TitleStudies on the Non-selective Cation Channels Involved in theCholinergic Contractile Responses in Smooth Muscles of UrinaryBladder and Small Intestine( 本文(Fulltext) )

Author(s) ALOM FIROJ

Report No.(DoctoralDegree) 博士(獣医学) 甲第527号

Issue Date 2019-03-13

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/77958

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

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1. Introduction 7

2. Materials and Methods . 8

3. Results 12

4. .. 19

Figures .. 24

1. Introduc 36

2. Materials and Methods 38

3. Resul 43

4. Discus .. 49

Figures .. 53

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Contraction of the detrusor smooth muscle (DSM) is responsible for the voiding of urine

from the urinary bladder, while abnormal contractility of DSM can produces various diseases

including overactive bladder (OAB) (42, 80). OAB is characterized with symptoms that include

urinary urgency, with or without incontinence, frequency, and nocturia (1, 3, 4). Among factors

contributing to OAB, detrusor overactivity is the most common factor which is caused by altered

activity of neurogenic or myogenic functions affecting contractility of DSM (16, 24, 86).

Currently, antimuscarinics are used as the primary pharmacotherapy for OAB aimed at reducing

detrusor overactivity, however, their clinical use is limited due to lack of efficacy and

undesirable side effects (3, 4). The current research efforts direct towards identifying alternative

pathways with the potential to reduce DSM contractility and therefore, detailed understanding of

the cholinergic signal transduction pathways involved in regulation of DSM contraction is

essential to identify novel therapeutic targets in order to improve the clinical treatment of OAB.

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Contraction of DSM is triggered by a network of signals involving various receptors and

ion channels. Targeting of the ion channel may be the promising alternative pharmacological

intervention for the treatment of OAB (68, 69). The precise mechanisms by which ion channels

regulate the function of DSM have not yet been fully revealed. Previous studies reported that

various types of ionic channels including K+, Ca

+, Cl

- and non-selective Na

+ permeable cation

channels control the resting membrane potential and contractility of the DSM (3, 4, 39, 40, 66-

68, 84). Depolarization of the cell membrane occurs due to the activation of nonselective cation

channels which leads to the opening of VDCC resulting in increase in intracellular Ca2+

concentration triggering contraction of the DSM (98). The suppression of these cation channels

or activation of K+ channels lead to membrane hyperpolarization causing relaxation of DSM (68,

84). The depolarization of cell membrane potential and increased excitability of DSM is

therefore responsible for the enhanced contractility of DSM.

The discovery of the TRP protein family has particular interest as a candidate for

molecular basis of non-selective cation channels in smooth muscle cells (65). Recently, TRPM4

channel is suggested to be involved in membrane excitability of a certain type of smooth muscle

(36, 60). This channel is Ca2+

activated non-selective cation channel and permeable to Na+ and

K+ but impermeable to Ca

2+ (35, 60). Experiments conducted in HEK-293 cells expressing

TRPM4 channels suggested that activation of this channels causes membrane depolarization via

Na2+

influx (23) and also in cerebral artery smooth muscle using TRPM4 antisense suggested

that TRPM4 channels are present in native cerebral artery smooth muscle cells and its activation

lead to membrane depolarization, activation of VDCC, and thereby producing contraction (18,

30). On the other hand, inhibition of TRPM4 channels causes cell membrane hyperpolarization

leading to relaxation of cerebral artery smooth muscles (29).

Recently, TRPM4 channels have been also suggested to express and involved in

contractions of rat, guinea pig and human DSM (41, 78, 79). Thus, selective modulation of

TRPM4 channels may also modulate membrane excitability and contractility, and may be

potential and novel target for the treatment of pathological condition of urinary bladder including

OAB. However, the precise roles of TRPM4 channels in contractile responses are not fully

clarified. Furthermore, there are few reports about the involvement of TRPM4 channels in the

cholinergic contractile responses in mice DSM.

A few studies reported to the presence of TRPM4 channels in gastrointestinal smooth

muscles such as colonic smooth muscles (17). Dwyer et al. (17) recorded -

current in human and monkey colonic smooth muscle cells and

suggested that TRPM4 channels contributes to the setting of resting membrane potential.

However, information about the physiological and pathophysiological role of TRPM4 channels

in gastrointestinal tract is limited. If TRPM4 channels play a role for the contraction in

gastrointestinal smooth muscles, same aforesaid side effects related to gastrointestinal system

such as constipation will occur if TRPM4 channels are targeted for the treatment of OAB.

Therefore, it is important to compare the functional relevance of TRPM4 channels between

intestinal and DSM preparations.

The studies in this chapter were conducted to investigate the involvement of TRPM4

channels in cholinergic contractile responses in DSM and ileal longitudinal smooth muscle

preparations in mice. I used 9-hydroxyphenanthrene (9-phenanthrol) as a selective blocker for

TRPM4 channels, which has been widely used in biological studies to reveal the physiological

roles of this channels (36). 9-phenanthrol effectively blocked TRPM4 channels expressed in

HEK-293 cells under whole-cell patch clamp conditions (31). However, 9-phenanthrol exerts no

inhibitory effect on the close family member of TRPM4 i.e., TRPM5 currents recorded after its

expression in HEK-293 cells and also TRPM7 currents recorded in mouse interstitial cells of

Cajal (31, 49). Moreover, 9-phenanthrol showed no changes in the activity of the transient

receptor potential canonical 3 and 6 channels, BK channels, voltage-gated K+ channels, VDCC

or inwardly rectifying K+ channels (29). These findings endorse that 9-phenanthrol is a selective

and potent inhibitor of the TRPM4 channels and is useful to investigate the involvement of

TRPM4 channels in the cholinergic contraction of detrusor and gastrointestinal smooth muscles.

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Total RNA was extracted from whole DSM or ileal tissue using TRI REAGENT

samples were treated by TURBO DNA-free Kit (Thermo Fisher Scientific, MA, USA) to remove

any contaminating genomic DNA. cDNA was synthetized from the extracted RNA with reverse

transcriptase and random primers of ReverTra Ace- - (Toyobo Life Science, Osaka, Japan),

according to the manufacturer's protocol. The TRPM4 channel specific primer pair was designed

by Primer-BLAST (National Center for Biotechnical Information) and synthesized by Invitrogen

-AGGATGGTGTTGAGTTCCCCCT-

- GCTCCCACAGGCTTTCACAA- -1234,

amplicon size=509), respectively. The cDNA produced was amplified using KOD FX Neo

(Toyobo Life Science) and the specific primers for the TRPM4 channel. The amplification

profile was as follows: 2 min at 94°C (Pre-denaturation), 10 s at 98°C (denaturation), 30 s at

62°C (annealing) and 15 s at 68°C (extension) for 45 cycles. Positive control experiments were

performed in the presence of a housekeeping gene primer (G3PDH) contained in ReverTra Ace-

- -ACCACAGTCCATGCCATCAC- -

TCCACCACCCTGTTGCTGTA-

min at 94°C (Pre-denaturation), 10 s at 98°C (denaturation), 30 s at 66°C (annealing) and 27 s at

68°C (extension) for 45 cycles. After separation of the PCR products by electrophoresis in 1.8%

agarose gel/Tris-Acetate-EDTA Buffer, images of the DNA bands stained with GelRed Nucleic

Acid Gel Stain (Biotium, CA, USA) were captured using ChemiDoc MP (Bio-Rad, CA, USA)

under Image Lab software (Bio-Rad).

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3. RESULTS

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heteromultimer combinations may underlie

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human and monkey colonic smooth muscle cells (17). Here, I investigated the effects of

9-phenanthrol (10 μM) on the resting membrane potential of the single detrusor and ileal smooth

muscle cells held under nystatin-perforated, whole-cell current-clamp mode.

As shown in Fig. 23, 9-phenanthrol (10 μM) hyperpolarized the resting membrane

potential in both detrusor and ileal preparations. In DSM preparations, 9-phenanthrol (10 μM)

significantly reduced the resting membrane potential from -20.9 ± 2.4 mV to -33.2 ± 3.3 mV

(n=8). Similarly, in ileal smooth muscle preparations, 9-phenanthrol significantly reduced the

resting membrane potential from -35.9 ± 1.8 mV to -42.7 ± 2.1 mV (n=8).

These results suggest that TRPM4 channels are constitutively active and involved in the

setting of resting membrane potential, and thereby regulate basal tone in detrusor and ileal

smooth muscle cells.

4. DISCUSSION

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General Discussion

In many visceral smooth muscle cells, including those of the gastrointestinal and urinary

tract, M2 and M3 muscarinic receptor subtypes are co-expressed in their cell membranes, which

mediate the physiological action of the parasympathetic neurotransmitter ACh in evoking

smooth muscle excitation and contraction (9, 13, 21, 37, 57, 76). Stimulation of muscarinic

receptors causes the opening of nonselective cationic channels in smooth muscle cells of the

gastrointestinal and urinary tract (2, 7, 9, 45, 51, 57, 76, 87, 95). Thereby depolarization and

facilitated action potential discharges are occurred, which in turn result in increased Ca2+

influx

via VDCC leading to contraction of smooth muscle. However, the precise activation mechanisms

and molecular components for the muscarinic non-selective cation channels are still unclear.

TRPC channels are downstream effectors of G-protein-coupled receptors (70, 72, 77, 93)

including muscarinic receptors (85). In smooth muscle cells, these channels are believed to

compose nonselective cation channels (5, 14, 46). Among TRP channel families,

85).

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genetic KO mouse of TRPC4 channels would be a useful tool to explore the functional

role of this channel, although it should be also noted a compensatory influence due to lacking of

the TRPC4 gene.

TRPC4 channels have been suggested to contribute ~50% of the cholinergic contraction in

intestinal smooth muscles by recording EFS-evoked contractile responses using TRPC4 KO

mice (85). More recently, TRPC4 channels have been also suggested to be responsible for the

same extent of contribution of the EFS-evoked cholinergic contraction in DSM from TRPC4 KO

mice (33). Therefore,

In chapter II, I studied the involvement of TRPM4 channels in the cholinergic contraction

of DSM and ileal smooth muscles by using a novel and potent selective blocker of TRPM4

channels, 9-phenanthrol. 9-phenanthrol inhibited EFS- and carbachol-evoked contractile

responses of mouse DSM. However, the agent has no inhibitory effect on the carbachol-evoked

contractile responses in ileal preparations, suggesting that TRPM4 channels have a significant

role in the cholinergic contractions in DSM.

Strikingly, the inhibition of EFS-evoked contractile responses by ML204 was frequency

dependent. This is explained by assuming that lower frequencies can release smaller amount of

ACh from cholinergic nerve-endings and therefore, ML204 can easily antagonize the action of

ACh at lower frequencies compared to higher ones, resulting in strong inhibition at lower

frequencies. However, 9-phenanthrol showed no frequency-dependency in inhibition of EFS-

evoked contractile responses probably due to lacking of such antagonistic action of 9-phenathrol

to muscarinic receptors.

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However, these

differences of the muscarinic signaling by TRPM4 channels in detrusor and ileal smooth muscles

may be important for the specific treatment of bladder pathologies leading to OAB conditions

to surmount side effects of presently available antimuscarinic drugs on the

gastrointestinal system

General Conclusion

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Results in this Chapter also provide evidence that TRPM4 channels are involved in the

cholinergic contractions in DSM but not, in ileal smooth muscles. Therefore, it is possible to

target TRPM4 channels for the treatment of OAB that will produce minimum side effects of the

presently available antimuscarinic drugs on the gastrointestinal system.

Acknowledgements

I would like to express my deep appreciation to the following individuals, who have given me

support and assistance during the PhD course:

Foremost, I would like to express my sincere gratitude to my main advisor, Dr. Toshihiro

Unno, Professor, Laboratory of Veterinary Pharmacology, Department of Veterinary Medicine,

Faculty of Applied Biological Sciences, Gifu University, for the continuous support of my PhD

study and research, for his patience, proper guidance, motivation, enthusiasm, and immense

knowledge. His guidance helped me in all the time of research and writing of this thesis. I could

not have imagined having a better advisor and mentor for my Ph.D study.

My sincere thanks goes to my primary assistant advisor, Dr. Hayato Matsuyama, Associate

Professor, Laboratory of Veterinary Pharmacology, Department of Veterinary Medicine, Faculty

of Applied Biological Sciences, Gifu University, for technical support, helpful advice,

encouragement, insightful comments, and hard questions which incented me to widen my

research from various perspectives.

I would like to express my heartfelt gratitude to Dr. Yasutake Shimuzi, Professor, Laboratory

of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Applied Biological

Sciences, Gifu University; Dr. Toshiaki Ishii, Professor, Department of Basic Veterinary

Medicine, Obihiro University of Agriculture and Veterinary Medicine; Dr. Hiroshi Satoh,

Professor, Veterinary Pharmacology and Toxicology Laboratories, Faculty of Agriculture, Iwate

University; for their effective suggestions and helpful comments in my work.

A special thanks to my secondary assistant advisors, Dr. Minoru Shimoda, Professor, and Dr.

Kazuaki Sasaki, Associate Professor, The Department of Veterinary Medicine, Faculty of

Agriculture, Tokyo University of Agriculture and Technology, Japan, for their helpful advices

and comments in my work.

I would like to express my gratitude to Dr. Yasuyuki Tanahashi, Associate Professor, and Ms.

Hitomi Kouzaki, Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto

Sangyo University for his helpful advice, technical support and counsel in the work.

I thank my fellow labmate, my good friend, Mr. Hiroshi Nagano and Mr. Masumi Miyakawa,

who encouraged and helped me at every stage of my personal and academic life and for all the

fun we have had in the last few years.

I am very much grateful to the MEXT (Ministry of Education, Culture, Sports, Science and

Technology), Japan for providing scholarship to complete the PhD work successfully.

Last but not the least, I would like to thank my parents, wife, daughter and son for all their

love and encouragement.

Above all, I owe it all to Almighty God for granting me the wisdom, health and strength to

undertake this research task and enabling me to its completion.

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