J. Physiol. (1977), 273, pp. 195-209 195With 3 text-figuremPrinted in Great Britain

KINETIC ANALYSIS OF SODIUM AND CHLORIDE INFLUXESACROSS THE GILLS OF THE TROUT IN FRESH WATER

BY J-P. GIRARD AND P. PAYANFrom the Groupe de Biologie marine du D'partement de Biologiedu Commisaariat a' l'Energie Atomique, Station Zoologique, 06230

Villefranche-sur-Mer, France

(Received 28 April 1977)

SUMMARY

1. Na and Cl intake through the gill of the perfused head of trout werestudied in fresh water with (10-5 M) or without adrenaline in the perfusingsolution.

2. Ionic influxes occur exclusively across the lamellae in fresh waterwhile in the sea-water adapted trout part of salt entry is extralamellar.

3. In absence of adrenaline, Na and Cl enter the gills at the same rate(respectively 6-9 + 1-30 and 6-6 + 1-55,ttequiv/hr. 100 g). Adrenaline (10-5M)increased the Na influx to 47-8 + 4-12 ,equiv/hr. 100 g, a value similarto that observed in vivo. The Cl influx remains unchanged however (6-3 +2-40 piequiv/hr. 100 g), a value much smaller than that found in vivo.

4. Radioactive loading experiments coupled with unloading experimentsallowed the determination of the relative permeabilities of the serosal andmucosal barrier for Na+ and C1. For both ions, the basal membrane isless permeable. Adrenaline by increasing the Na permeability across theapical barrier enhances the active Na pumping through the basal membrane.

5. Intracellular Na and Cl exchangeable pools were calculated. Theyrepresent less than 1 % of the total ionic content of the epithelium.Adrenaline increased by sixfold the Na pool without modifying the Cl pool.

INTRODUCTION

Krogh (1938) showed that salt uptake in a number of fresh-wateranimals is characterized by independent active transport systems forboth Na+ and Cl-. It is now accepted that Na+ is exchanged for NH4+ orH+ and Cl- for HCO3- (see review by Maetz, 1974).One question of particular interest concerns the relative movement of

ions across both mucosal and serosal barriers of the gills. Many investi-gations have been performed on isolated epithelia such as frog skin, toadbladder (see review by Motais & Garcia-Romeu, 1972) or mammalian gall

J-P. GIRARD AND P. PA YANbladder (Frizzel, 1976). Investigation of these transport mechanismsnecessitates a direct access to bathing media on both serosal and mucosalside of the membrane. The technique of the perfused head of trout (Payan& Matty, 1975) modified by Girard & Payan (1976) allows for the deter-mination of the relative permeabilities of the apical and basal barrier of theepithelium of the gills of sea-water adapted trout from the kinetics ofradioactive loading and unloading of the lamellar epithelium. In the pre-sent investigation an identical experimental protocol has been carried outwith fresh-water trout. In addition, the exchangeable ionic pools arecalculated and compared to the total sodium and chloride content of thegill epithelium.The effect of catecholamines administered in vivo and in vitro on passive

and active salt movement across epithelia have been extensively studied(see review by Foster, 1974). In fresh-water teleosts, adrenaline enhancesthe bronchial Na+ absorption pump (Richards & Fromm, 1970) by in-creasing the active influx ofNa+ while the Na+ efflux is unchanged (Payan,Matty & Maetz, 1975). On the other hand the vasodilatory effect of adrena-line on fish gills has been widely described (Keys & Bateman, 1932; Muir &Brown, 1971; Steen & Kruysse, 1964). Thus, the action of this catechol-amine on salt exchange may be mediated either through indirect effect viavascular change by increasing the functional surface (Bergman, Olson &Fromm, 1974) or by direct action of adrenaline on Na+ pumping mech-anism. In the present study, kinetic experiments have been performedwith (10-5M) and without adrenaline in the perfusing solution. Theseexperiments allowed one to get a further insight into the mechanism ofadrenaline action on salt uptake in fresh-water trout.

METHODS

Experimental technique and biological material

The rainbow trout (Salmo gairdneri) purchased from a local dealer were of approxi-mately uniform weight (171 ± 3-1 g, n = 13). They were kept in aerated running tapwater at a temperature of about 17 'C. Feeding was discontinued a few days beforethe experiment. The preparation of the isolated head as well as the perfusiontechniquehas been previously described (Payan & Matty, 1975; Girard & Payan, 1976). Theexternal medium was aerated tap water at 17 0C containing an additional 1-5 mm-NaCl. Constant flow rate perfusion (190 ml./hr. 100 g) was utilized as described byGirard (1976).Without adrenaline in the perfusing fluid, the observed arterial and venous flows

were respectively: 127 + 11-8 ml./hr. 100 g and 68 + 9-2 ml./hr. 100 g (n = 8) underthese conditions the average value of the afferent pressure of perfusion into the ven-tral aorta is 38 ± 3-4 mmHg (n = 8). In presence of 10-5 M of adrenaline, efferentarterial and venous flows were respectively 121 + 16-8 ml./hr. 100 g and 67 + 21-6 ml./hr. 100 g (n = 5) with an average value of the afferent pressure of perfusion of27+1-9mmHg(n= 5).

196

KINETICS OF SALT FLUXES ACROSS TROUT GILL 197

Experimental protocolKinetics of the Na+ and Cl- exchanges were compared in two different sets of

experiments without adrenaline (n = 8) or with adrenaline (n = 5) in the perfusionfluid. Loading and unloading experiments were carried out successively on the samepreparation.

(a) Loading experiments. This protocol has been previously described by Girard &Payan (1977). Loading period lasts 15 min with sampling of the external medium at0, 5, 10 and 15 min. Collection of the arterial and venous liquids started when theisotopes were introduced in the external medium. This collection is carried out every15 see during the first minute, every 30 see during the following 2 min and then everyminute for 12 additional min.

(b) Unloading experiments. They were performed as described by Girard & Payan(1977) but with a different timing. The rinsing period of the external medium (opencircuit) preceding the unloading experiment was shortened to 45 see in order toappreciate more accurately the fast compartment. The external medium being thenswitched to a closed circuit, the sampling was operated every 15 see during the firstminute, then every 30 see for 2 min and finally every minute for 12 additional min.24Na was measured by y counting in a well counter (Mecaserto). After allowing forthe decay of 24Na, 36Cl was determined with an Intertechnique SL 40 liquid scintil-lation counter. The efferent perfusion flow rates were determined gravimetricallyand are expressed as ml./hr. 100 g. The concentration of stable Na+ and Cl- in theexternal medium were measured by flame photometry (Eppendorf) and Techniconauto-analyser. Adrenaline was purchased from Merck.

Method of calculationThe external medium (1), the lamellar compartment (2) and the internal medium

(3) are considered as a system of three compartments in series. Mathematical treat-ment of such a system has been previously described by Girard & Payan (1977).The ratio r of transfer coefficients (K2,/K2I) was calculated for Na+ and Cl- from thequantities of radioactivities simultaneously appearing in the external medium andin the internal compartment during the first minute of unloading. Calculation ofinfluxes at steady state, transfer coefficients, and cellular pools for Na+ and Cl- wasmade as described in a previous paper (Girard & Payan, 1977).

Determination of the intracellular ion content

[14C]Manmitol (Amersham Radiochemical Centre) as a marker of the intracellularspace was added to the internal medium (0-25 /zc/ml.). The quantity of ions origina-nating from the external medium (fresh water) was considered negligible. The markerwas allowed to equilibrate during 10 min of perfusion of the preparation. The eightgill arches were then removed, gently blotted on filter paper and weighed. Thefilamental epithelium was separated from the underlying cartilage and placed into4 ml. HNO3 N/5 for 24 hr. AliquotE of the HNO3 solution were added to Braysolution and counted in a scintillation counter (Intertechnique SL 40). Stable Na+and Cl- were measured respectively in the solution of HN03. The quantity ofintracellular ion was calculated by deducting the amount of extracellular ion from thetotal measured. The concentration of the ion in the extracellular space was assumedto be identical to that existing in the Ringer solution.

J-P. GIRARD AND P. PAYAN

RESULTS

Table 1 clearly shows that the concentrations of radioactivity in efferentarteries and veins measured at equilibrium are identical for the twoisotopes employed. Contrary to observations in sea water (Girard &Payan, 1977), this result demonstrates that no extralamellar salt entrycan be shown in fresh water. Consequently, the sum of the radioactivitiesmeasured in the arterial (*JA) and venous (*Jv) exits per unit of timerepresents the radioactive influx across the lamellar epithelium: *JL =

VA + *Jv, (in c.p.m. h-1. 100 g-1). The unidirectional fluxes JL (in ,&equiv.hr-1. 100 g-1) are calculated by dividing *JL by the specific radioactivity ofthe isotope (in c.p.m./,uequiv) in the external medium.

TABEi 1. Lamellar influxes (JL) and ratio of the radioactive concentrations (*C) inthe two efferent exits: arterial (A) and venous (V). Adrenaline effect

No adrenaline 10b m adrenaline

JL JI.*CJ*CV (#equiv/hr. 100 g) *CA/*CV (,uequiv/hr. 100 g)

Na+ 1*0+ 0*14 6.9+ 1*30 1-0 ± 0-08 47.8 + 4 12*C1l 0*9 + 0*09 6*6+1*55 0*9 ± 0*11 6*3 ± 2-40

Means values + S.E. with n = 8 for no adrenaline, n = 5 for 10- M adrenaline.Adrenaline effect: *P < 0.001. Lamellar influxes and radioactive concentrationsare calculated at steady state 15 min after adding isotope into the external medium.

Loading experiments. Fig. 1 (A and C) shows the kinetics of appearanceof 24Na and 36Cl in the internal lamellar compartment in the presence andabsence of adrenaline in the perfusion medium. Na+ and Cl- influxes cal-culated at equilibrium (the mean of the values obtained during the last3 min of radioactive loading) are shown in Table 1. The cumulative curvesof radioactivity appearance enable the sum of transfer coefficients K21+K23 to be calculated (Table 2) from the determination of 0 (see appendix:Girard & Payan, 1977). An example of the graphical evaluation of 0 forNa+ and Cl- is shown in Fig. 1 B and D. The analysis of Fig. 1A shows thatin the absence of adrenaline, the curves of appearance of 24Na and of 36CIare superimposed, consistent with the not significantly different values ofO (Fig. 1B). When 10-5M-adrenaline is present in the perfusion medium(Fig. 1 C), a significant increase ( x 7) of Na+ influx is provoked, whereasCl- influx remains unchanged (Table 1). Nevertheless, the sum of thetransfer coefficient under these conditions remains not significantlydifferent for Na+ and Cl- (Table 2).

Unloading experiments. Figs. 2 and 3 show data obtained from experi-ments where 24Na and M"Cl were rinsed after the loading period. Semi-

198

KINETICS OF SALT FLUXES ACROSS TROUT GILL 199

A CNo adrenaline 10-5 M adrenaline

c.p.m./hr. 100 g - c.p.m./hr. 100 gx10-3 x1O-3 12-----200 -C.P.m.

ONa=0-82 min / c.p.m.8c, =096 min /1 400 6,,=0N 65 min// J ~~~~~~~~~Oc,=075mn

20 L 20 j D

0 5 min 0 5 min

oldI 044

0 10 min 0 10 min

Fig. 1. Loading experiment: appearance of 24Na (M) and 36C1 (0) in theinternal lamellar compartment (*JL) in c.p.m./hr. 100 g. A, without adren-aline in the Ringer; C, in presence of 10 OM adrenaline. Each pointrepresents the mean + S.E. of eight experiments (A) and five experiments(C). The values are normalized to a specific radioactivity of theseions in the external medium (106 c.p.m.//uequiv). Inserts represent experi-mental curves of cumulative appearance of 2MNa (@) and 36C1 (0) in theinternal medium. B, without adrenaline in the Ringer; D, in presence of10- M of adrenaline.

TABLE 2. Sum oftransfer coefficient (K21 +K23) calculated from loading and unloadingexperiments concerning the fast exchangeable compartment. Adrenaline effect

Loadingexperiment Unloading experiment

InternalInternal External lamellar

compartment compartment compartment)

Na+ No adrenaline 112+ 7-9 120+ 15*2 104+ 6*210- M adrenaline 140 +26-6 188± 32-3 141+ 18-8

Cl- No adrenaline 97+ 53 135 +24*4 84 + 4-410- M adrenaline 150+ 27-4 144 +27-2 148 + 8-4

Means values ± S.E. with n = 8 for no adrenaline, n = 5 for 106 M adrenaline.K21 + K231 expressed in % . min-". Loading experiments: K21 + K23 obtained from thegraphical determination of 0 = I/K21 + K2 (min) (see Fig. 1). Unloading experimentK2, + K2 was calculated from the half-time (min) of radioactive unloading of thelamellar compartment into the internal and external compartment.

J-P. GIRARD AND P. PA YAN

ANo adrenaline

) 10510-

O O-

D10-5 M adrenalinec.p.m./min

atc.p.m.5x0,a External

Lamellar

1 m 01 0 min

104jB 104:External Lamellar

io Lamlla 103 xternal

104 ~N 104-

0 5 min 0 5 min

03 103

C~~ ~ ~ ~ ~F

0 2 min 0 2 min

Fig. 2. Left hand 8ide: 24Na radioactive unloading experiment and itsmathematical treatment without adrenaline in the Ringer. A, appearanceof 24Na in the external medium in c.p.m. and in the internal lamellar com-partment in c.p.m./min. Graphical analysis. B, in ordinate logarithm of thequantity of radioactivity appearing per unit of time in the external (El) andthe internal lamellar compartment (N). C, experimental curve representingthe fast exchangeable compartment, same symbols as B. Right hand 8ide:24Na radioactive unloading experiment and its mathematical treatment inpresence of 10- M adrenaline. D, E, F, same legend as A, B, C.

200

KINETICS OF SALT FLUXES ACROSS TROUT GILL 201logarithmic plots were constructed which compared the rates of appearanceof isotopes in the external medium and in the internal compartments.This analysis demonstrates that for Na+ and Cl-, two exponentials are

ANo adrenaline

103 C.P.M./min cp~External

Lamellar

0 0l in

D10-5 M adrenalinec.p.m./min c.p.m.l

10~4

1

0 2 min 0 2 min

Fig. 3. 36CI radioactive unloading experiment and its mathematical treat-ment without adrenaline in the perfusing solution (A, B, C) and in presenceof 105 M adrenaline (D, E, F). Same legend as for Fig. 2.

obtained having different slopes for the external and internal compart-ments (Table 3). While the tj for the rapidly turning over compartmentsare identical in both the internal and external media, differences in the tj

- 2x105

0

I

J-P. GIRARD AND P. PAYAN

values were observed for the slowly turning over components of both Na+and Cl- (Table 3). In Table 4 is shown the ratio of the transfer coefficients,r = K21/K23, which represents the ratio of the quantities of radioactivitieswhich appear in the external medium and in lamellar compartment duringthe first minute of unloading. When 10-5 M adrenaline is present in theperfusion medium, a non-significant increase of the sum of the transfercoefficients is observed for Na+ as well as for C1- (Table 2). Adrenalinesignificantly diminishes the ratio 'r' for Na+ by a factor of 4 withoutmodifying the corresponding value for C1-. This result is illustrated bycomparing the rapid exponentials of Figs. 2C and 2F as well as 3Cand 3F.

TABLE 3. Effect of adrenaline on the calculated half time of radioactive unloading ofthe lamellar compartment into external and internal lamellar compartment

Fast exchangeable Slow exchangeablecompartment compartment

External Internal External Internal

Na+ No adrenaline 0-6+0-07 07+0-04 1-8+0X13 5.9+ 1.09**10- M adrenaline 0-4+ 0-06 0-5+ 0-07 1-7 + 0-11 10-2 + 1-42***

Cl- No adrenaline 0-5+ 0-09 0-8 +0-04 3-1+ 0-62 6-9+ 1-54*10- M adrenaline 0-5+ 0-09 0-5+0-03 3-0+ 0-40 11-6 + 1-64**

Means values + s.E. with n = 8 for no adrenaline, n = 5 for 10-5 M adrenaline.Half-times are expressed in mn. Statistical comparison between external and inter-nal compartments: *P < 0-05, **P < 0-01, ***P < 0-001.

TABLE 4. Adrenaline effect on transfer coefficient (K) for Na+ and Cl-into the fast exchangeable compartment

K21 +K2 K21IKwJ = r K21 K23Na+ No adrenaline 112+6-9 7+0-8 101+5-1 11±2-8

10-5 M adrenaline 156± 30-6 2+0.1** 100 + 20-3 56 + 11.2**Cl- No adrenaline 105+8-3 12+4-1 91+7-2 14±3-1

104 M adrenaline 146 + 13.3* 12 + 2.5* 132 ± 13.6* 14+ 3-6

Means values + s.E. with n = 8 for no adrenaline, n = 5 for 10- M adrenaline.Adrenaline effect: *P < 0-02, **P < 0-001. K21 +K2, (expressed in % .min-") valueswere the average of the values obtained from loading and unloading experiments(see Table 2). r = K21/K2 were obtained from unloading experiments.

Characteristic of the lamellar compartment. In the two cases studied,with and without adrenaline, the sum of the transfer coefficients whichcorresponds to the rapid exponentials calculated during loading and un-loading experiments, is identical (Table 2). Consequently, it is possible tocompare the preparation to a system of three compartments in series

202

KINETICS OF SALT FLUXES ACROSS TROUT GILL 203

+1 -

0~~~~

+ ~~~~6

0 V

0 C)0~ ~

A ~~~~~~IOI,-

0 H+I +1+ *

! o oodo tz * "*O =

S ~~~~H-H -H-

coc

0~~~~~~

0~~~~

0 0

00

_ ~~~~~~~~~~~~~~~~~e.

- X- w-0 '

o *a o - o

*QC

-0Z p~~~~~~~: + 3~~~~b ~ ~~A 0

J-P. GIRARD AND P. PA YAN(Girard & Payan, 1977). An identical mathematical treatment of the slowexponential is not possible, since they have different tj values, dependingon whether they are measured in the external or internal lamellar com-partments (Table 3). Using this three compartment model, it is possibleto calculate the basal and apical fluxes across the lamellar epithelium, aswell as the size of the ionic pools participating in the exchanges. The sumof the transfer coefficients which is used for this calculation (Table 4) is themean of the values obtained with the loading and unloading experiments(Table 2).Table 4 shows the values of the transfer coefficient across the apical and

basal barriers of the lamellar epithelium. It appears that the basal mem-brane represents the limiting factor for salt entry into the gill. Uni-directional flux values across the membranes which limit the lamellarepithelium are shown in Table 5. It appears that adrenaline increases Na+flux across the basal membrane primarily by increasing the transfer coeffi-cient of this ion as well as its unidirectional flux F.3 (Tables 4 and 5). Novariation of these parameters was observed for Cl in the presence ofadrenaline.

Table 5 shows that the size of the exchangeable Na+ and Cl- pools aremuch smaller than the total intracellular Na+ and Cl- content of the gillepithelium. Adrenaline significantly increases the size of the exchangeableNa+ pool ( x 6) without noticeably modifying the size of the exchangeableCl- pool.

DISCUSSION

The absence of anastomoses between the afferent arteries and the venoussinus in the trout (Vogel, Vogel & Pfautsch, 1976; Laurent & Dunel, 1976)suggests that the totality of perfusion liquid entering via the ventral aortatraverses the lamellar exchange zone. In fresh water, the radioactive con-centrations of arterial and venous efferent liquids are equal (Table 1). Thus,NaCl entry into the gills occurs uniquely across the lamellae, in contrastto observations in sea water, where a negligible portion of influx( 25 %) follows an extralamellar path (Girard & Payan, 1977). In freshwater as in sea water, the 'ensemble' consisting of the external medium,the lamellar epithelium and the internal lamellar compartment may becompared to a system of three compartments in series. The mathematicaltreatment of this system allows the determination of the characteristicsof the limiting barriers of the lamellar epithelium, the site of ionicexchanges.

Comparison of equilibrium flux values obtained in vivo and in vitro. TheNa+ and Cl- influx values obtained in the course of this study (Table 1),in the absence and presence of 10-5 M-adrenaline, can be compared to the

204

KINETICS OF SALT FLUXES ACROSS TROUT GILL 205

corresponding values obtained in vivo in our laboratory. These latterexperiments were performed on animals of the same origin and in thepresence of 1 mM-NaCl in the external medium. Under these particularconditions, we can consider that the plasma concentrations of adrenalinevary between 3 x 10-8 and 106 M, depending on the stressed state of theanimal (Nakano & Tomlison, 1967). Influxes measured in the trout in vivoare 37-2 + 4418 uequiv.hr-'. 100 g-1 (n = 8) (unpublished results). Thisvalue is intermediate between that obtained in vitro with and withoutadrenaline in the perfusion Ringer. In the case of chloride influxes, thevalue obtained in vitro, with or without adrenaline (Table 1), is significantlysmaller than that obtained in our laboratory in vivo: 25-0 + 4*66 #tequiv.hr-1. 100 g-1 (n = 8) (G. De Renzis, personal communication). In additionthe values obtained in vitro are to be compared to those obtained byKerstetter & Kirschner (1972) utilizing the irrigated gill method: 20/uequiv. hr-'. 100 g-1. This difference in the values of chloride influxmeasured in vivo and in vitro will be discussed below.

Interdependence of trans-epithelial influxes of Na+ and Cl-. The kineticsof the appearance of 24Na and 36Cl in the internal medium as shown inFig. I are identical in absence of adrenaline. This catecholamine con-siderably modifies the appearance rate of Na+, while that of ClO remainsunchanged. Thus in the presence of adrenaline, and important componentof the Na+ influx, representing about 85% of the maximal flux, is inde-pendent of the Cl- influx (Table 1). Whether the 15% of Na+ influx in theabsence of adrenaline is linked to the Cl- influx remains to be discussed.In favour of such a coupled NaCl influx the following arguments may beput forward. The similarity of kinetics of 24Na and 36Cl appearances isconsistent with such an hypothesis. Also previous observations obtainedin vivo or on the perfused head preparation demonstrate that part of theNa+ or Cl- influx cannot be dissociated from the transport of the co-ion.For instance, if one of the two ions is eliminated from the external medium,then the influx of the other ion decreases significantly (Maetz & Garcia-Romeu, 1964; Kerstetter, Kirschner & Rafuse, 1970; De Renzis & Maetz,1973). Under the influence of specific inhibitors such as amiloride orthiocyanate the corresponding Na+ and Cl- influxes are inhibited by80-85 %. Thus a 15-20% component of either the Na+ or Cl- influxamounting to 6-10 /tequiv. hr. 100 g-1 is unaffected by these drugs in bothgoldfish and trout (Kirschner et al. 1973; De Renzis, 1975). Such a coupledentry mechanism for Na+ and Cl- has been demonstrated in other epitheliasuch as small intestine and gall-bladder (see review by Frizzel, 1976).

Relative permeability of apical and basal membranes. Ion movementacross the gill epithelium can be visualized in terms of two barriers, theouter cell membrane and the inner membrane (blood border). It is certain

J-P. GIRARD AND P. PAYANthat teleosts absorb both Na+ and Cl- actively in fresh water (Garcia-Romeu & Maetz, 1964; Kirschner, 1970). In spite of the observations ofKerstetter & Keller (1976), it is generally admitted that a coupled Na+/NH4+ or Na+/H+ exchange occurs at the outer membrane (Kerstetteret al. 1970; Maetz, Payan & De Renzis, 1976). Furthermore, Kerstetter etal. (1970) have suggested the presence of a Na+/K+ exchange pump locatedat the basal membranes of the gill epithelium of fresh-water teleosts. Thishypothesis has been confirmed by autoradiographic studies with the helpof [3H]ouabain in the chloride cells of Fundutlu heteroclitus adapted to lowsalinity media (Karnaky et al. 1976). Furthermore, addition of ouabain tothe perfusion fluid inhibits Na+ entry in perfused gills preparation (Richards& Fromm, 1970; Payan et al. 1975). Similar conclusion can be drawn fromthe effect of K+ removal in the perfusion fluid of eel gills studied byShuttleworth & Freeman (1974). Thus the flux across the baso-lateralmembrane is probably the energy dependent step of the Na+ transport.The present results show that the basal (or baso-lateral) membraneconstitutes the limiting step for Na+ entry across the epithelium.

Adrenaline acts in the apical membrane by increasing F21, therebyleading to a sixfold increase in the size of the exchangeable Na+ pool(Table 5). In addition, this catecholamine significantly increases the fluxacross the basal membrane, F23 (Table 5). This suggests that the mech-anism of active Na+ transport across the basal membrane is probably notsaturated in the absence of adrenaline. By increasing the size of theexchangeable Na+ pool, adrenaline would lead to an increase of the activepumping ofNa+ across the basal membrane. The increase of transepithelialNa+ fluxes may be associated with an increased permeability of the apicalmembrane towards this ion. The hypothesis according to which the rateof active transport is strongly dependent on the effective permeabilitytowards Na+ of the outer membranes has been previously suggested forfrog skin (Curran, Herrera & Flanigan, 1963; Morel & Leblanc, 1975).Active Na+ transport by isolated frog skin epithelium is increased when ,-adrenergic receptors are stimulated by low adrenaline concentration(Rajerison, Montegut, Jard & Morel, 1972). It is believed that the catechol-amine effects are mediated by modification in the levels of intracellularcyclic AMP. In fact, recent investigations (Pic, personal communication)have shown that adrenaline increases the level of cyclic AMP in the gillepithelium of the fresh water mullet. It is thus possible that the increasedactive transport ofNa+ observed after the stimulation of, receptors in thetrout (Payan et al. 1975) is dependent upon an increased intracellularconcentration of cyclic AMP.

Concerning Cl- movement, it has been suggested above that totalityof the Cl- influx measured in our preparation occurs through a coupled

206

KINETICS OF SALT FLUXES ACROSS TROUT GILL 207

NaCi transport. This would mean that Cl-/HCO3- exchange described byMaetz & Garcia-Romeu (1964), Kerstetter & Kirschner (1972, 1974) andDe Renzis (1975) in fresh water teleosts and localized on the apicalmembrane of the epithelium (De Renzis, 1975) is not functional in the per-fused head preparation. This deficiency is corroborated by the discrepancybetween the Cl- fluxes measured in vivo and in vitro.

Localization of the rapidly exchangeable ionic pools. The comparison ofthe lamellar epithelium to a three compartment system enables thedemonstration of rapidly exchangeable Na+ and Cl- pools which aredirectly implicated in ionic transfer. These pools represent 0 4% of thetotal Na+ and Cl- content of the gill epithelium, as measured in the presentwork (Table 5), and which confirms previously obtained results (Shuttle-worth & Freeman, 1973). While the total Na+ and Cl- content of the gillsis the same in fresh-water (Table 5) and sea-water adapted trout (J. P.Girard & P. Payan unpublished data), the exchangeable ionic pool is 10times larger in sea-water adapted fish (Girard & Payan, 1977).The comparative study of the ionic pools involved in transmembrane

exchanges in various epithelia shows that these pools are always smallerthan the total epithelial content of the corresponding ions. This is truefor the gills (present study and Girard & Payan, 1977) and for frog skin andtoad bladder epithelium (see review by Motais & Garcia-Romeu, 1972).This suggests that the cells involved in the ionic transport are a smallfraction of the epithelial cells. Evidence that active processing of Na+ andCl- occurs in the chloride cells is furnished by the presence of energy-yielding mitochondria (Sargent, Thomson & Bornancin, 1975). Therefore,the rapidly exhangeable chloride pool may be associated with thesechloride cells, a hypothesis which has already been advanced for salt-wateradapted trout (Girard & Payan, 1977). In frog skin, Morel & Leblanc (1975)have suggested that the rapidly exchanging Na+ pool is located in the outer-most living cell layers, whereas the slowly exchanging Na+ pool might belocated in underlying cell layers.

Our thanks are due to Dr J. Maetz for reading the manuscript and for his criticalcomments.

P. Payan is 'maitre-assistant' at the Comparative Physiological Laboratory,J-P. Girard is 'assistant' at the Cellular Physiological Laboratory, University ofNice, 06034 Nice, France.

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CuBRAN, P. F., HERRERA, F. C. & FIANIGAN, W. J. (1963). The effect ofCa and anti-diuretic hormone on Na transport. J. gen. Physiol. 46, 1011-1020.

J-P. GIRARD AND P. PA YANDE RENZIS, G. (1975). The bronchial chloride pump in the goldfish Carassiu8 auratuw:

relationship between Cl-jHCO- and Cl-/Cl- exchanges and the effect of thio-cyanate. J. exp. Biol. 63, 587-602.

DE RENZIS, G. & MAETZ, J. (1973). Studies on the mechanism of chloride absorptionby the goldfish gill: relation with acid-base regulation. J. exp. Physiol. 59, 339-358.

FOSTER, R. W. (1974). Adrenoceptors and salt and water movement in epithelia.Naunyn-Schmiedebergs Arch. exp. path. Pharmac. 281, 315-326.

FRIZZEL, R. A. (1976). Coupled sodium-chloride transport by small intestine andgallbladder. In Intestinal Ion Transport, ed. ROBINSON, J. W. L. pp. 101-109.Lancaster: MTP Press Ltd.

GARcIA RoMEu, J. & MAETZ, J. (1964). The mechanism of sodium and chloride up-take by the gills of a fresh water fish, Caramiius auratus I. Evidence for an inde-pendent uptake of sodium and chloride ions. J. gen. Physiol. 47, 1195-1207.

GIRARD, J. P. (1976). Salt excretion by the perfused head of trout adapted to seawater and its inhibition by adrenaline. J. comp. Physiol. 111, 77-91.

GIRARD, J. P. & PAYAN, P. (1976). Effect of epinephrine on vascular space of gillsand head of rainbow trout. Am. J. Physiol. 230, 1555-1560.

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