Cardiac effects ..........................................................................................38 2.6.1.6 Other effects .............................................................................................38

2.6.2 Effects of CNP.................................................................................................39 2.6.2.1 Vascular and cardiac effects......................................................................39 2.6.2.2 Other effects .............................................................................................40

2.7 Natriuretic peptide system in nonmammalian vertebrates......................................41 2.7.1 Avian, reptile and amphibian natriuretic peptides and their receptors .............41 2.7.2 Fish natriuretic peptide system ........................................................................42

2.7.2.1 ANP in fish ...............................................................................................42 2.7.2.2 CNP in fish ...............................................................................................42 2.7.2.3 Ventricular natriuretic peptide ..................................................................43 2.7.2.4 Salmon cardiac peptide.............................................................................44 2.7.2.5 Fish natriuretic peptide receptors..............................................................45

2.8 Evolutionary history of the natriuretic peptide system ...........................................46 2.9 Endothelin-1 in cardiovascular physiology ............................................................46

2.9.1 Endothelins ......................................................................................................46 2.9.2 Endothelin receptors in mammals ...................................................................47 2.9.3 Cardiovascular effects of endothelin-1 in mammals........................................48

2.9.3.1 Vascular effects .........................................................................................48 2.9.3.2 Inotropic effects ........................................................................................48 2.9.3.3 Effect of endothelin-1 on natriuretic peptide release ................................50 2.9.3.4 Other cardiovascular effects .....................................................................51

2.9.4 Endothelins in fish...........................................................................................52 3 Aims of the research ...................................................................................................... 54 4 Materials and methods................................................................................................... 55

4.1 Experimental animals .............................................................................................55 4.2 Experimental models ..............................................................................................56

4.2.1 Myocardial strip preparation (IV)....................................................................56 4.2.2 Isolated perfused ventricle preparation (I – IV)...............................................56

4.2.2.1 Mechanical load experiments (I, III) ........................................................58 4.2.2.2 Ambient temperature experiments (II)......................................................58 4.2.2.3 Endothelin-1 experiments (IV) .................................................................58 4.2.2.4 Endothelin-1 and β-adrenergic activation experiments (IV).....................59

4.2.3 Seasonal sampling of salmon (II) ....................................................................59 4.2.4 Chronic cannulation of salmon (III) ................................................................60

4.3 Extraction of sCP from serum and tissues ..............................................................60 4.4 Hormone assays......................................................................................................61

4.4.1 Radioimmunoassay of sCP..............................................................................61 4.4.2 Radioimmunoassay of NT-pro-sCP (II, III) .....................................................61

4.5 Immunoelectron microscopy (I) .............................................................................62 4.6 Gel filtration high performance liquid chromatography (I, III) ..............................62 4.7 Isolation of mRNA and Northern blot analysis (I, II) .............................................63 4.8 Extraction of total RNA and quantitative PCR (IV) ...............................................63 4.9 Statistical analyses..................................................................................................64

5 Results ........................................................................................................................... 65 5.1 Molecular forms of stored and secreted sCP (I) .....................................................65 5.2 Radioimmunoassay for measuring the aminoterminal fragment of pro-sCP

(NT-pro-sCP) (III) .................................................................................................65

5.3 Effects of mechanical load on sCP secretion (I) .....................................................66 5.3.1 Effect of mechanical load on the release of sCP from the isolated

salmon ventricle ..............................................................................................67 5.3.2 Effect of mechanical load on the ventricular immunoreactive sCP and

sCP mRNA level..............................................................................................67 5.4 Effects of different temperatures on sCP secretion (II)...........................................68

5.4.1 Seasonal levels of plasma and cardiac immunoreactive sCP...........................68 5.4.2 Seasonal levels of cardiac sCP mRNA ............................................................69 5.4.3 Seasonal relative ventricle mass ......................................................................70 5.4.4 Effects of different acclimation temperatures on sCP secretion ......................70

5.5 Effects of endothelin-1 on sCP secretion................................................................71 5.5.1 Effects of endothelin-1 in sCP release from salmon ventricle in vitro

(IV) ..................................................................................................................71 5.5.2 Effects of endothelin-1 on sCP secretion in vivo (III) .....................................72 5.5.3 Effects of endothelin-1 on sCP mRNA levels (IV)..........................................74

5.6 Interactions between endothelin-1 and β-adrenergic activation on the regulation of salmon cardiac contractile and endocrine function (IV) ..................74

5.6.1 Effects of endothelin-1 and β-adrenergic activation on salmon cardiac contractile function..........................................................................................75

5.6.2 Effects of endothelin-1 and β-adrenergic activation on sCP secretion from isolated perfused salmon ventricle..........................................................76

6 Discussion ..................................................................................................................... 78 6.1 Stored and secreted sCP..........................................................................................78 6.2 NT-pro-sCP as a marker of sCP activity .................................................................79 6.3 Factors regulating sCP............................................................................................80

6.3.1 Effects of mechanical load on sCP secretion ...................................................80 6.3.2 Effects of temperature on sCP secretion ..........................................................81

6.3.2.1 Effects of temperature on secreted sCP levels ..........................................81 6.3.2.2 Effects of temperature on sCP production in cardiac tissue......................82

6.3.3 Effects of endothelin-1 on sCP secretion in vivo.............................................83 6.3.4 Effects of endothelin-1 in vitro on sCP secretion and its synergistic

action with β-adrenergic activation .................................................................83 6.3.4.1 Effects of endothelin-1 and β-adrenergic activation on salmon

cardiac contractile function ......................................................................84 6.3.4.2 Effects of endothelin-1 and β-adrenergic activation on

sCP secretion............................................................................................85 6.3.4.3 Effects of endothelin-1 on sCP production ...............................................86

7 Summary and conclusions ............................................................................................. 87 References Original articles

1 Introduction

The natriuretic peptides are a family of hormones or paracrine factors that play a fundamental role in salt and water balance and blood pressure regulation. Natriuretic peptides exhibit a wide spectrum of effects directed largely towards pressure and volume homeostasis, and they protect the cardiovascular system from volume and pressure overload (Brenner et al. 1990). Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are the known members of the mammalian natriuretic peptide family. The close relationship between plasma levels of natriuretic peptides and cardiac load has led to their use as diagnostic markers of cardiac health in cardiovascular disorders (for review see Ruskoaho 2003). In addition, due to their favourable biological properties natriuretic peptides may be useful therapeutic agents (for review see Rademaker & Richards 2005).

The cardiac natriuretic peptides, ANP and BNP, are predominatly secreted by the heart. ANP is mainly produced in the atria, where it is stored in specific granules as a prohormone (for review see Rosenzweig & Seidman 1991, Ruskoaho 1992, McDowell et al. 1995). Final processing to mature hormone takes place during exocytosis of the hormone (for review see Ruskoaho 1992). BNP is primarily a ventricular hormone, which is secreted constitutively from myocytes as the mature peptide (Wei et al. 1993b). CNP is produced in several tissues, such as the central nervous system and endothelium (for review see e.g. Potter et al. 2006).

The most important stimulus for the secretion of cardiac natriuretic peptides is myocyte stretch (Lang et al. 1985, Ruskoaho et al. 1986, Kinnunen et al. 1993, Mäntymaa et al. 1993, Laine et al. 1994a, b). Therefore, secretion of natriuretic peptides is increased in response to elevated central blood volume resulting in an increase of cardiac pressure load. Secretion is also affected by many other factors including mechanical, physiological and neurohumoral stimuli (for review see Thibault et al. 1999, Dietz 2005). The activation of local paracrine factors, such as endothelin-1 (ET-1) and angiotensin II, has been invoked as being related to natriuretic peptide release following volume expansion (Donckier et al. 1992, Leskinen et al. 1997a). However, despite intensive research, the mechanisms involved in the processing and secretion of natriuretic peptides from myocyte are still largely unknown and require further studies.

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The aim of the present study was to determine the factors regulating natriuretic peptide secretion by using a novel cardiac peptide hormone, sCP or salmon cardiac peptide (Tervonen et al. 1998), as a model. Salmon (Salmo salar) was selected as an experimental model, since this euryhaline species has an excellent adaptability to wide variations in environmental salinity. Therefore, it must be able to handle both volume and salt loads. Especially, the effects of mechanical load of the heart, ambient temperature and ET-1 on sCP secretion were examined using in vitro (isolated perfused salmon ventricle preparation) and in vivo methods. Comparisons between the natriuretic peptide systems in phylogenetically distinct species such as salmon and mammalian species were done in order to reveal new aspects of the functional evolution of the natriuretic peptide system.

2 Review of the literature

2.1 Natriuretic peptide system in mammals

Natriuretic peptides are a family of structurally related hormones or paracrine factors. Atrial, brain and C-type natriuretic peptides (ANP, BNP and CNP, respectively) are the known members of the mammalian natriuretic peptide system. In 1981 de Bold et al. demonstrated that atrial extracts contained natriuretic activity, which led them to isolate the atrial natriuretic peptide (ANP) (de Bold et al. 1981). In 1988, Sudoh et al. isolated a peptide with similar biological activity to ANP from porcine brain (Sudoh et al. 1988). The peptide was named “brain natriuretic peptide” (BNP), although the heart was subsequently found to be the major source of circulating BNP (Minamino et al. 1988). Two years later, Sudoh et al. described a further, structurally similar peptide from porcine brain, which they named C-type natriuretic peptide (Sudoh et al. 1988, 1990). Accordingly, ANP and BNP are often referred to as A- and B-type natriuretic peptide

ANP and BNP are primarily synthesized in the heart and display the most variability in primary structures, whereas CNP is largely present in the brain, chondrocytes and endothelial cells and is highly conserved among species (for review see e.g. Potter et al. 2006). The effects of natriuretic peptides are widespread, including regulation of blood pressure, blood volume, ventricular hypertrophy and long bone growth (for review see e.g. Potter et al. 2006).

The natriuretic peptides are ligands for natriuretic peptide receptors (NPR). Three types of natriuretic peptide receptors exist, named NPR-A, NPR-B and NPR-C. Two receptors are transmembrane guanylyl cyclase receptors (NPR-A and NPR-B) that mediate the biological effects of natriuretic peptides and have cyclic GMP as their main second messenger. The third one (NPR-C) has no guanylyl cyclase domain, but may mediate some natriuretic peptide function via the modulation of intracellular cAMP concentrations (Anand-Srivastava et al. 1990, for review see e.g. Potter et al. 2006). NPR-C has an important role in the removal of natriuretic peptides from the circulation and is therefore also known as a clearance receptor.

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2.2 Structure and biosynthesis of mammalian natriuretic peptides

Each natriuretic peptide is coded for by a separate gene but shows similar exon-intron properties suggestive of a common evolutionary ancestor (for review see e.g. Suzuki et al. 2001). Each natriuretic peptide gene produces a precursor protein that is further processed to produce the mature hormones. The biologically active hormone sequence lies in the carboxy-terminal (C-terminal or COOH-terminal) portion of the prohormone. All three members of mature mammalian natriuretic peptides show structural homology due to a disulphide bridge formed by two cysteine residues flanking a sequence of 17 amino acids that shows homology across vertebrates (for review see Yandle 1994) (Fig. 1). The disulfide-looped sequence of 17 amino acids plus various COOH- and NH2-terminal (amino-terminal) extensions are important for receptor binding and necessary for biological activity (de Bold 1985).

Fig. 1. Structure of the natriuretic peptide family in man (modified from McGrath & de Bold 2005).

The biologically active form of human ANP comprises 28 amino acids (Fig. 1). The amino acid sequence of ANP is highly conserved across species, all mammals having an identical sequence except for a single amino acid substitution (for review see e.g. Yandle 1994, Pandey 2005). Human BNP is composed of 32 amino acids (Fig. 1). BNP displays the most variability in its primary structure (for review see e.g. Yandle 1994, Pandey 2005). The precursor forms of CNP are highly conserved between species (for review see e.g. Yandle 1994, Pandey 2005, Potter et al. 2006). It is highly homologous to ANP, but lacks the carboxy-terminal tail (for review see e.g. Pandey 2005). The biological activity of CNP has been attributed to the ring portion of the molecule with a disulfide bond and especially a Leu9-Lys10-Leu11 sequence (Furuya et al. 1992). In man, cleavage of proCNP results in a 53-amino acid peptide, which is further processed to a 22-amino acid form in some tissues (for review see Potter et al. 2006) (Fig. 1). Although CNP-22 and CNP-53 elicit similar functions, their tissue expression differs (for review see Potter et al. 2006).

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2.2.1 Biosynthesis of ANP

The ANP gene is located on chromosome 1 and the structure comprises three exons interrupted by two introns (for review see e.g. Rosenzweig & Seidman 1991, Yandle 1994, Scotland et al. 2005). ANP is mainly produced in cardiac atria. In the normal adult ventricle, ANP expression is low, about 1% of that in the atrium (Gardner et al. 1986a). However, much more ANP is present in the ventricular tissue of foetuses and neonates and in hypertrophied ventricles (Izumo et al. 1988, Lee et al. 1988, Gu et al. 1989). Cardiac myocytes are the predominant cells for ANP production in the heart (Argentin et al. 1994), although ANP may also be synthesized by fibroblasts (Cameron et al. 2000). The atrial natriuretic peptide gene is also expressed in the kidney, in which alternative processing of the precursor generates a 32-amino acid peptide called urodilatin (for review see Yandle 1994, Potter et al. 2006). Furthermore, many other tissues, such as the central nervous system, lung, adrenal, kidney, gastrointestinal tract and vascular system contain ANP mRNA or proANP–like immunoreactivity (Gardner et al. 1986a, Vuolteenaho et al. 1988, Ruskoaho 1992).

Human ANP is synthesized as a 151-amino acid preproANP that contains sequences of active peptides in its C-terminal region, and the major form of circulating ANP is a 28-residue molecule (for review see Potter et al. 2006) (Fig. 2). ProANP is formed by cleavage of a signal peptide from preproANP. The 126-amino acid propeptide is the predominant storage form of ANP in the atrial tissue and it is stored in high amounts in specific granules (Vuolteenaho et al. 1985, for review see Rosenzweig & Seidman 1991, McDowell et al. 1995). At the same time, very little ANP with only occasional secretory granules can be found in the ventricles in basal conditions (Thibault et al. 1992). During or soon after exocytosis proANP is split further between Arg and Ser residues by a membrane-bound enzyme into an inactive N-terminal peptide (residues 1-98) and the biologically active hormone, the C-terminal peptide (residues 99-126) (Vuolteenaho et al. 1985). The mature ANP is produced by a cardiac serine protease, corin, which is considered a proANP-converting enzyme (Yan et al. 2000, Wu et al. 2002). Mice lacking corin have undetectable levels of the mature form of ANP in heart tissue and are hypertensive (Chan et al. 2005). ANP and the N-terminal fragment of proANP (NT-proANP) circulate in the plasma, and their concentrations are elevated by an increase of intravascular volume, in heart failure, and in cardiac hypertrophy (Lang et al. 1985, Ruskoaho 1992, Stein & Levin 1998). Therefore, measurements of NT-proANP can be used to characterize endogenous secretion of biologically active ANP (for review see e.g. Sagnella 1998, Ruskoaho 2003, Clerico & Emdin 2004). Normally, plasma levels of ANP in humans are approximately 3 – 16 pmol/l (Clerico et al. 1998).

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Fig. 2. The biosynthetic pathway of human ANP, BNP and CNP (modified from Nakao et al. 1992a). Solid black sections represent the region coding for the prohormone or the prohormone itself. Diagonal striped sections represent the region coding for the signal peptide or the signal peptide itself.

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2.2.2 Biosynthesis of BNP

BNP is predominantly synthesized in and secreted from the ventricle. When tissue weight is taken into account, the total content of BNP mRNA in the ventricle is 3-fold larger than that in the atrium (Ogawa et al. 1991). BNP expression also occurs in the central nervous system, adrenal gland and gut (for review see Rosenzweig & Seidman 1991, Espiner 1994, Yandle 1994). By contrast, the peptide (BNP) concentration in the ventricle is less than 1% of that in the atrium due to only a small capacity of storage in the ventricles (Ogawa et al. 1991). However, the BNP level is only 2-5% of the ANP level in the atria and 20-25% of the ANP level in the ventricle of a normal heart (Aburaya et al. 1989, Mukoyama et al. 1991, Ogawa et al. 1991). The plasma BNP level in normal subjects is approximately 0.35 – 6.4 pmol/l (Clerico et al. 1998, 2005).

The BNP gene is located close to the ANP gene on chromosome 1 in humans, and it comprises three exons separated by two introns (for review see e.g. Yandle 1994). The biosynthesis of BNP is similar to that of ANP. Human BNP is synthesized as a 134-amino acid prepropeptide that is processed by endoprotease cleaving to a 108-amino acid proBNP (for review see e.g. Yandle 1994, Potter et al. 2006) (Fig. 2). The prohormone is cleaved into the biologically active 32-amino acid C-terminal fragment and a 76-amino acid N-terminal fragment (Sudoh et al. 1988).

The posttranslational processing of BNP precursors seems to be different from that of ANP, and the processing sites are not conserved between species, resulting in various lengths of BNP (for review see Yandle 1994). The predominant circulating forms of BNP are 26-, 45- and 32-amino acid peptides in pigs, rats and humans, respectively (for review see Nakao et al. 1992a). Species specificity in the circulating forms occurs, since in man a considerable amount of proBNP can be found in the circulation (Tateyama et al. 1992). The major storage form of BNP differs among species as well. Cardiac myocytes contain either cleaved BNP and NT-proBNP, or a varying ratio of proBNP, BNP and NT-proBNP or proBNP alone (Minamino et al. 1988, Kambayashi et al. 1990, Tateyama et al. 1990, Thibault et al. 1992, Hira et al. 1993, Pemberton et al. 1997). The most abundant form of BNP in atrial myocardium in humans is the mature 32-amino acid peptide (Hino et al. 1990, Kambayashi et al. 1990). Therefore, the timing of the posttranslational processing of proBNP varies among species (Ogawa et al. 1991, Pemberton et al. 1997), occurring intracellularly or at the time of secretion, since processing of proBNP has not been observed in blood or plasma (Hunt et al. 1997). The cleavage of the proBNP occurs immediately after the Arg-X-X-Arg sequence, a cleavage site similar to that of the endoprotease furin (Sawada et al. 1997). A cardiac serine protease, corin, can also activate proBNP (Yan et al. 2000).

2.2.3 Biosynthesis of CNP

In man, the CNP gene is located on chromosome 2, and contains two exons and one intron (Tawaragi et al. 1991). The biosynthesis of CNP includes formation of preproCNP of 126 amino acids, from which proCNP is formed by cleavage of a signal peptide in humans (Barr et al. 1996) (Fig. 2). ProCNP of 103 amino acids is subsequently cleaved

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by a proprotein convertase furin to yield CNP-53 (Wu et al. 2003). This peptide can be secreted and/or processed further by an unknown mechanism to form the active 22-amino acid CNP. CNP-53 is also found in brain tissue (Totsune et al. 1994c), endothelial cells (Stingo et al. 1992b) and heart (Minamino et al. 1991). Although CNP-53 may also have biological functions, the 22-amino acid fragment may, however, be regarded as the more biologically active peptide. CNP-53 and CNP-22 have both been detected in the plasma (Stingo et al. 1992b, Totsune et al. 1994a). Normal plasma CNP concentration is in the low picomole per litre range (Stingo et al. 1992b).

CNP is widely distributed in mammalian tissues, although the concentrations are very low. CNP was first identified from porcine brain (Sudoh et al. 1990), after which it was described in the central nervous system in different mammalian species (for review see Barr et al. 1996). CNP is also a major natriuretic peptide in cerebrospinal fluid (Kaneko et al. 1993). Additionally, CNP is present in the cardiovascular system, most notably in vascular endothelial cells (Stingo et al. 1992b, Suga et al. 1992b, Yamada & Yokota 1996). However, very little if any CNP is present in the mammalian myocardium (Nakao et al. 1992a, Vollmar et al. 1993, Barr et al. 1996). A recent study, however, identified the human heart as a CNP-producing organ, at least in the pathophysiological condition of chronic heart failure (Kalra et al. 2003). Furthermore, CNP is expressed in cultured rat cardiac fibroblasts (Horio et al. 2003). CNP mRNA or CNP immunoreactivity have been detected in the kidney (Stingo et al. 1992b, Nir et al. 1994, Totsune et al. 1994b) and in most peripheral tissues (Minamino et al. 1993), such as cartilage (Hagiwara et al. 1994, Chusho et al. 2001). Moreover, CNP mRNA is present in reproductive tissues, such as the uterus and ovaries (Stepan et al. 2000) and in placenta in mice (Cameron et al. 1996).

2.3 Regulation of mammalian natriuretic peptides

The secretion of the cardiac natriuretic peptides, ANP and BNP, is enhanced by various stimuli. The regulation of CNP is not well understood. The most important factor governing ANP and BNP secretion is mechanical stretching of atria, which normally occurs when extracellular fluid volume is elevated. In addition, other stimuli such as endothelin-1 (ET-1) and other paracrine factors play an important role in modulating natriuretic peptide secretion from the heart. Endocrine responses of the heart to changes in pressure or volume overload result in acute as well as chronic responses. This review is mainly focused on acute regulation of natriuretic peptides.

2.3.1 Regulation of ANP

2.3.1.1 Hemodynamic factors

Myocyte stretch is widely believed to be the major stimulus in the regulation of ANP release. In vitro myocyte stretch increases ANP release from atrium (Lang et al. 1985, Ruskoaho et al. 1986, Laine et al. 1994a, b) and ventricle (Kinnunen et al. 1993,

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Mäntymaa et al. 1993). In vivo plasma volume expansion increases the circulating level of ANP (Lang et al. 1985, Anderson et al. 1986, Eskay et al. 1986, Salazar et al. 1986, Tulassay et al. 1988). The in vivo mechanism of ANP release may involve the activation of several local paracrine factors following volume expansion. It has been shown that ANP release during volume expansion in rats is almost completely blocked by the combined inhibition of endothelin (ET) and angiotensin II receptors (Leskinen et al. 1997a), while volume expansion in conscious dogs induced ANP release that was potentiated by infusion of ET-1 (Donckier et al. 1992). Moreover, a selective nitric oxide (NO) synthase inhibitor potentiated ANP release by volume expansion in conscious rats (Leskinen et al. 1995). Therefore, induction of these local factors by volume expansion may regulate ANP release.

The mechanism underlying the mechanotransduction process responsible for stretch-induced ANP release is unclear. One possible initiator in stretch-induced ANP release could be a mechanosensitive ion channel (Fig. 3). It has been shown that inhibition of the gadolinium-sensitive stretch-activated (SA) ion channels by gadolinium inhibited stretch-induced ANP release from superfused atria (Laine et al. 1994a). Ca2+ also seems to be involved in the stretch-induced ANP secretion. It has been shown that during stretch-induced ANP release in isolated perfused rat atria, ANP release was enhanced by high extracellular Ca2+ concentration (Laine et al. 1996). The entry of Ca2+ into the cell can originate from several pathways such as voltage-dependent Ca2+ channels or SA channels (for review see Thibault et al. 1999). The release of Ca2+ from an intracellular storage pool may also be important in the stretch-secretion coupling process, since blocking of ryanodine-sensitive intracellular Ca2+ stores has been shown to inhibit stretch-induced ANP secretion (Laine et al. 1994b). In addition, recent evidence suggests that G proteins may act as important transducers of stretch-activated ANP secretion (Fig. 3). It has been shown that Gi/o inhibition by pertussis toxin abolished stretch-secretion coupling without affecting baseline secretion and that Gi/o agonist increased ANP secretion (Bensimon et al. 2004).

Increase in the atrial contraction rate is a potent stimulus for ANP release. In vivo, increased pacing or tachycardia increases plasma ANP concentrations in sheep and dog (Rademaker et al. 1996, Grantham et al. 1997, Pemberton et al. 2000) as well as in humans (Espiner et al. 1985, Tikkanen et al. 1985b, Roy et al. 1987). The secretory response is partially explained by an increased mean atrial pressure. However, it has been shown that during pacing tachycardia in open-chest pig models with unchanged atrial dimensions, ANP release was stimulated by an increased atrial pressure during atrial systole (Christensen & Leistad 1997). It is also shown that the enhanced ANP release during atrial pacing is coincident with an increased atrial stroke volume and an increase in translocation of extracellular fluid (Cho et al. 1995). This indicates that increases in wall tension and wall stress could be the most important factors regulating ANP release during tachycardia (for review see Thibault et al. 1999). In neonatal rat atrial myocytes, pace-induced ANP release was found to be associated with a cytoplasmic Ca2+ elevation and inhibited by nifedipine and ryanodine and by a Ca2+/calmodulin-dependent protein kinase II inhibitor (McDonough et al. 1994). This indicates that regulation of ANP secretion by contraction rate depends on the calcium transients and calmodulin.

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Fig. 3. Proposed scheme for stimulation of ANP secretion from cardiomyocyte by mechanical stretch and ET-1 and inhibition by nitric oxide (modified from Dietz 2005). SA channel = stretch-activated ion channel, ET-1 = endothelin-1, NO = nitric oxide, ETA = endothelin type A receptor, G = G protein, PLC = phospholipase C, PIP2 = phosphatidylinositol 4,5-diphosphate, DAG = diacylglycerol, IP3 = inositol triphosphate, PKC = protein kinase C, AA = arachidonic acid, PG = prostaglandins.

Since ANP is stored at high concentrations in secretory granules and thereby becomes available for bolus release in response to appropriate secretagogues, the storage and secretion of atrial ANP is suggested to utilize the regulated pathway (Bloch et al. 1986). However, ventricular cells secrete ANP rapidly after synthesis and lack secretory granules. Therefore, ventricular ANP is proposed to utilize a constitutive pathway (Bloch et al. 1986, De Young et al. 1994).

Myocyte stretch produces only transient changes in natriuretic peptide secretion, and the activation of ANP gene expression in response to elevated mechanical load takes hours or days to develop (for review see de Bold et al. 1996). In perfused rat heart, mechanical stretching for up to two hours does not change the ANP mRNA levels

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(Mäntymaa et al. 1993). A more rapid induction of ANP gene expression has been reported in rat cardiac papillary muscles after five hours of stretching (Jarygin et al. 1994). In neonatal rat cardiac myocytes stretch-induced changes in ANP mRNA level occur after 24 hours (Gardner et al. 1992, Sadoshima et al. 1992). Thus, acute regulation of atrial ANP mostly occurs at the level of hormone release, not at the level of ANP gene transcription. The induction of the ANP gene involves transcription factors which need to be translated before up-regulation of ANP transcription is possible (for review see Ruskoaho 1992, de Bold et al. 1996).

Chronically enhanced release and synthesis of ANP is mediated by haemodynamically-induced muscle stretch. In chronic stimulation the cardiac foetal programme is activated (for review see Vuolteenaho et al. 2005), and stimulation of the ANP gene takes place in both atria and ventricles (for review see de Bold et al. 1996). Cardiac ANP gene expression increases in response to cardiac overload and myocardial hypertrophy in several animal models (Lee et al. 1988, Ruskoaho et al. 1989a, Rockman et al. 1994, Marttila et al. 1996, Ogawa et al. 1996, Gidh-Jain et al. 1998). Increased plasma and cardiac levels have been measured in patients with congestive heart failure (Tikkanen et al. 1985a, Burnett et al. 1986, Wei et al. 1993b, Yasue et al. 1994, de Boer et al. 2001). Interestingly, in hypertrophied heart the ANP gene expression and the level of immunoreactive ANP are increased particularly in the ventricles (Lee et al. 1988, Ruskoaho et al. 1989a, Kinnunen et al. 1992, Ogawa et al. 1996). However, due to the limited capacity of the normal ventricle to store the hormone, the elevated level of ventricular ANP secretion is not sustained (for review see Ruskoaho 1992). Myocardial hypertrophy seems to be a requirement for enhanced ANP gene expression and, thus, elevated secretion of ANP from the ventricles. Ventricles therefore become a major source of circulating ANP in hypertrophied heart.

In conclusion, it seems that atrial stretch is the main factor involved in the release of ANP induced by haemodynamic changes. Locally induced vasoactive factors could be the paracrine mediators of ANP secretion, and Ca2+ seems to be an important signal in ANP secretion. The acute response to stretch is based on stretch-secretion coupling, resulting in enhanced secretion of ANP stored in the atria. In chronic stimulation the cardiac foetal programme is activated and stimulation of the ANP gene takes place in both the atria and ventricles.

2.3.1.2 Vasoactive agents

Several vasoactive agents stimulate the rate of ANP secretion. Among the vasoactive factors that are involved in the modulation of the endocrine heart, endothelin-1 (ET-1) appears to be one of the most potent stimuli for ANP secretion. The role of ET-1 in natriuretic peptide secretion is discussed later (see section 2.9.3.3.)

Angiotensin II is able to induce ANP release. In vivo, the ability of angiotensin II to increase plasma ANP concentration may partially be due to the effect of this hormone on atrial pressure (Dietz 1988, Ruskoaho et al. 1989b). However, it has been shown that angiotensin II also has the ability to directly stimulate ANP release in cultured neonatal rat myocytes (Church et al. 1994a), isolated rat atrial appendages (Veress et al. 1988) and

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isolated rabbit heart independently of haemodynamic changes (Focaccio et al. 1993). The response is mediated via angiotensin type 1 (AT1) receptor, and the activation of protein kinase C (PKC) and rise of intracellular Ca2+ are important (Soualmia et al. 1996, 1997). A cyclooxygenase product may be involved in this pathway (Church et al. 1994a). The effect of angiotensin II on ANP secretion may be modulated by nitric oxide (NO), likely via the cGMP pathway (Soualmia et al. 2001). However, several investigators have failed to find any effect of angiotensin II on ANP secretion in isolated tissues or primary cell cultures (Dietz 1988, Matsubara et al. 1988b, Glembotski et al. 1991). Therefore, it has not been established conclusively whether angiotensin II possesses a direct inhibitory or stimulatory effect on ANP secretion.

Arginine vasopressin appears to be one of the regulators in atrial stretch-induced ANP release. Hypophysectomy reduces plasma ANP levels (Zamir et al. 1987, Arjamaa et al. 1988, Dietz & Nazian 1988), and pithing, which interrupts the humoral influences of the brain and pituitary gland on the heart, reduces the basal and volume load-induced ANP release in rats (Eskay et al. 1986, Ruskoaho et al. 1989b). Vasopressin also increases ANP release from isolated rat atrial appendages (Veress et al. 1988), and in vivo it induces the basal and stretch-activated ANP release (Katsube et al. 1985, Manning et al. 1985). In intact animals the release of ANP may be due to increased arterial pressure (Katsube et al. 1985, Manning et al. 1985, Dietz 1988). On the other hand, arginine vasopressin can inhibit ANP release through the production of NO (Melo & Sonnenberg 1996). Thus, the physiological role of vasopressin in natriuretic peptide secretion is unclear.

In addition to the above-mentioned hormones, several other vasoactive factors may be involved in the regulation of ANP. Inhibitory effects of several vasoactive agents, such as ET-1, arginine vasopressin and acetylcholine, on ANP release may result from a production of NO (Melo & Sonnenberg 1996, Skvorak & Dietz 1997). Furthermore, stretch-induced ANP secretion is modulated by NO, since inhibition of NO synthase enhances volume load-induced ANP release in conscious rats (Leskinen et al. 1995). The basal ANP release is also increased by NO synthase inhibition (Leskinen et al. 1995). Adrenomedullin has been shown to increase cAMP accumulation and subsequent inhibition of ANP secretion and gene expression (Sato et al. 1997). Prostaglandins seem to modulate ANP release as well. Prostaglandins have been shown to stimulate ANP release in rat cardiocytes (Gardner & Schultz 1990).

2.3.1.3 Adrenergic stimulation

The effect of adrenergic stimulation on natriuretic peptide secretion is complex and still in part unknown. In vivo noradrenaline produces an increase in plasma ANP concentration, which is related to the increase in atrial pressure (Katsube et al. 1985, Manning et al. 1985, Haass et al. 1987). In addition, noradrenaline directly stimulates ANP secretion in perfused heart (Schiebinger et al. 1987, Ruskoaho & Leppäluoto 1988, Wong et al. 1988) and atrial cardiocytes (Ambler & Leite 1994), although other studies show that it had no effect on ANP release (Arjamaa & Vuolteenaho 1985, Gibbs 1987). The full secretory response seems to be dependent upon PKC and an influx of

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extracellular Ca2+ (Ambler & Leite 1994). ANP secretion is also rapidly increased with α1-specific agonists (Schiebinger et al. 1987, Matsubara et al. 1988a, Ruskoaho & Leppäluoto 1988, Bruneau et al. 1996). Moreover, α1-specific agonist phenylephrine caused an increase in ANP mRNA levels after 6 hours (Bruneau et al. 1996).

In isolated rat heart, adrenaline infusion produces a rise in heart rate and contractile force, as well as an increase in ANP release (Toth et al. 1986). Adrenaline administration also induces ANP release from perfused atrium (Wong et al. 1988) and perifused atrial cardiocytes (Gibbs 1987). The β-adrenergic agonist, isoproterenol, has been reported to increase ANP secretion in atrial cardiocytes (Gibbs 1987) and atria (Schiebinger et al. 1987, Ruskoaho & Leppäluoto 1988, Agnoletti et al. 1992, Azizi et al. 1993, Bruneau et al. 1996). In other studies, it had either no effect (Matsubara et al. 1988a) or an inhibitory effect (Ambler & Leite 1994) on ANP secretion in atrial myocytes. In isolated mouse cardiomyocytes, isoproterenol reduced the expression of BNP mRNA but not of ANP, an effect that was prevented by β1-antagonist (Ander et al. 2004). The secretory response could be mediated by cAMP and, at least in part, by prostaglandins, but it did not require extracellular calcium (Azizi et al. 1993). cAMP is formed intracellularly upon adenylyl cyclase by agonists to G-protein-coupled receptors. cAMP can then activate cAMP-dependent protein kinases (PKA) that phosphorylate several proteins. Nevertheless, chronic treatment with β-blockers is usually associated with a significant fall in BNP and its related peptides in heart failure (Clerico & Emdin 2004, Frantz et al. 2005). To sum up, it is unclear which receptor is mainly responsible for adrenergic effects on ANP secretion, although most evidence is in favour of a predominant effect of the α1-adrenergic receptor subtype.

2.3.1.4 Hormones and other factors

Several circulating hormones are also involved in the stimulation of ANP secretion. An in vivo increase in ANP secretion has been observed in response to glucocorticoids (Gardner et al. 1986b), mineralocorticoids (Ballermann et al. 1986) and thyroid hormones (Gardner et al. 1987). Glucocorticoids (Matsubara et al. 1987a, b, Gardner et al. 1988), testosterone (Matsubara et al. 1987a, b) and thyroid hormones (Gardner et al. 1987, Matsubara et al. 1987a, b) have been shown to stimulate ANP release in vitro from cell cultures. In isolated perfused rat heart, corticotropin releasing factor (CRF) induced an increase in ANP release as a consequence of the haemodynamic effects of CRF (Grunt et al. 1992). However, CRF may also have a direct effect on ANP release, since it produced a secretory response, probably mediated by the PKC pathway, in cultured neonatal rat cardiac myocytes (Tojo et al. 1996). CRF was shown to increase both ANP and BNP release and protein synthesis was also observed (Tojo et al. 1996). Thus, CRF may affect natriuretic peptide release through protein synthesis rather than by regulated secretion.

Opioids have been shown to increase ANP secretion in vivo and in isolated atria (Crum & Brown 1988, Kim et al. 2002). Calcitonin gene-related peptide (CGRP) is present in nerve endings within atrial tissue. It was first shown to increase ANP secretion from isolated rat atria (Schiebinger & Santora 1989), but more recently CGRP was found to suppress ANP secretion in vitro (Piao et al. 2004). Platelet-activating factor (PAF) is an

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antihypertensive vasorelaxant factor that has been shown to produce a potent secretion of ANP, possibly mediated by prostaglandins (Rayner et al. 1991, 1993, Church et al. 1994b). In addition, relaxin, a reproductive peptide, produced a marked increase in natriuretic peptide release in isolated perfused heart (Toth et al. 1996).

2.3.1.5 Hypoxia

Exposure to hypoxia stimulates ANP secretion effectively. The main source of enhanced circulating ANP in hypoxia-adapted animals is the heart (Stockmann et al. 1988). It has been reported that chronic reduction in oxygen tension plays a role in up-regulating cardiac ANP synthesis and raising plasma ANP levels independent of changes in pulmonary artery pressure and cardiac tissue remodelling (Baertschi et al. 1986, 1988, Stockmann et al. 1988, Baertschi & Teague 1989). Acute hypoxia causes a marked increase in ANP release in rabbits and lambs in vivo and stimulates the release of ANP from isolated rabbit and rat hearts in vitro in the absence of atrial distension and changes in heart rate, suggesting that hypoxia may be able to stimulate cardiac ANP release directly, independent of its effect on right heart afterload (Baertschi et al. 1988, Baertschi & Teague 1989, Lew & Baertschi 1989b). In addition, in cultured atrial myocytes, in the absence of haemodynamic, neural or hormonal influences, hypoxic exposure increases ANP expression and stimulates ANP release (Chen et al. 1997, Klinger et al. 2001). Studies have shown that hypoxia activates ANP gene promoter activity through direct action of hypoxia-inducible factor-1 (HIF-1) in rat ventricular myoblast cell line (Chun et al. 2003). Therefore, stimulation of the ANP promoter by HIF-1 may be responsible for the induction of the ANP gene in ischaemic ventricular myocardium. HIF-1 is a hypoxia-responsive transcriptional factor that regulates the expression of multiple genes in response to changes in cellular oxygen tension (Semenza 2001). Stimulation of ANP synthesis and release during hypoxia plays an important role in the normal adaptation to hypoxia and in the pathogenesis of cardiopulmonary diseases and right ventricular hypertrophy and right heart failure (for review see Chen 2005).

2.3.2 Regulation of BNP

BNP is produced by both the atria and the ventricles in the normal human heart (for review see Ruskoaho 2003, Vuolteenaho et al. 2005). The regulation of storage of BNP differs from that of ANP. Although BNP is stored with ANP in granules in the atria (Nakamura et al. 1991), it is not stored in granules in the ventricles (for review see e.g. Potter et al. 2006). BNP is released mainly constitutively after secretion (Wei et al. 1993b), although a portion of BNP, stored in the atrial granules, is secreted utilizing the regulated pathway (for review see de Bold et al. 1996). Healthy individuals have plasma concentrations of approximately 1 pmol/l (Mukoyama et al. 1991). However, plasma BNP concentrations of patients with congestive heart failure are elevated between 200- and 300-fold (for review see e.g. Potter et al. 2006). Although both ANP and BNP levels may be increased in patients with heart failure, hypertension or chronic renal failure (see

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e.g. Burnett et al. 1986, Yandle et al. 1993, Ala-Kopsala et al. 2004, Richards et al. 2004), BNP plasma levels correlate more closely than ANP levels with left ventricular function, a common indicator of heart disease (for review see e.g. de Lemos et al. 2003, Potter et al. 2006). Therefore, BNP is considered a better diagnostic marker of heart failure than ANP.

The major stimulus for BNP release from the atria and ventricles is myocyte stretch (Bruneau et al. 1997, Mizuno et al. 2000, Wiese et al. 2000). The BNP gene is induced by pressure and volume overload similarly to the ANP gene (Ogawa et al. 1991, Brown et al. 1993). In neonatal rat ventricular cells, it has been shown that cardiac myocytes are able to respond to mechanical stretch by increasing BNP secretion and gene expression without neurohumoral control (Liang et al. 1997, Sawada et al. 1997). Results suggest that the regulation of the BNP gene differs from that of ANP. Atrial stretch induces rapid stimulation of secretion as well as synthesis of BNP. In contrast to ANP, BNP gene expression can increase very rapidly in response to an appropriate stimulus. Indeed, a 1.9-fold increase was found in right auricular BNP mRNA levels after 1 hours’ stretching (Mäntymaa et al. 1993). The induction of BNP gene expression in the very early stages of cardiac overload mimics the induction of the immediate early genes. The early genetic response to mechanical load in cardiomyocytes includes transcription of immediate early response genes such as c-fos, c-myc, c-jun, and Egr-1. Therefore, whereas regulation of ANP seems to occur at the level of release from storage granules, BNP regulation takes place during gene expression. In addition, the brain natriuretic peptide can be regarded as an early responsive “emergency” gene to stress in myocardium (Nakagawa et al. 1995), because it is expressed earlier and in greater amounts than ANP in response to different stimuli.

The BNP mRNA is rapidly transcribed in response to stimuli, but it is also rapidly degraded. This may be due to the structure of BNP mRNA. BNP mRNA differs from that of ANP by virtue of the presence of four AUUUA repeat sequences in the untranslated 3’ region (for review see Nakao et al. 1992a, Yandle 1994). This feature is conserved among species. The AUUUA motif is generally found in the mRNAs of transiently expressed genes for cytokines, growth factors, or cellular proto-oncoproteins, and it is involved in the translation-dependent mRNA-degrading mechanism (Shaw & Kamen 1986, Wilson & Treisman 1988, Brawerman 1989). These AU-rich sequences are thought to be involved in “the selective degradation of transiently expressed messengers” (Shaw & Kamen 1986) by directing the removal of the poly(A) tail and thus destabilizing mRNA (Wilson & Treisman 1988).

BNP is produced in the atrium and ventricles in different cardiac disorders. BNP synthesis and secretion are enhanced in the ventricle of the hypertrophied heart (for review see Yandle 1994, Ruskoaho 2003, Vuolteenaho et al. 2005). Ventricular levels of BNP mRNA are substantially increased in response to chronic cardiac overload in the human heart (Takahashi et al. 1992) and in experimental models of cardiac overload including SHR (Ogawa et al. 1991, Dagnino et al. 1992, Kinnunen et al. 1993) and rats with myocardial infarction (Hama et al. 1995). In patients with congestive heart failure, BNP release is a biochemical marker of left ventricular function, and the secretion of BNP is augmented much more than that of ANP (Mukoyama et al. 1990, Maeda et al. 1998, Tsutamoto et al. 2001). The predominant source of circulating BNP may be

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different (the atria or ventricles) depending on the severity and cause of the cardiac disorder (for review see Ruskoaho 2003).

Since cardiac BNP is not stored to the same extent as ANP (Thibault et al. 1992), increased release may require a longer stimulus to increase its rate of synthesis and subsequent secretion. Indeed, an increase in BNP secretion is preceded by an increase in mRNA in response to atrial stretch, in contrast to ANP, which is immediately released from stores (Mäntymaa et al. 1993). In addition, in a canine model of early left ventricular dysfunction, BNP mRNA and tissue BNP remain low in the ventricle, whereas in severe congestive heart failure, BNP synthesis is notably elevated in the left ventricle and contributes significantly to the further increase in plasma BNP (Luchner et al. 1998). Moreover, acute intravenous saline loading (Lang et al. 1993) and changes in posture (Richards et al. 1993a) that modify atrial pressure and acutely increase plasma ANP values (Ruskoaho 1992) do not increase plasma BNP levels. Instead, several days of dietary salt loading results in an increase in plasma BNP (Lang et al. 1991, Buckley et al. 1994). Therefore, it seems that ANP is an acutely responsive hormone, whereas BNP levels better reflect sustained cardiac pressure and volume overload (for review see Ruskoaho 2003).

In addition to primary regulation via myocyte stretch, BNP secretion can be augmented by ET-1. ET-1 increases the secretion of BNP from rat atrial myocytes (Horio et al. 1992, Suzuki et al. 1992a) and from rat atrium preparation (Bruneau & de Bold 1994, Bruneau et al. 1997). In addition, BNP gene expression is modulated by ET-1. The level of BNP mRNA increases in the rat atria within 2 h in response to ET-1 (Bruneau & de Bold 1994, Bruneau et al. 1997). Conversely, perfusion of rat atria with the mixed ETA/ETB receptor antagonist, bosentan, decreases the mechanical stretch-induced increase in BNP mRNA level (Magga et al. 1997). However, injection of bosentan had no effect on the pressure-overload-induced increase in ventricular BNP mRNA levels in vivo (Magga et al. 1997), suggesting that ET-1 is not required in pressure load-induced increase in BNP mRNA levels in rat left ventricle. In cultured neonatal ventricular cells, blockade of ET receptors suppressed stretch-induced BNP gene transcription by approximately 50% (Liang & Gardner 1998). The ET-1-dependent component of mechanical stretch-induced increase in BNP mRNA appeared to need the interaction of cardiac nonmyocytes and myocytes, because stretch did not stimulate production of BNP in pure myocyte culture (Harada et al. 1998).

Other vasoactive agents also modulate BNP secretion. Arginine vasopressin infusion has been reported to increase plasma BNP levels as well as ventricular BNP synthesis (Magga et al. 1994). Administration of angiotensin II has been shown to increase plasma BNP concentration dose-dependently in rats (Horio et al. 1992). However, injection of the angiotensin type 1 (AT1) receptor antagonist losartan had no effect on the pressure-overload-induced increase in ventricular BNP mRNA levels in vivo (Magga et al. 1997). In some studies in vitro, arginine vasopressin and angiotensin II have not been found to stimulate BNP expression (Horio et al. 1992, Magga et al. 1997). However, in human atrial and ventricular muscle preparations, BNP gene expression was increased by angiotensin II, and the effect could be blocked by losartan (Wiese et al. 2000). In addition, in cultured neonatal ventricular cells, blockade of AT1 receptors suppressed stretch-induced BNP gene transcription by approximately 50% (Liang & Gardner 1998). Recently, it has been shown that the dual angiotensin II and endothelin-1 blockade by

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losartan and bosentan, respectively, diminished BNP secretion in developing heart failure in sheep (Rademaker et al. 2004).

In addition, adrenergic stimulation may regulate BNP. The α1-adrenergic agonist phenylephrine has been shown to stimulate BNP secretion after 15 min in isolated rat atria (Bruneau et al. 1996). BNP mRNA levels were also significantly increased after 6 hours (Bruneau et al. 1996).

2.3.3 Regulation of CNP

In vitro studies using endothelial cells have shown marked augmentation of CNP mRNA production and CNP secretion by transforming growth factor (TGF) and shear stress (Suga et al. 1992b, Okahara et al. 1995, Chun et al. 1997). Several other cytokines, including tumour necrosis factor, interleukin-1 and basic fibroblast growth factor, also enhance CNP secretion from cultured cells (Suga et al. 1992b, 1993). In addition, lipopolysaccharide, a bacterial endotoxin, stimulates CNP secretion from the endothelial cells (Suga et al. 1993, 1998). It has also been shown in adult rat cardiac fibroblasts that TGF-β1, a basic fibroblast growth factor, and ET-1 significantly stimulated CNP secretion (Horio et al. 2003). Furthermore, it has been shown that CNP production is enhanced by activation of protein kinase C (Yamada & Yokota 1996), of which ET-1 is one of the activators. CNP is also strongly stimulated by ANP and BNP (Nazario et al. 1995).

Basal CNP release into circulation seems to occur in man. CNP-22 immunoreactivity was first detected at low concentration in human plasma by Stingo et al (Stingo et al. 1992b). Difficulty arises from the fact that CNP-22 has a short half-life of approximately 2.6 min (Hunt et al. 1994). Elevated levels of CNP have been found in various pathological states. Plasma CNP is significantly elevated in hypoxia and chronic renal failure (Barr et al. 1996) and in patients with septic shock (Hama et al. 1994). It is not entirely clear whether the raised levels of CNP represent leakage of the peptide from the damaged endothelium or whether they constitute a more specific response. On the other hand, no increase in plasma CNP concentration was found in essential hypertension (Cheung et al. 1994) or chronic heart failure (Totsune et al. 1994a). Whilst basal plasma levels appear to remain static, increased myocardial (Wei et al. 1993b, Kalra et al. 2003) and urinary levels (Mattingly et al. 1994) of CNP are reported in chronic heart failure. In the brain, CNP expression is regulated in response to acute challenges to water and salt balance and by central angiotensin II in olfactory regions (Cameron et al. 2001).

2.4 Natriuretic peptide receptors in mammals

Three subtypes of natriuretic peptide receptors (NPR) have been characterized in mammals, namely natriuretic peptide receptor A (NPR-A), receptor B (NPR-B) and receptor C (NPR-C). NPR-A and NPR-B are particulate guanylate cyclase-linked receptors. NPR-A and NPR-B contain an extracellular ligand-binding domain, a single transmembrane spanning region, and an intracellular protein kinase-like homology

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domain and guanylyl cyclase catalytic domain (for review see e.g. Pandey 2005, Potter et al. 2006). The kinase homology domain normally inhibits guanylate cyclase. When an appropriate natriuretic peptide binds to the external domain, inhibition of guanylate cyclase ceases, enabling the formation of cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP) (Chinkers & Garbers 1989). The elevation in intracellular cGMP is thought to mediate the various biological actions of the natriuretic peptides. In biological assays natriuretic peptides stimulate cGMP accumulation via NPR-A with a rank order potency of ANP ≥ BNP >> CNP, whereas that via NPR-B is CNP > ANP ≥ BNP (Suga et al. 1992a).

NPR-A and NPR-B mediate most of the known physiological actions of natriuretic peptides. Genetically modified animals exemplify the importance of natriuretic peptide receptors in cardiovascular homeostasis. NPR-A knockout mice are afflicted by salt-resistant hypertension, cardiac hypertrophy and increased susceptibility to hypoxia-induced pulmonary hypertension (Lopez et al. 1995, Oliver et al. 1997, Zhao et al. 1999). The NPR-A receptor has been found in kidney, adrenal, terminal ileum, vascular smooth muscle cells, endothelial cells, adipose tissue and the central nervous system (for review see Maack 1992, Nakao et al. 1992b, Potter et al. 2006). NPR-B receptors predominate in the brain, but they are also found in chondrocytes, lung, vascular smooth muscle and uterus (for review see Barr et al. 1996, Levin et al. 1998, Horio et al. 2003, Potter et al. 2006). In mice lacking NPR-B a dramatic impairment of endochondral ossification and attenuation of longitudinal vertebrae or limb-bone growth are seen (Tamura et al. 2004). A loss-of-function mutation of the Npr2 gene (coding NPR-B) is responsible for dwarfism in mice (Tsuji & Kunieda 2005). Additionally, female mice lacking NPR-B are infertile (Tamura et al. 2004).

The third receptor, NPR-C, has also been identified and characterized (Maack et al. 1987, He et al. 2001). Unlike the other receptor subtypes, NPR-C is not linked to guanylate cyclase but possesses a short cytoplasmic tail that contains a pertussis toxin-sensitive Gi-binding domain (Fuller et al. 1988, Anand-Srivastava et al. 1996, Murthy & Makhlouf 1999). NPR-C exists on the cell membrane in a homodimer form. NPR-C is the most abundantly expressed NPR and binds all natriuretic peptides with equal affinity (Maack 1992). NPR-C is widely distributed in several tissues, such as kidney and pulmonary vascular bed, and cell types, including vascular, endothelial, and smooth muscle cells (Barr et al. 1996). In rat, water deprivation and high salt intake significantly increase NPR-C density in the kidney glomerular membranes (Kollenda et al. 1990, Michel et al. 1998). In addition, cardiac hypertrophy leads to the disappearance of NPR-C mRNA in the atrium and ventricle and to a significant decrease in the receptor level in the endocardium (Brown et al. 1993, 1997).

The primary role of NPR-C appears to be to regulate the plasma levels of natriuretic peptides and to function as a clearance receptor. This is achieved by binding, internalizing and targeting the peptides for lysosomal degradation (Maack et al. 1987). G-protein coupling might confer a positive signalling role to NPR-C in some tissues, including inhibition of adenylyl cyclase activity (Pagano & Anand-Srivastava 2001) and stimulation of phosphoinositide hydrolysis (Murthy et al. 2000). The half-life of ANP in the circulation of mice lacking NPR-C is longer than in the wild type, and they have a reduced ability to concentrate urine and exhibit mild diuresis (Matsukawa et al. 1999). In

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addition, NPR-C activation is linked to smooth muscle cell hyperpolarization induced by CNP in resistance arteries (Chauhan et al. 2003, Hobbs et al. 2004).

2.5 Elimination of natriuretic peptides in mammals

Natriuretic peptides are metabolized by two major mechanisms: cleavage by neutral endopeptidase 24.11 (NEP) and internalization by the clearance receptor NPR-C. Binding of a natriuretic peptide to the NPR-C results in endocytosis and subsequent lysosomal hydrolysis of a receptor-ligand complex. NEP has a wide tissue distribution and is in particular located in the vascular endothelium and at high levels in the kidney (Soleilhac et al. 1992).

ANP has a short half-life in plasma, 2-5 minutes in man (Yandle et al. 1986, Tonolo et al. 1988). ANP is cleared from circulation by enzymatic degradation and binding to the clearance receptor (for review see Ruskoaho 2003). ANP is eliminated by NEP mainly in the kidney, lung, and endothelium (Maack 1992). It binds to NPR-C principally in the vascular endothelium (for review see e.g. Ruskoaho 1992, McDowell et al. 1995). NT-proANP has a longer half-life in plasma compared with ANP and its plasma concentration is up to 10-50 times that of ANP (Ruskoaho 2003). NT-proANP appears to be passively eliminated by excretion by the kidney (Katsube et al. 1985, Franz et al. 2000, Hartter et al. 2000).

BNP appears to be cleared by similar mechanisms as ANP. However, BNP is more stable than ANP in plasma (McNairy et al. 2002) and has a longer half-life (approximately 20 min in man) (Richards et al. 1993b). The difference in the clearance rates between BNP and ANP may be due to lesser affinity of BNP to NPR-C and neutral endopeptidases (for review see Yandle 1994, Ruskoaho 2003). NT-proBNP may be even more stable in plasma, since at least in sheep, the half-life of NT-proBNP is 15 times longer than that of BNP (Pemberton et al. 2000). In humans, however, circulating NT-proBNP is very heterogeneous, and the full-length peptide is exposed to truncation at both termini (Ala-Kopsala et al. 2004).

CNP is eliminated by similar mechanisms as other natriuretic peptides. In vitro CNP appears to be more rapidly hydrolyzed by NEP than the other natriuretic peptides. NEP hydrolyzes natriuretic peptides in vitro with the rank order potency CNP > ANP > BNP (Kenny et al. 1993, Kishimoto et al. 2001a). Targeted deletion of NEP 24.11 (Lu et al. 1995) does not lead to skeletal overgrowth like the targeted deletion of NPR-C (Matsukawa et al. 1999), which suggests that CNP concentrations in the growth plate are primary controlled by NPR-C in mice. In addition, CNP is cleared from the circulation by the kidney, since both CNP-22 and CNP-53 have been identified in the urine (Mattingly et al. 1994).

2.6 Physiological effects of natriuretic peptides in mammals

Natriuretic peptides play diverse roles in mammals. ANP and BNP act as circulating endocrine hormones and are secreted by the heart in response to increased myocardial

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stretch. Their main actions are natriuresis, diuresis, vasorelaxation, and inhibition of the renin-angiotensin-aldosterone system. The physiological effects of ANP and BNP are mediated through the guanylate cyclase receptor, NPR-A. C-type natriuretic peptide acts via NPR-B to elicit widespread effects such as regulation of vascular tone and bone growth. Even if the results of a number of studies support a pivotal role for natriuretic peptides in the maintenance of volume and electrolyte balance and cardiovascular homeostasis, the exact physiological role of natriuretic peptides remains to be defined.

2.6.1 Effects of ANP and BNP

2.6.1.1 Hypotensive nature of ANP and BNP

Cardiac natriuretic peptides play an essential role in the regulation of blood pressure and extracellular fluid volume. Their combined effects on intravascular volume, natriuresis, diuresis and vasorelaxation mediate the hypotensive nature of ANP and BNP. For the induction of hypotensive action relatively high doses of natriuretic peptides are needed. Several studies have demonstrated that low-dose infusions of ANP (Soejima et al. 1988, Solomon et al. 1988, Shen et al. 1990, Woods & Anderson 1990) or BNP (McGregor et al. 1990, Florkowski et al. 1997, Jensen et al. 1998) did not affect blood pressure. However, it has been shown in conscious animals that blood pressure was decreased by long-term infusions of ANP (Granger et al. 1986, Harrison-Bernard et al. 1991). In sheep, short-term infusion of BNP has been shown to decrease blood pressure (Pidgeon et al. 1997). Similarly, mice completely lacking ANP (John et al. 1995) or NPR-A (Lopez et al. 1995, Oliver et al. 1997) have higher blood pressures than normal and conversely, transgenic mice overexpressing ANP (Steinhelper et al. 1990, Melo et al. 1999, 2000) or BNP (Ogawa et al. 1994) are markedly hypotensive.

2.6.1.2 Renal effects

ANP action is perceived to enhance the excretion of water and salt with an increase in glomerular filtration rate. ANP also inhibits sodium and water reabsorption. Diuresis and natriuresis are mediated by NPR-A in mice because these effects are lost in genetic mouse models with disruption in NPR-A (Kishimoto et al. 1996, Shi et al. 2003). ANP increases the glomerular filtration rate by elevating the blood pressure in the glomerular capillaries through coordinated afferent arteriolar dilation and efferent arteriolar constriction (Marin-Grez et al. 1986). Furthermore, ANP inhibits sodium and water reabsorption throughout the nephron. In the proximal tubules, ANP inhibits angiotensin-stimulated water and sodium transport (Harris et al. 1987), and in collecting ducts, it suppresses sodium absorption by inhibiting an amiloride-sensitive cation channel (Light et al. 1990). It has been shown that binding of ANP to its receptor requires the presence of chloride (Misono 2000). Thus, it has been suggested that chloride-mediated feedback control of NPR-A occurs in the kidney and plays a role in ANP-mediated sodium excretion (Misono 2000). BNP causes natriuresis and diuresis as well when injected into

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rats (Gunning et al. 1990) or infused at physiological doses in humans (Cheung et al. 1994, Florkowski et al. 1997). However, the natriuretic effect of BNP in humans is less potent than that induced by ANP (Pidgeon et al. 1996).

2.6.1.3 Inhibition of renin-angiotensin-aldosterone system

Studies have shown that ANP through NPR-A receptor suppresses renin and decreases blood pressure (Burnett et al. 1984, Melo et al. 1999). BNP also attenuates renin secretion in dog (Akabane et al. 1991) and in humans (Cheung et al. 1994, Florkowski et al. 1997). In humans, high doses of ANP do not reduce renin levels because of compensatory responses associated with the marked decreases in arterial blood pressure (for review see Potter et al. 2006). Instead, physiological doses of ANP decrease both renin and aldosterone levels (Richards et al. 1988). Interestingly, at birth, NPR-A null mice have elevated kidney renin and angiotensin II levels but in adult mice, the renal and systemic levels of renin and angiotensin II are decreased (Shi et al. 2001). This decrease in renin activity is probably due to progressive elevation in arterial pressure leading to inhibition of renin release. ANP also directly inhibits aldosterone synthesis and release from the adrenal gland (Atarashi et al. 1984, Brenner et al. 1990). BNP also inhibits aldosterone secretion and decreases plasma aldosterone levels in sheep and in humans (McGregor et al. 1990, Yoshimura et al. 1991, Charles et al. 1996, Hunt et al. 1996). Therefore, ANP and BNP regulate blood pressure, in part, through the inhibition of the renin-angiotensin–aldosterone system.

2.6.1.4 Vascular effects and effects on intravascular volume

ANP and BNP have the ability to relax vascular smooth muscle preparations (Currie et al. 1983, Rapoport et al. 1985, Sudoh et al. 1988, Kambayashi et al. 1990). The response requires NPR-A because preparations lacking it are unresponsive to ANP and BNP (Lopez et al. 1997). Furthermore, mice selectively lacking NPR-A in vascular smooth muscle cells do not undergo an acute reduction in blood pressure in response to a bolus injection of ANP (Holtwick et al. 2002).

Early and recent studies have demonstrated (de Bold et al. 1981, Sabrane et al. 2005) that ANP increases haematocrit levels and therefore affects intravascular volume. Furthermore, it has also been shown that infusion of ANP causes a shift of plasma and intravascular albumin to the interstitial space in mice and humans (Wijeyaratne & Moult 1993, Sabrane et al. 2005). Therefore, the ANP-induced increase in haematocrit value is due to increased capillary permeability. Moreover, mice lacking NPR-A in their vascular endothelium are slightly hypertensive and volume-expanded (Sabrane et al. 2005). Thus, ANP increases vascular permeability through endothelial NPR-A. This action is involved in hypovolemic and hypotensive effects of ANP.

Both ANP and BNP inhibit the production and release of ET-1 from vasculature. It has been shown that ANP and BNP inhibit the synthesis and release of ET-1 in cultured rat

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aortic endothelial cells (Emori et al. 1993). Both hormones also stimulate the secretion of CNP from bovine aortic endothelial cells (Nazario et al. 1995).

2.6.1.5 Cardiac effects

ANP and BNP have direct effects on the heart. Mice lacking ANP (John et al. 1995) or NPR-A (Oliver et al. 1997) have enlarged hearts, whereas animals overexpressing ANP (Barbee et al. 1994) have smaller hearts. Studies in primary cultures of cardiac myocytes (Calderone et al. 1998, Silberbach et al. 1999, Horio et al. 2000) and NPR-A deficient mice in vivo (Kishimoto et al. 2001b, Knowles et al. 2001, Holtwick et al. 2003) have shown that ANP inhibits cardiac hypertrophy. The expression of ANP and BNP genes is greatly augmented in cardiac hypertrophy, which supports the notion that autocrine or paracrine effects of ANP or BNP play an important cardioprotective role against pathological hypertrophy (Masciotra et al. 1999, Silberbach et al. 1999).

Natriuretic peptides also inhibit the proliferation of cardiac fibroblasts in culture (Cao & Gardner 1995), and in vivo mice lacking BNP display pressure-sensitive ventricular fibrosis (Tamura et al. 2000). It has been suggested that BNP plays a role as an autocrine or paracrine ventricular protective mechanism in the heart against myocyte damage and fibrosis (Tamura et al. 2000). Several studies indicate that the ANP/BNP/NPR-A system inhibits pressure-induced cardiac remodelling as well (Knowles et al. 2001, Holtwick et al. 2003, Tsuneyoshi et al. 2004). In addition to its antimitogenic effect, ANP has been shown to induce apoptosis in neonatal rat cardiac myocytes (Wu et al. 1997).

2.6.1.6 Other effects

All natriuretic peptides and their receptors have been found in the brain. Consistent with the systemic volume-depleting effects, injection of ANP into the third ventricle of the hypothalamus inhibits water intake induced by dehydration or angiotensin II in conscious rat (Antunes-Rodrigues et al. 1985). Intracerebroventricular infusion of ANP suppresses salt appetite (Itoh et al. 1986) as well as AVP release from the hypothalamus (Samson et al. 1987). ANP-mediated suppression of sympathetic activity in the brain stem has been observed (Steele et al. 1991). Specifically, ANP was shown to sensitize vagal afferent activity and dampen the arterial baroreceptor response (Schultz et al. 1990, Steele et al. 1991, Yang et al. 1992).

Natriuretic peptides and their receptors are found in many immune cells. ANP have an anti-inflammatory effect by reducing the production of proinflammatory cytokines (Kiemer et al. 2000). ANP increases neutrofil migration (Elferink & De Koster 1995), and NPR-A knockout mice exhibit decreased neutrophil infiltration to cardiac tissue after injury (Izumi et al. 2001) as well as decreased eosinophil accumulation in the lungs after allergic challenge with ovalbumin (Mohapatra et al. 2004). Furthermore, ANP has been shown to act as a growth suppressor in several cell types including kidney, heart, neurons, thymus and vasculature (for review see Pandey 2005). BNP transgenic mice

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overexpressing increased levels of BNP exhibited pronounced skeletal overgrowth (Suda et al. 1998).

In addition, several studies have shown that ANP and BNP as well as CNP are locally synthesized and may play important physiological roles in both male and female gonads. For example, ANP and BNP stimulate the synthesis and release of testosterone in Leydig cells (Bex & Corbin 1985, Khurana & Pandey 1993, Pandey et al. 1999) and oestradiol in the female gonad (Gutkowska et al. 1993). Finally, widespread actions of natriuretic peptides include several other effects such as regulation of fat metabolism (for review see Potter et al. 2006).

2.6.2 Effects of CNP

Biological functions of CNP differ notably from those of ANP and BNP. In contrast to these cardiac natriuretic peptides, which are expressed mainly in the heart and interact primarily with NPR-A receptor, CNP is expressed in non-cardiac tissues such as central nervous system and vasculature and elicits its effects mainly via NPR-B and possibly via NPR-C receptors. CNP exhibits various local paracrine or autocrine functions and is not usually considered an endocrine hormone.

2.6.2.1 Vascular and cardiac effects

CNP has the capacity to affect vascular tone. It is produced by endothelial cells and is able to relax vascular preparations with and without endothelium (Suga et al. 1992b, Wei et al. 1993a, 1994a, Drewett et al. 1995, Ikeda et al. 1996). NPR-B is present in aortic vascular smooth muscle and mediates CNP relaxation of precontracted rat aorta (Drewett et al. 1995). CNP is more potent than ANP in eliciting smooth muscle relaxation, but is less potent at inducing diuresis and natriuresis (Clavell et al. 1993). In vivo, infusion of CNP has had diverse results on blood pressure, some studies showing vasorelaxation and others finding no effect (Stingo et al. 1992a, Clavell et al. 1993, Charles et al. 1996). However, the high plasma levels of CNP achieved by infusion can affect plasma ANP and BNP levels, for example by competing for binding to NPR-C and reducing clearance, and may thus actually stimulate NPR-A receptors (Lopez et al. 1997, Schulz 2005). Studies in NPR-A-deficient mouse have shown that CNP infusion in vivo lowers blood pressure and causes vasorelaxation (Lopez et al. 1997). Even then, CNP acting via NPR-B is unable to compensate for the hypertension observed in NPR-A-deficient mouse. This confirms the hypothesis that CNP acts locally rather than systemically. CNP is also able to relax smooth muscle from trachea (Takagi et al. 1992), colon (Kim et al. 2001a) and oviduct (Kim et al. 2001b), suggesting potential roles for CNP in the respiratory, digestive and reproductive systems. The fact that CNP and its receptor NPR-B are expressed in the endothelium and underlying vascular smooth muscle, respectively, has led to the hypothesis of a vascular natriuretic peptide system and designation of CNP as an endothelium-derived relaxing factor (Chen & Burnett 1998). New findings indicate that CNP acts as “an endothelium-derived hyperpolarizing factor”, perhaps acting via NPR-C

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receptors. Indeed, the CNP/NPR-C dilator pathway has been shown to exist in different vascular beds (Chauhan et al. 2003, Hobbs et al. 2004).

CNP has been shown to increase cardiac contractility in animal preparations (Beaulieu et al. 1996, 1997, Hirose et al. 1998, Brusq et al. 1999). In perfused mouse heart CNP had a biphasic effect on contractile function: an immediate increase in the maximal rate of contraction and relaxation followed by a slowly developing decrease in both (Pierkes et al. 2002). In addition, CNP produced by cardiac fibroblasts has a suppressive effect on fibroblast proliferation and extracellular matrix production and it may therefore play a role as an autocrine regulator against excessive cardiac fibrosis (Horio et al. 2003). However, due to its low expression in the heart CNP is not considered a cardiac natri-uretic peptide.

2.6.2.2 Other effects

The most obvious physiological effect of CNP is to stimulate long bone growth. It regulates many types of bone cells, but chondrocytes appear to be its major target (for review see e.g. Potter et al. 2006). In a number of studies (see e.g. Hagiwara et al. 1996, Yasoda et al. 1998, Miyazawa et al. 2002) CNP has been demonstrated to regulate bone development and endochondral ossification. Inactivating mutations in the gene coding for CNP (Chusho et al. 2001) cause dwarfism, whereas supraphysiological levels of CNP resulting from over-expression (Miyazawa et al. 2002, Yasoda et al. 2004) or reduced clearance (Matsukawa et al. 1999) cause skeletal overgrowth.

CNP is the most abundant natriuretic peptide in cerebrospinal fluid (Kaneko et al. 1993), and CNP and NPR-B mRNAs have been detected in various regions of the brain (Minamino et al. 1993, Brown & Zuo 1995, Fulle et al. 1995). In the olfactory epithelium it causes an antiproliferative, prodifferentation effect of neuronal precursors (Simpson et al. 2002). The pituitary and hypothalamus are rich sources of CNP and NPR-B (Komatsu et al. 1991, Herman et al. 1993, 1996). These findings suggest possible roles of CNP as a neuromodulator rather than a cardiac hormone. CNP appears to regulate gonadotropin-releasing hormone (for review see Schulz 2005) and therefore to play a role in neuroendocrine regulation of reproduction. CNP and NPR-B are expressed in male and female reproductive organs as well as in placenta (Chrisman et al. 1993, dos Reis et al. 1995, Cameron et al. 1996, Stepan et al. 2000). There is a lot of other evidence for a biologically relevant role of CNP in reproduction and foetal development (Cameron et al. 1996, Huang et al. 1996, Acuff et al. 1997, Stepan et al. 2000, Kuthe et al. 2003).

CNP has antiproliferative and anti-inflammatory properties as well. It inhibits cell proliferation in rat vascular smooth muscle (Furuya et al. 1991) and mesangial cells (Segawa et al. 1998). CNP also inhibits DNA and collagen synthesis in cardiac fibroblasts (Horio et al. 2003) and it may, therefore, act as an autocrine regulator of fibroblast proliferation.

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2.7 Natriuretic peptide system in nonmammalian vertebrates

Natriuretic peptides and their receptors have been identified in a large number of vertebrate species. Recently, a novel natriuretic peptide (EbuNP) cDNA was cloned from the heart and brain of hagfish (Eptatretus burgeri) (Kawakoshi et al. 2003). Therefore, natriuretic peptides exist even in evolutionarily the oldest vertebrate group, the cyclostomes. While natriuretic peptides are hormones important for volume regulation in mammals, in non-mammalian vertebrates they may have highly diverse functions.

2.7.1 Avian, reptile and amphibian natriuretic peptides and their receptors

In chicken, CNP-22 and a 29-amino acid peptide belonging to the B-type natriuretic peptide subgroup have been isolated from the brain and heart, respectively (Akizuki et al. 1991, Arimura et al. 1991, Mifune et al. 1996). Although binding sites have been localized in duck kidney (Schutz et al. 1992), natriuretic peptide receptors have not been cloned in birds. Natriuretic peptides seem to play a role in fluid and ion homeostasis in birds (for review see Toop & Donald 2004).

In reptiles, peptides structurally belonging to the natriuretic peptide family have been characterized in various snake species (Schweitz et al. 1992, Ho et al. 1997, Murayama et al. 1997, Michel et al. 2000). DNP or Dendroaspis natriuretic peptide is a natriuretic peptide purified from green mamba snake (Dendroaspis angusticeps) venom (Schweitz et al. 1992). DNP is a 38-amino acid peptide with a 17-amino acid disulfide ring structure resembling ANP, BNP and CNP (Schweitz et al. 1992). This peptide possesses an 8-amino acid aminoterminal and a 15-amino acid carboxy-terminal extension. Like other known natriuretic peptides, DNP also acts via a specific natriuretic peptide receptor, NPR-A, mediating a vasorelaxant action (Schweitz et al. 1992, Best et al. 2002). However, its role has been poorly described to date. C-type natriuretic peptides are also present in snakes, in which the venom gland expresses CNP mRNA (Murayama et al. 2000).

The natriuretic peptide system in amphibians consists of ANP, BNP and CNP. They have all been identified in frog (Lazure et al. 1988, Sakata et al. 1988, Yoshihara et al. 1990, Kojima et al. 1994, Fukuzawa et al. 1996). Interestingly, frogs express two distinct natriuretic peptide precursors for CNP, and thus the mature forms are named CNP I and CNP II (Yoshihara et al. 1990, Kojima et al. 1994). In addition to the peptides, NPR-A, NPR-B, and NPR-C receptors have also been cloned from amphibians (Sekiguchi et al. 2001). However, the role of natriuretic peptides in overall volume regulation in amphibians is not known.

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2.7.2 Fish natriuretic peptide system

In elasmobranch fishes, CNP is the only natriuretic peptide. It is the most conserved peptide in the natriuretic peptide family, and despite the major phylogenetical distance between elasmobranchs and mammals, the sequence homology of elasmobranch shark and mammalian CNP is high.

In teleost fish, the natriuretic peptides include ANP, CNP, and ventricular natriuretic peptide (VNP) and salmon cardiac peptide (sCP). It now seems clear that teleost fishes also have BNP (Kawakoshi et al. 2004). Recently, BNP-like cDNA was cloned from the atrium of a primitive teleost fish, the sturgeon (Acipenser transmontanus) (Kawakoshi et al. 2004). In addition, BNP-like cDNA was cloned from the heart of two other species, pufferfish (Takifugu rubripes) and tilapia (Oreochromis mossambicus) (Kawakoshi et al. 2004). It is evident that natriuretic peptides have some role in osmoregulation in fish (for review see Evans & Takei 1992, Loretz & Pollina 2000).

2.7.2.1 ANP in fish

ANP has been characterized in eel (Anguilla japonica) (Takei et al. 1989). Eel ANP is unique in that its carboxy-terminal end is amidated (Takei & Hirose 2002). It has been suggested that osmotic stimulus is a primary regulator for ANP secretion in eel. In this species the level of circulating ANP increases acutely after exposure to hyperosmotic environment (Kaiya & Takei 1997). Unlike in mammals, blood volume expansion is only a weak stimulus for ANP secretion (Kaiya & Takei 1996b). Therefore, the major target of ANP for its biological action is plasma sodium concentration, not blood volume in eel (Takei & Hirose 2002). Recent data show that tilapia and pufferfish as well as sturgeon also have amidated ANP (Kawakoshi et al. 2004). In addition to these teleost species, in which homologous natriuretic peptides have been identified, immunoreactive natriuretic peptides have been detected in many fish species using heterologous assays.

2.7.2.2 CNP in fish

Studies have demonstrated CNP to be present in the cardiovascular system of different fish species. In contrast to mammals, in elasmobranch sharks the main site of CNP synthesis is the heart. A high molecular weight CNP and its cDNA have been found in the myocardium of the sharks Scyliorhinus canicula (common dogfish), Squalus acanthias (spiny dogfish) and Triakis scyllia (Schofield et al. 1991, Suzuki et al. 1991, 1992b, 1994). In sharks, the molecular forms of CNP differ among tissues. In the brain, a substantial proportion is CNP-22, with a small quantity of the prohormone, CNP-115 (Suzuki et al. 1992b). In contrast to mammals, the molecular form of CNP in shark plasma and secreted from the heart appears to be proCNP, which circulates at high concentrations of about 2 nmol/l (Suzuki et al. 1992b, 1994). The low molecular forms 38 or 39 amino acids long are also present in small amounts in the heart (Suzuki et al. 1992b, 1994). Interestingly, the typical proteolytic processing signal, Lys-Lys, for CNP-

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22 remains uncut in proCNP, CNP-38, and CNP-39 in the heart (Suzuki et al. 1992b). CNP is thus processed differently in the brain and heart of elasmobranchs.

In sharks, CNP has a major site of action in the rectal gland. Shark CNP stimulates potently chloride secretion from the rectal gland of spiny dogfish (Solomon et al. 1992, Gunning et al. 1993, 1997). CNP exerts its action via NPR-B receptor to activate chloride channels on the apical surface of epithelial cells of the rectal gland (Aller et al. 1999). As shown in mammals, CNP has been shown to act as a powerful vasorelaxant in elasmobranches as well. Shark CNP is a potent vasodilator of the aortic ring in spiny dogfish (Evans et al. 1993). Shark CNP also causes a relaxation of the branchial artery, and in vivo, a dose-dependent reduction in mean arterial blood pressure in common dogfish (Bjenning et al. 1992).

The published sequences of CNP in teleosts include that of killifish (Fundulus heteroclitus) (Price et al. 1990), Japanese eel (Anguilla japonica) (Takei et al. 1990) and rainbow trout (Oncorhynchus mykiss) (Inoue et al. 2003b). In addition, four distinct CNP cDNAs from separate genes have been cloned from medaka (Oryzias latipes) and pufferfish (Takifugu rupripes) (Inoue et al. 2003a). CNP was recently identified from a primitive teleost, the sturgeon (Acipenser transmontanus) (Kawakoshi et al. 2004).

The natriuretic peptide system of the eel has been studied extensively. The CNP level in eel plasma is fairly high, from 15 pmol/l to 125 pmol/l (Takei et al. 2001). The plasma CNP concentration is higher in animals adapted to fresh water than in those adapted to seawater. However, the adaptation to high environmental salinity does not have an impact on the expression level of CNP in the eel brain (Takei et al. 2001). CNP infused at doses within physiological range increases plasma sodium concentration in freshwater eels without changing the arterial blood pressure (Takei et al. 1990). This increase is not detectable in seawater eels. In addition, CNP infused into the circulation of freshwater eels facilitates the uptake of 22Na added to the environmental freshwater (Takei & Hirose 2002). These results suggest that CNP stimulates the uptake of sodium by the gills, thereby promoting freshwater adaptation in eels. Therefore, ANP and CNP have opposite effects on adaptation to environmental salinity in eels; ANP is obviously a sodium-extruding hormone and CNP a sodium-retaining hormone. Whereas ANP acts on various sites responsible for osmoregulation, such as the brain, intestine, gill and kidney through NPR-A to promote seawater adaptation, CNP acts on the gill through NPR-B to promote freshwater adaptation in eel (Takei & Hirose 2002).

In summary, whereas in mammals CNP is mainly a paracrine factor acting locally in the tissues, in fish CNP is an active and important component of the natriuretic peptides and acts as an endocrine hormone as well as paracrine factor.

2.7.2.3 Ventricular natriuretic peptide

Ventricular natriuretic peptide (VNP) has only been identified in teleosts. Eel VNP cDNA encodes a preproVNP of 150 amino acids, containing a signal sequence of 22 amino acids at its N-terminal end and mature VNP at its C-terminal end (Takei et al. 1994c). A long C-terminal tail sequence with 14 amino acids that extend from the intramolecular ring is typical for mature forms of VNP (Takei et al. 1991). The major circulating form of eel

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VNP is 36 amino acids, but VNP-25 has also been detected in plasma (Takei et al. 1994a). VNP has also been isolated from rainbow trout ventricle (Takei et al. 1994b). Trout VNP consists of 35 amino acids containing a C-terminal tail, similar to that in eel VNP (Takei et al. 1994b). The VNP sequence is highly conserved in two phylogenetically distant teleost species, the eel and rainbow trout (Takei 2000). Recently, VNP was also identified from a primitive teleost fish species, the sturgeon (Kawakoshi et al. 2004).

VNP is synthesized mainly in the heart, at least in eel, so that the VNP concentration in the ventricle is three times higher than in the atrium (Takei et al. 1994a). In freshwater eel the peripheral VNP concentration in plasma is approximately 35 pmol/l (Kaiya & Takei 1996a). In addition to mature forms of VNP, also proVNP circulates in the plasma, contributing to one-third of the total immunoreactivity. The secretion of VNP is regulated by both osmotic and volemic stimulus in vivo in eels (Kaiya & Takei 1996b).

VNP has unique biological functions, since it has high affinity to both NPR-A and NPR-B receptors (Katafuchi et al. 1994). VNP has both vasoactive and renal effects. Trout VNP exerts a biphasic vasopressor and depressor effect in trout. In rat, trout VNP causes almost equipotent vasodepressor and natriuretic effects as human ANP (Takei et al. 1994b). In eel, VNP causes vasodepressor and natriuretic effects (Takei et al. 1994a) and in dog, infusion of eel VNP produces diuresis and natriuresis (Arai et al. 1996). However, the role of VNP in osmoregulation of fishes is currently not well understood.

2.7.2.4 Salmon cardiac peptide

Natriuretic peptides play a significant role in the regulation of fluid and salt balance in many fish species. Salmon (Salmo salar) is especially interesting because of its ability to osmoregulate in different salinities during its natural life cycle. Salmon develops in fresh water and migrates to the sea for maturation before returning to fresh water for spawning. Tervonen et al. (Tervonen et al. 1998) cloned and characterized from salmon a novel cardiac hormone (sCP) structurally related to mammalian natriuretic peptides. In spite of the fact that the peptide and gene of sCP contain features of both ANP and BNP (Majalahti-Palviainen et al. 2000), structural and functional comparisons (Tervonen et al. 1998, Takei 2001, Takei & Hirose 2002) place it in the group of ANP-type peptides. sCP mRNA and immunoreactivity can be found in the heart of several fish species such as salmon, rainbow trout (Oncorhynchus mykiss) and vendace (Coregonus albula) (Tervonen et al. 2000). There is only one amino acid substitution between sCP and a similar peptide of rainbow trout (Takei et al. 1997). The distribution of sCP mRNA and the peptide is strictly restricted to the heart, with high levels in both the atrium and the ventricle (Tervonen et al. 2000). sCP appears to be the most heart-specific hormone among the natriuretic peptide family found thus far. Despite the very small overall sequence homology, caused by the extensive phylogenetic distance, its promoter is as active as that of rat ANP in driving expression in neonatal rat atrial myocytes (Majalahti-Palviainen et al. 2000). The biological effects of sCP include diuresis and natriuresis, as well as relaxation of preconstricted aortic smooth muscle in salmon (Tervonen et al. 2002). The receptor-binding elements appear to be conserved in sCP and mammalian natriuretic peptides, since sCP is able to significantly induce excretion of water and

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electrolytes, and to dilate preconstricted blood vessel preparation in rats as well (Tervonen et al. 2002). The plasma sCP level is decreased after a short exposure to hyperosmotic environment in which salmon is challenged by the threat of dehydration (Tervonen et al. 2002). This indicates that sCP has an important role in fish volume regulation (Tervonen et al. 2002). Recent results also suggest that hypoxia-induced haemoconcentration partially achieved by increased diuresis is caused by sCP in rainbow trout (Tervonen et al. 2006).

2.7.2.5 Fish natriuretic peptide receptors

The bony fish display two guanylate cyclase receptors, NPR-A and NPR-B. In the teleost eel (Anguilla japonica) NPR-A mRNA is widely expressed in various osmoregulatory organs, such as the gill, kidney, intestine and urinary bladder (Kashiwagi et al. 1999), whereas the NPR-B receptor is predominantly expressed in the liver and atrium as well as the gill. Smaller amounts of NPR-B mRNA are found in the eel brain, ventricle, oesophageal sphincter, stomach, posterior intestine and kidney (Katafuchi et al. 1994). The NPR-B mRNA levels in the liver, atrium and gill decrease markedly when eels are transferred from fresh water to seawater (Katafuchi et al. 1994). Since similar changes are known to occur in the CNP - the ligand for the NPR-B receptor - levels when eels face osmotic challenges, the CNP/NPR-B system appears to play an important role in their adaptation to salinity changes (Katafuchi et al. 1994).

Two natriuretic peptide clearance receptors have been identified in eel. NPR-C mRNA is highly expressed in the gill and heart and, to a much lesser extent, in other tissues including the brain and intestine (Takashima et al. 1995). The NPR-C mRNA levels are down-regulated in most tissues when eels are transferred from fresh water to seawater, but in the anterior intestine the levels are, however, up-regulated (Takashima et al. 1995). This suggests that NPR-C plays a role in the adaptation to salinity changes in the euryhaline eel.

The D-type natriuretic peptide receptor (NPR-D) has only been found in eel (Kashiwagi et al. 1995). The NPR-D receptor does not have a domain corresponding to a kinase-like or guanylate cyclase region, retaining only the extracellular domain connected to a short cytoplasmic tail (Kashiwagi et al. 1995). However, the receptor is tetrameric similarly to GC-coupled receptors (Takei 2001). Eel NPR-D shows sequence and structural similarity to the NPR-C receptor and also acts as a clearance receptor (Kashiwagi et al. 1995). The NPR-D mRNA is predominantly expressed in the brain and is also present in the gill (Kashiwagi et al. 1995). It is interesting to note that the natriuretic peptide clearance receptors are highly expressed in the gills. The reason for this is that the gills are ideally located to serve as a buffer to regulate the plasma concentration of hormones secreted by the heart, since they receive all the blood expelled from the heart.

In cartilaginous fishes, two types of natriuretic peptide receptor seem to exist in the rectal gland and gills. In spiny dogfish rectal gland, one receptor type stimulated cGMP production and was thus presumably NPR-B, and the other receptor bound all natriuretic peptides and thus resembled the NPR-C-type (Gunning et al. 1993). The presence of

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these receptors was confirmed by the partial cloning of the NPR-C from the gills (Donald et al. 1997) and by sequencing of the NPR-B cDNA from the rectal gland of spiny dogfish (Aller et al. 1999).

2.8 Evolutionary history of the natriuretic peptide system

Tetrapods generally have three subtypes of natriuretic peptides: ANP, BNP and CNP. Instead, some teleost species lack BNP, while elasmobranch sharks and the more primitive hagfish have only one natriuretic peptide. Thus, natriuretic peptides have diverged during fish evolution, probably reflecting alterations in osmoregulatory systems. Recently, four distinct CNPs (CNP-1, CNP-2, CNP-3 and CNP-4) were found from the teleost species medaka and pufferfish (Inoue et al. 2003a). It is suggested that four CNP genes were generated by chromosomal duplications before the divergence of elasmobranches (Inoue et al. 2003a). The CNP-3 gene generated ANP and BNP genes through tandem duplication before the branching of tetrapods and teleosts (Inoue et al. 2003a). Specific lineages may have been lost in each vertebrate class during evolution. CNP-1 and -2 genes were retained in the teleost lineage but not in tetrapods (Inoue et al. 2003a). After divergence of amphibians, tetrapods may have lost the CNP-3 gene, although mammals maintain the ANP and BNP genes derived from the CNP-3 lineage and a CNP gene derived from the CNP-4 lineage (Inoue et al. 2003a).

2.9 Endothelin-1 in cardiovascular physiology

Endothelins are a family of three peptides (ET-1, ET-2 and ET-3), which are involved in the regulation of cardiovascular function. In the vessels endothelins have a basal vasoconstricting role, and in the heart they affect inotropy and chronotropy. Endothelins are produced by endothelial cells and many other cell types as well. Circulating levels of endothelins are very low and they are therefore considered local autocrine or paracrine factors rather than circulating hormones. In addition, endothelins have emerged as important participants in the pathophysiology of a variety of cardiovascular diseases.

2.9.1 Endothelins

Endothelin-1 (ET-1) was identified in 1988 as a potent vasoconstrictor peptide from cultured porcine aortic endothelial cells (Yanagisawa et al. 1988). Besides endothelial cells, a wide variety of cells such as airway epithelial cells, macrophages, fibroblasts, cardiomyocytes and neurons can produce and secrete ET-1 (for review see Kedzierski & Yanagisawa 2001). ET-1 has several physiological functions, with roles in basal vascular tone, sodium balance, development, and neurotransmission (for review see Kedzierski & Yanagisawa 2001). In addition, ET-1 has a crucial role in cardiovascular function and cardiovascular diseases. The endothelins include three peptides, namely ET-1, ET-2, and

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ET-3, encoded by three different genes. ET-1 is the isoform mainly responsible for cardiovascular effects, whereas ET-2 is involved in neonatal growth and intestinal functions, and ET-3 plays a role in the central nervous system and intestinal tract (for review see Kedzierski & Yanagisawa 2001, Yang et al. 2005).

The product of the human ET-1 gene is preproendothelin-1. Preproendothelin-1 is processed by a furin-like enzyme to produce an inactive intermediate termed big ET-1. Formation of the mature 21-amino-acid peptide ET-1 requires cleavage of the Trp21-Val22 in big ET-1 by endothelin-converting enzymes (ECEs), which are membrane-bound zinc metalloproteases (Emoto & Yanagisawa 1995). The mature hormone can be released by at least two mechanisms. ET-1 can be continuously transported into and released from secretory vesicles by a cAMP-independent mechanism (Benigni 2000), or it can be stored in endothelial cells and released at the cell surface upon appropriate stimuli (Russell et al. 1998). The concentration of ET-1 in plasma is low. Under normal physiological conditions, ET-1 appears to act as an autocrine and paracrine factor instead of a circulating hormone (for review see Yang et al. 2005).

The clearance of endothelins is regulated by neutral endopeptidase (EC 3.4.24.11) in renal tissue, because the inhibitors of this enzyme increase urinary endothelin levels markedly (Abassi et al. 1992). In addition, endothelin B receptor (ETB) likely functions as a clearance receptor. Indeed, the ETB-selective antagonist BQ-788 inhibits accumulation of intravenously administered, radiolabelled ET-1 in lungs and kidneys, thereby slowing its clearance (Fukuroda et al. 1994).

2.9.2 Endothelin receptors in mammals

In mammals, two G-protein-coupled endothelin receptors (ETA and ETB) have been identified (Arai et al. 1990, Sakurai et al. 1990). Each receptor activates an overlapping set of G proteins. Stimulation of ETA and (or) ETB results in the activation and dissociation of Gq proteins, leading to an increase in the activity of phosphoinositide-specific phospholipase C (PLC) and subsequent phosphatidylinositol 4,5-diphosphate (PIP2) hydrolysis (Simonson et al. 1989, Takuwa et al. 1989, Ambar & Sokolovsky 1993). The resulting second messengers inositol triphosphate (IP3) and diacylglycerol (DAG) (Nishizuka 1988) then trigger the mobilization of intracellular Ca2+ and activation of protein kinase C (PKC) (Simonson & Herman 1993). In addition, ETA is coupled to a stimulatory G protein, leading to accumulation of second messenger cAMP by activation of adenylate cyclase, whereas ETB is coupled to an inhibitory G protein through which it limits the levels of cAMP (Aramori & Nakanishi 1992).

ETA is found on vessel and airway smooth muscle cells, cardiomyocytes, liver stellate cells and hepatocytes, brain neurons, osteoblasts, melanocytes, keratinocytes, adipocytes, and various cells in the reproductive tract (for review see Kedzierski & Yanagisawa 2001). ETB exists on vessel endothelial cells and smooth muscle cells, liver hepatocytes, renal collecting duct epithelial cells, airway smooth muscle cells, osteoblasts, neurons, and various cells of the reproductive tract (for review see Kedzierski & Yanagisawa 2001).

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2.9.3 Cardiovascular effects of endothelin-1 in mammals

2.9.3.1 Vascular effects

ET-1 was originally discovered as a vasoconstrictor substance. The only vascular endothelin identified is ET-1 (Kedzierski & Yanagisawa 2001). The local ET-1 concentration within vascular wall is ≥ 100-fold that of plasma level, stemming in part from the fact that 80% of the hormone is secreted on the basal side of endothelial cells (Howard et al. 1992, Wagner et al. 1992). ET-1 interacts with ETA found on underlying smooth muscle cells and ETB found on endothelial cells and on some smooth muscle cells (Arai et al. 1990, Sakurai et al. 1990, Batra et al. 1993).

Intravenous bolus administration of ET-1 in different species leads to a short-term decrease, followed by a long-term increase in vascular resistance (Wright & Fozard 1988). The initial increase in perfusion is caused via nitric oxide and prostacyclin release by ETB-stimulated endothelial cells (Berti et al. 1993, Filep et al. 1993), whereas the decrease in perfusion is mediated by ETA on smooth muscle cells (Bird et al. 1993). Furthermore, administration of the ETA-selective antagonist into the human vasculature leads to an increase in local blood flow (Haynes & Webb 1994). In addition, hypotensive effects are noted in humans and mammalian species when the ETA/ETB-combined antagonist or the ETA-selective antagonist or ECE inhibitors are administered (McMahon et al. 1991, Veniant et al. 1994, Haynes et al. 1996). Similarly, mice lacking ET-1 in endothelial cells have reduced blood pressures (for review see Kedzierski & Yanagisawa 2001). However, not all studies replicate the hypotensive effect of ETA inhibition in normotensive animals (Gardiner et al. 1991). Despite its participation in the regulation of vascular tone, the role of ET-1 in human hypertension is not clearly defined. Instead, the endothelin system has been implicated in a number of vascular pathophysiologies such as atherosclerosis (for review see Kedzierski & Yanagisawa 2001).

2.9.3.2 Inotropic effects

ET-1 elicits a positive inotropic effect in mammalian cardiac muscle of most species including rat, guinea pig, rabbit and ferret (Takanashi & Endoh 1991, Wang et al. 1991). In man, ET-1 has been shown to increase the contractile force and the duration of contraction in atrial and ventricular strips (Li et al. 1991, Meyer et al. 1996, Burrell et al. 2000). The inotropic effect is likely mediated by the ETA receptor because the ETA-selective receptor antagonist BQ-123, but not the ETB-selective receptor antagonist BQ-788, prevents the effects of ET-1 on myocyte shortening and partially reduces the duration of contraction (Burrell et al. 2000). ET-1 also modifies cardiac contractility in the human heart in vivo (Kaumann et al. 1999). Infusion of BQ-123 also caused a marked reduction in systolic contractility, suggesting a role of endogenous ET-1 in the regulation of cardiac contractility (MacCarthy et al. 2000). Additionally, ET-1 may have chronotropic effects in that it induces hyperpolarization and shortens action potential duration in isolated guinea-pig myocyte cultures (James et al. 1994, Ono et al. 1994).

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In rabbit myocardium, both ET-1 and ET-3 were reported to induce similar contractility-enhancing effects, suggesting that they might be mediated by ETB receptors (Takanashi & Endoh 1991). Moreover, two selective ETB receptor stimulants promoted positive and negative inotropic effects, and the negative inotropic effect seemed to be mediated by NO and prostaglandins (Leite-Moreira & Bras-Silva 2004). In rabbit cardiomyocytes, the positive cell-shortening effect has been linked to ETA receptors (Kelso et al. 2000), whereas in open-chest rats, the positive inotropic effect seems to be mediated mainly via ETB receptors (Beyer et al. 1996, 1999). In mouse, ETB receptor antagonist augmented the load-induced contractile response, whereas ETA or mixed ETA/ETB antagonist attenuated it, suggesting that the ETA receptor-mediated increase in contractile force is suppressed by ETB receptor stimulation (Piuhola et al. 2003). In human right atrium, the cardiostimulant effect of ET receptor agonists is generally attributed to ETA receptors (Meyer et al. 1996).

The binding of ET-1 to ETA receptors on the myocyte surface stimulates hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP2) to diacylglycerol (DAG) and inositol triphosphate (IP3) (for review see Brunner et al. 2006) (Fig. 4). IP3 formation and changes in myofibrillar Ca2+ responsiveness have been observed in association with the positive inotropic effect of ET-1 in human left ventricle (Pönicke et al. 1998, Pieske et al. 1999). The inotropic effect may be partially due to Ca2+ release mediated by IP3 receptors in atrial myocytes, since they express functional IP3 receptors and IP3 ester evokes an increase in the amplitudes of action potential-evoked Ca2+ transients (Lipp et al. 2000). A central mechanism in the positive inotropic response of ET-1 is activation of phospholipase C (PLC), with or without concomitant activation of L-type Ca2+ channels (Yanagisawa et al. 1988) leading to an increase in intracellular Ca2+ transient. In addition, the changes in Ca2+ movements induced by ET-1 can be brought about by stimulation of Na+/H+ exchanger 1 (NHE-1) (for review see Brunner et al. 2006). Stimulation of NHE-1 leads to intracellular alkalinization and an increase in intracellular Na+ (Krämer et al. 1991, Moor & Fliegel 1999). This reverses the normal direction of sarcolemmal Na+/Ca2+ exchange, leading to an increase in intracellular Ca2+ (Endoh et al. 1998). The intracellular alkalinization increases the affinity of troponin C for Ca2+. This sensitizes the actomyosin ATPase to Ca2+ and decreases the rate of dissociation of Ca2+ from the troponin C-Ca2+ complex (for review see Sugden 2003). In rabbit, ET-1 was found to activate a PLC pathway without the participation of protein kinase C (PKC) (Kelso et al. 2000), and in guinea-pig myocardium an integrated approach uncovered a combination of mechanisms including an increase in transmembrane Ca2+ influx through L-type Ca2+ channels and the stimulation of Na+/H+ exchange via PKC activation (Woo & Lee 1999).

Besides eliciting positive inotropic effects, ET-1 may depress myocardial contractility in certain experimental conditions and preparations. In strips of right mouse ventricle, high concentrations of exogenous ET-1 exerted a negative inotropic effect (Izumi et al. 2000). ET-1 mediated by ETA receptors also caused a marked reduction in active tension in isolated rabbit right atria and dog ventricular trabeculae prestimulated with isoprenaline (Zhu et al. 1997). Additionally, it has been demonstrated that ET-1 has the potential to elicit a positive or negative inotropic effect by interacting with endogenous noradrenaline whose tissue concentration determines the direction of the net effect via activation of concentration-dependent signalling pathways (Chu et al. 2003). The main mechanisms that mediate the observed negative inotropic actions of ET-1 are effects on

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membrane currents and reductions in the level of cellular cAMP (for review see Brunner et al. 2006). The transient negative inotropic effect demonstrated in human atria with exogenous ET-1 was attributed to inhibition of adenylyl cyclase (Dhein et al. 2000). Likewise, the ET-induced negative inotropic effect after prestimulation with isoprenaline in rabbit atria was accompanied by markedly reduced cAMP levels, whereas the reduced contractile force induced in dog ventricle was primarily exerted via pertussis toxin-sensitive G proteins at unchanged cAMP levels (Zhu et al. 1997).

Fig. 4. Proposed signal transduction mechanisms involved in ET-1-mediated positive inotropic effect (modified from Brunner et al. 2006). NCX = Na+/Ca2+ exchanger, NHE-1 = Na+/H+ exchanger, PIP2 = phosphatidylinositol 4,5-diphosphate, DAG = diacylglycerol, IP3 = inositol triphosphate, PKC = protein kinase C, PLC = phospholipase C, ET-1 = endothelin-1, ETA = endothelin type A receptor, ETB = endothelin type B receptor, Gq = G protein.

2.9.3.3 Effect of endothelin-1 on natriuretic peptide release

ET-1 is one of the most powerful stimulants of natriuretic peptide secretion and gene expression (de Bold et al. 2001, McGrath & de Bold 2005). It enhances the secretion of ANP in co-culture of endothelial and atrial myocytes (Lew & Baertschi 1989a, 1992) and in cultured rat atrial and ventricular myocytes (Fukuda et al. 1988, Sei & Glembotski 1990, Uusimaa et al. 1992). ET-1 has also been shown to increase ANP release from isolated rat atria (Hu et al. 1988, Stasch et al. 1989). BNP secretion is also increased by ET-1 from rat atrial myocytes (Horio et al. 1992, Suzuki et al. 1992a) and rat atrial preparation (Bruneau & de Bold 1994, Bruneau et al. 1997). In vivo ET-1 administration significantly increases the plasma ANP levels in rats and dogs (Miller et al. 1989, Stasch

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et al. 1989). However, in vivo exogenously administered ET-1 may increase ANP secretion in part indirectly because of its vasoconstrictive and hypertensive action.

In isolated rat heart ET-1 enhanced basal as well as atrial stretch-stimulated ANP secretion, suggesting that ET-1 is involved in the regulation of stretch-induced ANP release (Mäntymaa et al. 1990, Skvorak et al. 1995). Furthermore, passive immunization of rats with antiserum raised against ET-1 (Fyhrquist et al. 1993) and inhibition of ETA/B receptors decreased the ANP response to acute volume load (Leskinen et al. 1997a). ET-1 appears also to be required for the stretch-induced activation of BNP gene expression in the atria but not in the ventricle. Administration of the ET antagonist bosentan decreased the stretch-induced increase in BNP mRNA level in perfused rat atria, but in ventricle ET-1 is not required in pressure load-induced increase in BNP mRNA levels (Magga et al. 1997). Therefore, ET-1 appears to stimulate natriuretic peptide release by a direct action on cardiac myocytes, and seems to be one of the factors required for a full response of the endocrine heart to load.

The stimulation of ANP secretion from the rat atrial preparation by ET-1 is rapid and potent (Bruneau & de Bold 1994, Bruneau et al. 1997). ET-stimulated ANP secretion by atrial myocytes was associated with activation of the ETA receptor (Irons et al. 1993, Leite et al. 1994, Thibault et al. 1994). This G-protein coupled receptor is linked to hydrolysis of phosphatidyl inositol (Sokolovsky 1993). Inhibition of the ETA receptor with the antagonist BQ-123 was associated with a marked decrease of IP3 formation and parallel inhibition of ANP secretion (Irons et al. 1993). Increase in ANP secretion after ET-1 stimulation is transient and might possibly be due to ETA desensitization as well as the down-regulation of its effector molecule, phospholipase C (Leite et al. 1994). Moreover, it has been reported that the cyclooxygenase inhibitor indomethacin abolished ET-1-stimulated ANP secretion in anoxic conditioned atria (Skvorak et al. 1996), and that the cytochrome P-450 arachidonic acid metabolite inhibitor decreased ET-induced ANP secretion from perfused rat atria and cultured ventricular myocytes (Lee et al. 2004). Therefore, prostaglandins play a role in the ANP secretion produced by ET-1 as well.

Acute secretion of ANP by ET-1 is not, however, accompanied by changes in ANP mRNA levels, whereas BNP mRNA levels increase within 2 hours in response to ET-1 (Bruneau & de Bold 1994). Nevertheless, chronic blockade of ETA receptors in deoxycorticosterone acetate-salt hypertensive rats prevented the rise in blood pressure along with circulating ANP levels (Bianciotti & De Bold 2000). ANP gene expression was also reduced in response to chronic ETA blockade in the ventricles but not in the atria (Bianciotti & De Bold 2000), suggesting that there are differences between atrial and ventricular natriuretic peptide gene expression in terms of their sensitivity to the endocrine environment (McGrath & de Bold 2005).

2.9.3.4 Other cardiovascular effects

Several studies have shown that endothelins play an important role in cardiovascular development (for review see Kedzierski & Yanagisawa 2001, Yang et al. 2005). For example, functional disruption of ET-1, ETA, or ECE-1 resulted in malformations in

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arterial structures and defects in ventricular septae (Kurihara et al. 1995, Clouthier et al. 1998, Yanagisawa et al. 1998, 2000).

ET-1 also has pathological roles in the heart, and the ET-1 system has been implicated in many cardiac diseases. Indeed, ET-1 concentrations in plasma are elevated in severe cardiac ischaemia (Stewart et al. 1991) and in modest to severe heart failure (McMurray et al. 1992, Stewart et al. 1992, Wei et al. 1994b, Pousset et al. 1997). In addition, local production of ET-1 is increased in the failing myocardium of patients with idiopathic and ischaemic dilated cardiomyopathy (Pieske et al. 1999, Serneri et al. 2000). Similar results are found in experimental animal models of heart failure (Sakai et al. 1996a, b). There are also a lot of data to support a role for ET-1 as a pro-inflammatory cytokine and fibrotic factor (for review see Yang et al. 2005). ET-1 has been implicated in atherosclerosis (Babaei et al. 2000) and myocardial inflammation and fibrosis in hearts of deoxycorticosterone acetate hypertensive rats (Ammarguellat et al. 2001, 2002). In addition to its inflammatory and fibrotic roles in the heart, ET-1 exerts trophic effects in cardiomyocytes (Ito et al. 1991, Barton et al. 1998) and therefore, induces cardiac hypertrophy.

2.9.4 Endothelins in fish

The first evidence for endothelin-like immunoreactivity in fish came from studies in which immunolabelled axons were detected in the brain of medaka (Oryzias latipes) and immunoreactive cells were observed in its gills (Kasuya et al. 1991). Later, endothelin-immunoreactivity has been found in neuroendocrine cells in fish gills of several species (Zaccone et al. 1996). Moreover, a ET-2-like peptide has been purified from rainbow trout (Oncorhynchus mykiss) kidney (Wang et al. 1999). It differs from human ET-2 in three and from human ET-1 in four amino acid residues.

In rainbow trout, mammalian ET-1 was found to increase gill vascular resistance and to cause transient, dose-dependent reduction in the dorsal aortic pressure in vivo (Olson et al. 1991). Intracerebroventricular and intra-arterial administration of very low doses of mammalian ET-1 produced hypertension in conscious trout (le Mevel et al. 1999). Homologous trout endothelin (Wang et al. 1999) has been shown to increase ventral and dorsal aortic pressures, gill and systemic resistance and to decrease cardiac output, heart rate and stroke volume (Hoagland et al. 2000). Interestingly, trout ET showed a weaker cardiovascular potency than mammalian ET-1 in rainbow trout (Hoagland et al. 2000). However, trout ET reminds human ET-2 more than ET-1, and an ET-1-like peptide has not been found in trout. It may therefore be possible that salmonids also have an ET-1-like hormone, which has higher cardiovascular potency than the known trout ET. Endothelins seem to play an important role in gill vasculature. ET-1 has been shown to increase pillar cell contraction and redistribute blood flow through gills (Sundin & Nilsson 1998, Stenslokken et al. 1999).

Endothelin receptors have been detected in rainbow trout gills (Lodhi et al. 1995). Binding affinity studies suggest that an ETA receptor is the main subtype in the branchial vasculature in rainbow trout (Lodhi et al. 1995). In addition, the lack of effect of intracerebroventricular and intra-arterial administration of ET-3 in conscious trout

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indicates that the haemodynamic actions of ET-1 are mediated both centrally and peripherally through ETA receptors (le Mevel et al. 1999). However, in spiny dogfish shark (Squalus acanthias) there is evidence for ETB-like receptors in the gill (Evans & Gunderson 1999) and aorta (Evans et al. 1996). Also in the eel (Anguilla rostrata) an ETB-like receptor seems to be involved in bulbus arteriosus contraction (Evans et al. 2003). Recently in Atlantic cod (Gadus morhua), it was shown that the effects of ET-1 on branchial circulation are mediated by an ETB-like receptor (Stenslokken et al. 2006). Therefore, both ETA and ETB receptors can mediate the effects of endothelins in fish.

3 Aims of the research

The aim of this study was to characterize factors affecting natriuretic peptide secretion using salmon cardiac peptide (sCP) as a model. Specifically, the role of mechanical load, temperature and endothelin-1 (ET-1) in salmon cardiac peptide secretion and production was studied. Salmon (Salmo salar) was selected for use as a model since this euryhaline species has an excellent adaptability to wide variations in environmental salinity. Therefore, sCP and other natriuretic peptides can be expected to have vital physiological regulatory significance.

Specific aims of this study were:

1. to characterize sCP release from salmon ventricle in vitro 2. to find out the effects of mechanical load on sCP release and gene expression 3. to evaluate the role of ambient temperature in regulating sCP 4. to identify the role of ET-1 in sCP release and gene expression and in cardiac

contractile function 5. to characterize the interactions between ET-1 and β-adrenergic activation

(isoprenaline) in salmon cardiac endocrine and contractile function

4 Materials and methods

4.1 Experimental animals

The experiments were approved by the Animal Experimentation Review Board of the University of Oulu. Mature salmon (Salmo salar) of both sexes were obtained from the Game and Fisheries Research Institute, Taivalkoski, Muonio and Laukaa, Finland. Salmon parrs were provided by the hatchery of Montan lohi (Imatran Voima power plant) in Muhos, Finland, and Voimalohi Oy fish hatchery in Ii, Finland. The fish were kept in fresh river water and under natural photoperiod and temperature in the hatcheries. A summary of experimental animals and protocols is presented in Table 1.

Table 1. Summary of experimental protocols

Study Salmon Experimental models Methods I Adults Isolated perfused ventricle preparation

Effects of mechanical load Immunoassay HPLC Northern blot Immunoelectron microscopy

II Adults Parrs

Seasonal sampling of salmon Effect of ambient temperature

Isolated perfused ventricle preparation Effect of ambient temperature

Immunoassay Northern blot

III Adults Isolated perfused ventricle preparation Effect of ET-1

Chronic cannulation of salmon Effect of ET-1

Production of recombinant protein Immunoassay HPLC Injections and sampling of salmon in vivo

IV Adults Parrs

Myocardial strip preparation Isolated perfused ventricle preparation

Effect of ET-1 + β-adrenergic activation

Cardiac contractile function measurements Immunoassay Quantitative RT-PCR

The animals were transported from the hatchery to the laboratory of the Department of Physiology, University of Oulu, in plastic packages containing oxygenated water. In the laboratory the fish were kept in a 350-litre or 1500-litre tank containing dechlorinated,

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recirculated and aerated tap water. The tank water temperature was adjusted and maintained using a cooling unit (Aqua Medic, Melle, Germany). The fish were kept under a light-dark cycle appropriate for the season.

4.2 Experimental models

4.2.1 Myocardial strip preparation (IV)

The contractile function of salmon ventricle was studied using a simple ventricular strip preparation. The salmon were stunned and placed on an operation table. The pericardium and ventral aorta were exposed by incising the ventral skin and retracting the muscle and connective tissues around the pericardium and ventral aorta. The ventral aorta was cut to expose the beating heart, which was removed. Myocardial strips were prepared by cutting the salmon ventricle in half. The preparation was mounted in an organ chamber, attached to a force transducer (F10 type 375, Hugo Sachs Electronics, March-Hugstetten, Germany) and superfused with modified Cortland saline (pH 7.9, 240 mOsm/kg H

2O)

(Farrell et al. 1988) containing 124 mmol NaCl, 5 mmol CaCl2•2H2O, 3 mmol KCl, 0.09 mmol NaH2PO4•H2O, 1.8 mmol Na2HPO4•2H2O, 1.1 mmol MgSO4•7H2O, 5.6 mmol dextrose, and 12 mmol NaHCO3 at 1.5 ml/min. The buffer was cooled to the temperature at which the fish had been acclimated (10°C) using a K15 refrigerated circulator (Haake, Karlsruhe, Germany) and gassed with 95% O2, 5% CO2. The flow rate was controlled by a peristaltic pump (7553-85, Cole-Parmer Instrument Co., USA). The muscle strips were electrically stimulated by field stimulation (0.25 Hz). The preparations were allowed to equilibrate for 30 min at low preload. After that, they were gradually stretched to the optimal length, at which the developed force attained maximum. Final preload was then adjusted to 50% of the force at the optimal length. In the first group, the concentration of human ET-1 (Peninsula Laboratories Europe, Merseyside, UK) in the buffer was cumulatively increased (1–100 nM) and in the second group, similar experiments were performed in the presence of the ETA receptor antagonist BQ-610 (125 nM) (Bachem, Bubendorff, Switzerland). In the third group (positive control), the concentration of isoprenaline (isoproterenol, Sigma) in the buffer was cumulatively increased (0.1–10000 nM) and finally, the fourth group acted as a control group.

4.2.2 Isolated perfused ventricle preparation (I – IV)

The salmon ventricle perfusion setup was constructed on the basis of the rat atrial perfusion system described previously (Laine et al. 1994a, b). Isolated perfused salmon ventricle preparation was used to study the release of sCP and the contractile function of a ventricle. The effects of mechanical load, ambient temperature, ET-1 and β-adrenergic activation were examined. The scheme of isolated perfused salmon ventricle preparation is shown in Fig. 5.

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Fig. 5. Scheme of isolated perfused salmon ventricle preparation.

Salmon were stunned and the beating heart was removed as described in section 4.2.1. The atrium was cut off. An X-branch polyethylene adapter was inserted into the lumen of the ventricle and the ventricle was mounted in an organ bath formed by a glass vessel. A tube with a smaller diameter was inserted inside the adapter in order to carry perfusate inflow into the lumen of the ventricle. The outflow from the ventricle came from one cross branch of the X-cannula. The ventricle was perfused with a gassed modified version of Cortland saline (see section 4.2.1), with a constant flow rate. The buffer was cooled to the temperature at which the fish had been acclimated. The flow rate was controlled by a peristaltic pump. The ventricle was paced by a focal supramaximal stimulus near the atrio-ventricular node. The other cross branch of the X-cannula was connected to a pressure transducer (TCB 100, Millar instruments, Inc., USA), so that the pressure in the lumen of the ventricle could be recorded. The pressure transducer was connected to a Grass multichannel recorder (I–III) or computer (IV). The contraction force (pressure generated by the contraction) was recorded concomitantly with the collection of perfusate samples (IV).

Experiments were started after an equilibration period (30 or 60 min). Mechanical load was produced by adjusting the outflow height of the perfusate. The perfusate samples were collected in order to measure the concentration of immunoreactive sCP and/or NT-pro-sCP in the perfusate. The samples were collected in polyethylene tubes and stored at -20°C for use in radioimmunoassays and chromatography. The ventricles were stored at -70°C after experiments for use in radioimmunoassays, mRNA isolation and chromatography.

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4.2.2.1 Mechanical load experiments (I, III)

The effect of mechanical load on sCP and NT-pro-sCP secretion from salmon ventricle was studied using isolated ventricle preparation. The ventricle was perfused with a modified version of Cortland saline (see section 4.2.1), with a flow rate of 3.8-4 ml/min. The buffer was cooled to the temperature at which the fish had been acclimated (6°C) and gassed with 95% O2, 5% CO2. The preparation was paced by a focal stimulus (0.2 Hz). The sampling of perfusate was started after an equilibration period. Basal-level samples were collected without mechanical load. The mechanical loads of 4, 8 and 13 cm H20 were tested. The samples were collected at 1-minute fractions in order to measure the concentration of immunoreactive sCP (I, III) or NT-pro-sCP (III) secreted to the perfusion fluid.

4.2.2.2 Ambient temperature experiments (II)

This set of experiments was designed to study the influence of different temperatures on sCP secretion from ventricles. The experiments were performed during the winter, when the fish were acclimatized to cold water (0.2°C). The acclimation temperatures in the laboratory were 6°C and 12°C. The stock of 30 fish was kept at the set temperature for 10 days, after which half of the fish were used for experiments. The ventricles were perfused at the same temperatures at which the fish had been acclimated. Due to the limitation of the perfusion system, the preparates from the salmon acclimatized to near zero temperatures were studied at 6°C. The buffer was gassed with 95% O2, 5% CO2. The preparation was paced by a focal stimulus (0.2 Hz). The perfusate flow rate was 1.5 ml/min. First, basal level samples were collected without mechanical load and then, mechanical load of 13 cm H20 was applied. The samples were collected at 5-minute fractions.

4.2.2.3 Endothelin-1 experiments (IV)

To characterize the effects of ET-1 on the release of sCP from salmon ventricle, human ET-1 was added to the perfusion fluid. The ventricle was paced by a focal stimulus (0.25 Hz) and perfused with a flow rate of 4 ml/min with a modified version of Cortland saline (gassed with 95% O2, 5% CO2). The perfusate was collected in one-minute fractions. In the first and second group, after the equilibration period mechanical load (13 cm H2O) was applied with or without ET-1 (5 nM). In the third group, ET-1 was added without any mechanical load. ET-1 was present in the perfusion buffer during the whole experiment. In the fourth group, the selective endothelin receptor A (ETA) antagonist BQ-610 (125 nM) was added in the perfusion buffer at the time the mechanical load was applied.

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4.2.2.4 Endothelin-1 and β-adrenergic activation experiments (IV)

The last set of perfusion experiments was planned to study the role of ET-1 and possible interactions of ET-1 and β-adrenergic stimulation (isoprenaline) in both cardiac endocrine function and the contribution of ETA and ETB receptors. Because of the expected chronotropic effect of isoprenaline the ventricle preparation was paced at a higher physiological frequency (0.6 Hz focal stimulus) and perfused with a flow rate of 2 ml/min with a modified version of Cortland saline (gassed with 99.5% O2, 0.5% CO2). The perfusate was collected in 5-minute fractions. Basal samples were collected during the first period with a mechanical load of 10 cm H

2O. Thereafter ET-1 (10 nM),

isoprenaline (0.5 nM) or the selective ETA antagonist BQ-610 (1 µM) or endothelin receptor B (ETB) antagonist BQ-788 (100 nM) (Bachem, Bubendorff, Switzerland) was applied to the buffer. After this second period, ET-1 or isoprenaline concentration in the perfusion buffer was cumulatively increased every 30 min from 1 to 3, 10, 30 and 100 nM (ET-1) or from 0.1 to 1, 10, 100 and 1000 nM (isoprenaline). Thus, the groups were as follows: dose-response for ET-1, dose-response for ET-1 in the presence of BQ-610, dose-response for ET-1 in the presence of isoprenaline, dose-response for ET-1 in the presence of isoprenaline and BQ-610, dose-response for ET-1 in the presence of BQ-788, dose-response for isoprenaline, dose-response for isoprenaline in the presence of ET-1, dose-response for isoprenaline in the presence of BQ-610, dose-response for isoprenaline in the presence of ET-1 and BQ-610, dose-response for isoprenaline in the presence of ET-1 and BQ-788, BQ-610, BQ-788, and control.

4.2.3 Seasonal sampling of salmon (II)

Ventricle tissue and plasma samples were collected monthly for one year from fish kept under field conditions in natural temperature. In order to find out whether sCP gene expression is similar during different developmental stages samples were collected from young salmon parrs and mature salmon. Samples from parrs were collected in the hatchery. Adult salmon were transported from Taivalkoski to the laboratory of Department of Physiology for sampling. When the sampling was started, the parrs were two years of age. During the investigation their body weight increased from 61 ± 8 g to 262 ± 40 g. Adults were 5 years of age at the beginning of the study and weighed 464 ± 51 g. In the last sampling month they weighed 590 ± 78 g. The salmon were stunned and weighed and body length was measured. Blood was drawn from the caudal vein into chilled EDTA tubes and centrifuged. The body cavity was opened, the heart was removed and the atrium and ventricle separated, weighed and frozen in liquid N2. The samples were stored at -70°C for use in radioimmunoassays and mRNA isolation.

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4.2.4 Chronic cannulation of salmon (III)

The effect of ET-1 on sCP secretion in vivo was studied using cannulated conscious mature salmon. The salmon were kept in the laboratory as described in section 4.1. Bolus injections of synthetic human ET-1 were used to induce the secretion of pro-sCP-derived peptides, sCP and N-terminal fragment of pro-sCP (NT-pro-sCP). The salmon were anaesthetized by 100 mg/l of tricaine (ethyl m-aminobenzoate methanesulfonate, MS-222, Sigma) before surgery. The dorsal aorta was cannulated by a previously described method (Soivio et al. 1975) through the roof of the buccal cavity using polyethylene tubing. The cannula was filled with heparinized saline (100 USP units/ml heparin in 9.0 g/l NaCl). The fish were then revived and placed in plastic tubes (length 72 cm, diameter 20 cm) that were immersed in the tank. The experiments were conducted 48 h after the surgery. Basal blood samples of 0.8 ml were drawn through the cannula before the injection. A bolus of 1 µg of ET-1 was injected into the dorsal aorta during 5 s followed by a 0.1 ml heparinized saline flush. Blood samples of 0.3 ml were drawn at 3 min, 7 min, 12 min and 30 min after the administration of ET-1, and samples of 0.8 ml at 0 min, 20 min and 40 min. Each blood sample was replaced with the same volume of saline and the cannula was filled with heparinized saline. Control experiments were performed identically except that vehicle (saline) was administered instead of ET-1. The effect of ET blockade was also studied. A 25-molar excess (relative to ET-1) of either BQ-123 (a specific ETA receptor antagonist, Sigma) or BQ-788 (ETB receptor antagonist) was administered as a bolus injection 5 min before the administration of ET-1. The blood samples were centrifuged at 2000 g for 15 min. The serum samples were stored at -20°C for NT-pro-sCP and sCP radioimmunoassays.

4.3 Extraction of sCP from serum and tissues

Extraction of the hormone from blood and tissue samples was done in order to concentrate it and to prevent non-specific interference. Serum samples (0.5–1 ml) were extracted with SepPak C18 cartridges (Waters, Milford, MA, USA) for use in sCP RIA. The serum was acidified with 1 M HCl/0.2 M glycine (200 µl per ml of serum), and the samples were loaded into the cartridges that had been preactivated with isopropanol and aqueous 0.1% TFA, and washed with 10 ml of 0.1% TFA. The peptide fraction was then eluted with 2 ml of 60% acetonitrile in aqueous 0.1% TFA, dried in SpeedVac concentrator (Savant Instruments, Hicksville, NY) and stored at -20°C.

Aliquots from the guanidine isothiocyanate/2-mercaptoethanol homogenates obtained from the RNA isolation were used to determine the concentration of tissue immunoreactive sCP. In experiments in which the RNA was not isolated, the cardiac tissue samples were pulverized in liquid N2 and homogenized in 4.5 vol of boiling H2O and 4.5 vol of 2 M acetic acid/40 mM HCl. The homogenates were centrifuged (15000 g, 30 min at 4°C) and the supernatants were lyophilized and used for RIA.

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4.4 Hormone assays

4.4.1 Radioimmunoassay of sCP

The immunoreactivity of sCP from perfusate and blood samples was determined by radioimmunoassay (RIA). The sCP RIA was performed using procedures described previously (Vuolteenaho et al. 1992, Tervonen et al. 1998). Briefly, duplicate samples (100 µl) were incubated overnight with the specific goat anti-sCP antiserum used at the final dilution of 1/8000, followed by overnight incubation with 5000-10000 cpm radioiodinated sCP. Tyr0-sCP was radioiodinated with the chloramine-T technique (Greenwood et al. 1963) and purified by gel filtration using a 2-ml Sephadex G-25 M column (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK), followed by reverse phase HPLC with a 4.6 x 300 mm Vydac C18 column (Separations Group, Hesperia, CA, USA) and a 30-min 10% to 40% acetonitrile gradient in aqueous TFA. Synthetic sCP-29 ranging from 8 to 2000 pg/tube was used as a standard. The bound and free fractions were separated by precipication with 0.5 ml of 8% polyethylene glycol 6000 followed by centrifugation (4000 g, 20 min). The radioactivity in the precipitates was counted in a Wallac Clinigamma 1272 gamma-counter. Sensitivity of an assay was 10 pg/tube. Inter- and intra-assay coefficients of variation were < 10% and < 15%, respectively. Rat ANP, BNP and CNP cross-reacted less than 0.1% in the assay.

4.4.2 Radioimmunoassay of NT-pro-sCP (II, III)

Because of the lower concentration and lability of sCP, an extraction step appears to be mandatory for the reliable measurement of sCP from plasma or serum. The sample volume (at least 0.2 ml) required for the sCP RIA sets a limit to the number of serial samples that can be drawn from the same animal. To circumvent these problems, we hypothesized that the 97-amino acid aminoterminal fragment of pro-sCP (NT-pro-sCP) (Majalahti-Palviainen et al. 2000) could be used to assess the release of sCP in salmon analogously with the utilization of NT-proANP and NT-proBNP measurements in mammals. Thus, we developed a specific RIA for NT-pro-sCP suitable for the direct measurement of NT-pro-sCP in salmon serum.

Full-length cDNA (Tervonen et al. 1998) was used as the template to amplify the sequence encoding the NT-pro-sCP (pro-sCP 1-97) (Majalahti-Palviainen et al. 2000). A recombinant plasmid expressing GST-NT-pro-sCP was prepared. Thrombin digestion (Chang 1985) was used to cleave GST from NT-pro-sCP. The purified recombinant protein was used for development of specific RIA for NT-pro-sCP. Recombinant GST-NT-pro-sCP fusion protein was emulsified with Freund's complete adjuvant (Difco Laboratories, Detroit, MI, USA) and injected at multiple sites at the back of a goat. Two booster injections with one-half the amount of antigen emulsified in incomplete adjuvant were given at monthly intervals.

Recombinant NT-pro-sCP was radioiodinated with the chloramine-T technique (Greenwood et al. 1963) and purified by desalting in a 2-ml Sephadex G-25 M column,

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followed by final purification by reverse-phase-HPLC using a 4.6 x 250-mm Vydac C18 column eluted with a 40-min 10-50% acetonitrile gradient in aqueous 0.1% TFA. The flow rate was 1 ml/min. Fractions of 1 ml were collected and monitored for radioactivity in a Multi-Gamma gamma counter (Wallac, Turku, Finland). When stored at -20°C the radioiodinated peptide was usable for approximately six weeks.

Recombinant NT-pro-sCP ranging from 5 to 1250 pg/tube was pipetted in duplicates of 100 µl for use as standard. The serum samples (2 µl) were diluted 1/50 with assay buffer, and duplicates of 100 µl were used directly in NT-pro-sCP radioimmunoassay. Goat anti-NT-pro-sCP serum (1/75000 final dilution) was added (100 µl), and the tubes were incubated overnight at 4°C. 125I-NT-pro-sCP (100 µl) was added, followed by another overnight incubation at 4°C. The bound and free fractions were separated by precipitation with 0.5 ml of 8% polyethylene glycol 6000 containing donkey anti-goat IgG antiserum (Scantibodies Laboratory, Santee, CA, USA) and normal goat serum (1 µl) as carrier. After centrifugation (2000g for 15 min at 8°C) and decantation of the supernatants, the precipitates were counted in a Wallac Clinigamma 1272 gamma-counter.

4.5 Immunoelectron microscopy (I)

Immunoelectron microscopic methods were used in order to localize sCP in salmon cardiac tissue. Standard methods described previously for the localization of rat cardiac ANP (Rinne et al. 1986) were utilized, with the exception that the antiserum was the goat anti-sCP used in the radioimmunoassay, and the antigen used in the specificity studies to displace the staining was synthetic sCP-29.

4.6 Gel filtration high performance liquid chromatography (I, III)

Gel filtration high performance liquid chromatography and the radioimmunoassays of sCP and NT-pro-sCP were used to characterize the molecular form of salmon cardiac peptide in the cardiac tissue, blood and the perfusates from salmon ventricle. The tissue samples were pulverized in liquid N2 and homogenized in 4.5 vol of boiling H2O and 4.5 vol of 2 M acetic acid/40 mM HCl. The homogenates were centrifuged (15000 g, 30 min at 4°C), and the supernatants were lyophilized and dissolved in 300 µl of 40% acetonitrile in aqueous TFA for use in chromatography. The molecular form of immunoreactive sCP was also characterized from aliquots of the guanidine isothiocyanate supernatants obtained from RNA extraction, which were diluted in 400 µl 40% acetonitrile in aqueous 0.1% TFA.

Samples (3 ml) of the ventricular perfusates were first extracted with SepPak C18 cartridges as follows. The perfusate samples were acidified with 600 µl 1 M HCl, 1.6% glycine and applied into the cartridges preactivated with isopropanol and aqueous 0.1% TFA. After a wash with 10 ml 0.1% TFA, the peptide fraction was eluted with 2 ml 80% acetonitrile in aqueous 0.1% TFA. The extracts were dried in a SpeedVac concentrator and reconstituted in 400 µl 40% acetonitrile in aqueous 0.1 % TFA.

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A salmon serum sample (300 µl) was mixed with 200 µl 0.25% TFA in acetonitrile. The precipitate was removed by centrifugation at 10000 g for 15 min, and the supernatant was passed through 0.45-µm Millipore filters. The clear sample was applied through a Rheodyne loop injector to a Waters Proteinpak 125 HPLC gel filtration column (3.9 x 300 mm), eluted with 40% acetonitrile in aqueous 0.1% TFA at the flow rate of 1 ml/min. Fractions of 0.5 ml were collected and dried in a SpeedVac concentrator. The dried residues were dissolved in 0.5 ml assay buffer for use in sCP and NT-pro-sCP radioimmunoassays. The column was calibrated with BSA (68 kD, void volume), rat or human proANP (14 kD), synthetic sCP-29 (3 kD) and 125I (total volume).

4.7 Isolation of mRNA and Northern blot analysis (I, II)

Northern blot analysis was used to detect sCP mRNA in the tissue samples. The ventricles were frozen and stored at -70°C. For RNA isolation, the ventricular samples were ground to powder in liquid N2 and homogenized in 4 M guanidine isothiocyanate, 0.025 M sodium citrate, pH 7, 0.1 M 2-mercaptoethanol, 0.5% N-lauroylsarcosine. An aliquot of each homogenate was stored at -70°C for use in RIA. Total RNA was isolated from the remaining homogenates with the acidic phenol method (Chomczynski & Sacchi 1987). The RNA was size-fractionated on 1% formaldehyde agarose gels, blotted to nylon membrane and fixed by baking at 80°C for 2 h. The membrane was hybridized overnight with a [32P]-dCTP-sCP cDNA probe (Tervonen et al. 1998) labelled to a specific activity of > 108 dpm/µg by random-primed labelling and purified by Sephadex G-50 gel filtration. The hybridization membranes were washed with 0.2 x saline-sodium citrate (SSC) and 0.2% sodium dodecyl sulfate (SDS) for 60 min at 65°C and used to expose X-OMat films (Kodak, Rochester, USA) with an intensifying screen at -70°C for 3.5 h. To control potential differences in the RNA load, a 482 bp cDNA probe, corresponding to the sequence 922-1403 of the rat gene for 18S ribosomal RNA (Torczynski et al. 1983), was prepared by RT-PCR, cloned to the EcoRI site of pBlueScript SKII+, and the sequence was confirmed using the dideoxy method. The insert was labelled and used in the hybridizations as described for sCP.

4.8 Extraction of total RNA and quantitative PCR (IV)

For quantitative PCR, total RNA was extracted using a kit (GenEluteTM Mammalian Total RNA Miniprep Kit, Sigma). The first strand cDNA was synthesized from RNA using MMLV reverse transcriptase. The quantitative PCR reactions were performed with ABI 7700 Sequence Detection System using TaqMan chemistry, primers and bifunctional probes for sCP and 18S RNA as described (Majalahti-Palviainen et al. 2000).

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4.9 Statistical analyses

Results are expressed as mean ± SE. The data from repeated measurements were analysed with one-way or two-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test, Holm-Sidak post-hoc test or Dunnett’s test (SPSS Inc., Chicago, USA or SigmaStat 3.11, Systat Software Inc, Point Richmond, USA). For the comparison of statistical significance between two groups, Student t-test for paired and unpaired data was used (InStat, GraphPad Software, Inc., CA, USA). In the case the variances were non-homogeneous, the non-parametric Mann-Whitney test was used (II). Correlation coefficients were calculated by linear regression analysis. The statistical test results are expressed as the two-sided P-value, and P < 0.05 was considered statistically significant.

5 Results

5.1 Molecular forms of stored and secreted sCP (I)

To characterize the nature of immunoreactive sCP stored in the heart and secreted to the perfusion fluid or blood circulation, the extracts were separated with gel filtration HPLC and the fractions analysed by sCP radioimmunoassay. In cardiac tissue extracts the immunoreactivity consisted mainly of high-molecular-weight material (10–15 kDa), which corresponds to the size of pro-sCP (Fig. 6A). On the other hand, the immunoreactive sCP present in the perfusates was due to approximately 3-kDa material, which coelutes with the 29-amino acid synthetic sCP standard (Fig. 6B). The difference in the molecular forms between the stored and secreted peptide was not an artefact caused by the extraction of the perfusate samples, because gel filtration of unextracted perfusate samples from mechanically loaded ventricles displayed the same chromatographic pattern as that of samples with SepPak extraction. Mechanical load of 13 cm H2O subjected onto ventricular tissue did not alter the gel filtration pattern of immunoreactive sCP in the perfusate (Fig. 6).

5.2 Radioimmunoassay for measuring the aminoterminal fragment of pro-sCP (NT-pro-sCP) (III)

A specific radioimmunoassay for the aminoterminal fragment of pro-sCP (NT-pro-sCP) suitable for the direct measurement of NT-pro-sCP in salmon serum was developed. A recombinant expression plasmid was constructed, containing the sequences encoding GST and NT-pro-sCP. The fusion protein was used as such as an immunogen to raise the anti-NT-pro-sCP antiserum. GST was removed from the fusion protein by thrombin digestion, and NT-pro-sCP was purified to homogeneity by RP-HPLC. The apparent molecular weight (10–15 kDa), as assessed by gel filtration HPLC, was consistent with the expected molecular weight (11,026 kDa) of NT-pro-sCP. The authenticity of recombinant NT-pro-sCP was further confirmed by 10 cycles of NH2-terminal sequence analysis.

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Fig. 6. HPLC gel filtration analyses of immunoreactive sCP present in acid extracts of salmon ventricles (A) and SepPak C18 extracts of ventricle perfusates (B). Open circles = tissue or perfusate sample obtained from experiment without mechanical load, closed circles = sample obtained during (perfusate) or after (tissue) mechanical load of 13 cm H2O. The elution positions of BSA (V0), rat proANP, sCP-29, and 125I (Vtot), used for calibration of the column, are shown by arrows.

The goat anti-NT-pro-sCP antiserum was specific for the NT-pro-sCP sequence and did not cross-react (molar cross-reaction < 0.1%) with synthetic sCP, human ANP, human BNP, human CNP, eel ANP, rainbow trout VNP or human ET-1, or with recombinant human NT-pro-ANP or human NT-pro-BNP. In the RIA the least detectable dose varied from 0.500 to 1.400 fmol/tube, which corresponds to circulating concentrations of 0.250-0.700 nmol/l when serum was assayed according to the incubation protocol described in Materials and methods (see section 4.4.2). The high sensitivity resulted in two advantages. Serum NT-pro-sCP could be assayed without extraction, and only a very small amount of serum (2 x 2 µl) was required for the assay. Serial dilutions of salmon serum diluted in parallel with the NT-pro-sCP standard, and thus the assay fulfils the basic requirement for use in quantification of NT-pro-sCP. The recovery of NT-pro-sCP added to salmon serum (99.8 + 3.2%, n = 3) was linear in the concentration range 1-11 nmol/l (y = 1.06x-0.38, r2 = 0.999). The intra- and inter-assay coefficients of variation were 7 (n = 10) and 14% (n = 7), respectively.

Since mechanical load is a potent stimulator of sCP release from salmon heart, the effect of loading on the release of NT-pro-sCP from ventricle was studied using an isolated salmon ventricle preparation in vitro. We found that the secretion of immunoreactive NT-pro-sCP increased dose-dependently with load, in parallel with sCP. This finding provides strong functional validation for the novel NT-pro-sCP assay as a marker of the activity of the sCP system.

5.3 Effects of mechanical load on sCP secretion (I)

Mammalian studies have shown that stretching of atrial and ventricular heart cells stimulates the secretion of cardiac natriuretic peptides (Lang et al. 1985, Kinnunen et al.

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1993, Mäntymaa et al. 1993). In order to study sCP release from salmon ventricle, a perfusion system of isolated salmon ventricle was set up (see Fig. 5). This in vitro set-up enables manipulation of the mechanical load imposed on the tissue and the collection of samples of the perfusate for hormone assays. In addition to the sCP measurements from perfusates, ventricular immunoreactive sCP and sCP mRNA levels were also determined.

5.3.1 Effect of mechanical load on the release of sCP from the isolated salmon ventricle

When isolated salmon ventricles were perfused at a constant temperature (6°C), the level of immunoreactive sCP released from the ventricle remained stable at 80 ± 9.2 pmol/l (mean ± SE) during the 30-min study period (Fig. 7) when no mechanical load was applied. When the mechanical load of the ventricle was increased, an almost instantaneous load-dependent elevation of the perfusate sCP levels was observed. The load of 4 cm H2O caused a 2.0 ± 0.3-fold increase in the perfusate sCP level (Fig. 7, P < 0.01), and the 8 cm H2O load a 3.0 ± 0.6-fold increase (Fig. 7, P < 0.01). Further increase of the load to 13 cm H2O resulted in a 3.3 ± 0.8-fold increase (Fig. 7, P < 0.01).

5.3.2 Effect of mechanical load on the ventricular immunoreactive sCP and sCP mRNA level

The results showed that mechanical load is a potent inducer of sCP release from isolated salmon ventricle. Therefore, the effect of mechanical load on ventricular storage was studied. Immunoelectron microscopic results demonstrated the presence of a large number of granules containing immunoreactive sCP in salmon ventricle. In addition, a high level of immunoreactive sCP was detectable in the ventricle in the basal condition (5.03 ± 0.50 nmol/g, n = 6), as well as after it had been mechanically loaded with 13 cm H2O for 20 min (6.95 ± 0.62 nmol/g, n = 8), with no statistically significant difference between the two.

In order to estimate the sCP gene expression activity, the ventricular sCP mRNA level was analysed using the Northern blot method. When mechanically loaded for up to 2 hours, the sCP mRNA level was not significantly increased.

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Fig. 7. Release of sCP from isolated perfused salmon ventricle in response to mechanical load (time points 5–25 min) produced by raising perfusate outflow height by 0 (A, n = 4), 4 (B, n = 6), 8 (C, n = 7), or 13 cm H2O (D, n = 8). Results (mean ± SE) were calculated by dividing sCP level at each time point by mean level during 0–5 min (basal level).

5.4 Effects of different temperatures on sCP secretion (II)

We hypothesized that sCP may be important for the adaptation of the salmon cardiovascular system to the large annual variations in water temperature. Therefore, the effects of different temperatures on sCP levels in plasma and cardiac tissue and its mRNA levels in cardiac tissue were determined. The possible effect of the stage of maturation was taken into account by sampling young parrs and adult salmon separately.

5.4.1 Seasonal levels of plasma and cardiac immunoreactive sCP

The circulating levels of sCP showed remarkable seasonal changes, with elevated levels during the summer and low levels during the winter (n = 5 per group in each sampling

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month). The plasma levels of sCP correlated positively with the ambient water temperature (Table 2, r = 0.65, P < 0.001 in parrs and r = 0.58, P < 0.001 in adults). The increase in plasma sCP level during the summer period was especially prominent in parrs (from 157 ± 25 pmol/l in March to 2805 ± 998 pmol/l in September). In adult salmon the levels increased from 250 ± 46 pmol/ in May to 663 ± 46 pmol/l in June. As a result, plasma sCP levels were higher in parrs than in adults during the months of June, July and September (P < 0.001, P < 0.01 and P < 0.01, respectively).

The fluctuations of atrial and ventricular immunoreactive sCP levels were similar to those observed in plasma sCP. In adults, the tissue sCP levels were significantly higher (P < 0.01) during the late summer and early autumn (July–September) than those during the remaining months. The concentration of sCP in the parr atrium ranged from 24 ± 3 nmol/g in May to 54 ± 6 nmol/g in September. In adult salmon the atrial sCP concentrations were higher than those in the parrs, varying from 36 ± 9 nmol/g in February to 73 ± nmol/g in September. The ventricular sCP concentrations in parrs varied from 5.3 ± 0.7 nmol/g in July to 7.8 ± 0.8 nmol/g in August and in adults from 4.3 ± 1 nmol/g in December to 14.7 ± 1.9 nmol/g in August. Although the seasonal variations were similar in parrs and in adults, sCP levels in adult salmon were higher in the atrium in June and July (P < 0.05) and in the ventricle in August and September (P < 0.01) compared with those in the parrs.

Table 2. Summary of the correlation of the seasonal level of cardiac sCP mRNA, and plasma and cardiac sCP with water temperature. + indicates a positive correlation, - a negative correlation, and ns no significant correlation.

sCP mRNA sCP Parr Adult salmon Atrium - ns Ventricle - - Plasma + + Atrium ns ns Ventricle ns ns

5.4.2 Seasonal levels of cardiac sCP mRNA

In addition to the significant seasonal variations in circulating sCP levels, marked seasonal variations were also observed in cardiac sCP mRNA levels (n = 5 per group in each sampling month). However, the mRNA levels tended to be higher during the winter when plasma and cardiac hormone levels were low. There was an inverse correlation between the plasma levels of sCP and those of sCP mRNA in the ventricles of parrs and adults (r = - 0.3, P < 0.05 for both), and between temperature and ventricular sCP mRNA concentration of parrs and adults (Table 2, r = - 0.53, P < 0.001 and r = - 0.5, P < 0.01, respectively). In the parrs, the atrial sCP mRNA level was inversely correlated with water temperature as well (Table 2, r = - 0.49, P < 0.001).

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5.4.3 Seasonal relative ventricle mass

The lowest relative ventricle mass (ventricle mass/BW * 100, n = 5 per group in each sampling month) was detected in the warm-acclimatized salmon, in August (0.065 ± 0.003 in parrs, 0.099 ± 0.008 in adults), and the highest in March in salmon that had acclimatized to temperatures near 0°C for 5 months (0.12 ± 0.0018 in parrs, 0.13 ± 0.007 in adults). In both groups, the increase was significant (P < 0.05).

5.4.4 Effects of different acclimation temperatures on sCP secretion

The effect of temperature on secretion of sCP was studied using the in vitro perfused ventricle model (see section 4.2.2). Isolated salmon ventricles, obtained from salmon acclimatized to 0°C and subsequently acclimated to 6°C and 12°C, were perfused in corresponding temperatures. The amount of sCP secreted from the ventricle without any extra mechanical load was not different in salmon acclimated to the different temperatures (Table 3, 460 ± 94 pmol/l/g ventricle tissue at 0.2°C, n = 10; 459 ± 16 pmol/l/g ventricle tissue at 6°C, n = 10; 314 ± 66 pmol/l/g ventricle tissue at 12°C, n = 10). In ventricles obtained from fish acclimatized to 0.2°C, the load of 13 cm H2O increased the release of sCP 5.3 ± 0.6-fold, compared with 3.5 ± 0.5–fold and 3.7 ± 0.7–fold in ventricles acclimated to 6 and 12°C, respectively (Table 3, n = 6 per for loaded ventricles, n = 4 for unloaded ventricles).

Even if the sCP secretion remained at similar levels in different temperatures, the blood level of sCP, however, was higher in the warm-acclimated salmon used for ventricle perfusion studies than in cold-acclimatized fish (Table 3). In salmon acclimatized at 0.2°C, the plasma sCP level was 148 ± 16 pmol/l, and after 10 days at 6°C it was 244 ± 46 pmol/l. In salmon in which the temperature was further increased to 12°C, the plasma level of 471 ± 102 pmol/l was higher than that in 6°C (P < 0.05). The circulating sCP correlated with the acclimation temperature (r = 0.63, P < 0.001). Since the N-terminal fragment is released to the circulation in equimolar amounts with sCP, corresponding responses were expected for NT-pro-sCP. Interestingly, plasma levels of NT-pro-sCP were, however, not elevated in response to the increase in temperature (Table 3).

The sCP mRNA level in the ventricles used for the perfusion experiments was lower when the tissue was obtained from salmon acclimated to 12°C rather than from those acclimated to 6°C or acclimatized to 0.2°C (Table 3). The difference was found to be associated with the temperature (r = -0.68, P < 0.01 in control experiments; r = -0.91, P < 0.001 in experiments with mechanical load). However, the peptide level was higher in the ventricles obtained from warm-acclimated salmon than in those from cold-acclimated salmon (10.2 ± 1.9 nmol/l/g ventricle tissue at 12°C, n = 6; 8.9 ± 1.0 nmol/l/g ventricle tissue at 6°C, n = 6; and 6.4 ± 0.7 nmol/l/g ventricle tissue at 0.2°C, n = 4). The sCP level correlated significantly with temperature in non-loaded ventricles (Table 3, r = 0.55, P < 0.05).

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Table 3. Summary of the effects of increased temperature on the secretion or plasma concentration of sCP, and ventricular hormone and mRNA level. An arrow indicates increase or decrease, and a dash no change.

Treatment Ventricular sCP mRNA

Ventricular sCP sCP secretion Plasma sCP Plasma NT-pro-sCP

Increased temperature

↑ -

loaded ↓ - ↓ non-loaded ↓ ↑ -

5.5 Effects of endothelin-1 on sCP secretion

In mammalian experiments, ET-1 has been found to be the potential paracrine factor inducing cardiac ANP and BNP secretion (Mäntymaa et al. 1990, Bruneau & de Bold 1994, Bruneau et al. 1997, Magga et al. 1997). To determine whether also sCP release can be induced by ET-1 the effects of human ET-1 on sCP release were examined in vitro in perfused salmon ventricle and in vivo using chronically cannulated salmon.

5.5.1 Effects of endothelin-1 in sCP release from salmon ventricle in vitro (IV)

To find out whether ET-1 is able to stimulate sCP secretion from salmon ventricle, the effects of ET-1 on sCP release from isolated perfused salmon ventricle in basal conditions and during mechanical load were studied. In the control experiments sCP release declined steadily during the 40-min perfusion period to approximately 50% of the starting level (Fig. 8A). This decline was counteracted by 5 nM

human ET-1 (P < 0.01), which was

therefore able to stimulate sCP secretion (Fig. 8B). Mechanical load (13 cm H2O) resulted in a 3.40 ± 0.081-fold increase of sCP release (Fig. 8C, P < 0.001). ET-1 did not affect the peak sCP release during mechanical load, but it did result in an elevated sCP secretion compared to control after the load was released (Fig. 8C). A specific type A endothelin receptor (ETA) antagonist BQ-610 (125 nM) inhibited significantly the stimulatory effect of mechanical load on sCP release (Fig. 8D, P < 0.01).

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Fig. 8. Release of sCP from isolated perfused salmon ventricle in response to mechanical load of 13 cm H2O (A), 5 nM ET-1 (B), mechanical load in the presence of ET-1 (C), and mechanical load in the presence of 125 nM ETA antagonist BQ-610. Results (means ± SE) were calculated by dividing sCP level at each time point by mean level during 0–5 min (basal level).*P < 0.05, **P < 0.01, ***P < 0.001.

5.5.2 Effects of endothelin-1 on sCP secretion in vivo (III)

The effects of ET-1 on the sCP system in vivo were studied using chronically cannulated salmon. This also served as a test of the usefulness of the novel NT-pro-sCP assay for monitoring the endocrine function of salmon heart. sCP was assayed simultaneously to serve as a reference. However, because of the need for a larger sample volume and extraction, sCP could only be assayed in a subset of the samples.

Serum levels of immunoreactive NT-pro-sCP remained relatively stable during the control experiments, varying between 0.552 + 0.038 nmol/l and 0.506 + 0.040 nmol/l (Fig. 9A). ET-1 administration caused a relatively slow but large increase of serum NT-pro-sCP levels from 0.987 + 0.112 nmol/l at 0 min to 4.61 + 1.49 nmol/l at the end of experiment (Fig. 9A, P < 0.001). A parallel increase of serum sCP was observed in response to the ET-1 bolus, with levels increasing from 0.063 + 0.009 nmol/l to 0.949 + 0.459 nmol/l (Fig. 9B, P < 0.01). In control experiments, serum levels of sCP remained stable throughout the experiment, varying from 0.042 + 0.001 nmol/l to 0.053 + 0.005

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nmol/l (Fig. 9B). Levels of NT-pro-sCP showed a good linear correlation (r2 = 0.75) with those of sCP in the same samples. The levels of NT-pro-sCP were on the average 1.70 nmol/l higher than those of sCP in the same samples.

Fig. 9. Effect of intra-arterial bolus injection of human ET-1 (1 µg) on serum immunoreactive NT-pro-sCP (A) and sCP (B). Open circles = injection of saline vehicle (n = 3), closed circles = injection of human ET-1 (n = 7). ). *P < 0.05, **P < 0.01, ***P < 0.001.

To find out how the effect of ET-1 on the sCP system is mediated, the specific antagonists BQ-123 and BQ-788 of the ETA and ETB receptors, respectively, were used. In vehicle control experiments, the ET-1 bolus injection caused a 2.3-fold elevation of serum NT-pro-sCP levels (Fig. 10, P < 0.05). Neither of the antagonists appeared to affect the basal serum level of NT-pro-sCP (Fig. 10). BQ-123, however, almost completely abolished the stimulatory effect of ET-1 (Fig. 10, NT-pro-sCP levels were 0.57 ± 0.13 nmol/l vs. 0.69 ± 17 nmol/l in samples taken before the ET-1 administration and 40 min after it, respectively, P < 0.05). On the other hand, BQ-788 did not have any effect on the NT-pro-sCP response to ET-1 (Fig. 10, 0.42 ± 0.06 nmol/l and 1.58 ± 0.51 nmol/l before and at 40 min after ET-1, respectively).

Gel filtration HPLC analyses and NT-pro-sCP radioimmunoassay were performed to characterize the molecular size of the immunoreactive material present in salmon serum. One peak of immunoreactivity, with sizes between those of proANP and sCP, was detected in both samples obtained in basal conditions and after stimulation by human ET-1. This immunoreactive material corresponds to NT-pro-sCP. However, the immunoreactivity in the cardiac tissue extracts consisted of material with a higher molecular weight than NT-pro-sCP, which probably corresponds to pro-sCP (in agreement with the results shown in Fig. 7A).

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Fig. 10. Effect of ETA antagonist BQ-123 and ETB antagonist BQ-788 on human ET-1-induced secretion of NT-pro-sCP in salmon in vivo. Closed circles = ET-1 alone (1 µg, n = 5), open circles = ET-1 and BQ-123 (n = 6), closed squares = ET-1 and BQ-788 (n = 6). Antagonists were used in 25–fold molar excess over ET-1. *P < 0.05, **P < 0.01, ***P < 0.001 compared with 0 min.

5.5.3 Effects of endothelin-1 on sCP mRNA levels (IV)

ET-1 has a stimulatory effect on sCP release and mammalian natriuretic peptide gene expression (Bruneau & de Bold 1994). Therefore, the effects of ET-1 on salmon cardiac sCP gene expression were studied. In isolated perfused salmon ventricle, three-hour treatment with cumulatively increasing doses of human ET-1 (from 1 to 100 nM) failed to have any effect on ventricular sCP mRNA levels, as tested by real time quantitative RT-PCR.

5.6 Interactions between endothelin-1 and β-adrenergic activation on the regulation of salmon cardiac contractile and

endocrine function (IV)

ET-1 and the β-adrenergic system are major regulators of cardiac function in mammals. It seems likely that cardiac function is regulated by crosstalk of ET-1 and β-adrenergic activation as well as several other endogenous regulators. Therefore, the role of ET-1 in cardiac function and the possible interactions between ET-1 and β-adrenergic activation were examined in a salmon model. These ideas were tested by monitoring myocardial strip preparation and perfused isolated salmon ventricle contractile function and by measuring sCP secretion from salmon ventricle in response to human ET-1 and isoprenaline.

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5.6.1 Effects of endothelin-1 and β-adrenergic activation on salmon cardiac contractile function

Frank-Starling responses were determined to characterize the contractile function of the salmon ventricular muscle strip preparations (see section 4.2.1). The change in developed force at a given resting fibre length was measured directly in vitro on the basis of the peak isometric tension developed at a fixed initial fibre length. Stepwise increases of stretch (0.5 mm/step) resulted in well-developed Frank-Starling responses.

To find out the effects of ET-1 on salmon cardiac contractile function, varying doses of ET-1 were added to the perfusion medium of the myocardial strip preparations and intact salmon ventricle preparations (see section 4.2.2). The receptor subtype responsible for the inotropic effect of ET-1 and the possible interaction of the effects of ET-1 and isoprenaline (β-adrenergic stimulation) on cardiac function were also studied.

In myocardial strip preparations ET-1 increased the contraction amplitude in a dose-dependent manner. The maximal response, 17 ± 1.3% compared to the basal level (P < 0.001, n = 5), was attained with the highest dose tested (100 nM ET-1). An approximately 10-percent increase (P < 0.05) was observed with as little as 1 nM ET-1. To define whether the inotropic effect is mediated by ETA receptors, similar experiments were performed in the presence of the selective ETA receptor antagonist BQ-610 (125 nM, n = 7). At this dose BQ-610 shifted the ET-1 on contractility dose-response curve to the right, and was able to completely abolish the effect of 1 nM ET-1. As expected, isoprenaline increased markedly (47 ± 9.8%, P < 0.001, n = 6) the contraction amplitude of salmon myocardial strip preparation.

In intact isolated perfused ventricles the contraction force was recorded concomitantly with the sampling of perfusate by measuring the pressure in the lumen of the ventricle. The maximal response, a 17 ± 4.7% increase in contraction amplitude compared to the basal level (P < 0.01), was attained with the concentration of 30 nM ET-1 (Fig. 11A). Isoprenaline (0.5 nM) treatment did not have any significant effect on the response induced by 10 nM ET-1 (Fig. 11A, 25 ± 4.0% increase in contraction amplitude, P > 0.05 compared to ET-1 alone). ETA antagonist (BQ-610) was able to inhibit this response caused by ET-1 and isoprenaline (Fig. 11A, P < 0.01). On the other hand, ETB

receptor antagonist (BQ-788) did not have any effect on the ET-1 response (data not shown).

The maximal response of isoprenaline (100 nM) was a 30 ± 13.1% increase in contraction amplitude compared to the control experiments (Fig. 11B, P < 0.05). ET-1 or ET receptor antagonists did not have any significant effect on the responses caused by isoprenaline (data not shown). ETA or ETB

receptor antagonist alone had no significant effect on contraction amplitude (data not shown), nor did ET-1 or isoprenaline have any significant effect on time parameters of isometric twitch compared to control experiments.

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Fig. 11. A) Dose-response relationship of ET-1 and contraction amplitude of isolated salmon ventricle (exposed to each dose for 30 min, mean ± SE). Control (n = 3), ET-1 alone, ET-1 in the presence of isoprenaline (ISO) (n = 4), ET-1 in the presence of ISO and BQ-610 (n = 6) ETA antagonist (BQ-610 1 µM) and ISO (0.5 nM) were added 30 min before the first ET dose. B) Dose-response relationship of ISO and contraction amplitude of isolated salmon ventricle. Control (n = 3), ISO alone (n = 5), and ISO in the presence of ET-1 (n = 5). ETA antagonist and ET-1 (10 nM) were added 30 min before the first ISO dose. *P < 0.05, **P < 0.01, ***P < 0.001.

5.6.2 Effects of endothelin-1 and β-adrenergic activation on sCP secretion from isolated perfused salmon ventricle

In the control experiments sCP release declined steadily during the 210-min perfusion period to approximately 30% of the starting level (Fig. 12A). This decline was counteracted by 10 – 100 nM ET-1. ET-1 (10 nM) increased the release of sCP 3.3 ± 0.14 times over the control (Fig. 12A, P < 0.05). The ETA antagonist BQ-610 did not block the effects of ET-1, whereas the ETB antagonist BQ-788 (100 nM) enhanced the effect of ET-1 (Fig. 12D, P < 0.01). Isoprenaline (0.1–1000 nM) did not affect sCP secretion when tested alone (Fig. 12E). However, isoprenaline (0.1 nM) caused a marked increase of sCP release (a 5.4 ± 0.07 time increase over the control) in the presence of 10 nM ET-1 (Fig. 12E, P < 0.01 compared to isoprenaline alone), which was partly blocked by BQ-610 (Fig. 12G, P < 0.05), but not BQ-788 (Fig. 12H). BQ-610 alone did not affect sCP release (Fig. 12A). BQ-788, however, markedly stimulated the basal sCP release compared to control experiments (Fig. 12A, P < 0.05).

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Fig. 12. A-D) Effects of ET-1 on sCP secretion from isolated salmon ventricle. A) Control, ET-1 alone, ET-1 in the presence of isoprenaline (ISO), BQ-610 alone, and BQ-788 alone, B) ET-1 in the presence BQ-610, C) ET-1 in the presence of ISO and BQ-610, and D) ET-1 in the presence of BQ-788. ETA antagonist (BQ-610 1 µM), ETB antagonist (BQ-788 100 nM) and ISO (0.5 nM) were added 30 min before the first ET dose. E-H) Effects of ISO on sCP secretion from isolated salmon ventricle. E) Control, ISO alone, and ISO in the presence of ET-1, F) ISO in the presence of BQ-610, G) ISO in the presence of ET-1, and ISO in the presence of ET-1 and BQ-610, and H) ISO in the presence of ET-1 and BQ-788. ETA antagonist, ETB antagonist and ET-1 (10 nM) were added 30 min before the first ISO dose. Ventricles were exposed to each dose for 30 min. *P < 0.05, **P < 0.01, ***P < 0.001.

6 Discussion

The aim of this study was to elucidate the factors regulating the novel cardiac peptide hormone, sCP, and to characterize its secretion process. The results showed that salmon ventricle stores large amounts of the prohormone of sCP, whereas the secreted circulating form is the biologically active form of the peptide. The N-terminal fragment of pro-sCP (NT-pro-sCP) is co-secreted with sCP in equimolar amounts. sCP is released rapidly in response to appropriate stimulus, whereas induction of its gene expression is a slower process.

Mechanical load increases dose-dependently the release of sCP from salmon ventricle and is therefore an important regulator of sCP secretion. Temperature also plays a major role in regulating sCP by affecting its elimination from circulation. ET-1 turned out to be a potent secretagogue of the sCP system and an inotropic agent in salmon heart. Furthermore, the present results reveal interesting synergism between the cardiac effects of ET-1 and β-adrenergic stimulation.

Thus, the sCP system in salmon ventricle resembles largely the ANP system in mammalian atrium and can be used for studying general mechanisms in natriuretic peptide biology. On the other hand, due to their specific characteristics salmon ventricle and sCP offer a unique model for comparative and evolutionary studies.

6.1 Stored and secreted sCP

The results of this study showed that large amounts of immunoreactive sCP and sCP mRNA can be found in salmon ventricle. The concentration of sCP in salmon ventricle (5030 ± 500 pmol/g) is much higher than that of ANP (30 pmol /g), BNP (3 pmol/g) (Minamino et al. 1993) or CNP (0.6 pmol/g) in rat ventricle (Minamino et al. 1993), and of VNP (1600 pmol/g) in eel ventricle (Takei et al. 1994a). In addition, a large number of vesicles staining with anti-sCP antiserum and with typical morphologic features of secretory granules were detected in salmon ventricular myocytes. Thus, salmon ventricle, with high basal levels of mRNA and hormone stores, produces sCP in a similar manner as mammalian atrium, or as foetal ventricle produces ANP. This differs significantly from

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the production of ANP and BNP in postnatal mammalian ventricle, which appears to be made on demand in non-pathological situations (Nakao et al. 1992a).

Gel filtration HPLC analyses showed that the molecular weight of immunoreactive sCP stored in salmon ventricle (10 kDa) is considerably higher than that of the secreted peptide (3 kDa). The large-molecular-weight form probably corresponds to pro-sCP, and the small-molecular-weight form is the biologically active 29-amino acid form of sCP. One peak of immunoreactivity, with sizes between those of proANP and sCP (which corresponds to NT-pro-sCP), was also detected in salmon serum. It seems that salmon ventricle processes sCP in a manner resembling that of ANP in mammalian atrium. ProANP is processed during the exocytosis of the secretory granules (Vuolteenaho et al. 1985, Dietz 2005, McGrath & de Bold 2005), presumably catalysed by the membrane-bound serine protease corin (Yan et al. 2000).

Hence, as is the case with ANP and NT-proANP, NT-pro-sCP appears to be co-secreted from salmon cardiomyocytes in equimolar quantities with sCP. As a result, the circulating levels of NT-pro-sCP can be utilized to estimate the rate of secretion of sCP from the salmon heart. The basal serum levels of NT-pro-sCP were more than five times higher than those of sCP on a molar basis, which suggests that the elimination of NT-pro-sCP differs markedly from that of sCP. It appears that the fates of circulating sCP and NT-pro-sCP resemble those of mammalian ANP and NT-proANP. ANP is removed quickly from the circulation by neutral endopeptidase-mediated proteolysis and by binding to the A- and C-type natriuretic peptide receptors, whereas NT-proANP appears to be eliminated only slowly via the kidneys (Thibault et al. 1994).

On the other hand, at least in man and rat, proBNP appears to be processed while stored in the heart (Sudoh et al. 1989, Ogawa et al. 1990). The ventricular secretion of mammalian natriuretic peptides, the foremost of which is BNP, is considered to take place via the constitutive pathway (Bloch et al. 1986). The endoprotease furin is considered one of the major processing enzymes of the constitutive secretory pathway (Sawada et al. 1997). With regard to prepro-sCP, however, the lack of an arginine residue at position -4 from the cleavage site of prepro-sCP (Tervonen et al. 1998) makes it an unlikely substrate for furin. While in mammals the ventricular secretion of the natriuretic peptides is believed to take place via the constitutive pathway (Bloch et al. 1986), the present results show that this is not the case with sCP production in the salmon ventricle. The secretion of sCP from the ventricle clearly utilizes the regulated pathway. Therefore, the constitutive pathway is not an inherent property of ventricular hormone release.

6.2 NT-pro-sCP as a marker of sCP activity

When starting the present project, a radioimmunoassay for sCP (Tervonen et al. 1998) was available, usable for both in vitro and in vivo studies. However, because the clearance of circulating sCP is rapid (Tervonen et al. 1998) and may vary considerably in different environmental conditions, the secretion rate of pro-sCP-derived peptides is difficult to assess from the serum levels of sCP. In addition, the instability of sCP necessitates an extraction step, resulting in a relatively large sample volume. In order to study the physiology of the sCP system also in in vivo models, where repeated serum sampling is

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required, the first objective was therefore to set up, with the assays of the N-terminal fragments of mammalian ANP (Sundsfjord et al. 1988) and BNP (Hunt et al. 1997) as models, a RIA for NT-pro-sCP, which could be used with minute amounts of unextracted serum. It also provides a way to assess the contribution of specific elimination pathways to the circulating levels of the biologically active peptide, sCP.

A specific RIA for NT-pro-sCP was developed utilizing the GST-NT-pro-sCP fusion protein for immunization and the purified peptide for radioiodination and standardization. The sensitivity of the assay, combined with the high circulating levels, made it possible to measure NT-pro-sCP levels in very small sample volumes (2 µl serum). In actual experiments this means that a practically unlimited number of serial samples can be drawn from the same animal without any significant volume loss. Parallel measurements, both in basal conditions and when stimulated by ET-1, showed a good correlation between serum NT-pro-sCP and sCP, demonstrating that circulating NT-pro-sCP can be used as an indicator of serum sCP activity. Moreover, in isolated perfused salmon ventricle preparation, mechanical load caused a parallel increase of both sCP and NT-pro-sCP. Thus, in this sense, NT-pro-sCP resembles mammalian NT-proANP, widely used as a useful and practical marker of plasma ANP activity, due to its stability in the circulation (Hall et al. 1994, Kettunen et al. 1994, Schirmer & Omland 1999).

6.3 Factors regulating sCP

6.3.1 Effects of mechanical load on sCP secretion

Unlike mammalian ventricles, teleost ventricular tissue is not dependent on coronary circulation (Farrell 1993). Therefore it can be readily maintained on simple external perfusion. In this perfusion system the load can be controlled by adjusting the height of the outflow path (see Fig. 5). Considering the ease of use of salmon ventricle for in vitro perfusion experiments, sCP is an excellent model to investigate the secretion mechanisms of natriuretic peptides as well as the cellular mechanisms and transduction pathways coupling mechanical stimuli to exocytosis of cellular secretory products.

The results showed that the secretion of ventricular sCP is dependent on mechanical load. A modest increase in load resulted in high induction of the release. In previous studies with rat ventricle, a relatively large load was required in order to induce a moderate increase in ANP secretion (Kinnunen et al. 1992). In salmon ventricle, the maximal response was more than threefold with a 13-cm H2O load, whereas in the rat study a load of 18.2 cm H2O only resulted in a 1.5-fold response (Kinnunen et al. 1992). Thus, sCP appears to be a highly mechanosensitive hormone.

The acute sensitivity of the secretion of sCP to mechanical load resembles that of mammalian atrial ANP. The ventricular stores of sCP are sufficiently large for short-term sCP need, since no significant change can be detected in the ventricular sCP or its mRNA levels after a 20-min stimulation of secretion by mechanical load. Numerous studies have shown that stretching of atrial myocytes in vitro or by volume or pressure load in vivo stimulates the secretion of ANP within minutes, while the activation of ANP gene expression takes hours or days to develop (for review see Ruskoaho 1992, de Bold et al.

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1996). In the case of BNP, on the other hand, mechanical load causes a rapid induction of gene expression, whereas the secretory response is slower to evolve (for review see Ruskoaho 2003). A significant increase in right auricular BNP mRNA levels was found after 1 h of stretching (Mäntymaa et al. 1993).

6.3.2 Effects of temperature on sCP secretion

Physiological functions are strongly influenced by temperature in mammals, and in ectothermic fishes in particular. In this study the regulation of cardiac endocrine function using salmon exposed to extensive seasonal variation in ambient temperature was investigated. The results showed that the plasma levels and production of sCP display significant seasonal variation, with elevated activity associated with high ambient temperature. The responses could be due to increased temperature, or the sCP levels may vary seasonally regardless of changes in temperature. While the level of the expression of the sCP gene appears to be differently regulated in salmon acclimated to cold and warm, the major part of the up-regulation of the plasma peptide levels can be explained by decreased elimination, rather than increased secretion of the peptide at high ambient temperature. According to the present results, salmon maturation does not appear to be associated with major qualitative changes in the regulation of the sCP system, but it may affect the sensitivity of sCP regulation.

6.3.2.1 Effects of temperature on secreted sCP levels

The present results showed that the plasma concentration of sCP is significantly higher in salmon adapted to warm ambient temperature than in those adapted to cold. Elevated sCP plasma levels associated with increasing water temperature can be detected both in animals studied under field conditions and in those acclimated in the laboratory. Elevated sCP plasma levels can result from an increased secretion of the peptide from the heart. On the other hand, increased natriuretic peptide levels may result from a decreased rate of their elimination, since inhibition of the major routes of elimination of ANP, the natriuretic peptide receptor C or neutral endopeptidase have been shown to result in elevated basal plasma ANP levels and a diuretic response in mammalian studies (Kukkonen et al. 1992, Matsukawa et al. 1999).

The results show that the release of immunoreactive sCP from perfused salmon ventricle is not augmented by increased temperature. In fact, there appears to be a slight decrease in the basal release and accelerated fading out of the response to constant loading with increased temperature. In mammalian ventricle (at 37°C) the response to stretch has been found to level out even faster (Kinnunen et al. 1993). The apparent temperature dependence indicates that the levelling off is an active and regulated process, rather than a depletion of peptide stores, as has previously been suggested (Bloch et al. 1986). Thus, the results with the isolated perfused ventricle preparation were inconsistent with increased secretion as an explanation for the high plasma sCP levels in salmon during the warm period of the year. However, the results showed that while warm-

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acclimation of salmon is associated with clearly elevated plasma sCP levels, the levels of the NT-pro-sCP remain stable. N-terminal fragments of natriuretic peptides are produced and secreted in equimolar concentrations with the respective biologically active peptides originated from the same precursors. The elimination of at least the N-terminal fragment of proANP, however, is much slower than that of ANP (Thibault et al. 1988). Therefore, the results of this study indicate that the high plasma levels of sCP in salmon during the warm summer months result primarily from a decreased rate of elimination, rather than increased secretion.

The clearance receptors have an important role in regulating the half-life and the bioavailability of the natriuretic peptides. In fish, the gills have been found to express clearance type and other natriuretic peptide receptors (Katafuchi et al. 1994, Kashiwagi et al. 1995, Takashima et al. 1995, Donald et al. 1997, Callahan et al. 2000). It has been shown that the plasma concentrations of sCP in salmon are much higher in the ventral aorta, receiving blood directly from the heart, than in the peripheral circulation, which receives its blood from the branchial circulation (Tervonen et al. 2000). Therefore, it seems that in salmon, the expression of the clearance-type receptors is down-regulated by acclimation to higher environmental temperatures. This would in turn cause an increased circulating half-life for natriuretic peptides secreted from the heart.

6.3.2.2 Effects of temperature on sCP production in cardiac tissue

Increased ambient temperature was found to be associated with a decrease in sCP mRNA levels in seasonally collected samples as well as in the experiments with in vitro perfused salmon ventricles. However, the cardiac immunoreactive sCP levels were not decreased concomitantly with sCP mRNA levels. The lack of synchronous decrease in the peptide levels could indicate that the sCP mRNA is translated more efficiently at higher temperatures. One explanation could be the variable stability of sCP mRNA according to different temperatures. For example, in winter flounder a significant seasonal variation of antifreeze protein mRNA level seems to be associated with the decreased half-life of mRNA at elevated temperatures, resulting in low tissue and plasma antifreeze protein concentrations (Duncker et al. 1995). In this study possible changes in mRNA stability cannot explain the divergent changes in ventricular and plasma sCP levels, which appear not to have a connection with those of cardiac sCP mRNA. The findings suggest that in low temperatures the production of pro-sCP is actively inhibited at the translational level, or that the half-life of pro-sCP in ventricular tissue is very short. The high circulating sCP levels in warm temperature may also exert a feedback inhibition on the transcription of the sCP gene. In rat it has been shown that ANP can inhibit its own secretion by a NPR-A-mediated mechanism (Leskinen et al. 1997b). Interestingly, the relative ventricle mass also increased in salmon acclimatized to cold. Therefore, the increase in sCP mRNA levels coincides with low temperature-associated hypertrophy of the heart, indicating that sCP might serve as a marker of cardiac hypertrophy in salmon, analogously to ANP and BNP in mammals (for review see Ruskoaho 2003, McGrath et al. 2005).

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6.3.3 Effects of endothelin-1 on sCP secretion in vivo

ET-1 is a potent stimulator of natriuretic peptide release in mammals. Therefore, the efficacy of human ET-1 on sCP system in vivo was examined. The human ET-1 was used because the peptide has not been characterized in fish species. A peptide homologous to human ET-2 has been purified from trout, but trout vessels were found to be more sensitive to mammalian ET-1 than the trout ET-like peptide (Wang et al. 1999). Cardiovascular effects of mammalian ET-1 and the trout ET-2-like peptide have been studied previously (Olson et al. 1991, le Mevel et al. 1999, Wang et al. 1999, Hoagland et al. 2000), but the endocrine and inotropic effects of ET-1 on fish heart are not known.

According to the present results, human ET-1 is a powerful secretagogue of pro-sCP peptides in vivo. The secretion of pro-sCP-derived peptides was monitored with the measurement of NT-pro-sCP in unextracted serum samples. The level of immunoreactive sCP in a subset of serum samples was also determined for comparative purposes. However, in vivo experiments the potent haemodynamic effects of ET-1, such as an increase in blood pressure (Yanagisawa et al. 1988, Yamamoto et al. 1991), could explain at least part of the response. In previous studies with the teleost fish rainbow trout, ET-1 has been found to cause several short-term oscillations in blood pressure followed by a sustained hypertensive period (Olson et al. 1991, le Mevel et al. 1999, Hoagland et al. 2000). Therefore, action of ET-1 on sCP secretion in vivo may be, in part, indirect.

Previous studies in mammals have shown that ET-1 stimulates atrial ANP and BNP secretion by an ETA-mediated mechanism (Irons et al. 1993, Leite et al. 1994, Thibault et al. 1994). In the present study a specific ETA antagonist, but not an ETB antagonist, was capable of inhibiting the ET-1-induced activation of the sCP system in vivo. Neither of the antagonists had any significant effect on the basal levels of the sCP peptides. The results demonstrate that ET-1 has similar potent effects and mechanism of action on the cardiac natriuretic peptides in such phylogenetically distant species as rat and salmon. This indicates a fundamental role for ET-1 in regulating the endocrine function of the heart.

6.3.4 Effects of endothelin-1 in vitro on sCP secretion and its synergistic action with β-adrenergic activation

ET-1 turned out to be a potent secretagogue of the sCP system in vivo. To find out whether ET-1 has direct cardiac effects, the effects of human ET-1 were studied in an isolated perfused salmon ventricle preparation setup. In addition, the interactions between two important regulators of heart function, ET-1 and β-adrenergic stimulation, both of which are activated in many pathophysiologic conditions, were studied.

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6.3.4.1 Effects of endothelin-1 and β-adrenergic activation on salmon cardiac contractile function

ET-1 was found to have a positive inotropic effect, suggesting that it has a role in the regulation of the contractile function of the salmon heart. The maximal increase in the contraction amplitude was, however, less than that produced by isoprenaline that served as a positive control in this study. While the maximal response in salmon intact ventricle was observed with 30 nM ET-1, a dose as low as 1 nM ET-1 was able to produce an about 11–13% increase in the contraction amplitude. The ratio of potency of ET-1 over that of isoprenaline appears to be highly variable, depending on the animal species studied. For example, in the rat the effect of ET-1 reached the maximum at 3 nM, and the maximum response to ET-1 amounted to less than 20% of that of isoprenaline (Pieske et al. 1999). The present results show that, in salmon, the maximum response to ET-1 is 36% of that of isoprenaline. Therefore, ET-1 seems to be a fairly potent inotropic agent in fish.

The present results indicate that ETA receptors mediate the inotropic effect of ET-1 in salmon, as is the case in man (Pieske et al. 1999). The dose-response curve of the positive inotropic effect of ET-1 on salmon myocardium shifted to the right with a selective antagonist of the ETA receptor, a finding that is consistent with competitive inhibition. The inability of 1 µM BQ-610 to completely inhibit the inotropic effect of large doses of ET-1 is probably dose-related. The selective ETB receptor antagonist BQ-788 did not modify the cardiac contractile effects of ET-1 in the salmon. The result is consistent with a study in Atlantic cod in which the ETB agonist BQ-3020 did not cause any significant change in cardiac output (Stenslokken et al. 2006). In rat heart, however, ETB receptor-mediated inotropic effects of ET-1 have been reported (Beyer et al. 1995). Results in mouse have indicated opposite roles for ETA and ETB receptors in the regulation of contractile force (Piuhola et al. 2003). ETA receptor activation accounts for ET-1-mediated enhancement of contractile force during elevated load, whereas ETB receptor activation has an inhibitory action on contractile function (Piuhola et al. 2003). Activation of endothelial cell ETB receptors can produce vasodilatation by releasing vasorelaxing factors such as NO (Verhaar et al. 1998). It has been shown that the positive inotropic effect of ET-1 in rat heart is augmented with inhibition of nitric oxide synthase (Kinnunen et al. 2000). Therefore, ETB receptor activation-induced NO release may play an inhibitory role in the regulation of contractile force during loading. Another possible mechanism for the ETB blockade-induced augmentation of contractility could be the decreased clearance of locally acting ET-1, thus indicating an increase in ET-1 binding to ETA receptors (Piuhola et al. 2003).

The subcellular mechanism that is responsible for the positive inotropic effect of ET-1 in mammals includes a considerable increase in myofibrillar sensitivity to Ca2+ ions and a moderate increase in the intracellular Ca2+ transient in mammals (Moravec et al. 1989, Takanashi & Endoh 1992). Stimulation of the ETA or ETB receptors activates the phosphoinositol pathway via phospholipase C, causing the activation of protein kinase C and the release of inositol triphosphate and a rise in the intracellular Ca2+ concentration (Cramer et al. 1998, Sugden 2003, Brunner et al. 2006). Stimulation of β-adrenoceptors activates adenylate cyclase via Gs, initiating the protein kinase A-dependent phosphorylation of ion channels and regulatory proteins, such as L-type Ca2+ channels,

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phospholamban and myofibrillar proteins involved in cardiac excitation-contraction coupling and energy metabolism in mammals (for review see Dzimiri 2002). The subcellular mechanisms for the inotropic effects of ET-1 or isoprenaline in salmon myocardium are not known.

6.3.4.2 Effects of endothelin-1 and β-adrenergic activation on sCP secretion

ET-1 is a major ANP and BNP secretagogue in mammals (Fukuda et al. 1988, Hu et al. 1988, Bruneau & de Bold 1994, Bruneau et al. 1997). The present results showed that ET-1 stimulates sCP secretion from salmon ventricle, but does not augment its release induced by a 13-cm H2O mechanical load, a stimulus previously found to cause a marked and consistent increase of sCP release. This was unexpected, because it has been shown that ET-1 markedly enhances load-induced cardiac natriuretic peptide release in vivo and in vitro in the rat (Mäntymaa et al. 1990, Leskinen et al. 1997a). Moreover, passive immunization with an antiserum against ET-1 has been found to reduce both the basal and volume load-induced levels of plasma ANP in rats (Fyhrquist et al. 1993). Considering the endothelin receptor antagonist data (see below), a likely explanation for the discrepant result is that the stimulus used (13 cm H2O) was a near-maximal one, and that ET-1 is able to shift the dose-response curve of mechanical load vs. natriuretic peptide release to the left, but does not affect the maximal response. This conclusion is supported by the finding that an increase of sCP release was indeed observed in the presence of ET-1, but only after the 13-cm H2O load was released (see Fig. 8C).

The present results showed that an ETA receptor antagonist was able to inhibit the ET-1-induced release of sCP. Previously, it has been shown that the antagonism of ETA/B receptors by bosentan decreases, and the combined blockage of ETA/B and angiotensin II receptor type 1 almost completely abolishes the response of ANP secretion to volume load in the rat (Leskinen et al. 1997a). The proximity of ET-1-producing endothelial cells and cardiomyocytes, and the presence of endothelin receptors on the cardiomyocytes (Davenport et al. 1989) suggest that ET-1 regulates ANP secretion, possibly by modulation of Ca

2+ (Uusimaa et al. 1992). ET-1 has been proposed to have a permissive

role, facilitating the release of ANP from cardiac myocytes. It may act as a tonic regulator of intracellular Ca

2+, which, if interrupted by an endothelin antagonist, may render the

ANP-secreting myocytes less sensitive to stretch (Fyhrquist et al. 1993). It has been reported that enhanced Ca

2+ influx plays a significant role in ET-1-stimulated ANP

secretion, but that the release of intracellular Ca2+

from the sarcoplasmic reticulum does not participate in the secretory response (Schiebinger & Gomez-Sanchez 1990). In the present study an ETA antagonist markedly attenuated the load-induced secretion of sCP. Thus, ET-1, via ETA receptors, appears to have an important physiological role as a mediator of volume load-induced sCP secretion from the salmon ventricle.

Both α- and β-adrenergic agonists stimulate ANP secretion in isolated rat atria (Schiebinger et al. 1987). Noradrenaline stimulates ANP secretion by both α- and β-adrenergic mechanisms but the secretory response pattern of noradrenaline reflects a predominance of β-adrenergic activity (Schiebinger et al. 1987). It has also been shown

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in superfused rabbit atria that isoproterenol-stimulated release is mediated by cAMP, and the effect does not require extracellular calcium (Azizi et al. 1993). The present results showed that β-adrenergic activation alone does not stimulate sCP secretion but has a strong synergistic effect with ET-1. These findings indicate that there is a synergism between the cellular signalling pathways of ET-1 and β-adrenergic stimulation. The mechanism for the synergism is not known, but it may involve the concurrent activation of two separate but interconnected signalling pathways. The effects of both are mediated by G-protein coupled receptors, Gq activating phospholipase C in the case of ET-1, and Gs activating adelylate cyclase-protein kinase C pathway in the case of β-adrenergic stimulation. Similar synergism has previously been described with corticotropin-releasing hormone and arginine vasotocin stimulating the release of ACTH (Baker et al. 1996). In addition, it has been found that ET-1 alone does not affect the contractile function of canine ventricular myocardium, but clear effects on contractility can be observed in the presence of noradrenaline (Chu et al. 2003). This effect again required the simultaneous activation of protein kinase A and protein kinase C signalling pathways (Chu et al. 2003). Furthermore, it has been shown that while ET-1 alone does not affect intracellular cAMP concentrations, it enhances isoprenaline-stimulated cAMP accumulation (Wu-Wong & Opgenorth 1998). It thus seems that ET-1 and β-adrenergic activation are engaged in crosstalk at different levels of their respective signalling pathways (Chu et al. 2003). For instance, a positive feedback mechanism seems to exist at the level of the synthesis of noradrenaline by which ET-1 increases the blood concentration of noradrenaline (Miller et al. 1989). In addition, noradrenaline facilitates the expression of mRNA that encodes the prepro-ET-1 (Masaki et al. 1991) and the production of ET-1 (Yanagisawa et al. 1988).

The results showed that the inhibition of ETB receptors with the selective antagonist increased the release of sCP. A possible mechanism for the ETB inhibition-induced stimulation could be the decreased clearance of ET-1, inducing an increase in ET-1 binding to ETA receptors. Especially in the lungs, ETB receptors have been implicated in the clearance of ET- 1 from plasma (Fukuroda et al. 1994).

6.3.4.3 Effects of endothelin-1 on sCP production

The results of this study showed that ET-1 does not affect sCP gene expression, at least in the short term. The finding is therefore in accordance with the results obtained with mammalian ANP. ET-induced secretion of ANP by an isolated rat atrial preparation is not associated with acute changes in ANP mRNA levels (Bruneau & de Bold 1994). In contrast to ANP, ET-1 induces a marked increase in BNP gene expression in isolated rat atria (Bruneau et al. 1997).

7 Summary and conclusions

1. Salmon ventricle stores large amounts of the prohormone of the novel cardiac peptide (sCP) belonging to the family of natriuretic peptides. The ventricular levels of mRNA encoding sCP are also correspondingly high. An appropriate stimulus, such as mechanical load, induces acute increase in the secreted sCP without causing corresponding rapid changes in the tissue peptide or mRNA levels. On the contrary to mammalian cardiocytes in which the ventricular secretion of the natriuretic peptides takes place via the constitutive pathway, the secretion of sCP from the ventricle clearly utilizes the regulated pathway. The constitutive pathway appears not to be an inherent property of ventricular hormone release and salmon ventricle; therefore it offers a unique model for studying regulated ventricular natriuretic hormone release.

2. Immunoreactive sCP stored in the ventricle has a high molecular weight and is therefore stored in the prohormone form. However, the secreted form is a 29-amino acid mature hormone. An inert N-terminal fragment of pro-sCP (NT-pro-sCP) seems to be co-secreted in parallel with sCP. In the present study, a specific radioimmunoassay for measuring circulating NT-pro-sCP was set up, and using this new RIA it was shown that serum NT-pro-sCP levels can be used to estimate the levels of the biologically active peptide sCP. Thus, the posttranslational processing to mature sCP appears to take place in conjunction with exocytosis of the secretory granules and resembles that of mammalian ANP.

3. The cardiac hormone sCP is highly sensitive to myocyte stretch. Salmon ventricle offers a useful model for studying the effects of mechanical stimuli on cardiac secretion, since it can readily be maintained on simple external perfusion. A modest increase in load results in high induction of the release, whereas induction of sCP gene expression appears to be a slower response. Thus, the acute sensitivity of the secretion of sCP to mechanical load resembles that of mammalian atrial ANP and therefore, mechanosensitive sCP serves a model for studying general mechanisms in load-induced natriuretic peptide secretion.

4. Temperature appears to play a major role in sCP biology. The circulatory levels of sCP are up-regulated in salmon in the warm season. While similar changes can be detected in cardiac tissue sCP levels, the sCP mRNA fluctuates in the opposite direction, with low levels during the summer. On the contrary, the release of sCP from perfused

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salmon ventricle is not increased by acclimation to higher temperatures. Interestingly, concurrent measurements of circulating sCP and NT-pro-sCP revealed that the increased sCP levels at warm ambient temperature result from decreased elimination rather than increased secretion of sCP. Thus, the present study demonstrates that not only induction of the gene expression, but also a change in the elimination rate of the natriuretic peptide can be used for the regulation of the natriuretic peptide hormone system.

5. The present study provides the first evidence for an important direct role of ET-1 as a cardiac secretagogue and inotropic agent in fish. It also reveals interesting synergism between the cardiac effects of the two important regulators of heart function, ET-1 and β-adrenergic stimulation. The positive inotropic and secretory responses appear to be mainly mediated by ETA receptors, as is the case with the regulation of mammalian natriuretic peptides, whereas the role of ETB receptors in cardiac function remains uncertain. The results demonstrate that ET-1 has similar potent effects and mechanism of action on the cardiac natriuretic peptides in mammals and salmon. This indicates a fundamental and phylogenetically highly conserved role for ET-1 in the maintenance of cardiac function.

Fig. 13. Summary of the factors regulating the sCP system.

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Original articles

I Kokkonen K, Vierimaa H, Bergström S, Tervonen V, Arjamaa O, Ruskoaho H, Järvilehto M & Vuolteenaho O (2000) Novel salmon cardiac peptide hormone is released from the ventricle by regulated secretory pathway. American Journal of Physiology Endocrinology and Metabolism 278: E285-E292.

II Tervonen V, Kokkonen K, Vierimaa H, Ruskoaho H & Vuolteenaho O (2001) Temperature has a major influence on cardiac natriuretic peptide in salmon. Journal of Physiology 536: 199-209.

III Vierimaa H, Hirvinen M, Tervonen V, Arjamaa O, Ruskoaho H & Vuolteenaho O (2002) Pronatriuretic peptide is a sensitive marker of the endocrine function of teleost heart. American Journal of Physiology Endocrinology and Metabolism 282: E843-E850.

IV Vierimaa H, Ronkainen J, Ruskoaho H & Vuolteenaho O (2006) Synergistic activation of salmon cardiac function by endothelin and beta-adrenergic stimulation. American Journal of Physiology Heart and Circulatory Physiology 291: H1360-H1370.

The permission of the following copyright owners to reproduce the original articles has been granted

(I, III, IV) Reprinted with the permission from The American Physiological Society (II) Reprinted with kind permission from the Blackwell Publishing Ltd.

Original articles are not included in the electronic version of the dissertation.


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