(Received 14 November 1978) (0.95 cm2 pieces) was measured at different external sodium concentrations. With a

Transcription

1 J. Phy8iol. (1979), 295, pp With 5 text-ftgurem Printed in Great Britain UPTAKE OF [3H]BENZAMIL AT DIFFERENT SODIUM CONCENTRATIONS. INFERENCES REGARDING THE REGULATION OF SODIUM PERMEABILITY BY J. ACEVES* AND A. W. CUTHBERT From the Department of Pharmacology, University of Cambridge, Hills Road, Cambridge CB2 2QD (Received 14 November 1978) SUMMARY 1. The effect of benzamil on short-circuit current in frog skin was measured at different external sodium concentrations. A linear relationship exists between the concentration of benzamil reducing short-circuit current by 50 % and the external sodium concentration, indicative of some form of competitive antagonism between sodium and benzamil. 2. Uptake of [3H]benzamil into isolated frog skin epithelium and whole skin (0.95 cm2 pieces) was measured at different external sodium concentrations. With a sodium concentration of 111 mm in the external medium the uptake of [3H]benzamil is linear with concentration. Uptake amounted to 8-8 f-mole nm-1, a value similar to the linear component of the uptake measured at low (1 1 mm) sodium concentration. 3. Using a variety of other conditions the maximal number of specific binding sites for [3H]benzamil was calculated from displaceable binding and the fractional occupancy, the latter being derived from the inhibition of short-circuit current. This approach gave similar binding site densities to those reported previously at low sodium concentrations. 4. The reduction in specific [3H]benzamil uptake at high sodium may result from two mechanisms, competition of sodium with the ligand for an external binding site and a reduction in the site density as the intracellular sodium concentration increases. 5. It is concluded that the saturation of sodium transport which occurs at high sodium concentration is likely a consequence of the reduced availability of entry sites, rather than saturation of the uptake process. INTRODUCTION In sodium transporting epithelia the relation between the sodium transport and the sodium concentration is hyperbolic (Cereijido, Herrera, Flanigan & Curran, 1964; Biber & Curran, 1970). Explanation for this type of saturating behaviour is that sodium moves into the epithelium using a carrier system, which at high sodium concentration becomes saturated. * Present address: Departmento de Fisiologia, Centro de Investigacion del JIPN, Mexico 14, DF, Mexico /79/ $ The Physiological Society.

2 492 J. ACEVES AND A. W. CUTHBERT In recent years ideas have been changing about the control of sodium entry, and the experiments to be described here were designed to investigate further how sodium itself may regulate sodium entry into frog skin epithelium. The regulation of sodium entry at the outer or mucosal face may be dependent on the intracellular sodium concentration. For example, Erlij & Smith (1973) found that sodium uptake through the mucosal surface of frog skin was inhibited when epithelia had been preincubated with ouabain in the presence of sodium. Later it was found (Cuthbert & Shum, 1978) that when epithelia were preincubated with ouabain the amount ofdisplaceable [14C] amiloride measured under low sodium conditions was reduced, but only when the tissue had been exposed to high sodium immediately before labelling. It was suggested that when the intracellular sodium concentration was raised the entry sites were modified in a way which prevented sodium entry. Similar conclusions on the effects of raised intracellular sodium have been arrived at with rabbit urinary bladder (Lewis, Eaton & Diamond, 1976) and rabbit colon (Turnheim, Frizzell & Schultz, 1978) using different experimental approaches. Evidence for the regulation of sodium permeability by sodium acting from outside the mucosal membrane of frog skin has also been described. For example, when the mucosal sodium concentration is suddenly increased the short circuit current increases rapidly and then declines, which it was suggested was due to some entry sites being switched off by an action of sodium at an external regulator site (Zeiske & Lindemann, 1974; Fuchs, Hvid Larsen & Lindemann, 1977). In addition Lindemann & van Dreissche (1977) showed from current fluctuation analysis that the number of entry sites contributing to the noise was decreased when the sodium concentration was raised. With [3H]benzamil (Aceves, Cuthbert & Edwardson, 1979) of high specific activity and affinity it was possible to consider binding measurements to frog skin epithelium at other than low sodium concentrations, the only situation which could be attempted with [14C]amiloride. METHODS The methods used in this paper are essentially the same as those of the previous paper (Aceves et al. 1979). Uptake of [3H]benzamil by the mucosal surface of frog skin epithelium and whole skin was measured during min in pieces 0 95 cm2. The serosal (inner) solution was always normal Ringer, ph 7-6. The mucosal (outer) solution contained sodium at either 1 1, 9 9, 19.8, 39-8 or 111 mm with the ph adjusted to 6-5. The new feature in this paper was the use of whole skin rather than isolated epithelium. When whole skin was dissolved in soluene the resulting solution varied from dark green to yellow. The counting efficiency of tritium in scintillants containing dissolved skin was low (> 1 %). Attempts to decolorize these solutions with peroxide or perchloric acid did not improve the counting efficiency. As an alternative we decided to leave the skin intact and extract the radioactivity with solvents which did not remove colour. To investigate this possibility discs of skin (0.95 cm2) were placed in scintillation vials together with a small aliquot (100,ll.) of [3H]- benzamil and allowed to stand for 30 min. Scintillator was then added and the activity compared with identical vials but without added tissue. In spite of the high lipid solubility of [3H]benzamil only 60 % of the radioactivity entered the scintillant when the solvent was toluene and ethoxyethanol. However, following the suggestion of Dr J. Cerbon, it was found that if skins with radioactivity were treated first with a small volume of 12 % sodium dodecyl sulphate solution practically all the added radioactivity was counted (see Table 1). This method, using 0 5 ml. 12 % solution of sodium dodecyl sulphate, was subsequently employed for those experiments in which whole skin was used.

3 REGULATION OF SODIUM PERMEABILITY The Ringer solution used throughout had the following composition (mm): NaCl, 111; KCI, 2; CaC12, 1; Tris buffer ph 7 6, 5 and glucose I 1 1. The solution was gassed with air. Solutions used for the mucosal surface sometimes contained less NaCi, and the ph was adjusted to 6-5. No compensation was made for tonicity when the NaCl concentration was reduced C i5o 100 ~~~~~~~~~mm-na Benzamil (M) Fig. 1. Log-concentration response curves to benzamil in a single skin. Inset shows the relation between s.c.c. and the sodium concentration in the absence of benzamil. The serosal solution was normal Ringer at ph 7-6 throughout. The mucosal solutions were adjusted to ph 6-5. The sodium concentrations of the mucosal solutions are indicated by each curve (mm). RESULTS Effects of benzamil on s.c.c. at different [Na]0 Measurements of the inhibition of short-circuit current (s.c.c.) by benzamil at ph 6-5 were made at the same times as the binding studies, using animals from the same batches. Fig. 1 illustrates the relation between s.c.c. inhibition and benzamil concentration at a variety of sodium concentrations. Clearly the concentration required to produce 50 % inhibition of the s.c.c. increased with the sodium concentration. The relation of s.c.c. to sodium concentration is also shown in the inset to Fig. 1 and assuming saturation type kinetics of Km of 5 mm for sodium is indicated. Defining the Km for benzamil as the concentration required to inhibit s.c.c. by 50 %, it can be seen that in this example increasing the sodium concentration of the mucosal bathing solution from 1-1 to 111 mm results in a fourteen-fold increase in Km. The relation between Km and sodium concentration is a linear one. Fig. 2A shows a plot of the data from Fig. 1 plus two further examples. It is clear that the change in Km with a 100-fold change in sodium concentration was not always as great as shown in Fig. 1. The minimal change in Km seen in these experiments was five-fold. In a previous study (Cuthbert & Fanelli, 1978) we were unable to discover any relation between the basal s.c.c. and the Km for amiloride at high sodium concentration, so it is unlikely that the variability in the change in Kmwith sodium concentration can be attributed to different basal s.c.c.s. The results of a number of experiments in this series are summarized in Fig. 2B. The linearity of the plots shown in Fig. 2 can be interpreted in terms of a competitive interaction between benzamil and sodium (see discussion). It is important to emphasize that the values were obtained at ph 6-5, the same condition as used for the binding studies given later in this paper. These values from Fig. 2B will be used in later calculations of occupancy and are given below for convenience. The apparent Km

4 494 J. ACEVES AND A. W. CUTHBERT values for benzamil were (nm): (1.1 mm-sodium), (9.9 mm-sodium), (19.8 mm-sodium), (39-8 mm-sodium) and (111 mmsodium). [3H]benzamil uptake into whole skin A method designed to measure uptake into whole skin was devised at a time (mid- March) when difficulty was found in preparing isolated epithelium. The method is 20 '5 10 E 20r B (7)(10)(7) (7) (11) 0 J0 L_, a I Na concentration (mm) Fig. 2. The relation for the Km for benzamil versu the sodium concentration of the mucosal bathing solution. A, curves are shown for three individual skins. B, a composite result for all experiments is given. The number of values contributing to each mean value is given above each point. E 0 L A B C D E 40. N C 1000 E~~~A [3H] benzamil (nm) Fig. 3. Uptake of [3H]benzamil with concentration in ten pieces of skin all from the same frog. The mucosal solution (ph 6.5) contained 1-1 mm-[na]. The curve corresponds to the formula: uptake = uptake maximum [[B]/[B] + KB] + b[b], where [B] and KB are the concentration of benzamil and the Km for benzamil (1-2 nm) respectively. b is the slope of the linear component of uptake. The values of uptake maximum and b were 1050 dpm 0-95 cm-2 and 140 dpm nm-1 respectively. The straight line is the asymptote to the curve. Maximal uptake corresponds to 150 binding sites /tm-2 at a specific activity of 19,600 c-mole-l.

5 REGULATION OF SODIUM PERMEABILITY 495 illustrated by the experiment shown in Fig. 3. Uptake was measured for periods of min, as with experiments with epithelia (Aceves et al. 1979) to ensure the mucosal surface was equilibrated with the ligand. Ten pieces of skin were taken from a single animal and pairs taken from adjacent areas were exposed to one of five ligand concentrations. Uptake is clearly non-linear with concentration and contiguous pieces of skin give similar uptakes. However, notice that maximal difference between paired pieces corresponds to 4 f-mole cm-2, that is 20 % of the maximal specific uptake demonstrated in the previous paper (Aceves et al. 1979). Thus in a later experiment described here (Table 3) we have used eleven paired pieces to obtain mean values. As the Km for benzamil under the conditions used for the experiment in Fig. 3 is 1x2 nm the data were fitted to U -Um[B] + b[b], [B]+Km where U and Um are uptake and maximal uptake into a saturable component with a Km of 1P2 nm, [B] is the benzamil concentration and b the proportionality constant for non-saturable uptake. The curve shown in Fig. 3 is intended only as an indication that specific binding site density is of the same order in whole skin as was found with epithelia. The value of Um here corresponds to 150 sites tm-2. Effects of 111 mm-sodium on [3H]benzamil uptake at ph 6-5 A few experiments were made with isolated epithelia to compare uptake of [3H]- benzamil at different concentrations and at ph 6-5 in the presence of sodium at 1P1 and 111 mm. The experiments are illustrated in Fig. 4A and show two distinct features. First there is less uptake at high sodium concentration, and secondly the uptake at high sodium concentration appears to be linear with concentration. Taking the Km of benzamil as 1 2 nm under low sodium conditions the fractional occupancy at 2 nm is 0X625 (from [B]/Km + [B]). Then from the difference in uptake at high and low sodium at a benzamil concentration of 2 nm it can be shown that high sodium displaces maximally the ligand bound to 146 sites sm-2. This value is consistent with other values reported here and in the previous paper (Aceves et al. 1979) for the maximal binding capacity in low sodium. To confirm that uptake was indeed linear at high sodium concentration uptake was examined in a further five experiments. The composite data for the measurements in high sodium are shown in Fig. 4 B. It was not necessary to make further measurements at 1 1 mm-sodium for comparison as these are given in the previous paper. However, the fitted curve for uptake in low sodium has been added to Fig. 4B to illustrate the extent of the displacement. In the presence of high sodium the occupancy at 7 nm benzamil is If the number of binding sites remains constant and at high sodium the reduced binding is due only to a reduction in affinity then we could expect the uptake to deviate from linearity by about 400 dpm 0'95 cm-2 at 7 nm-[3h]benzamil. This is probably not a very sensitive test, however the linearity of the uptake curve at high sodium, coupled with the equivalence of the maximal number of binding sites displaced by high sodium with the maximal number of specific binding sites at low sodium, suggest that few specific binding sites are available at high sodium. Benzamil uptake at high sodium was 8-8 f-mole nml1.

6 496 J. ACEVES AND A. W. CUTHBERT A E u 2000 Q Ci 0 0. X E c [3H]benzamil (nm) Fig. 4. A, uptake of [3H]benzamil with concentration at low sodium (1.1 mm) (filled symbols) and at high sodium (111 mm) (open symbols). Triangles are means for two pieces of epithelium from separate frogs, while the circles are values for single pieces from two frogs (the pieces from one frog were used for either low Na uptake (filled circles) or high sodium uptake (open circles). The value for one piece exposed to low sodium at 5-7 mm is not shown. Its value was 5430 dpm 0-95 cm-2 and this high value was probably due to label leaking to the serosal side. B, uptake of [3H]benzamil in the presence of 111 mm-sodium at ph 6-5. The results are shown for six experiments in which uptake was measured at five or six different concentrations into epithelia prepared from a single animal. The characteristics of the regression line are y = 407[B] + 105, where [B] is the benzamil concentration, 95 % confidence limits of the slope are , r = 0-97, P < The slope corresponds to 8-8 f-mole nm-1. The upper line corresponds to the total uptake at 1-1 mm-sodium and taken from Aceves et al. ( 1979). Relation of benzamil uptake to sodium concentration From Fig. 4A it is seen that the ratio of displaceable to total uptake is maximal over the concentration range of 1-2 nm-benzamil. It was decided to compare uptake at various sodium concentrations with that at 111 mm-sodium and at low ligand concentrations. Each experiment was performed with disks of skin prepared from a single animal to reduce any variation between animals. Five pairs of disks were cut as illustrated in Fig. 3, and one of each pair exposed to 111 mm-sodium, while the other was exposed to a low concentration. Each pair of disks was then labelled with one of five [3H]benzamil concentrations so that uptake curves were obtained as in Fig. 5. The results of these experiments were calculated as follows. The amount of [3H]- benzamil displaced by 111 mm-sodium was multiplied by the reciprocal of the fractional occupancy, obtained from [B]/[B] + K where B was the [3H]benzamil concentration and K was the Km for benzamil at the appropriate sodium concentration (and given earlier in the first section of the Results). In Fig. 5 the displaceable

7 REGULATION OF SODIUM PERMEABILITY 497 uptake at each ligand concentration is given as sites,um-2. The statistical significance of each result was assessed by a simple non-parametric sign test. The difference in uptake by each pair was positive if uptake was less at 111 mm-sodium. Only experiments in which all five differences were positive were significant (P = 0031) A A~~~~~~ I E u> 2000 B E N 0)~~~~~~~~~~~~" To~~~~~~~~~~9 6~~~~~~~~~ ~~~~ I D C _ _ 0 0O [3H]benzamil (nm) Fig. 5. Uptake of [3H]benzamil into whole frog skin at ph 6-5. The open symbols indicate the uptake of [3H]benzamil in the presence of (A) 1 1 mm-sodium, (B) 9 9 mmsodium, and (C) 39-8 mm-sodium in the mucosal bathing solution. The filled symbols indicate the uptake in the presence of 111 mm-sodium. Uptake is expressed as dpm 0-95 cm'. Displaceable uptake was used to calculate the maximal uptake making use of the occupancy. The mean site density (sites,czm-2) was in (A), 107 ± 22 in (B) and in (C). In each experiment the ten pieces of skin were derived from a single animal. Table 2 gives the results for 18 separate experiments. Four out of six experiments yielded significant values at a sodium concentration of 1 1 mm, three out of four were significant at 9-8 mm-sodium, but no significant displaceable binding could be detected at either 19-8 or 39-8 mm-sodium. This type of test is a severe one as a single anomalous value will make the result non-significant. For example, in the last experiment using 1.1 nm-sodium four out of the five paired measurements gave values between 50 and 117 sites /sm-2, but a single piece exposed to 111 mm-sodium gave an anomalously high uptake, and hence apparently large negative site density. In Fig. 5A the mean value for displaceable binding is 119 sites,sm 2, however when the plotted result is examined it may be supposed that the real result is probably

8 498 J. ACEVES AND A. W. CUTHBERT nearer 140 sites /um-2. Therefore all that can be concluded from this set of experiments is that a similar density of specific uptake sites is measured up to 10 mm-sodium compared with 1 1 mm, but that no significant specific uptake can be detected at higher sodium concentrations, even allowing for the reduced affinity of the ligand. TABLE 1. Recovery of radioactivity from whole skin Recovery of tritium (%) A B C D [3H]benzamil alone [3H]benzamil plus skin n The conditions for extraction were: A, toluene; 0-6 % butyl BPD (10 ml.) B, toluene, 66 %; ethoxyethanol, 30 %; 0.6% butyl BPD (10 ml.) C, 0*5 ml 12 % sodium dodecyl sulphate (SDS) followed by Triton X-100, 33 %; toluene, 66 %; 0-6 % butyl BPD (15 ml.) D, as C with 1.0 ml. 12 % SDS. TABLE 2. Antagonism of benzamil uptake by sodium [3H]benzamil Sodium displaced by concentration 111 mm-sodium Significance Occupancy (mm) (sites gm-2) P (range) *03 0* ns -1 ns ns ns ns 106 ns 68 ns ns ns 63 ns 80 ns Occupancy was calculated from [B]/[B] + K where [B] is the benzamil concentration and K is the Km for benzamil at the relevant sodium concentration. The occupancy ranges used are shown in the right hand column. To gain further insight into the effects of raised sodium a further type of experiment was devised to examine benzamil uptake at 39-8 mm-sodium but at higher occupancy than in the previous experiments. Also we were anxious to develop a method which would avoid the differences in the non-specific uptake between animals. In each experiment three adjacent pieces of skin from the same animal were used. Uptake of [3H]benzamil was measured over min at one of three conditions, namely 1-1 mm[na]o, ph 6-5; 39-8 mm[na]o ph 6-5 and 39-8 mm[na]0+amiloride,

9 REGULATION OF SODIUM PERMEABILITY /LM, ph 6-5. For convenience these conditions will be referred to as Low sodium, High sodium and High sodium plus amiloride. The experiment was repeated eleven times and the results are summarized in Table 3. TABLE 3. Uptake of benzamil under varying conditions dpm [3H]benzamil uptake 0-95 Cm-2 Low Na High Na High Na - high Na Low Na - high Na Expt. Low Na High Na + amiloride + amiloride - high Na + amiloride mean + S.E P < < 0005 > 01 The values in columns 2-4 indicate the uptake of [3H]benzamil in dpm 0.95 Cm-2 in frog skin. All values in a given row are for tissues from one animal. Columns 5-7 show the differences between pairs of conditions. The mean values of these differences were tested for significance from zero by a paired t test. Specific activity of [3H]benzamil was 19,600 c-mole-1 and concentration was 5-75 nm. In these experiments we are concerned with the amount of benzamil displaced by [Na] mm or [Na] mm plus 1 /um-amiloride from the condition obtaining at [Na]0 1 1 mm. We may ignore the variations between skins and concentrate on the mean values of these differences. Using a paired t test it is found that increasing the sodium concentration to 39f8 mm from 1*1 mm causes a highly significant displacement, as does increasing the sodium concentration to 39-8 mm plus addition of Although addition of amiloride,(1 /SM) to sodium, 39-8 mm causes amiloride (1 /um). further displacement, it fails to reach statistically significant proportions. The three mean values given in Table 3 can be considered as single points on three different uptake curves, but all measured at the same [3H]benzamil concentration of 5-75 nm (specific activity c-mole-1). From the earlier part of this paper we know that when benzamil is used to inhibit s.c.c. the Km is 1-2 nm at 1.1 mm [Na]o and 5*5 nim at 39-8 mm [Na]0. Thus the occupancy, calculated from [B]/Km + [B] is 0-83 at 1 1 mm [Na]O and 0 57 at 39-8 mm [Na]O respectively at a benzamil concentration of 5-75 nm. The occupancy of the specific binding sites by [3H]benzamil in the presence of 39-8 mm [Na]O and amiloride, 1 /tm can be estimated from [B]KB (2) 1+ [B]KB + [A]KA + [Na]KNa'(2 x, A

10 500 J. ACEVES AND A. W. CUTHBERT where KB, KA and KNa are the affinity constants of benzamil, amiloride and sodium for the entry sites in the absence of other ligands. Taking KB as 109 M-1, KA as 108 M-1 and KNa as 200 x-1 (from Fig. 1) then under the conditions of the experiments a = Consider the displacement which occurs between low sodium and high sodium plus amiloride. The difference in occupancy is = 0-78 corresponding to 1178 dpm 0o95 cm-2. These figures indicate that 100% occupancy at low sodium corresponds to 222 binding sites per /am2, a figure not unrepresentative of values given in this paper and the earlier ones (Aceves et al. 1979). If indeed high sodium causes sites to become unavailable this calculation will give a small over-estimate. The minimal value for the site density at 1.1 mm-sodium would be 209 sites jtm-2, arrived at by taking 0-83 as the maximal difference in occupancy. Now consider the displacement which occurs between low and high sodium. The difference in occupancy is = If no binding sites disappear on raising the sodium concentration, that is the reduction in binding of 848 dpm 095 cm-2 is due entirely to the change in affinity, then at full occupancy there are 390 site /Im-2. This value is not only inconsistent with those given above, but much greater than expected from the other results in this paper. The displacement caused by moving to high sodium from low sodium may be approached in a different way. A displacement of 848 dpm 0o95 cm-2 represents 125 sites tm-2. At any condition the number of sites actually bound will be given by the occupancy multiplied by the site density. Thus 125 sites /tm-2 represents the difference between the sites bound at low and high sodium. This may be stated explicitly as (0.83 x 215)- (0.51 x x) = 125, where x is the actual site density at high sodium (39.8 mm). The value 215 is the mean value of the upper and lower limits for the site density at low sodium (1 1 mm). Thus x is 104 sites /Zm-2 at 39-8 mm-sodium compared to 215 sites /tm-2 at low sodium. In other words half the sites have become unavailable for binding by increasing sodium from 1l to 398 mm. Thus far we have used only values of occupancy and the two statistically significant differences in binding determined by experiment. Addition of 1 /,LM-amiloride to 39-8 mm-sodium caused a further displacement of 329 dpm 0 95 cm-2. This value was not statistically significant, but we can consider what the magnitude of this difference should be. If there are 104 sites /Zm-2 at 39-8 mm-sodium then [3H]benzamil will be displaced from (0* ) x 104 sites /um-2 by addition of 1 /tm-amiloride, that is 48 sites,um-2, or 325 dpm 0o95 cm-2. Thus although the extra displacement caused by the addition of amiloride did not reach statistical significance it is, nevertheless, exactly of the predicted value. It must be concluded that this experiment can be interpreted within a consistent framework which requires that binding sites are reduced by 50 % when the sodium concentration is raised from 1.1 to 39-8 mm.

11 REGULATION OF SODIUM PERMEABILITY 501 DISCUSSION The change in apparent affinity of benzamil, or amiloride, with sodium concentration is indicative of competition. This is seen not only with frog skin but also with isolated toad bladder (Cuthbert & Shum, 1975) and the skin of the adult Mexican axolotl (Aceves & Cuthbert, 1979). Assuming mass action kinetics, then if sodium and benzamil compete for the same binding site the apparent Km for benzamil will be given by KB' where KB' = KB + [Na]KB' (3) KNa' where KB and KNa are the Km values for benzamil and sodium, each measured in the absence of the other. A linear relation between KB' and sodium concentration is thus expected. Recently Benos & Mandel (1978) have reported for another anuran species (Rana cateabiana) that the Km for amiloride did not change with sodium concentration. This might suggest that sodium and amiloride (or benzamil) interact with different sites which are allosterically coupled so that only one of the two sites can be occupied at any instant. A lack of coupling might then explain the result with R. cate8biana. These experiments have shown that increasing the sodium concentration to 111 from I1- mm reduces benzamil uptake, and more decisively the uptake is reduced to that obtained in the presence of amiloride or unlabelled benzamil at low sodium. This is an important result as it shows for the first time that sodium not only antagonizes the physiological effects of benzamil (Fig. 1) but also prevents uptake into that component which is thought to represent sodium entry sites (Aceves et al. 1979). If the effect of sodium on the availability of benzamil binding sites was due only to competition it might be expected that the uptake in the presence of high sodium would be greater than found in the presence of excess competing ligand. However, the uptakes were virtually identical, which may indicate that sites become unavailable at high external sodium concentrations. As amiloride does not penetrate the epithelium from the mucosal side (Salako & Smith, 1970) the binding site for amiloride, and presumably benzamil, must be on the outer surface of the cell membrane. With simple competition between sodium and benzamil the sodium binding site would be outside the cell also. Alternatively, if there are two sites coupled allosterically it is unlikely that the sodium site could be other than on the outer surface. For instance, if it was proposed that the sodium site was intracellular then there would have to be a linear relation between the sodium concentration outside and inside the cell to maintain the linear relationship between KB' and the external sodium concentration. This would not be so because of considerable changes in electrical gradient across the mucosal membrane in short circuited skins which occurs when external sodium is altered (Nagel, 1977). In an earlier study (Cuthbert, 1974) it was concluded that the site of amiloridesodium competition is not necessarily the same site as that for sodium translocation across the membrane. Fuchs et al. (1977) have concluded that sodium reduces apical membrane permeability by an action at an external regulator site. Lindemann & Vouete (1976) also suggest that amiloride interacts with the same site acting as a

12 502 J. ACEVES AND A. W. CUTHBERT 'super' sodium to regulate sodium entry. The competition of sodium and benzamil shown here by direct binding measurements would be in accord with these suggestions. In order to investigate further whether in addition to direct competition between benzamil and sodium, at either a common or coupled site, there was an overall change in the number of available binding sites at different sodium concentrations two further types of experiments have been made. First, partial binding curves comparing uptake at 111 mm-sodium with that at a lower concentration have been obtained using epithelia from single animals. In addition uptake was measured over a concentration range designed to maximize the difference between specific and nonspecific uptake. Significant specific uptake of the same order of magnitude as that given by the total uptake curve (Aceves et al. 1979) was found when binding at 1 1 and 111 mm-sodium were compared. This is an important result as the site density was calculated from the displaceable uptake and the fractional occupancy. This would suggest that calculation of fractional occupancy from s.c.c. measurements is appropriate, and confirms the usefulness of the earlier methods devised with [14C]- amiloride (Cuthbert, 1973; Cuthbert & Shum, 1974). Similar results were obtained when uptake was compared at 10 and 111 mm-sodium. Thus, although there was less displaceable binding (Fig. 5B), when allowance was made for reduced occupancy there was no indication that the total number of uptake sites was reduced. Comparison of uptake at 19-8 or 39-8 mm-sodium with that at 111 mm-sodium failed to show significant displaceable binding in any of the experiments (Table 2, Fig. 5C). This might suggest that even after allowing for the reduced occupancy a proportion of the sites had become unavailable. A further type of experiment was made comparing uptake at 39-8 mm-sodium with that at 1-1 mm-sodium and with that at 39*8 mm-sodium in the presence of excess amiloride. Using values of occupancy calculated from s.c.c. measurements the results were consistent with the notion that just over half the benzamil binding sites had become unavailable at 39-8 mm-sodium. On adding amiloride to 39-8 mm-sodium at a [3H]benzamil concentration of 5-75 nm the ligand was displaced from 48 sites /tm-2 instead of 96 sites um-2 (( ) x 209) if the original site density of 209 sites tm-2 is taken. However, in this experiment the site density in skin at low sodium is greater than found with isolated epithelia (130 sites /%m-2, Aceves et al. 1979). If this latter value is taken then the displacement by amiloride at 39-8 mm-sodium is only 20 % less than the expected value. Thus there is some evidence, but which is inconclusive, that at 39-8 mm-external sodium the availability of sites is reduced. This finding is in accord with two others made from binding measurements with [14C]amiloride. First, the reduction in transmucosal potential as the sodium concentration is raised (Nagel, 1977) might well be expected to reduce binding (Cuthbert & Shum, 1976). Secondly, it has been shown that when the extracellular sodium concentration is kept low (1 1 mm) but the intracellular concentration is raised the amount of displaceable [14C]amiloride binding is reduced by an effect which appears to be independent of potential changes (Cuthbert & Shum, 1978). In these experiments the serosal pump was inhibited by ouabain so that sodium exit was prevented. The reduction in amiloride binding only occurred if the ouabain poisoned skins were subject to high mucosal sodium immediately before returning to low sodium to measure binding. In this situation the intracellular

13 REGULATION OF SODIUM PERMEABILITY 503 sodium concentration may have risen to much higher values than achieved in these experiments at 39-8 mm-external sodium and with the pump intact. As indicated in the introduction evidence for the control of apical sodium permeability by intracellular sodium is available for two other sodium transporting epithelia (Lewis et al. 1976; Turnheim et al. 1978). Recent biochemical evidence (Rodriguez & Edelman, 1977) is also of interest. Using a technique to label apical membrane proteins in toad bladder with 1311 it was found that extra bands appeared on gels when labelling was carried out at low sodium concentrations, and it was suggested that these proteins may be involved in sodium transport. In these experiments we have described a way of measuring uptake into whole skin rather than epithelia. This has simplified the method which may be applicable to other epithelial tissues. In conclusion, our results support the notion that the saturation of sodium uptake at the apical surface of frog skin as the sodium concentration is raised may well represent a regulation by sodium, at both external and internal sites, rather than the saturation of a carrier type mechanism. This work was supported by grants from the National Kidney Research Fund and the Medical Research Council. J. A. is grateful for support from the Royal Society and the Mexican Academy of Sciences. REFERENCES ACEVES, J. & CUTHBERT, A. W. (1979). Chloride dependent sodium transport in the skin of Ambby8toma mexicanum. J. Physiol. 289, 79P. AcEvEs, J., CUTIBERT, A. W. & EDWARDSON, J. M. (1979). Estimation of the density of sodium entry sites in frog skin epithelium from the uptake of [3H]benzamil. J. Physiol. 295, BENoS, D. J. & MANDEL, L. J. (1978). Amiloride is a non-competitive inhibitor of Na-transport in isolated bullfrog skin. Biophy8. J. 21, 169a. BIBER, T. U. L. & CURRAN, P. F. (1970). Direct measurement of uptake of sodium at the outer surface of frog skin. J. gen. Phyeiol. 56, CEREIJIDO, M., HERRERA, F. C., FLANIGAN, J. W. & CURRAN, P. F. (1964). The influence of Na-concentration on Na transport across frog skin. J. gen. Physiol. 47, CUTHBERT, A. W. (1973). An upper limit to the number of sodium channels in frog skin epithelium. J. Physiol. 228, CUTHBERT, A. W. (1974). Interactions of sodium channels in transporting epithelia: a two state model. Molec. Pharmacol. 10, CUTHBERT, A. W. & FANELLI, G. M. (1978). Effects of some pyrazine carboxamides on sodium transport in frog skin. Br. J. Pharmac. 63, CUTrBERT, A.. W. & SAuM, W. K. (1974). Binding of amiloride to sodium channels in frog skin. Motec.Pharmacol. 10, CUTHBERT, A. W. & SHUM, W. K. (1975). Effects of vasopressin and aldosterone on amiloride binding in toad bladder epithelial cells. Proc. R. Soc. B 189, CUTHBERT, A. W. & SAuM, W. K. (1976). Characteristics of the entry process for sodium in transporting epithelia as revealed by amiloride. J. Physiol. 255, CUTrBERT, A. W. & SAuM, W. K. (1978). Interdependence of the two borders in a sodium transporting epithelium. Possible regulation by the transport pool. J. membrane Biol. (Special Issue), ERLIJ, D. & SMIrrH, M. W. (1973). Sodium uptake in frog skin and its modification by inhibitors of transepithelial sodium transport. J. Physiol. 228, Fucis, W., HVID LARSEN, E. & LINDEMANN, B. (1977). Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin. J. Physiol. 267,

14 504 J. ACEVES AND A. W. CUTHBERT LEWIS, S. A., EATON, D. C. & DIAMOND, J. M. (1976). The mechanism of Na+ transport by rabbit urinary bladder. J. membrane Biol. 28, LINDEMANN, B. & VAN DREISscHE, W. (1977). Sodium specific membrane channels of frog skin are pores: current fluctuations yield high turnover. Science, N.Y. 195, LINDEMANN, B. & VOftTE, C. (1976). Structure and function of the epidermis. In Frog Neurobiology, ed. R. LLINAS & W. PRECHT, chap. 5, pp Berlin: Springer. NAGEL, W. (1977). The dependence of the electrical potentials across the membranes of frog skin upon the concentration of sodium in the mucosal solution. J. Physiol. 269, RODRIGUEZ, H. J. & EDELMAN, I. S. (1977). Surface proteins of the luminal membrane of the toad bladder. Am. Soc. Nephrology, Abstracts 91 A. SALAKO, L. A. & SMITE, A. J. (1970). Changes in sodium pool and kinetics of sodium transport in frog skin produced by amiloride. Br. J. Pharmac. 39, TURNHEIM, K., FRIZZELL, R. A. & SCHULTZ, S. G. (1978). Interaction between cell sodium and the amiloride-sensitive sodium entry step in rabbit colon. J. membrane Biol. 39, ZEISKE, W. & LINDEMANN, B. (1974). Chemical stimulation of sodium current through the outer surface of frog skin epithelium. Biochim. biophy8. Acta 352,