proposed by Koefoed-Johnsen & Ussing (1958), suggested that Na+ passively entered directly.

Transcription

1 J. Physiol. (1981), 316, pp With 9 text-figures Printed in Great Britain INTRACELLULAR SODIUM ION ACTIVITY AND SODIUM TRANSPORT IN RABBIT URINARY BLADDER BY DOUGLAS C. EATON From the Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77550, U.S.A. (Received 28 July 1980) SUMMARY 1. Intracellular potentials and the intracellular activities of Na+ and K+ were examined using conventional and ion-selective micro-electrodes. 2. In animals on a normal diet, the intracellular Na+ activity was mm (mean + S.D.) with a mean short-circuit current of ,uA/cm2. 3. In animals on a low-na+ diet, the intracellular Na+ activity was mm with a short-circuit current of ItA/cm2 (mean+ S.D.). 4. There was a correlation between short-circuit current and intracellular Na+ activity which could be fitted by a saturating hyperbolic relationship. 5. Treatment of the tissue with ouabain and amiloride produced an increase and a decrease, respectively, in the intracellular Na+ activity. 6. Treatment with aldosterone produced a large increase in short-circuit current with a substantial increase in intracellular Na+ activity. 7. Intracellular Na+ activity does not seem to affect apical membrane permeability directly. INTRODUCTION All of the so-called tight epithelia transport Na+ from the apical surface to the basolateral surface. The original model of Na+ transport across tight epithelia, as proposed by Koefoed-Johnsen & Ussing (1958), suggested that Na+ passively entered the cell from the lumen across the apical membrane and was subsequently transported from the cell interior to the serosal solution by an active process across the basolateral membrane. Although this description appears to be fundamentally sound, the exact mechanisms that regulate the rate of transport are still unclear. Certain drugs, such as amiloride and ouabain, as well as naturally occurring hormones such as aldosterone are known to affect the transport. But what changes these induce in the properties of the epithelial membranes and in the intracellular ion gradients are not always evident. In particular, one of the major difficulties is a lack of knowledge about the intracellular ion activities under various transport conditions. In previous work (Lewis, Wills & Eaton, 1978), the K+ and Cl- activities inside mammalian urinary bladder cells were measured. These measurements yielded information about the permeability properties of the basolateral membrane.

2 528 D. C. EATON But in the final analysis, the important intracellular ion activity to measure is that of Na+ ion since its transport is the primary function of most tight epithelia. Several investigators have recognized this importance, and hypothesized regulation of transepithelial transport by intracellular Na+ (MacRobbie & Ussing, 1961; Lewis, Eaton & Diamond, 1976). Regardless of the importance, good estimates of intracellular Na+ are difficult to obtain, especially when one wishes to monitor dynamic changes in activity rather than the static levels obtainable from most chemical determination methods. The dynamic changes are particularly important in assessing the effects of transport modifying agents. However, with the advent of ion-specific micro-electrodes, several investigators have determined intracellular Na+ activities. (See Civan (1978) for a review of these measurements of intracellular ion activities.) I decided to examine the intracellular Na+ activity in rabbit urinary bladder since virtually all of the short-circuit current is due to Na+ transport (Lewis & Diamond, 1976). Therefore, by comparing the short-circuit current with the intracellular Na+ activity, I could determine the dependence of the basolateral transport process on intracellular Na+ as well as the effect of intracellular Na+ activity on apical membrane permeability. METHODS Urinary bladders were obtained from adult New Zealand white rabbits. The tissue was dissected, mounted according to the procedures of Lewis & Diamond (1976), and placed between modified Ussing chambers (15 ml./chamber) which were designed to eliminate edge damage and to reduce hydrostatic gradients during micro-electrode impalements (Lewis, Eaton, Clausen & Diamond, 1977). The exposed area of tissue was 1 cm2. Approximately half the rabbits in this study were placed on a special low-na+ diet (Purina) to induce aldosterone production with consequent increase in Na+ transport rate (Lewis & Diamond, 1976). These rabbits are identified as 'low-na+ diet animals'. In some cases, to induce maximal aldosterone production in vivo, 5 mm-kci was added to the distilled drinking water of the low-na+ diet animals. The remainder of the animals, fed on normal diet (Purina Rabbit Chow) and tap water, are identified as 'normal diet animals'. Electrical measurements Transepithelial voltage (VT) was monitored with two Ag-AgCl wires placed 10 mm apart on opposite sides of the tissue and referenced to the mucosal solution. Ag-AgCI electrodes placed at the end of each chamber led respectively to a current source and a virtual ground current to voltage converter used to determine short-circuit current(isc). The current source and voltage electrodes led to a voltage clamp. Transepithelial voltage and the signal proportional to short-circuit current were displayed on two digital voltmeters (Analogic) and a multi-channel chart recorder (Stoelting). Potentials from conventional electrodes were displayed on a digital voltmeter and chart recorder while the responses of the ion-specific electrodes were taken from digital voltmeters. Ordinarily the tissue was left in an open-circuit condition. The short-circuit was determined by voltage clamping the tissue for approximately 20 s at intervals of about 5 min. Micro-electrodes Conventional micro-electrodes were constructed from fibre-filled Pyrex tubing (outer diameter = 1 mm) which was pulled on a horizontal pipette puller (Industrial Associates). Micro-electrodes were filled with 3M-KCI and had tip resistances of MCI. They were connected to a differential electrometer (WP Instruments, Model 740) referenced to the mucosal solution. Fine positioning of the micro-electrode was achieved within +1 Ium with a hydraulic microdrive (Stoelting). K+-sensitive micro-electrodes were constructed according to methods described by Walker(1971) and Eaton, Russell & Brown (1975). Briefly, Pyrex capillary tubing was pulled to a tip of less than jsm diameter (mean resistance 30 MCI when filled with 3M-KCl). Micro-electrode tips were dipped

3 Na+ TRANSPORT AND INTRACELLULAR Na+ 529 in a 2-5 % solution of trichloro-n-butyl-silane in a-chloronaphthalene for about 15 s and then baked at 250 0C for 30 min. Afterwards, the tips were back-filled to a distance of approximately 250 utm from the tip with K+ exchanger (Corning Glass Works). The remaining portion of the electrode was filled with 0 5 M-KCI. The micro-electrode was then allowed to equilibrate in NaCl Ringer solution (see 'Solutions') for approximately 10 min. For calibration, the response of the micro-electrode was tested in 100, 10-1, 10-2 and 10-3 M-KC1 solution. Usable electrodes demonstrated a mv change per decade change in K+ activity with a response time of less than 1 s. Following the method of Walker (1971) the selectivity ratio of K+ to Na+ was calculated to be greater than 50: 1. Na+-sensitive micro-electrodes were constructed in a manner similar to the K+-sensitive electrodes. The primary difference was that the tips were dipped in a solution of trichloro-n-butyl silane in ac-chloronaphthalene for a period of time sufficient to draw up a column of silanizing agent (about mm). The amount of silane in the silanizing agent was varied between 5 and 50 % on a day to day basis depending upon the ambient relative humidity. When an appropriate quantity of silanizing agent was drawn up into the tip of the electrode, the solution at the tip was flash vaporized at a temperature of approximately 500 TC by heated air from a heat gun. The tip of the electrode was then back-filled with the Na+ exchanger (see below) to a distance of ,sum from the tip. The remaining portion of the electrode was then filled with 0 5 M-NaCl. Because of the high resistance of the exchanger, hydration of the electrode glass with concomitant shunting could have been a serious problem. To avoid the problem, electrodes were kept dry until use and between impalements. Average time in solution was less than 2 min for samples. In control experiments, immersion for this period of time did not degrade the response of the electrode. For calibration, the response of the electrode was tested in 100, 10-1, 10-2 and 10-3 M-NaCl solutions (Fig. 1). Electrodes with slopes less than 55 mv per decade change in Na+ activity were rejected at this point. To determine the electrode selectivity, the response of the electrode was again tested in solutions containing 100, 10-1, 10-2 and 10-3 M-NaCl plus 0-1 M-KCl. This solution represents a worst case situation since normal intracellular K+ activities are about 70 mm (Lewis et al. 1978). From these responses, the selectivity coefficients for Na+ over K+ could be calculated. The selectivities varied from electrode to electrode but in no case was an electrode with a selectivity ratio of less than 15: 1 Na+ over K+ used. The maximum selectivity observed was 35: 1 with typical values for the experimentally used electrodes lying between 15:1 and 25:1. All ion-selective electrodes were tested for response, slope, and, where appropriate, K+ selectivity both before and after an experiment. If either the slope or the selectivity to K+ had changed the experiment was discarded. Selectivity to other ions was also measured and found to be similar to that reported by Kraig & Nicholson (1976). At 1 M concentrations the selectivity ratios were 151:1 for Ca2+, 172: 1 for NH+4 17:1 for H+, and 8:1 for Mg2+. Thus only in the unlikely event of large changes in intracellular Mg2+ activity could there be a substantial error in estimation of Na+ activity. Na+ liquid ion exchangers The Na+ liquid ion exchanger was prepared by dissolving 10 mg of monensic acid and 10 mg of sodium tetraphenylboron in 80 mg of dry nitrobenzene. The monensic acid was derived from Na+-monensin and was prepared according to the method of Kraig & Nicholson (1976). The Na+-monensin was a generous gift from Dr Hammil of the Eli Lilly Pharmaceutical Corporation. In three experiments a Na+ exchanger of different composition (Steiner, Oehme & Simon, 1979) was used in the electrodes. This exchanger was a gift from Dr Kunze of the University of Texas Medical Branch. There was no significant difference in the selectivity or the experimental results from those obtained with the monensin exchanger. Calculation of activity To record intracellular ion activity, ion-sensitive micro-electrodes were juxtaposed to the apical membrane and a voltage reading in the mucosal solution noted (V.). The electrode was advanced across the membrane into the cell cytoplasm. Intracellular ion activity (ax) was calculated from the following equation (Eaton et al. 1975): ax = (ax + Kxj a?) explr ( 1K- Va)-Kxja1, (1) where ax and VO are defined above, Va is the apical membrane potential, Vi is the ion-sensitive micro-electrode reading inside the cell, n is a correction factor for non-ideal slope, al and aj are

4 530 D. C. EATON the internal and external concentrations of univalent competing ions, and Ki, is the selectivity ratio for the ion of interest and the competing ion. The last term on the right-hand side of the equation is unimportant for K+ measurements since KKNa is about 0-02 and aja is usually no more than 30 mm even in the worst cases, thus implying a correction of less than 1 mm in the intracellular K+ concentration. However, for intracellular Na+ measurements, KNaK may be as large as with an intracellular K+ concentration of mm. This implies a worst case correction of 3-5 mm in the intracellular Na+ concentration. Therefore, appropriate corrections are made for all intracellular Na+ calculations using the intracellular K+ concentrations determined with the K+-sensitive electrodes ~100 E NaCI zm/ 10 _ NaCI +0-1 M KCI 1 i' a *j Electrode voltage (mv) Fig. 1. Calibration and selectivity of Na+-sensitive electrodes. The straight line represents the response of a typical Na+ electrode to various Na+ activities in the absence of any interfering ions. The slope is 58 mv per 10-fold change in activity. The curved line represents the response in the presence of 100 mm-kci. The Na+ to K+ selectivity of this electrode is 19-5: 1. To evaluate the various components of eqn. (1), measurements with the three types of electrodes (conventional, K+ and Na+) were grouped. Three measurements with conventional electrodes were used to determine mean apical membrane potential (Va). These were followed by three measurements of intracellular K+, three measurements of Na+, followed by three more measurements of apical potential to bracket the ion-specific electrode measurements. To calculate intracellular Na+ the mean value of the six determinations of apical membrane potential was used for V. With this value of Va, the mean intracellular K+ concentration with mean Va can be used to calculate intracellular Na+. Control for impalement damage Several investigators have expressed concern about errors in measurement of both intracellular potentials and intracellular ion activities due to impalement damage (Lindemann, 1975; Suzuki & Fromter, 1977; Lewis et al. 1978; Graf & Giebisch, 1979). A method to assess impalement damage was developed by Lewis et al. (1978) and was subsequently expanded on by Lewis & Graf in an appendix to the work of Graf & Giebisch (1979). In this method the impalement damage was assessed from an examination of the voltage-divider ratio, i.e. the ratio of apical to basolateral resistance. If this ratio was abnormally low compared with the ratio obtained from electrodes known to produce insignificant impalement damage, the data associated with these impalements were discarded.

5 Na+ TRANSPORT AND INTRACELLULAR Na+ 531 In this work we used a similar method to select our ion-sensitive electrodes. Conventional electrodes that produce no detectable impalement damage could be selected on the basis of resistance (> 20 MC) and tip diameters (< 0-5,tm) (Lewis et al. 1977, 1978). Voltage-divider ratios for several cells could be determined with these electrodes. Voltage-divider ratios for the ion-specific electrodes could also be determined. In previous work (Lewis et al. 1978; Graf & Giebisch, 1979) the actual amount of impalement damage in terms of a conductance increase was calculated; however, the salient feature of the analysis is the comparison of voltage-divider ratios between suspect electrodes and electrodes previously shown to produce normally minimal impalement damage. If the mean value for the voltage-divider ratio obtained from the ion-selective electrodes was statistically different from the mean obtained with conventional electrodes (tested at a probability level of 0 05), the ion-specific electrode was discarded. Thus, the final recorded data were collected with electrodes that introduced no more impalement damage than the conventional electrodes. Solutions The composition (in mm) of the usual bathing solution (NaCl saline) was NaCl, 111 2; NaHCO3, 25; KCl, 5-8; CaCl2, 2-0; MgSO4, 1-2; KH2P04, 1-2; and glucose, This solution was gassed with 95 % 02-5 % CO2 and maintained at a ph of 7-4 and a temperature of 37 'C. The drugs, amiloride, ouabain and aldosterone, were added directly to this solution. In addition to the components of the normal saline, in the aldosterone experiments a mixture of amino acids (obtained from Grand Island Biological Company) was added that represents normal cellular requirements; final concentrations of amino acids (Jumol/l) were: alanine, 100; arginine, 725; asparagine, 100; aspartic acid, 100; cystine, 100; glutamic acid, 100; glutamine, 2000; glycine, 100; histidine, 200; isoleucine, 400; leucine, 400; lysine, 400; methionine, 100; phenylalanine, 200; proline, 100; serine, 100; threonine, 400; tryptophan, 50; tyrosine, 200; and valine, 400. The final total concentration of mixed amino acids was 6-27 mm. RESULTS The intracellular Na+ concentration of urinary bladder cells from twenty-nine rabbits was examined. The short-circuit current in these same bladders was measured as an estimate of transepithelial Na+ transport. The mean intracellular Na+ activity in normal diet animals was mm with a short-circuit current of ,uA/cm2 (mean+ S.D., n = 13). For low-na+ diet animals, the intracellular Na+ activity was mm with a short-circuit current of /ta/cm2 (mean + S.D._ n = 14). The intracellular Na+ activities are significantly different from one another (P < 0-01). This suggested that the rate of Na+ transport might be related to intracellular Na+ activity. To examine the relationship between transport and intracellular Na+ activities, the internal Na+ activities were plotted versus the short-circuit current (Fig. 2). Although there may be some variability in the data, the relationship does not appear to be linear (regression coefficient = 0 26); however, the data suggest that the rate of Na+ transport at the basolateral membrane is substrate (Nai) dependent. These results are consistent with the measurements of Nai concentration in Necturus gallbladder (Garcia-Diaz & Armstrong, 1980). Application of ouabain In the steady state, intracellular Na+ activity represents a balance between the rate of passive entry of Na+ at the apical membrane and the rate of active extrusion at the basolateral membrane. If this is the case, then any procedure which alters either of these processes should lead to qualitatively predictable changes in intracellular Na+. One such procedure would involve the blockage of the active extrusion step at the

6 532 D. C. EATON 16 14F_ 12 E 10 -~ /. * -.. U a * *.!. d a 6-0 4* 'B -i I l I I I I I I Na' activity (mm) Fig. 2. The relationship between intracellular Na+ activity and instantaneous short-circuit current (I,,). The curve drawn through the points is the best non-linear least-squares fit of the data to the equation Is= )n (Hagiwara & Takahashi, 1967). The values for Imax, KNa and n are 15-5,#A/cm2, 3-5 mm and 2-6, respectively. ;-) Ouabain ! 0000* A E i E I tb 0 4%~~9 I _~ ~ ~ ~ to -i E z : 01% Time (h) Fig. 3. The effect of ouabain on intracellular parameters. In A to D, short-circuit current (hc apical membrane potential (Ia) and intracellular K+ and Na+ activities ([K]i and [Na]j) were monitored before and after addition of 1O-4 M-ouabain to the serosal solution. Data are from a typical experiment on a low-na+ diet animal.

7 Na+ TRANSPORT AND INTRACELLULAR Na+ 533 basolateral membrane by the addition of ouabain to the serosal solution. We would qualitatively predict that, under these conditions, intracellular Na+ should increase and K+ should decrease. In Fig. 3 these predictions are confirmed. Using tissue from a low-na+ diet rabbit, 10-4 M-ouabain is applied after a stable level of short-circuit current is reached. As expected, short-circuit current begins to fall and continues to fall for the entire course ofthe experiment. Associated with the decrease in short-circuit current is (1) a depolarization of the apical membrane potential from a value near -40 mv to a value close to -15 mv, (2) a reduction of intracellular K+ activity from near 70 mm to a value less than 20 mm, (3) an increase in intracellular Na+ activity from about 16 mm to about 30 mm, (4) a decrease in voltage-divider ratio (ratio of apical to basolateral resistance) from to , and (5) an initial decrease in transepithelial conductance of variable magnitude followed by an increase in transepithelial conductance by a factor of 1-9 times. In five additional experiments using ouabain the results were qualitatively similar. Cemerikic & Giebisch (1980) in a preliminary report have described similar findings in Necturus proximal tubule. These results imply several things. First, even though ouabain has been applied for over 2 hr, the ion concentrations are not in electrochemical equilibrium across the apical membrane. Secondly, the sum of the cation activities in the cell decreases, suggesting that the cells swell. The magnitude of this decrease in total cation activities suggests an increase in volume of 50 % or more. Ouabain is known to produce similar swelling in a variety of other tissues (Tosteson & Hoffman, 1960; Eaton, 1972). From the magnitude of the changes in Na+ and K+ it appears that the short-circuit current after application of ouabain is not due to active transport, but rather follows from the selective permeabilities of the apical and basolateral membranes. Na+ enters the cell down its electrochemical gradient across the apical membrane. In response to this influx a K+ ion leaves the cell down its electrochemical gradient at the basolateral membrane. The net effect is a movement of cations across the tissue which appears as a short-circuit current. This process will continue until the electrochemical gradients are eliminated. This long-term net cation movement would explain the observations of a 'rapid' and 'slow' phase in ouabain action on frog skin (Helman, Nagel & Fisher, 1979) as well as the observation that when basolateral transport can be directly measured via its electrical effects, ouabain appears to act in 2-3 min (Lewis et al. 1978). Application of amiloride On the other hand, blockage of the entry step for Na+ on the apical membrane should produce a reduction in intracellular Na+ activity. In Fig. 4, 10-3 M-amiloride was added to the mucosal solution in an attempt to block a majority of Na+ entry at the apical membrane. In the experiment shown here, a low-na+ diet animal was used in order to obtain a preparation with high initial internal Na+ activity and relatively high short-circuit current. When amiloride at this concentration is first added to the solution bathing the apical membrane there is a rapid decrease in short-circuit current with a concomitant change in apical membrane potential, voltage-divider ratio, and transepithelial conductance. The initial voltagedivider ratio in the control case is This changes to after the addition of amiloride. The transepithelial conductance decreases by a factor of 1-52.

8 534 D. C. EATON These changes are consistent with the values obtained in previous work on this tissue (Lewis et al. 1976). Also, when amiloride is added there is a significant decrease in the intracellular Na+ activity (P < 0'01) from an initial value of mm to a final value of mm. The change in the intracellular K+ activity is not statistically significant. However, the level of remaining intracellular Na+ was more E 4*;Contvol * mm-. A" 5 0***..e..IoM-amiloridei NaCI A E4~~~~~~~~~~~ ou 3 A- 2 i 00.M _-10 I4zaB O-40.i IBi 100 E E10090~ _CoA~~~* 60 _ii _ Time (h) Fig. 4. The effect of amiloride on intracellular parameters. In A to D, short-circuit current (lkc) apical membrane potential (Va) and intracellular K+ and Na+ activities ([K]i and rnahi) were monitored before and after the addition of 1O-5 M-amilOride to the mucosal solution. Data are from a typical experiment on a low-na+ diet animal. than three times larger than the magnitude of the correction that was necessary for intracellular K+ (correction = 3S4 miw). Thus, blockage of the amiloride-sensitive pathways at the apical membrane is not able to reduce intracellular Na+. to very low levels. In four additional experiments with amiloride the results were qualitatively similar. Nagel et al. (1980) in a preliminary paper have reported similar results for frog skin. To try and determine whether there was a substantial measurement problem, we depleted intracellular Na+ by bathing the mucosal and serosal surface in a saline in which all but 1X33 mrm-na+ had been replaced with K+. This procedure caused a rapid decrease in intracellular Na+ levels with a concomitant increase in K+. With no substantial ionic gradients across either the apical or basolateral membranes, the membrane potential approached zero.

9 Na+ TRANSPORT AND INTRACELLULAR Na+ 535 Effect of aldosterone If blocking entry of Na+ reduces intracellular Na+ activity, then increasing the entry rate should produce an elevated intracellular Na+ activity. Aldosterone is believed to act on tight epithelia by enhancing the Na+ permeability of the apical membrane (Handler, Preston & Orloff, 1972). In Fig. 5 the effect of 10-6 M-aldosterone E A < Aldosterone 5 10 a E _ z 10 0 E E 15 _** * Tme 80~~~~~~~~~~~~~~~~ 7 * *. *. 8 * C * * ^-60_ 40_ Time (h) Fig. 5. Effect of aldosterone on intracellular parameters. In A to D, short-circuit current (I,,), apical membrane potential (Va) and intracellular K+ and Na+ activities ([K]i and [Na]i) were monitored before and after the addition of 10-6 M-aldosterone to the serosal solution. added to the serosal solution of a bladder from a normal diet rabbit is seen to be consistent with the idea of enhanced apical permeability. That is, apical membrane potential depolarizes, short-circuit current increases, intracellular Na+ activity increases from about 7 to about 20 mm, voltage-divider ratio decreases from to , and transepithelial conductance increases by a factor of 7'2. Also there seems to be some increase (about 10 mm) in the intracellular K+ concentrations. This could either be due to increased activity of the basolateral Na-K-ATPase or possibly to cell shrinkage. This preparation produced the largest increase in short-circuit current, but both of the other bladders examined after aldosterone displayed at least a 10-fold increase in short-circuit current and a doubling of intracellular Na+ activity. DISCUSSION Kinetics of basolateral transport All of the results are consistent with the classical model of epithelial function (Koefoed-Johnsen & Ussing, 1958), but in addition to confirming the model, the results can be used to determine some of the specific characteristics of the transport system.

10 536 D. C. EATON For a situation in which a specific number of Na+ ions, n, interact with surface sites of a membrane-bound ATPase and the number of sites is finite, the ratio of measured Na+ flux (as measured by the short-circuit current, Isc) to the maximal flux ('max) is given by the Langmuir equation (Hagiwara & Takahashi, 1967; Garay & Garrahan, 1973) 'se [Na]i2 imax ([Na]i + KNa)n (2) where [Na]i is the internal Na+ ion activity, and KNa is the internal activity of Na+ ion that leads to half the maximal current. Although this equation has recently been associated with certain types of enzyme kinetics, it is also useful in describing many saturating processes (Ainsworth, 1977). Although the actual reaction sequence associated with the Na-K-ATPase may be more complicated than several successive single-step reactions, investigators have had considerable success in describing the kinetics of Na-K-ATPase in terms of eqn. (2) if the reaction is examined under appropriate circumstances (for a review see Glynn & Karlish, 1975). Unless the ATPase found in this tissue is very much different from that found in a variety of other preparations, the same methods should apply. We need to worry about two complications: (1) variations in internal K+ altering the kinetics and (2) variations in electrochemical driving force for Na+ altering the kinetics. In our study we do have a variation in the control levels of intracellular K+; however, intracellular K+ changes within the range we observe produce no effect on Na+ transport in red blood cells because the internal K+ sites are completely occupied (Garay & Garrahan, 1973). We cannot rule out an effect of basolateral membrane potential on the transport rate ofthe ATPase; however, this effect cannot be large since the basolateral potential change in most experiments was usually less than 25 mv. Only in the experiments with ouabain did the basolateral potential change ever exceed 25 mv (but always less than 50 mv). Again, however, if this transport system is at all like similar systems, changes of potential of the magnitude of 50 mv have little effect on transport (Hodgkin & Keynes, 1955; Kostyuk, Krishtal & Pidoplichko, 1972; Brinley & Mullins, 1974). Since we felt these reservations to be relatively unimportant we attempted to describe the interaction of intracellular Na+ with an internal site in terms of eqn. (2). In Fig. 2 the broken line through the points represents the best non-linear least-squares fit of the data to eqn. (2) above (Colquahoun, 1971; Brown & Dennis, 1972). This line has an Imax of 15-5 ItA/cm2, a Na+ concentration at half saturation of 3-5 mm and a value for the number of sites, n, of 2-6 (regression coefficient = 0-81). Such a non-linear curve-fit does not introduce any of the biases associated with any of the various linearization methods described in the literature (Colquahoun, 1971; Ainsworth, 1977). However, we can represent the data in a more conventional way. In Fig. 6A and B the data of Fig. 2 are replotted as the reciprocal of the intracellular Na+ activity versus the reciprocal of the short-circuit current (for the case of n = 1) and the cube root of the reciprocal of the short-circuit current (for the case of n = 3), respectively. The continuous lines through the data are the best least-squares linear regression lines. The points in Fig. 6A are seen to deviate systematically from the regression line. Moreover the intercept (-0A159) is not

11 Na+ TRANSPORT AND INTRACELLULAR Na+ 537 physically meaningful. The systematic variation is emphasized by plotting the expectation for eqn. (2) for n = 3 (dashed line in A). Also, the fit of the data to the regression line appears better (regression coefficient = 0 97) in the plot of the cube root of the inverse short-circuit current versus reciprocal Na+ activity. The slope and intercept yield a value for KNa of 3 05 mm and for Imax of 14-2 pza/cm2. A 3 20 / E _-If E 1*20 - I ~05- E 1 60 E 0.9_ - A B * Reciprocal Nab activity Reciprocal Na' activity Fig. 6. In A, reciprocal short-circuit current (I.C) is plotted vs. the reciprocal of the intracellular Na+ activity. In B, the cube root of the reciprocal short-circuit current is plotted vs. the reciprocal of the intracellular Na+ activity. In this Figure the short-circuit current is used as a measure of Na-K-ATPase activity at the basolateral membrane. The first plot will yield a straight line if there is only one site with which Na+ interacts on the ATPase, while if there are three sites the plot in B should be linear. The continuous lines are least-squares regression lines. The dashed line in A is the expected curve for three sites. The data are pooled from the initial values of intracellular Na+ activity and instantaneous short-circuit current for all the tissues examined in this study. The maximal current predicted from these fits is comparable in its magnitude to the maximal transport that can be stimulated by treatment of the apical membrane with nystatin (Lewis et al. 1977). Also, the predicted KNa and Ima. for the system suggest that under normal conditions the system is operating at levels below its maximal rate but that many of the Na-K-ATPase molecules have one or two Na+ ions bound to the molecule. This is consistent with the observation that the electrogenic component of transport observed under certain conditions appears to saturate at levels of intracellular Na+ above 20 mm (Lewis et al. 1978). As to the question of multiple sites associated with the ATPase reaction, the regression coefficients of the multiple site versus the single site fits do not allow us strongly to support on a statistical basis one model over the other. Nonetheless, there are two reasons to suspect that a multiple site model might be present. First, other investigators have demonstrated multiple site processes: three sites in red blood cells (Garay & Garrahan, 1973) and probably two sites in frog skin (Nagel, 1979). Secondly, Lewis et al. (1978) in rabbit urinary bladder and Garcia-Diaz & Armstrong (1980) in Necturus gall-bladder have demonstrated a sigmoidal relationship between intracellular Na+ and short-circuit current when the apical membrane was treated with nystatin. The relationship in rabbit urinary bladder could be fitted fairly well by a

12 538 D. C. EATON model with two to four equivalent sites. Also, in a brief communication, Wills & Lewis (1980) report a value for intracellular Na+ activity in rabbit urinary bladder of approximately 7 mm while observing little or no variation of intracellular Na+ associated with a 4-fold change in short-circuit current. These results are somewhat at variance with those reported here. They are, however, best explained by the steep response of short-circuit current to intracellular Na+ activity found with multiple site models ' 0-80 _. E 75 0-,70 _-.. E _ 0-55 _, 0*50 045a Reciprocal Na' activity Fig. 7. The cube root of the reciprocal short-circuit current (I,,) vs. the reciprocal of the intracellular Na+ activity after amiloride treatment. The Figure is similar to Fig. 6 except that it represents variation in short-circuit current and intracellular Na+ activity in a single tissue treated with 1O-5 M-amiloride. The straight line is drawn from a fit of the original data to the Langmuir equation (see text). Similar values for Imax and KNa can be obtained from individual experiments in which the intracellular Na+ activity is caused to change by processes which do not affect the transport mechanism at the basolateral membrane directly. In particular, in experiments where amiloride is added to the mucosal solution and intracellular activity subsequently falls, double-reciprocal plots yield linear relationships. In Fig. 7, the data from such an experiment are plotted to demonstrate the relationship. In this particular case when values were obtained from a non-linear curve fit, Imax was 14-4,sA/cm2, KNa was 4-3 mm, and the probable number of sites was 3-3 (regression coefficient = 0 86). Values for KNa from seven experiments vary between 2-1 and 7-2 mm (mean = mm) with a current maximum between 14-2 and 24-5,A/cm2 (mean = ,A/cm2) and a value for the number of sites between 2-6 and 4-0 (mean = ). Mechanism of aldosterone action If the action of aldosterone is only due to an increase in the Na+ permeability of the apical membrane, then the relationship of the short-circuit current and the intracellular Na+ activity after aldosterone should also be described by the Langmuir equation. Fig. 8 shows that for prolonged exposure to aldosterone in vitro, this proved

13 Na+ TRANSPORT AND INTRACELLULAR Na+ 539 not to be the case, since for long exposures the short-circuit current increased more than would be expected for a situation described by eqn. (2). A possible cause of the increase is suggested by the fact that the maximal current reached after aldosterone stimulation far exceeds the typical values for Imax in unstimulated cells. This implies that aldosterone may not only be increasing the Na+ permeability of the apical membrane but also may be increasing the maximal transport capabilities of the basolateral membrane. The exact manner in which the aldosterone produces the increase is not clear from our experiments, although a simple increase in Imax with time after application of aldosterone may be sufficient to explain our results E 30 _ ;. 6 _.. *U Na4 activity (mm) Fig. 8. Short-circuit current (I,,) vs. intracellular Na+ activity after treatment with exogenous aldosterone. Although the relationship at low intracellular Na+ activities (corresponding to a short time after application of aldosterone) is similar to Fig. 2, there is a large increase in short-circuit current at higher Na+ activities (longer application times). I feel that the large changes observed in the short-circuit current and the necessity of postulating changes in the transport parameters may not normally be true. Only in animals given relatively high doses of exogenous aldosterone is there such a large increase in short-circuit current. Under normal, in vivo conditions, the primary effect of aldosterone is probably only on the apical membrane Na+ permeability. Permeability of the apical membrane Several investigators have suggested that intracellular Na+ activity may play an intimate role in regulating the Na+ permeability of the apical membrane (MacRobbie & Ussing, 1961; Lewis et al. 1976; Lewis & Wills, 1979). The hypothesis is very attractive since control of the basolateral transport system alone would be sufficient to regulate both Na+ efflux and Na+ influx. This hypothesis is difficult to formulate in its entirety. It is quite clear that when the Na+ transport mechanism is blocked by metabolic inhibitors the apical membrane resistance increases (Hviid Larsen, 1973; Lewis et al. 1976; Turnheim, Frizzell &

14 540 D. C. EATON Schultz, 1977). These investigators suspected and we observed in these experiments that the intracellular Na+ increased and intracellular K+ decreased at the same time as the resistance changed. The additional observation that is also quite clear is that the apical membrane resistance decreases as the short-circuit current increases (Lewis & Diamond, 1976; Lewis et al. 1976, 1978; Lewis & Wills, 1979). If intracellular Na+ activity is the primary mechanism for regulation of apical resistance, then we would expect that intracellular Na+ would necessarily decrease as short-circuit current increased. This would lead to the situation in which the ATPase transport would increase as the substrate concentration decreased: a property difficult to reconcile with current understanding of transport proteins. Our results may give us some information about the effect of intracellular Na+ on the apical membrane permeability. To do this we must make two assumptions. (1) The short-circuit current in this preparation is quantitatively described by the Na+ flux associated with active transport. That this assumption is very nearly true has been shown by Lewis & Diamond (1976) using unidirectional flux measurements. This means that for this tissue net Na+ flux across the apical membrane must equal the short-circuit current regardless of the apical entry pathway. (2) Our second assumption was that the apical membrane permeability could be described by the Goldman-Hodgkin-Katz model (Hodgkin & Katz, 1949; Hodgkin & Horowicz, 1959). If these two assumptions are correct,we can calculate the Na+ permeability of the apical membrane from the relationship (Hodgkin & Horowicz, 1959): RT exp(vaf/rt) -1 PNa = 'Na VF2 ala exp (Va F/RT)-a a' (3) where INa can be equated with the short-circuit current, Va is the apical membrane potential, aia and a a are the intracellular and mucosal Na+ activities, and the other symbols have their usual meaning. All the variables in this expression are known if we assume that the primary source of short-circuit current when the tissue is bathed in symmetrical solutions is due to movement of Na+ ion across the tissue (Lewis & Diamond, 1976). In Fig. 9 we have plotted the apical Na+ permeability for several different situations in which the intracellular Na+ activity varies. In one case, the Na+ permeability after application of aldosterone is calculated. Under the influence of aldosterone the permeability continues to increase with time and the intracellular Na+ activity also increases (see Fig. 5). Under these conditions, if intracellular Na+ is reducing actual Na+ permeability, the additional Na+ conductance induced by aldosterone more than compensates for the blockage. The substantial reduction in the voltage-divider ratio lends support to this idea. The PNa values from control cases (asterisks) in which variations of short-circuit current are probably due to in vivo variations in aldosterone production also fall near this curve although PNa and intracellular Na+ do not rise to levels as great as with large amounts of exogenous aldosterone. In the other two cases, one from a normal diet animal and one from a low-na+ diet animal, apical Na+ permeability is calculated after application of 10-4 M-ouabain in the serosal solution. In these situations the drug has no direct effect on the apical Na+ permeability so changes in the permeability may be attributed to the changes

15 Na+ TRANSPORT AND INTRACELLULAR Na+ 541 in intracellular Na+ activity. In neither case does there seem to be any substantial direct effect of intracellular Na+ on the apical Na+ permeability. In both tissues the Goldman permeability at high Na+ activities is virtually the same as the original permeability at the lowest values of intracellular Na+. In both cases there is also a slight decrease in the voltage-divider ratios; however, under conditions when the intracellular ion concentrations are changing so dramatically it is difficult to use the voltage-divider ratio by itself as an indication of the processes that are occurring at each membrane. The implication of this figure is that in normal situations the apical Na+ permeability, and consequently the steady-state level of intracellular Na+ activity, is set by the level of circulating aldosterone and the intracellular Na+ activity itself plays little role in directly controlling apical Na+ permeability E 12 - UJ.3 10 _ - x ~* 2. _ *** *.*.4 * * * Internal Na' activity (mm) Fig. 9. Apical Na+ permeability (PNa) vs. intracellular Na+ activity. The Figure represents changes in Na+ permeability (PNa) after various treatments that alter intracellular Na+ activity. The aldosterone-treated preparation reflects the ability of aldosterone to increase Na+ permeability of the apical membrane in vitro (@). That the effect is similar in vivo is implied by the fact that the initial apical Na+ permeability of a normal diet animal and a low-na+ diet animal lie on the aldosterone curve and that the apical permeabilities for control animals with various short-circuit currents also lie on this curve (*). The essentially zero slope of PNa vs. Na+ activity after treatment with ouabain, a drug that by itself probably does not affect apical Na+ permeability, suggests little direct effect of intracellular Na+ on apical Na+ permeability (PNa) after either a low-na+ diet (a) or a normal diet (*). Are there alternatives to our conclusion? Some of them are: (1) The Goldman equation does not apply to the apical Na+ permeability. We cannot rule out this possibility; however, several preparations appear to obey the relationship. (2) The resistance change after addition of metabolic blockers is not due to the associated increase of intracellular Na+, but rather to alterations of other factors. Eliminating the Na+ gradient could be expected to alter intracellular Ca2+ levels and possibly alter intracellular ph (Blaustein & Hodgkin, 1969; Thomas, 1974; Russell & Boron, 1976; Boron et al. 1978). Varying Ca2+, in particular, is known to affect membrane resistance (Frankenhaeuser & Hodgkin, 1957) and may affect apical permeability in toad bladder (Taylor, 1980). (3) The change in resistance is not associated with an alteration of Na+ conductance. Instead it may represent an alteration of an alternate conductive pathway which is sensitive to any of the intracellular factors which might be changing after application of metabolic blockers. Lewis & Wills (1979) suggest the existence of such a conductance.

16 542 D. C. EATON Mechanism of apical Na+ entry If all of the Na+ entry at the apical membrane were due to Na+ movement through amiloride-sensitive pathways, we would expect that, after application of amiloride, the short-circuit current should be very close to zero. Moreover, if all Na+ entry were blocked, the basolateral transport system might reduce intracellular Na+ to very low levels (the only substantial source of Na+ being leakage at the basolateral membrane). In fact, when the entire tissue is bathed in a solution in which all of the Na+ has been replaced with Tris+ or K+ as a Na+ substitute, the ouabain-blockable short-circuit current becomes small (< 0-2,A/cm2) and the intracellular Na+ falls to a level lower than can be accurately measured with the Na+ electrodes (< 3 mm). Possible explanations for the larger than anticipated intracellular Na+ activity after amiloride treatment are: (1) The amiloride is ineffective in completely blocking Na+ entry. (2) There are alternative pathways for Na+ entry into the cell (which may or may not contribute to the short-circuit current). (3) The energetics of the transport processes are such that reduction of Na+ activities below the levels measured here is not possible. Each of these possibilities appears somewhat unsatisfactory. The amiloride has been previously reported as an effective blocker of Na+ transport (Lewis & Diamond, 1976) at even lower concentrations than this. There is no additional evidence for additional Na+ permeability pathways other than a small finite basolateral permeability to Na+ (Lewis et al. 1978). And finally, it is quite clear from the literature that in red blood cells (Garay & Garrahan, 1973), snail and Aplysia neurones (Thomas, 1969; Eaton et at. 1975), and squid axons (Brinley & Mullins, 1968) the Na+ transport mechanism is capable of generating substantial fluxes in the face of larger electrochemical gradients than are present in urinary bladder. In this tissue the observed result is probably some combination of these three possibilities. In summary, we have shown that (a) the basolateral transport process appears to depend upon intracellular Na+ activity, that is, the transport ATPase is substratedependent; (b) the apical membrane permeability to Na+ ion is primarily dependent upon circulating aldosterone and does not seem to depend directly upon intracellular Na+ activity; and (c) there may possibly be entry pathways for Na+ at the apical membrane that are not sensitive to amiloride. I would like to acknowledge the excellent technical assistance of Ms Jan Scott, Mr A. Michael Frace and Mr Rudy Briner. I would also like to thank Ms Sandra Carreon and Mr Josef Bush for the preparation of the manuscript and Dr John Russell for helpful comments on the experiments and the content of the manuscript. The work was supported by Grant I RO1 AM and Research Career Development Award 1 -K04-AM from the National Institute of Arthritis, Metabolism and Digestive Disorders. REFERENCES AINSWORTH, S. (1977). Steady State Enzyme Kinetic8, p. 29ff. Baltimore: University Park Press. BLAUSTEIN, M. P. & HODGKIN, A. L. (1969). The effect of cyanide on the efflux of calcium from squid axons. J. Physiol. 200, BORON, W. F., RUSSELL, J. M., BRODWICK, M. S., KEIFER, D. W. & Roos, A. (1978). Influence of cyclic AMP on intracellular ph regulation and chloride fluxes in barnacle muscle fibres. Nature Lond. 276,

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