J. exp. Biol. (1978), 74, 227-237 2 2 7

Wrinted in Great Britain

EFFECTS OF EPINEPHRINE ON BRANCHIALNON-ELECTROLYTE PERMEABILITY IN RAINBOW TROUT

BY JACQUES ISAIA,* JEAN MAETZf AND GEOFFREY P. HAYWOODJGroupe de biologie marine, Departement de Biology, Commissariat a VEnergie

Atomique, Station Zoologique, .F-06230 Villefranche-sur-Mer

(Received 26 September 1977)

SUMMARY

Using isolated heads perfused at constant pressure, at rates close to thoseoccurring in vivo, the permeability of the gills of the trout Salmo gairdnerito a range of solutes was measured. Under epinephrine-free conditions,butanol and water showed similar high branchial permeability coefficients.Urea, inulin and dextrans (mol. wt 3000 and 20000) were 7-12 times lesspermeant, and mannitol 60-70 times less permeant than water or butanol.Epinephrine, at io~8 M, greatly increased the permeability of the gills to thesmall hydrophilic molecules, water and urea, and to the lipophilic substance,butanol, but did not affect the penetration of the large hydrophilic solutes,mannitol, inulin and dextrans.

In the presence of io~6 M propanolol, a /S-blocker, epinephrine had noeffect on the permeation of any of the test substances except that the per-meability to urea decreased somewhat.

The results suggest that epinephrine increases the permeability of themembranes of the branchial cells but does not affect the permeation ofsubstances that cross the gill walls by paracellular routes or via an intra-cellular ' bulk-transport' mechanism. Such an action would be expected toincrease the branchial transfer of oxygen.

INTRODUCTION

Recent investigations on non-electrolyte permeation across various epitheliasuggest that large polar solutes (e.g. mannitol, sucrose or inulin) pass through thetight junctions whereas small hydrophilic solutes (e.g. water or urea) and lipophilicmolecules (e.g. ethanol or butanol) pass through both the apical and the baso-lateralmembranes (Smulders & Wright, 1971; Smulders, Tormey & Wright, 1972; Wright& Pietras, 1974; Wright, Smulders & Tormey, 1972). Some study has been made ofthe effect of hormones on epithelial permeability. Antidiuretic hormone (ADH) hasbeen observed to increase the permeability of toad bladder to small hydrophilicsolutes, with a smaller augmentation of permeability to lipophilic substances, and tohave no effect at all on permeability to large hydrophilic molecules (Pietras & Wright,

• Present address: Laboratoire de Physiologie Cellulaire Faculty des Sciences de Nice.t This manuscript was completed after the death of Dr J. Maetz on 16 August 1977.t Present address: Department of Zoology, The University, Sheffield Sio aTN, England.

228 J. ISAIA, J. MAETZ AND G. P. HAYWOOD

1975). This suggests that ADH increases the fluidity of plasma membranes andno effect on the paracellular pathway.

In the present investigation we have investigated the effect and mode of action ofepinephrine on the permeability of trout gill epithelium. Fish gill epithelium exhibitsa low permeability to water (Evans, 1969; Motais & Isaia, 1972; Motais, Isaia, Rankin& Maetz, 1969) and to urea (Payan & Maetz, 1970), but has a relatively high perme-ability to very large molecules such as inulin (Lam, 1969; Masoni & Payan, 1974).Epinephrine increases branchial transfer of non-electrolytes. In particular, it increasesthe uptake of oxygen, a highly lipophilic solute. It also increases diffusional waterfluxes across the gills of sea-water- and freshwater-adapted Mugil capito (Pic, Mayer-Gostan & Maetz, 1974), and freshwater-adapted trout (Haywood, Isaia & Maetz,1977). Branchial influx and efflux of urea are also enhanced in freshwater-adaptedtrout (Bergman, Olson & Fromm, 1974; Haywood et al. 1977). Although this neuro-hormone produces alterations in the branchial irrigation which may entail changes inthe surface available for diffusion (Bergman et al. 1974) it has been shown to producea true change of permeability to water in the branchial epithelium, via /?-adrenoceptors(Haywood et al. 1977).

METHODS

Rainbow trout (Sabno gairdneri), obtained from a local trout farm, were kept intanks of running aerated fresh water (Ca concentration: 1-5 ITIM), at 15 ± 3 °C. Bothsexes, weighing from 130 to 200 g, were used.

The isolated perfused head preparation was carried out after Haywood et al. (1977)with similar experimental protocol. Labelled test substances were added to the per-fusion fluid and effluxes were measured for two 30 min periods with the externalmedium in a closed circuit of 240 ml. These two periods were separated by a 5 mininterval allowing for addition of epinephrine (L-epinephrine, Merck: io~6 M), rinsingoff the external medium and replacing it by fresh medium. The /^-blocking agentpropranolol (ICI: io~9 M) was present during both experimental periods in oneseries of investigations. Throughout these experiments, the perfusion rate wasmonitored by a drop counter and by weighing collected perfusion fluid in each period.

Table 1 gives details of the various substances tested. In most of the experiments,THO was used simultaneously with another test-substance, which had either a 14Cor a fluorescent label. Concentrations of 3H and 14C in the external and internal mediawere measured in an Intertechnique SL 40 scintillation counter with automaticquench correction. Fluorescence was measured in an Aminco SPF 125 spectro-photometer in samples made alkaline (pH 9-0) by the addition of NaOH.

During the early part of these investigations, control experiments were performedto verify that the gill is indeed permeable to inulin and dextran 20 and that the 14Cradioactivity appearing in the external medium corresponded effectively to inulinand dextran 20 and not to small breakdown products, in particular to i>fructose orD-glucose (mol. wt: 180). Diaflo R ultrafiltration was used to check for inulin anddextran 20 appearing into the external medium. Inulin was also checked by thin-layerchromatography. Aminco membranes (UM 05 for inulin and UM 10 for dextran 20)were employed to filter both internal and external media at the end of a 30 minefflux period. These filters, permeable to molecules of less than 500 mol. wt (UM 05J

Branchial permeability of test substances in trout

Table i. Non-electrolytes used in the present study

229

Component

n-Butanol (i-14C)

Water (THO)Urea (»C)

D-mannitol (i-14Q

FITC dextran 3Inulin (14C)

FITC dextran 20Dextran carboxyl(carboxyl-14C)

Molecularweight

(g)

74

1860

18a

30005300

2000020000

Stokesmolarradius

(A)

3-a

1-2

i-8

3 7

1 2

1 3 4

33

Partitioncoefficient

0 4 4

7 x io"4

1-5 x io-1

1-ax 10-*

InsolubleInsoluble

InsolubleInsoluble

Concentration(mole:l)

5 x io~'

10-4

o-8 x io- f

io-'io-«

2X IO-*io-'

OriginICN Isotopes NuclearDivision

CEN SaclayRadiochemical Centre,Ameraham

Radiochemical Centre,Amersham

Pharmacia, UppsalaRadiochemical Centre,Amersham

Pharmacia, UppsalaNew-England Nuclear

or 10000 mol. wt (UM 10) was used under 7 kg/cm2 and 4 kg/cm2 nitrogen pressurerespectively. Kieselgel (Merck F 254), 250 /tm thick, was used for thin layer two-dimensional chromatography of the external medium. Two solvents were usedsuccessively: ethanol/water (30/70), and ethylacetate/isopropanol/water (65/20/15).The first produced a parallel migration of D-fructose and inulin to Rf 0-5, while thesecond produced migration of D-fructose along to Rf 0-3. The medium was filteredthrough a millipore filter (0-22/4) and vacuum concentrated to about one-hundredth ofits original volume at room temperature. The sugars were revealed by spraying onanisaldehyde reagent after oven-heating the plate to 100 °C for 5 min (Stahl & Kalten-bach, 1964). The radioactivities associated with the inulin and fructose spots werecompared to that of a 100 /A aliquot of the concentrated external medium identical tothe aliquot placed on the silica gel.

Permeability coefficients were computed from the fluxes of the various testsubstances. Successive 5 min samples were taken from the external medium and thecorresponding regression line was used to calculate the appearance rate of radioactivity.For the most permeant solutes (butanol and THO) the mean radioactivities of theafferent and efferent fluids was used to calculate the specific radioactivity. With thepresent high perfusion rates, the difference between afferent and efferent fluid radio-activities amounted to no more than 10% of the input radioactivity. Fluxes (mol/100 gh)were calculated by dividing the rate of appearance of the label (fluorescent or radio-active) in the external medium by the specific activity of the test-substance in theperfusion fluid (Haywood et al. 1977). Permeability coefficients in cm/s were obtainedby dividing the flux in mol/100 g s by the gill surface in cm2 per 100 g body weight(i.e. 400 cm2) and by the concentration of the test-substance in the perfusion fluid inmol/cm3.

RESULTS

(1) Branchial permeability characteristics

Permeability coefficients measured in the epinephrine-free preparation, are listedin decreasing order in Table 2. Three groups may be recognized. Butanol and water,with almost identical coefficients, head the list. A second group of substances, about

230 J. ISAIA, J. MAETZ AND G. P. HAYWOOD

Table 2. Mean ±s.E. values for non-electrolyte permeability coefficients in thefreshwater trout gill

SubstanceButanolWaterUreaDextran 30InulinDextran 3Mannitol

n

6Si1 0

1 3I I

66

Poii-o±a-j9-o±o-61-a ±0-31-a ± 0 3

0-9210-38087 ±020o-i6±o-os

p./pTHO

I-I ±0-3—

0-i9±o-o40-14 ±007o-33±o-i5008±003o-oa±o-o8

P, Permeability coefficient in cm s"1 x io*.P*/Pmo, see text.

7-12 times less permeant, includes small hydrophilic solutes, such as urea, andhydrophilic macromolecules with an 18-fold variation in molar radii, but withsimilar permeability coefficients. Finally, mannitol, about 60-70 times less permeantthan water or butanol, has a permeability coefficient significantly lower than that ofurea. There is no significant difference between dextran 3, dextran 20 and inulinvalues.

Inulin itself, and not breakdown products, penetrates through the epithelium.Ultrafiltration showed that in the inulin used in the perfusion fluid there was 5 ± 1 %(n = 3) of metabolite with molecular weights less than 500, compared with 6 ± 2 %(n = 3) in the external medium. After thin-layer chromatography, 86 ± 6 % (n = 3)of the external medium radioactivity corresponds to the Rf of inulin.

For the dextrans, molecular weight distributions examined by gel filtration (samplesand data by Pharmacia) indicated no overlap in the molecular weights but we havefound similar permeability coefficients of the two populations. Concerning dextran 20the ultrafiltration showed 6% ± 2 % (n = 3) of metabolite with molecular weightssmaller than 10000 in the perfusion fluid compared to 4-0% + o-6% (n = 3) of meta-bolite in the external medium. The permeability of the gills to dextran 20 wasmeasured by means of a fluorimetric technique and using 14C-dextran 20. The valuesare respectively: Po = i-2±o-7 io~acm/s (n = 8) and Po = I - I ± O - 7 io~° cm/s(« = 4) in the absence of epinephrine and Pep = i-7±o-6 io~6cm/s (n = 8) andPep = 2-o± i-o io"6 cm/s (n = 4) when io~* M of epinephrine were added to theperfusion fluid. The values obtained by the two experimental techniques were notsignificantly different. These results are depicted in Tables 2 and 3. Clearly dextransof very high molecular weight cross the gill.

(2) Effect of epinephrine

Table 3 summarizes the effects of epinephrine. The permeabilities of the testsubstances measured after the addition of epinephrine, Pep, are given. Epinephrinesignificantly and uniformly increased the permeabilities to butanol, water and ureabut had no effect on the permeability to inulin, dextrans 3 and 20 and mannitol.Simultaneous measurement showed that water transfer increased, so the lack of effecton mannitol, inulin and dextrans is not due to tissue insensitivity. For instance, inthe experiments measuring the water and mannitol permeabilities with and withoutepinephrine, the ratio Pep/P0 for water was 1-71 ±0-17.

Branchial permeability of test substances in trout 231

Table 3. Effect of epinephrine on the permeability coefficients of the freshwater troutgills {mean ± S.E.)

Test substanceButanolWaterUreaDertran 20InulinDextran 3Mannitol

Units

n

6511 0

1 311

66

as in Table 3.

•P«p

24'9±6-9163 ±093-3 ±0-5

i-8±o-s3-2 ± 1 - 4

0-6o±o-i3Oi4±o-O3

p P

+ i39±4-4+ 7'3±°7+ I-I ±°'3+ O'6±o-5+ I - 3 ± I - I

— o-37±oo8— ooa±oo3

i-8±o-5

o-is±o-O30-13 ±006033 ±0140-04 ±o-oi

Pv, Permeability observed under epinephrine stimulation.Pa, Control permeability (see Table 2).

THOj gee text.

Table 4. Permeability effect of epinephrine in the presence of propranolol{mean ± S.E.)

Test substanceButanolWaterUrea

71

61 2

6

P1

5-8±i-4IO-S±I-42-7 ±0-9

P'JP'1-5 ±06o-8o±o-iao-6 ±o-i

P'cg — P'+ i-S±3-8-a-8±i-3-i-4±o-7

Units as in Table a.P1, Permeability observed during the control period with propranolol.P"^, Permeability observed after addition of epinephrine.

The ratio pypTHO calculated for the periods under epinephrine stimulationsignificantly increased in the experiments with butanol (P < o-oi using the pairedratio differences). Such changes did not occur with the other test molecules, includingmannitol. In the butanol experiments, increased water permeability {Pej)/P0 =I-35±O-IO), although significant, is definitely lower than that in all the otherexperiments {Pep/P0 = 2-01 ±0-15, n = 51) (P < o-oi). Butanol may interact withmembrane lipids to affect P™ 0 .

The perfusion flow rate, 113 ± 7-4 ml/100 g h under control conditions, increasedby 10% (+14-5 + 6-5 ml/100 g h) after addition of epinephrine.

(3) Effect of propranolol

Haywood et al. (1977) showed that the increased branchial water permeabilityproduced by epinephrine involved a /fl-adrenergic receptor. Table 4 summarizes datafrom experiments in which propranolol was present throughout the flux periods. Theeffectiveness of this blocker is verified by the considerably decreased perfusion rateobserved after addition of epinephrine, from 122 ± 12-2 ml/100 g h to 48 ± 6-7 ml/100 g h (n = 12; P < 001). Such an effect is to be expected from stimulation ofa-adrenoceptors, causing vasoconstriction in this preparation (Payan & Girard, 1977).

The mean permeabilities observed from the various test-substances during thecontrol period with propranolol, are not statistically different from the comparablevalues shown in Table 2. In the presence of the /?-blocker, epinephrine no longermgments effluxes of the various test-substances, and indeed permeability to ureadecreases significantly.

232 J. ISAIA, J. MAETZ AND G. P. HAYWOOD

DISCUSSION

(1) Validity of the branchial permeability measurements

The perfused head preparation offers two methods for measuring the efflux rateof test-substances. First, clearance (difference in concentration of the molecule in theafferent and efferent perfusion fluid) and the rate of perfusion may be used, providingthe difference between afferent and efferent concentrations exceeds 3 % of the input.To increase the difference, it is necessary to reduce the perfusion rate. Steen &Stray-Pedersen (1975) used perfusion rates attaining only 2-5-10% of the cardiacoutput normally observed in eels. At such low perfusion rates, the effective exchangearea may be drastically reduced. For these reasons, the clearance method, mostadequate for measuring the permeability of the most permeant solutes (lipophilic andsmall hydrophilic molecules) is almost certainly inadequate for evaluating thepermeabilities of larger hydrophilic solutes (sugars, dextran or inulin).

The method used during the present investigation, however, consists of the directmeasurement of the appearance rate of the solutes in the external medium, and thusavoids these difficulties. In addition, the present perfusion rate of about 2 ml/100 g min, is of a physiological order.

The interference of unstirred layers within the epithelium cannot be overlooked,and the relative thickness of the epithelia and of their corresponding connectivetissue is a good indicator of the importance of unstirred layers in impeding solutemovement. Thus for the rabbit gall bladder and the amphibian choroid plexus andurinary bladder values of 200—900 fim have been considered (Smulders & Wright,1971; Wright & Pietras, 1974). From morphological studies of the gill (Conte, 1964;Hughes & Morgan, 1973; Morgan & Tovell, 1973; Steen, 1971), unstirred layers areof minor importance in view of the absence or extreme thinness of the connectivetissue (< 1 /im), of the respiratory epithelium (< 5 fim), or even of the mitochondria-rich cells (< 20 /im). Moreover, the importance of the unstirred layers is inverselyrelated to the magnitude of the fluxes crossing the epithelium (Smulders et al. 1972).The gill is relatively impermeable to lipophilic and small hydrophilic solutes forwhich corrections are important in other epithelia. An important factor, however, israpid equilibration of the branchial blood supply. Under various circumstances,changes in perfusion rate, pressure or vascular resistance may cause blood to beshunted from the lamellae and channelled elsewhere (Girard & Payan, 1976, 1977;Haywood et al. 1977; Hughes, 1972; Hughes & Morgan, 1973; Payan & Girard,1977; Steen & Kruysse, 1964; Vogel et al. 1973, 1974)- Whether these changes cor-respond to all-or-none effects will be discussed later in relation to the effects ofepinephrine.

(2) Non-electrolyte permeability of gills compared with epithelial and red cell mem-branes (Table 5)

Except for the human skin, the gills are among the most impermeable epithelia,especially with respect to lipophilic and small hydrophilic solutes. There is goodagreement for water and urea permeabilities in the gills of the eel and trout under theinfluence of epinephrine. The value found for water in the perfused head of the

Tab

le 5

. Per

mea

bilit

y co

efic

tknt

s for

sm

all

test

mol

ecul

es in

var

ious

epi

thel

ia a

nd r

ed

Bio

logi

cal

mat

eria

l T

rou

t gi

ll

ep.

no e

p.

Eel

gil

l ep

. T

oad

uri

nar

y b

ladd

er

AD

H

no

AD

H

Rab

bit

gall

bla

dder

F

rog

Cho

roid

ple

xus

Hu

man

epi

derm

is

Hu

man

red

cel

ls

But

anol

0.

44

24'9

11'0

-

r 640

9x

0 -

-

0.37

41

cell

mem

bran

es

Ant

i-

pyri

ne

Eth

anol

W

ater

U

rea

Man

nito

l S

ucro

se

Inu

lin

3.

2 x

10-a

2.

6 x

IO

-~

7

x 10-a

1.

5 x

10-a

1

.2 x

10-a

I

x 1

0-6

o

Per

mea

bili

ty c

oeff

icie

nts

Ref

eren

ce

-

16.3

2.

3 0.

14

-

0.16

0.

16

-

2.2

}

Pre

sent

exp

erim

ent

9'0

0.

92

7'2

14

6

14.6

2.2

-

-

-

Ste

en &

Str

ay-P

eder

sen

(197

5)

h

13

-

> 1

300

4.9

0.17

0.

14

I0

-

O

} P

ietr

ss &

Wri

ght

(197

5)

1.4

0.19

0

.14

-

2 13

0 -

202

-

0.6

Wri

ght

& P

ietr

as (

1974

) r

76

89

4 -

68

12

2.

1 I .6

-

Wri

ght

& P

ietr

as (

1974

) 6

zq

3

-

-

-

-

-

Sch

eupl

ein

& B

lank

(19

7 I)

0.

08

-

h

-

88

-

-

-

Nac

cach

e &

Sha

'afi

(19

73)

2 91

.50

239

N.

All

val

ues

corr

ecte

d fo

r un

stir

red

laye

rs.

Uni

ts a

s in

pre

cedi

ng t

able

s.

a B

0 R

234 J- ISAIA, J. MAETZ AND G. P. HAYWOOD

freshwater eel is however somewhat lower than that reported previously for in vivostudies at 19 or 15 °C, respectively 18 and 25 x io~6 cm s"1.

To characterize the gill permeability to lipophilic solutes, a small range of soluteswould have to be examined to obtain the slope of the regression line between log Pand log Kon or to study the effect of increasing the chain length of the solute on P.We have pooled our data for butanol with the data given by Steen & Stray-Pedersen(1975) for ethanol and antipyrine and find that the slope of the line relating P andKoll (about 0-29) indicates that the gills are much less hydrophilic than the urinarybladder of the toad and about as hydrophilic as frog choroid plexus or rabbit gallbladder (Wright & Pietras, 1974). (With high branchial permeability molecules, theclearance technique is available.) This suggestion is substantiated by the increase ofP by 1-7, observed when butanol and ethanol permeabilities are compared. Anincrease in chain length of two methylene groups (butanediol hexanediol) producesa P increase of 1-9 in the gall bladder and of 4-5 in the urinary bladder (Wright &Pietras, 1974).

The branchial permeability to small polar solutes (water, urea) resembles per-meability of other epithelia and single-cell membranes insofar as it is obviouslygreater than is to be predicted on the basis of partition coefficients. It may be notedthat the por«ypTHO r a t j 0 j s m u c n greater for gills than for the toad urinary bladderor for red cells, and is similar to that observed in the choroid plexus. It may be thatpores, membrane carriers or a highly ordered membrane lipid configuration explainthe high permeability observed for the small polar solutes.

For large polar solutes, permeation is thought to occur via a few large pores in thetight junction as shown for the rabbit gall bladder (Smulders & Wright, 1971). Smallpolar solutes exhibit lower apparent activation energies than larger solutes. Moreover,the ratio of inulin to sucrose branchial permeabilities is similar to that of their free-solution diffusion coefficients and the apparent activation energy for sucrose is notdistinguishable from that reported for diffusion in aqueous solution. The branchialpenetration of large polar solutes has paradoxical properties, in that the permeabilityto mannitol is of an order of magnitude smaller than in the rather leaky gall bladderand choroid plexus and identical to that reported for the urinary bladder which is con-sidered to be a tight membrane. On the other hand the trout gill is twice as permeableto inulin as is the rabbit gall bladder. Permeability to mannitol is, in fact, ten timessmaller than for inulin, whereas the free-diffusion coefficient of mannitol is aboutfive times higher. Two permeation routes for large polar solutes across the branchialepithelium must be considered: a paracellular route for mannitol and possibly sucrose,and an intracellular 'bulk-transport' route for macromolecules like inulin anddextrans. Histochemical and light microscopic autoradiography reveal that variousorganic solutes including mannitol and inulin are accumulated in the mitochondria-rich cells (Masoni & Garcia-Romeu, 1972; Masoni & Isaia, 1973). The tubularsystem on the baso-lateral membrane is probably the accumulation site of thesesubstances. Whether this tubular system makes contact with the external medium bymeans of clefts or infoldings of the apical membrane (Bierther, 1970; Philpott &Copeland, 1963; Shirai & Utida, 1970) or by a process of vesicular transport andexocytosis (Isaia & Masoni, 1976; Shirai, 1972) remains to be elucidated. Maetz & Pic(1977) discussed the possible role of vesicular transport in salt excretion by the gill in

Branchial permeability of test substances in trout 235

sea-water. The presence of numerous microfilaments and microtubules in the apexof the mitochondria-rich cells suggests a role of cell motility in the permeationprocesses of these cells. Addition of colchicine (an inhibitor of microtubular poly-merization) to the external medium rapidly blocks salt secretion in the sea-water-adapted mullet. Water permeability, however, remains unchanged (Maetz &Pic, 1977).It would be of interest to study permeability to large polar solutes in animals treatedwith colchicine, and to examine why permeability to mannitol is smaller than that forinulin or dextran. Obviously, with non-specific bulk transport, the vesicles shouldhandle mannitol and dextran indiscriminately. It may be that the walls of the vesiclesare relatively permeable to the smallest of these molecules and these would leak outduring transport across the cell. If the apical cell membrane were impermeable tomannitol, its only exit route would be the tight junction.

(3) Effects of epinephrine

Both neurohypophysial hormones and epinephrine increase water permeability infrog skin (Maetz, 1968; Rajerison et al. 1972) by processes involving the adenylcyclase system. Epinephrine's effect is mediated through a /?-adrenoceptor and themechanism appears similar for increased gill permeability to water, since the responseis blocked by propranolol (Pic et al. 1974). As a ^-receptor is similarly involved indecreased branchial resistance to blood flow, variations in regional blood distributionmay indirectly change the flux, either by more efficiently mixing the internal mediumor by increasing available exchange surface via lamellar recruitment. By ruling outchanges in surface area, Haywood et al. (1977) showed that epinephrine induces a trueincrease in gill permeability to water, although it is appreciated that in most effluxexperiments, changes in the available surface often cannot be completely ruled out.Present results show that epinephrine increases the apparent permeability to butanol,water and urea in a rather non-specific manner, by about 100%, suggesting a possibleincrease in the available surface. This is not supported, however, by the absence ofeffect on permeability to inulin, dextrans and mannitol. Antidiuretic hormoneincreases the permeability of amphibian bladder to lipophilic solutes in a rather non-specific manner, by about 30% (Pietras & Wright, 1975). Transfer of small hydro-philic solutes is greatly increased, however, by a factor of 3-5 for urea, and more than10 for water. Permeability to larger hydrophilic solutes such as mannitol remainsunchanged (Pietras & Wright, 1975). ADH possibly has no effect on the paracellularpathway but increases membrane lipid fluidity, which is the preferred pathway forlipophilic and small hydrophilic molecules. This may also apply to epinephrine actionon the branchial epithelium, although with some differences. In the urinary bladderthe abnormally high permeability of small polar solutes, as compared with lipophilicsolutes, is considerably accentuated by ADH. This is not observed in the gill underepinephrine. The hypothetical paracellular route of mannitol permeation remainsinsensitive to epinephrine in the gill and to ADH in the urinary bladder.

Our observations with propranolol confirm observations (Haywood et al. 1977)that /?-adrenergic receptors mediate increases in permeability under epinephrine,irrespective of whether small hydrophilic or lipophilic solutes are considered. Thisagain suggests a common mechanism such as increased membrane lipid mobility.

In simultaneous studies, epinephrine has been shown to increase branchial per-

236 J. ISAIA, J. MAETZ AND G. P. HAYWOOD

meability more for butanol than for water. If transfer of lipophilic solutes ispreferentially stimulated, it is pertinent to examine the actions of epinephrine on thepermeability to the lipophilic respiratory gases. To date increased oxygen uptake dueto epinephrine has been interpreted purely in terms of vascular effects. RecentlyP. Payan, J. P. Girard, C. Peyraud and M. Peyraud-Waitzenegger (unpublishedobservations) have suggested that epinephrine actually increased membrane per-meability to oxygen in perfused trout heads.

The authors thank the Royal Society, for financial support to G. P. Haywood bymeans of a European Post-Doctoral Fellowship.

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surface area of isolated perfused gills of rainbow trout J. eomp. Pkysiol. 94, 267-286.BIERTHER, M. (1970). Die Chloridzellen des Stichlings. Z. Zellfortch. mikrosk. Anat. 107, 421-446.CONTE, F. P. (1969). Salt secretion. In Fish Physiology, vol. 1 (ed. W. S. Hoar and D. J. Randall),

pp. 241-292. New York: Academic Press.EVANS, D. H. (1069). Studies on the permeability to water of selected marine fresh water and euryhalin

teleost. J. exp. Biol. 50, 689-703.GIRARD, J. P. & PAYAN, P. (1976). Effect of epinephrine on vascular space of gills and head of rainbow

trout. Am. J. Pkysiol. 330, 1555-1560.GIRARD, J. P. & PAYAN. P. (1977). Kinetic analysis and partitioning of sodium and chloride influxes

across the gills of sea water adapted trout. J. PhysioL, Land. 367, 519-536.HAYWOOD, G. P., ISAIA, J. & MAETZ, J. (1977). Epinephrine effects on branchial water and urea in

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