Effect of amiloride on sodium and water ... - Wiley Online Library

J. Phy8iol. (1983), 335, pp. 75-88 With 7 text-figure8 Printed in Great Britain

75

EFFECT OF AMILORIDE ON SODIUM AND WATER REABSORPTION IN THE RABBIT GALL-BLADDER

BY 0. FREDERIKSEN From the University Institute of Experimental Medicine, 71 Norre Alle, DK-2100 Copenhagen, Denmark

(Received 5 February 1982) SUMMARY

1. The effects of the Na+-channel-blocking diuretic agent amiloride were assessed in the rabbit gall-bladder epithelium, a low-resistance epithelium with an isosmotic, coupled NaCl transport mechanism. 2. Amiloride caused a rapid, reversible, and dose-dependent decrease in fluid absorption when applied from the mucosal side in concentrations between 8-8 x 10-5 and 1P76 x 10-3 M. These concentrations were without effect from the serosal side, suggesting an action of amiloride in the luminal cell membrane as in high-resistance epithelia. 3. Amiloride did not affect the epithelial resistance or the passive serosa-to-mucosa Na+ flux, while net Na+ and water reabsorption were inhibited in parallel. Thus, amiloride did not affect the paracellular tight junction pathway, but inhibited a transcellular, coupled salt and water transport mechanism. 4. The kinetics of the amiloride effect were of a Michaelis-Menten type. The dose of amiloride giving 50 % inhibition of fluid absorption (ID50) was 4 x 10-4 M, a value about three orders of magnitude higher than in high-resistance, Na+-retaining epithelia. 5. The percentage inhibitory effect at each concentration of amiloride increased with increasing rate of spontaneous (control) fluid transport, reaching maximal responses fitting a Michaelis-Menten kinetic with an ID50 of 1-5 x 10-4 M. 6. No effects of changing the extracellular Na+ concentration between 51 and 145 mequiv/l on the maximal inhibitory effect of amiloride on Na+ and water reabsorption were observed. This suggests a non-competitive type of action of amiloride on a Na+-dependent isosmotic fluid transport mechanism. 7. Removal of mucosal Ca2+ did not alter the effect of amiloride. 8. The implications of these findings are discussed in relation to concepts concerning the mechanism of isosmotic salt and water transport. The data are compatible with the concept that amiloride interferes with a Na+-dependent formation and transcellular transport of isosmotic fluid volumes in a sequestered compartment in the epithelial cells.

76

0. FREDERIKSEN INTRODUCTION

Theories about the mechanism of transport of Na+ ion across isosmotically transporting epithelia were initially developed as conceptual analogues of the two-membrane hypothesis formulated by Koefoed-Johnsen & Ussing (1958) from studies in frog skin. Thus, Na+ was supposed to enter the cytoplasm across the apical (luminal) cell membrane by a passive (electrodiffusional) process as a consequence of a favourable electrochemical gradient, while Na+ exit from the cytoplasmic transport pool across the basolateral cell membrane was assumed to involve an active, neutral NaCl pump (see, e.g. review by Diamond, 1968). Recent studies have suggested that Na+ uptake across the apical cell barrier into the cellular transport compartment in isosomotically transporting epithelia such as gall-bladder and small intestine is governed by an apparently neutral carrier-mediated NaCl uptake mechanism driven by the electrochemical gradient for Na+ (see e.g. Frizzell, Field & Schultz, 1979). The diuretic agent amiloride has emerged as a very useful probe for the elucidation of the mechanism of Na+ transport in frog skin and other Na+-retaining, highresistance epithelia. In these epithelia amiloride is known to be a potent inhibitor of the passive Na+ flux through the Na+-selective outer (apical) cell membrane (for a review see Cuthbert & Shum, 1974b; Cuthbert, 1981). In an attempt to evaluate further the mechanism of Na+ transport in low-resistance, isosmotically transporting epithelia the possible effects of amiloride on water and Na+ transport in rabbit gall-bladders were tested. The results show that amiloride applied to the luminal side of the epithelium inhibits Na+ and water reabsorption in rabbit gall-bladders as in the Na+-retaining epithelia, probably by inhibiting Na+ uptake across the luminal cell barrier. Preliminary results of part of this investigation have been reported previously (Frederiksen, 1973; Frederiksen & Eldrup, 1981). MATERIALS AND METHODS

Female white rabbits weighing about 3 kg were killed by a blow on the neck. The gall-bladder was removed, rinsed and prepared for further studies as described previously (Frederiksen & Leyssac, 1969, 1977).

Fluid transport mea8urement8. Fluid transport rates were measured gravimetrically in cannulated, non-everted sac preparations (Diamond, 1964; Frederiksen & Leyssac, 1969, 1977). All experiments were carried out at 37 00 with identical Ringer solutions (see below) on both sides unless otherwise stated. Weighing periods of 5 or 10 min were used, and luminal (mucosal) content was renewed between weighing periods to ensure constant composition. Solution and chemical. The composition of the Ringer solution used in most of the experiments was (mM): 114-7, Na+; 7, K+; 2, Ca2+; 1-3, Mg2+; 102, Cl-; 17-5, HC03-; 1-2, S042-; 1-2, H2P047; 5, monoglutamate; and 11, glucose. In some experiments Na+ concentrations in the bathing Ringer other than 114-7 mequiv/l were used. Thus, concentrations or 51, 75 or 145 mequiv/l were obtained by varying the NaCl concentration. The osmolarity of the Ringer solution varied in parallel with these concentration changes. The pH was adjusted to 7-4 by equilibration with 96% 02 and 4% C02 at 37 0C. In a series of experiments Ca2+ was removed from the luminal medium by removing the 2 mM-CaCl2 with or without addition of 1 mM-EDTA. EDTA (ethylenediaminetetraacetic acid) was obtained from Merck (Darmstadt), and amiloride was a generous gift from Merck, Sharp and Dohme (Copenhagen, Denmark). Measurement8 of tran8epithelial electrical potential difference (p.d.) and resistance (RJ). These

AMILORIDE AND ISOSMOTIC FLUID TRANSPORT

77

measurements were performed with gall-bladders cut open and mounted between two Ussing half-chambers as described in detail previously (Leyssac, Bukhave & Frederiksen, 1974). Exposed gall-bladder surface area was 0-9 cm2; each half-chamber contained 10 0 ml Ringer solution; and temperature was kept at 37 'C. Rt was calculated from the change in p.d. after a short-lasting passage of 100 1IA of d.c. current (from the mucosal to the serosal side). All p.d. and Rt values were

corrected for values measured without the gall-bladder mounted. Measurements of Na+ fluxe8. Mucosa-to-serosa Na+ flux (JmN) was determined in gall-bladder sac preparations as described in detail previously (Leyssac et al. 1974). Briefly, the gall-bladder was filled with Ringer solution containing 2-4,uCi 22Na+/ml (22Na+ was obtained from Amersham International Ltd, England, as a carrier-free solution of NaCl). The bladder was then weighed and incubated at 37 0C in a beaker containing 25-0 ml Ringer solution (serosal medium) without labelled Na+. Samples (100 1I) of serosal fluid were taken every 30 s during the flux period (10-15 min). The preparation was then reweighed. JNa was calculated from the increment in serosal 22Na+ content and the mean specific activity of 22Na+ on the mucosal side. No correction was made for serosa-to-mucosa flux (Js) of 22Na+, as the specific activity of the serosal medium never exceeded 2 % of that of the mucosal medium. Counts of radioactivity were done to at least 104 counts in a gamma-well scintillation counter (Selektronik, Copenhagen, Denmark). JmN became rectilinear within 2-3 min. Net Na+ flux (JNet) was calculated from the loss of weight of the gall-bladder sac preparation during the flux measurement period, assuming the transported fluid to be isosmotic and Na+ to constitute 95% of the transported cations (Diamond, 1964). Finally, Jm could be calculated from the relation: JNm - JNa-neatIn a series of experiments Jm was measured directly in gall-bladders mounted in the Ussing chambers described above. The method has been described previously (Frederiksen & Leyssac, 1977). Briefly, 22Na+ was added to the serosal side and 100 pl samples were collected every 5 min from the mucosal side; the sample volume was replaced with 100 j1 cold Ringer solution. Statistic8. The data are presented as the means of values from individual gall-bladders or the means of differences of paired observations in individual experiments +s.E. of means. Student's t-test was used to determine statistical significance. RESULTS

Inhibition of fluid absorption by amiloride The average value of net fluid transport rate in the control period in gall-bladders incubated with Ringer solution containing 114-7 mequiv Na+/l was 32-2 + 1-86 mg/ min per mg tissue dry weight, in agreement with previous observations (Frederiksen, 1978). Application of amiloride in concentrations between 0-88 x 10-4 and 1-76 x 10-3 M to the mucosal Ringer solution for 60 min caused a dose-dependent decrease in fluid transport rate. This is shown in Fig. 1; the line drawn in this Figure is the theoretical one assuming a Michaelis-Menten-type reaction. The luminal concentration of amiloride to give 50 % inhibition of fluid absorption (ID50) was 4 x 10-4 M. Fig. 2 shows the time course of the effect of mucosal application of amiloride. The onset of inhibition was instantaneous, but the half-time for obtaining maximal effect was rather long (5-10 min). The effect of amiloride was reversible. This applied even to the highest concentration used (1-76 x 10-3 M). Application of amiloride (up to 1'76 x 10-3 M) from the serosal side was without any effect on fluid transport rate, and application from both sides of the epithelium elicited an effect similar to that of mucosal application.

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Fig. 1. Dose-response relationship for the effect of amiloride applied from the luminal side on fluid absorption by rabbit gall-bladder. Extracellular Na+ concentration was 114-7 mequiv/l. The drawn curve gives the theoretical relationship for Michaelis-Mententype kinetics assuming a maximal inhibitory effect of 100% and an ID60 of 4 x 10-4 M. Numbers indicate the number of experiments. Values are given as means + s.E. of means (bars).

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Fig. 2. Time course of the effect of amiloride applied from the luminal side on gall-bladder fluid absorption at 114-7 mequiv Na+/l. Values are given as means + s.E. of means (bars) (n = 6 for 0-44 mM-amiloride (open circles) and n 4 for 1-76 mM-amiloride (filled circles)). =


79

Dependence of fluid transport inhibition on the functional state The percentage inhibition of fluid absorption at a given concentration of amiloride varied somewhat from experiment to experiment. An analysis of the results demonstrates that the percentage inhibition was a function of the functional state of the epithelium before the application of amiloride, i.e. the lower the initial control fluid transport rate the lower the percentage inhibition of fluid transport by amiloride and 60 50

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Fig. 3. Plot of absolute decrease in gall-bladder fluid transport induced by mucosal amiloride as a function of control fluid transport rate, both expressed in terms of mg/h per mg tissue dry wt. Each point is the result from one gall-bladder. The effects of three concentrations of amiloride at an extracellular Na+ concentration of 1 14-7 mequiv/l are shown. El, 1-76 x 1O-3 M; 0, 8-8 x 1O-4 M; A, 2-0 x 1O-4 M-amiloride. Lines through data points were calculated by the method of least squares. Results with a fourth concentration of amiloride (4 4 x 1O-4 M) gave points and a slope of the regression line between those for 2-0 x 1O-4 M and 8-8 x 1O-4 M-amiloride (not shown for the sake of clarity). The slope of a line indicates the maximal inhibitory effect of amiloride at that particular concentration.

vice versa. In Fig. 3 the absolute decrease in fluid transport rate is plotted as a function of control fluid transport; data for three concentrations of amiloride are shown. Straight lines can be drawn through the data points but these lines intercept the abscissa (control transport rate) at values above zero, thus demonstrating that percentage inhibition of fluid transport rate increases with increasing control fluid transport rates in the epithelium. The maximal percentage inhibition of fluid transport rate at a given concentration of amiloride is determined by the slopes of the lines shown in Fig. 3. Plotting these maximal inhibitory values in a log dose-response diagram gives a relationship which, like the data in Fig. 1, indicate a Michaelis-Menten-type interaction of amiloride with the fluid transport mechanism, but the curve is shifted to the left (ID50 = 1.5 x 10-4 M).

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Fig. 4. Decrease in gall-bladder fluid absorption as a function of control fluid transport per mg tissue dry wt) in response to mucosal application of 2-0 x 10-4 M-amiloride (A) and 8-8 x 10-4 M-amiloride (B). The effects of amiloride at four different extracellular Na+ concentrations between 51 and 145 mequiv/l are shown: V, 51; 0, 75; *, 114-7; A, 145 mequiv/l. Each point is the result from one gall-bladder. Regression lines calculated for each Na+ concentration were not statistically significantly different at one particular concentration of amiloride. The lines shown in both panels are the regression lines for all data points; these lines are not significantly different from the corresponding lines in Fig. 3 obtained with 114-7 mequiv Na+/l only. rate (both in terms of mg/h

Effect of extracellular Na+ concentration

on

the effect of amiloride

In the abdominal skin from Rana temporaria (Salako & Smith, 1970; Cuthbert & Shum, 1974 a, b) it has been demonstrated that a reduction in Na+ concentration of the outer bathing solution potentiates the effect of amiloride. The kinetics of this effect indicate that amiloride competes directly with Na+ for the entry via the

AMILORIDE AND ISOSMOTIC FLUID TRANSPORT 81 Na+-selective channels ofthe apical cell membrane, with a stoichiometry of one-for-one for the reaction between Na+, amiloride and the channel (Cuthbert & Shum, 1974b). A possible similar mechanism of action in the gall-bladder epithelium was tested by comparing the inhibitory effect of mucosal amiloride (2-0 x 10-4 M and 88 x 1O-4 M) at extracellular concentrations of Na+ of 51, 75 and 145 mequiv/l with the abovementioned results at 114-7 mequiv Na+/l. Small but inconsistent and insignificant changes in the percentage inhibitory effect of amiloride on fluid transport rates were observed. These small changes could probably be attributed to differences in control fluid transport rates at different extracellubhr Na+ concentrations (Frederiksen & Leyssac, 1969). In any case, when the absolute inhibition of fluid transport by amiloride at Na+ concentrations of 51, 75, 114-7 and 145 mequiv/l was plotted as a function of control transport rates (see Fig. 4) the data obtained fitted straight lines not significantly different from those in Fig. 3 obtained with 114-7 mequiv Na+/l. Since net Na+ and water reabsorption rates were equally inhibited by amiloride (see below), these results indicate that the maximal percentage inhibitory effect of amiloride on Na+ and water reabsorption is independent of the extracellular Na+ concentration within the range of concentrations studied. Importance of Ca2+ for the effect of amiloride In frog skin removal of Ca2+ from the outer solution increases Na+ transport by increasing Na+ entry through the outer cell membrane (Curran & Gill, 1962; Curran, Herrera & Flanigan, 1963); it also reduces or abolishes the inhibitory effect of amiloride (Cuthbert & Wong, 1972). A similar effect of mucosal Ca2+ removal on Na+ and water reabsorption by rabbit gall-bladder cannot be demonsatrated

(0. Frederiksen, unpublished observation). Fig. 5 shows that the inhibitory effect of 8-8 x 1O-4 M-amiloride on gall-bladder fluid transport rate was not affected when mucosal Ca2+ was reduced from 2 mm to zero (in the presence or absence of 1O-3 M-EDTA). Thus, the data fitted the same relationship between control fluid transport rate and transport inhibition by amiloride as obtained in the presence of Ca2+ (dashed line in Fig. 5). Effect of amiloride on Na+ ftuxes In a series of experiments (n = 5) the effect of mucosal 8-8 x 10-4 M-amiloride on JNwas measured in gall-bladder sac preparations. The results of these experiments are shown in Table 1. JNat was calculated from the fluid transport rate measured simultaneously with the JNa assuming the transported fluid to be isosmotic with the bathing Ringer solution both in the absence and presence of amiloride, and assuming Na+ to constitute 95 % of the transported cations (Diamond, 1964). JNa was calculated from the difference between JNs and JnNt. As seen from Table 1, JNa was unaffected by amiloride. In a second series of experiments (n = 5) the effect of mucosal 8-8 x 1O-4 M-amiloride on JNm was measured directly in gall-bladders mounted in Ussing chambers. The results are shown in Fig. 6. It is seen that J~N was completely unaffected by amiloride, thus confirming the fluxes calculated from sac preparations. Consequently, the assumption of isotonicity of the transported fluid after partial inhibition of the fluid transport by amiloride is correct, indicating that amiloride inhibits net Na+ and net water reabsorption in parallel.

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TABLE 1. Effects of mucosal 8-8 x 10-4 M-amiloride on transepithelial Na' fluxes in rabbit gall-bladder sac preparations jNa jNa iNa Condition net sm ms Control 3-10+0-47 1-76 +0-26 1*34 +0-22 Amiloride 1-81 +0-22 0-51 +0-08 1-30+0-16 Inhibition by amiloride 1-29 + 0-42 1-25+0-24 004+0-24 All values are in /tequiv/min in whole sac preparations, and represent means+ S.E. of means of five experiments. JNa was measured as 22Na+ efflux; JNa was calculated from the gravimetrically measured fluid transport rate assuming isotonicity; and Jsm was calculated from the differences between JNI and JNa in the single experiments. For further explanation see Materials and Methods.

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10 20 30 40 5060 Control fluid transport (mg/h per mg dry wt) Fig. 5. Inhibition of gall-bladder fluid absorption by mucosal 8-8 x 10-4 M-amiloride as a function of control fluid transport rate in the absence of mucosal Ca2+. Each point is the result from one gall-bladder. Zero mucosal Ca2+ was obtained either by simple removal of mucosal CaCI2 (open circles) or by adding 1 mM-EDTA in the absence of Ca2+ on the mucosal side (filled circles). The dashed line gives the corresponding inhibitory effect of amiloride in the presence of mucosal 2 mM-Ca2+ (obtained from Fig. 3). Extracellular Na+ concentration was 114-7 mM.

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83

The control value of JNa in Ussing chambers was 173-3+7-1 nequiv/min cm2 (n = 5). In sac preparations with a serosal surface of 5-6 cm2 the average value of JNa of 1-34 mequiv/min corresponds to about 250 nequiv/min . cm2. This difference in control JNa may wholly or partially be due to the fact that gall-bladders are slightly stretched when mounted in Ussing chambers. Effects of amiloride on p.d. and Rt Stable values of p.d. and Rt in the control period were 3-4 + 02 mV (mucosal side positive) and 48-2 + 3 0 ohm . cm2 (n = 13), respectively. These values are in agreement with previous observations (Frederiksen, 1978; Frederiksen, M0llga'rd & Rostgaard, 1979). Application of 8-8 x 10-4 M-amiloride to the mucosal side for 60 min (see Fig. 7) had no effect on Rt; and the small change in the mucosa-positive p.d. of -0 15+0*35 mV (n = 13) was not statistically significant. Amiloride

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Fig. 7. Effects of mucosal 8-8 x 10-4 M-amiloride on transepithelial resistance (Rt; open circles) and potential difference (p.d., mucosal side positive; filled circles). Values are given as means+ S.E. of means (n = 13). DISCUSSION

The present results demonstrate that amiloride, as well as being an inhibitor of Na+ transport in high-resistance, Na+-retaining epithelia, also inhibits isosmotic Na+-salt and water transport in a low-resistance epithelium such as the gall-bladder. In high-resistance epithelia amiloride selectively inhibits passive cellular Na+ uptake across the apical cell barrier. The present study suggests a similar site of action of amiloride in the gall-bladder epithelium. Thus, amiloride reversibly inhibits fluid absorption in a dose-dependent manner when applied from the luminal side, while serosal application of the drug was without effect. It further seems that amiloride does not affect the high-conductance paracellular tight junction pathway through which the majority of passive, diffusional ion flux takes place (Frdmter, 1972). Thus, amiloride did not affect either Rt (Fig. 7) or JNa (Fig. 6). Since the paracellular route in rabbit gall-bladders does not serve as a pathway for net fluid absorption (Frederiksen et al. 1979) the present demonstration of a parallel decrease in net Na+ and water transport upon addition of amiloride must

0. FREDERIKSEN represent a decrease in active, coupled salt and water transport through a cellular

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pathway. Very few conclusive data are available regarding a possible effect of amiloride in low-resistance epithelia showing an isosmotic coupled NaCl transport mechanism. Amiloride administered intravenously in diuretic doses exerts its effects on the distal part of the nephron without any effect on proximal tubular isosmotic salt and water reabsorption (Duarte, Chomety & Giebisch, 1971; Wilczewski, Olson & Carrasquer, 1974). Attempts to disclose an effect on fluid reabsorption of high concentrations of amiloride (10-4 to 1O-3 M) from the luminal side of proximal tubules in rats, using split oil-drop or microperfusion techniques (Carrasquer, Fravert & Olson, 1974; Meng, 1975), led to inconsistent results possibly due to the numerous sources of errors with both methods (Gottschalk & Lasssiter, 1973). During free-flow perfusion of rat proximal tubules Fromter & Gessner (1975) were unable to detect any effects of luminal 1O-3 M-amiloride on transepithelial potential and resistance; this was interpreted as a lack of effect of amiloride on net ion movements and paracellular conductance. In the Necturu8 gall-bladder mucosal 1O-4 M-amiloride failed to interfere with the Na+ conductance of the luminal cell membrane and of the paracellular pathway (van Os, 1974), but a possible effect of amiloride on net Na+ and water transport was not investigated. Evidence has been presented that amiloride is a highly specific blocker of Na+ channels in the outer (apical) cell membrane of a wide variety of high-resistance, Na+-retaining epithelia. Since isosmotic fluid absorption in the gall-bladder requires the presence of Na+ in the mucosal bathing solution (Diamond, 1962; Wheeler, 1963) and, further, the site of action of amiloride resides in the apical cell membrane, it is likely that amiloride also in this epithelium interferes in some way with a Na+-specific site or channel in the luminal cell membrane preventing uptake of Na+-salts and water in isosmotic proportions from the luminal bathing medium into the cellular transport compartment. However, the concentration of amiloride necessary to give 50 % inhibition (ID50) of fluid absorption by gall-bladder is about three orders of magnitude higher than the ID50 of amiloride on Na+ transport in Na+-retaining epithelia. This might suggest that the affinity of amiloride for its binding site is much smaller in the gall-bladder than, for example, in frog skin, and/or that binding of amiloride has a much smaller effect on Na+ uptake in gall-bladders than in anuran epithelia. The present demonstration that the percentage effect of a given concentration of amiloride on gall-bladder isosmotic fluid absorption depends on the transport rate before application of the drug suggests either that Na+ uptake takes place both by an amiloride-sensitive and an amiloride-insensitive mechanism, or that the binding of amiloride to the luminal membrane depends on the functional state of the epithelium. The following arguments seem to exclude the former possibility: (1) the dose-response relationship (Fig. 1) fitted a Michaelis-Menten-type kinetic with 100 % maximal inhibition; and (2) the entire luminal Na+ uptake in rabbit gall-bladder takes place by a single coupled Na+/CI- translocation process (Frizzell, Dugas & Schultz, 1975; Cremaschi & Henin, 1975). The latter possibility, in turn, would be in agreement with the suggestion that the rate-limiting step in the process of transcellular transport of fluid is the luminal uptake of NaCl and water (Frederiksen & Leyssac,


85

1969), a notion which is further supported by the above-mentioned MichaelisMenten-type kinetic of the interaction of amiloride with the entire transepithelial fluid transport process. Salako & Smith (1970) and Cuthbert & Shum (1974a) have reported that the ID50 of the effect of amiloride on Na+ transport in the abdominal skin of Rana temporaria was shifted towards lower values when the Na+ concentration in the outer bathing solution was lowered. This suggests that amiloride competes with Na+ for entry via the Na+ channel in a one-for-one manner. A similar effect of changing the extracellular Na+ concentration (between 51 and 145 mequiv/l) on the effect of amiloride on fluid absorption by gall-bladder could not be demonstrated in the present study. Thus, the maximal effect of a given concentration of amiloride was independent of the Na+ concentration in the external medium (Fig. 4). This result suggests that the interaction of amiloride with a Na+ site or channel in the apical cell membrane is not of a competitive type. Non-competitive types of inhibition kinetics have been reported in toad bladder (Bentley, 1968) and in skin from frogs other than Rana temporaria (Benos, Mandel & Balaban, 1979). In anuran epithelia species differences exist regarding the requirement of external Ca2+ ions for the inhibitory action of amiloride (Benos et al. 1979), indicating that binding of amiloride does not necessarily involve complex formation with Ca2+ as was suggested by Cuthbert & Wong (1972). This notion is supported by results in the present study demonstrating that the effect of amiloride on gall-bladder isosmotic transport is independent of the presence of Ca2+ in the luminal medium. A generally accepted basic concept regarding the mechanism of isosmotic fluid transport is that water transport is a passive consequence of active Na+ (or NaCl) transport from the cytoplasm into the lateral intercellular spaces where the osmotic equilibration is held to take place (see e.g. Diamond & Bossert, 1967). Recent studies of epithelial geometry (Rostgaard & Frederiksen, 1981), water permeability (Hill, 1980), and lateral space hypertonicity (Simon, Curci, Gebler & Fr6mter, 1981), however, have led to a critical re-evaluation of this hypothesis. Previous results from rabbit gall-bladder suggest, in fact, that the route for most if not all of the net salt and water transport is through the epithelial cells (Frederiksen et al. 1979). Transport-linked 02 consumption in rabbit gall-bladders was found to be linearly related to the transfer rate of fluid volumes rather than to the net transport of Na+ (Frederiksen & Leyssac, 1969). These data were interpreted as evidence for a mechanical fluid transport mechanism rather than an ion-pump or salt-pump mechanism. A model was proposed which involves formation of isosmotic fluid volumes beneath the luminal cell membrane, dependent on specific Na+ (plus anion) binding and translocation. Fluid volumes thus formed were assumed to be transported mechanically through the cells sequestered from the cytoplasm (Frederiksen & Leyssac, 1969; Leyssac & Frederiksen, 1974). The model is kinetically (but not structurally) similar to pinocytosis with regard to the solutes being transported. The contractile rature of the solute and water transport mechanism in gall-bladder is supported by its sensitivity to cytochalasin B (Frederiksen & Leyssac, 1977). Recently, the mechanical nature of gall-bladder isosmotic fluid transport was further supported by the demonstration of its sensitivity to a small serosal hydrostatic pressure (Eldrup, Frederiksen, M0llgard & Rostgaard, 1982). A serosal pressure as

86

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small as 4 5 cm H20 nearly abolished the cellular active Na+ and water transport. decrease in JNat resulted primarily from a decrease in JNa. A small increase in JNa induced by the serosal pressure was completely prevented when 10-3 M-amiloride was added to the mucosal medium. In view of the present demonstration that the site of action of amiloride resides in the luminal cell membrane the former observation suggests that the pressure-induced small increase in JsN is transcellular, and that a serosal pressure in some way is transmitted through the epithelial cells to the luminal cell membrane. It has been suggested (Eldrup et al. 1982) that transcellular fluid absorption takes place via the cellular tubulo-cisternal endoplasmic reticulum (t.e.r.), thus allowing the transported fluid to by-pass the cytoplasm, as suggested previously (Frederiksen & Leyssac. 1969). The t.e.r. is highly developed in isosmotic transporting epithelia (including gall-bladders), and it makes contact with both the apical and the basolateral cell membranes (M0llgard & Rostgaard, 1981). Although mechanisms for entry into and exit from this possible route for transcellular fluid movement remain unsolved, a possible site of action of amiloride could be the very small areas of contact between the t.e.r. and the apical cell membrane. At these areas amiloride might block a fast uptake of Na+ (together with Cl- and water) which has the character of an electroneutral NaCl transfer (Frizzell et al. 1975), leaving the rest of the apical cell membrane, which (from an electrophysiological point of view) has a low Na+ permeability (Reuss & Finn, 1975; Henin & Cremaschi, 1975; van Os & Slegers, 1975), unaffected. Further, the observation that the degree and pattern of inhibition of isosmotic fluid absorption at each concentration of amiloride are independent of the extracellular Na+ concentration (Fig. 4) might suggest that amiloride inhibits a Na+-dependent formation of isosmotic fluid volumes at the luminal cell membrane; this would be in agreement with the proposed transport model (Frederiksen & Leyssac, 1969). Thus, the

The present study was supported by grants from the Danish Medical Research Council. The skilful technical assistance of Mrs Conni Temdrup and Mrs Lisbeth Wybrandt, and the valuable criticism of the manuscript given by Dr P. P. Leyssac and I)r L. Baumbach are gratefully acknowledged. REFERENCES BENOS,I). J., MANDEL, L. ,J. & BALABAN, R.S. (1979). On the mechanism of the amiloride-sodium entry site interaction in anuran skin epithelia. J.gen. Physiol. 73, 307-326. BENTLEY, P. J. (1968). Amiloride: a potent inhibitor of sodium transport across the toad bladder. J. Physiol. 195, 317-330. CARRASQUER, G., FRAVERT, D. G. & OLSON, A. K. (1974). Effect of intraluminal amiloride on Na transport in the rat proximal tubule. Proc.Soc. exp. Riol. Med. 146, 478-480. CREMASCHI, 1). & H9NIN, S. (1975). Na+ and Cl- transepithelial routes in rabbit gallbladder. Tracer analysis of the transports. Pfluyers Arch. 361, 33-42. CURRAN, P. F. &GJIL, J. R. (1962). The effect of calcium on sodium transport by frog skin. J.yen. Physiol. 45, 625-649. CURRAN, P. F., HERRERA, F. C. & FLANIGAN, W. J. (1963). The effect of Ca and antidiuretic hormone on Na transport across frog skin.II. Sites and mechanisms of action. J.gen. Physiol. 46, 1011-1027. A. W. (1981). Sodium entry step in transporting epithelia: results of ligand-binding studies. In Ion Transport by IEpithelia, ed. SCHITTZ, S. G., pp. 181-195. New York: Raven Press. A. W. & SHUM, W. K. (1974a). Binding of amiloride to sodium channels in frog skin. Molec. Pharmac. 10, 880-891.

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