manipulating serosal solution K+ concentration and measuring changes in the

Transcription

1 J. Physiol. (1983), 341, pp With 4 text-figures Printed in Great Britain APICAL MEMBRANE PERMEABILITY AND KINETIC PROPERTIES OF THE SODIUM PUMP IN RABBIT URINARY BLADDER BY S. A. LEWIS AND N. K. WILLS From the Department of Physiology, Yale Medical School, 333, Cedar Street, New Haven, CT U.S.A. (Received 30 March 1982) SUMMARY 1. Previous studies have shown that aldosterone stimulates the rate of Na+ transport across the rabbit urinary bladder epithelium by increasing the apical membrane permeability to Na+. Paradoxically, ion-sensitive and conventional micro-electrode measurements demonstrated that intracellular Na+ activity ana+ was essentially unchanged by aldosterone, i.e. aka+ was constant regardless of the rate of Na+ transport. The present study was designed to resolve this apparent contradiction. 2. The effects of elevated, endogenous aldosterone levels produced by low-na+ diet (Lewis & Diamond, 1976) on urinary bladder Na+ transport were investigated in vitro using Ussing-type chambers and intracellular conventional and ion-sensitive microelectrodes. Apical membrane selectivity and the kinetics of the Na+ pump were assessed as a function of hormone stimulation. 3. The aldosterone-stimulated increase in Na+ transport was accounted for by increases in both the relative selective permeability of the apical membrane to Na+ and an increase in its absolute Na+ permeability. 4. The kinetics of the Na+ pump were evaluated electrically by loading the cells with Na+ (monitored with Na+-sensitive micro-electrodes) or alternatively by manipulating serosal solution K+ concentration and measuring changes in the basolateral membrane electromotive forces and resistance. From these measurements the current generated by the pump was calculated as a function of intracellular Na+ or extracellular K+. The kinetics of the pump were not altered by aldosterone. A model of highly co-operative binding estimated Km for Na+ as 14-2 mm and 2-3 mm for K+. Hill coefficients for these ions were 2-8 and 1-8, respectively, consistent with a pump stoichiometry of 3 Na+ to 2 K+. The kinetic properties of the Na-K pump indicate that physiological levels of aka+ are poised at the foot of a step kinetic curve which energetically favours Na+ extrusion. INTRODUCTION Many of the tight epithelia have amiloride-sensitive, aldosterone-stimulated Na+ transport systems. The original transport model for such epithelia was devised by Koefoed-Johnsen & Ussing in This model proposed passive Na+ entry down

2 170 S. A. LEWIS AND N. K. WILLS a net electrochemical gradient across the apical membrane, and active extrusion of Na+ in exchange for K+ across the basolateral or blood-side membrane. K+ was thought to diffuse back into the blood down a net electrochemical gradient. The diffusion of K+ was responsible for the resting potential of the basolateral membrane with the K+ gradient being maintained by the Na+-K+ pump. This reciprocating active exchange of Na+ for K+ results in a net Na+ current traversing the epithelial cells. Although the model was originally developed for frog skin, it has been extended and modified for the rabbit urinary bladder (Lewis, Wills & Eaton, 1978). Aldosterone is known to increase Na+ transport across rabbit urinary bladder. Micro-electrode measurements revealed that this hormone stimulates Na+ entry across the apical membrane by increasing its Na+ conductance (Lewis, Eaton & Diamond, 1976). Such an increase in Na+ entry might be expected to cause an increase in cell Na+ activity a a+. However, the amount of increase in a a+ will depend on the kinetic properties of the pump as well as the density. In a recent paper Wills & Lewis (1980) reported that, although Na+ transport was increased by up to 4 times after endogenous aldosterone stimulation (produced by placing rabbits on a low-na+ diet for two weeks), there was no significant change in ai a+. There are a number of possible explanations for the above finding. Aldosterone might alter not only apical Na+ permeability but also pump kinetics and density. Rather than altering pump properties, the kinetics of the pump might be very steep for the transport rate studied and consequently any change in aka+ may be within the measuring accuracy of the Na+ micro-electrodes. Alternatively, the Na+ electrode might measure the activity of a separate Na+ pool which does not originate from the mucosal solution. In this paper we demonstrate, using electrophysiological methods, that only very small changes in ai a+ are necessary to have a marked effect on the rate of Na+ translocation. In addition, increases in endogenous aldosterone levels by low-na+ diet does not appear to directly affect Na+-pump kinetics or density. METHODS Rabbit urinary bladders were dissected and after removal of underlying muscle layers were mounted in modified Ussing chambers designed to reduce edge damage (see Lewis & Diamond, 1976). Electrical mea8urement8 The transepithelial potential (VT, serosa ground) was measured by Ag-AgCl electrodes placed close to and on either side of the preparation. Transepithelial conductance (GT) was determined by passing a square current pulse (I) across the epithelium from Ag-AgCl electrodes placed in the rear of each half chamber. Both current-passing and voltage-measuring micro-electrodes were connected to an automatic voltage clamp. Basolateral membrane potentials (Vbl) and intracellular ion activities were measured using conventional and ion-specific micro-electrodes. These electrodes were placed in Ag-AgCl half cells which were mounted on hydraulic drive micromanipulators and connected to a high input impedance differential electrometer (Model 750 for the conventional and a F223A for the ion specific, W.P. Instruments Hamden, CT, U.S.A.). Cell impalements were from the mucosal solution across the apical membrane. All micro-electrode measurements were referenced to the Ag-AgCI voltage-measuring electrode in the serosal chamber. VT, I, Vbl and the potential measured by the ion-specific micro-electrode (JVi) were displayed on a storage oscilloscope (Tektronix, Beaverton, OR, U.S.A.) and paper chart recorder. In addition, all four measurements

3 Na+ PERMEABILITY AND PUMP KINETICS were digitized (Analog Devices A/D converter) and stored on floppy disks by a small laboratory computer (Horizon II North Star Computers, CA, U.S.A.) for future data analysis. Micro -electrodes Both conventional and ion-specific micro-electrodes were made from thin-walled Corning redrawn Pyrex glass tubing no. 7740) with an o.d. of 1-2 mm and an i.d. of 0 9 mm. The glass tubing was pulled to a tip diameter of less than 1 jam on a Narishige horizontal puller. Conventional micro-electrodes were filled with a 3 M-KCl solution and had tip resistances ranging between 10 and 30 MCI. Micropipettes to be used for ion-sensitive micro-electrodes were first silanized using the method described by Wills & Lewis (1980). After silanization, either a K+ exchanger resin (Corning Medical Products Div., Medfield, MA, U.S.A. no ) or a Na+-exchanger resin and tetraphenylborate mixture (Steiner, Oehme, Ammann & Simon, 1979) was incorporated into the tip. The remainder of the electrode was back-filled with either a 0-5 M-KCl or NaCl solution, respectively. Because the resistance of the Na+ resin was comparable to that of the glass wall of the electrode, all Na+ electrodes were shielded according to the method of Lewis & Wills (1980). Ion-sensitive micro-electrodes were calibrated before and after each use for response to decade changes in pure and mixed salt solutions to determine both the slope and selectivity to competing ions. Intracellular K+ and Na+ activities (ak + and a'a+) were calculated from the Nicolsky equation (Wills & Lewis, 1980) using potentials measured by the conventional and ion-specific micro-electrodes and the slope and selectivity from the calibration procedure. In addition to measuring the membrane or ion activity plus membrane potential signal, we routinely measured the resistance ratio (a = ratio of apical to basolateral membrane resistance Ra/Rbl) as an indicator of a successful cell impalement (see Lewis, Wills & Eaton, 1978). Solutions The composition (in mm) of the usual bathing solution (NaCl Ringer solution) was: NaCl, 11 12; NaHCO3, 25; KCI, 5-8; CaCl2, 2; MgSO4, 1-2; KH2PO4, 1-2 and glucose 111. This solution was aerated with 95 % 02-5 % CO2 and maintained at a ph of 7-4 at 37 'C. In one series of experiments a K2SO4 Ringer solution was used, which had a composition (in mm) of: K2SO4, 58-5; KHCO3, 25; Ca (methanesulphonate), 10; MgSO4, 1-2; KH2PO4, 1-2; glucose, 111 and sucrose, 80. In these experiments voltage-measuring electrodes consisted of 0-5 M-KCl agar bridges. Unless otherwise stated NaCl Ringer solution was routinely used. Schwartz & Burg (1978) measured a significant increase in plasma aldosterone levels after rabbits were fed a low-na+ diet for 6 to 21 days. To study the long-term effects of increased, endogenous plasma aldosterone levels on bladders, we placed rabbits on a low-na+ diet (Ralston-Purina) and distilled water for 14 days prior to the experiment, the tacit assumption being that changes in bladder transport properties are only a consequence of increasing plasma aldosterone levels and not other physiological responses to the dietary regime. All data are expressed as the mean +S.E. of the mean. Calculation of equivalent circuit parameters Apical, basolateral membranes and junctional resistances (Ra, Rbl, Rj) as well as apical and basolateral membrane electromotive forces (Ea and Ebl) were calculated as described below. In order to determine the membrane electromotive forces (e.m.f.s) the three epithelial resistances were first calculated. The method of Lewis, Eaton & Diamond (1976) was used. In brief, the resistance ratio (a = Ra/Rbl) and transepithelial resistance was measured before and after amiloride addition (10-4 M) to the mucosal solution. Since amiloride is assumed to increase only apical resistance (Lewis, Eaton & Diamond, 1976), one can calculate the apical, basolateral membrane and junctional resistances before and after amiloride addition. Once the individual resistances had been determined, we next calculated the e.m.f. of the apical and basolateral membranes. This calculation required a measurement of the transepithelial potential (VT) and basolateral potential (VbJ) before and after amiloride addition. The basolateral e.m.f. (Ebl) was calculated using the following equation (see Reuss, 1979): 171 EbI = Vb1 + VT-X (1) Rj

4 172 S. A. LEWIS AND N. K. WILLS where Vbl is the spontaneous basolateral membrane potential, VT the transepithelial potential, and Rbl and Rj the basolateral and junctional resistance respectively. A comparable equation can be derived to calculate the apical membrane e.m.f. (Ea): Ea T(1+f)Vbli (2) where Ra is the apical membrane resistance and the other values as above. RESULTS The results are presented in three sections. First we assessed the source of the Na+ pool measured by ion-sensitive micro-electrodes. Next the selectivity properties of the amiloride-sensitive channel were considered as a function of hormone stimulation. Lastly, the electrogenic nature of the Na+ pump and its kinetic properties were characterized. Intracellular Na+ activity Wills & Lewis (1980) reported that after stimulation of Na+ transport there was no measureable change in the intracellular Na+ activity. In order to determine whetherthe Na+ micro-electrode was measuring Na+ ofmucosal origin (i.e. the so-called Na+ transport pool), we measured a'a+ before and after replacement of all mucosal Na+ with K+. This replacement caused a'a+ to fall from a value of 7 +1 mm (n = 4) to mm (n = 4) while intracellular K+ (ak+) was not significantly changed. Replacement of Na+ by choline in both bathing solutions caused ana+ to decrease to a value not significantly different from zero ( , n = 4). These data indicate that the Na+-specific micro-electrode is measuring cell Na+ which is entering the cell not only from the mucosal solution across the apical membrane but also from the serosal solution across the basolateral membrane. This latter Na+ source confirms previous measurements of a finite permeability of the basolateral membrane to Na+ reported by Lewis, Wills & Eaton (1978) and is a crucial feature in the calculation of pump stoichiometry presented below. Apical selectivity of amiloride-sensitive pathways Lewis & Diamond (1976) demonstrated that placing rabbits on a low-na+ diet for two weeks resulted in a doubling of the net Na+ transport across the urinary bladder. Such an increase in Na+ transport in the absence of a measurable change in intracellular Na+ activity indicates an increase in the permeability of the apical membrane to Na+ as previously suggested by Lewis & Diamond (1976) and Lewis, Eaton & Diamond (1976). These authors were unable to directly confirm this hypothesis because the value of the net driving force for Na+ for the control and stimulated conditions was not known. With the present measured values for a'na+, membrane potentials and resistances, we are now able to directly calculate not only the absolute permeability to Na+ but also the selectivity of the amiloride inhabitable pathway. Using eqn. (1) and (2) (see Methods) we calculated both the apical and basolateral membrane e.m.f.s as a function of diet and before and after amiloride action. These are shown in Table 1. From these values we estimated Eamil (i.e. the apical membrane amiloride-sensitive e.m.f.) using the following relationship: Ea

5 before amiloride is given by: Na+ PERMEABILITY AND PUMP KINETICS Raami RL =Raami+ RJaRmil + RL' where EL and RL represent the e.m.f. and resistances of a leak ionic pathway, respectively and Ramil is the resistance of the apical membrane Na+ pathway. After amiloride action, Ramil is essentially infinite thus Ra = RL and the new apical e.m.f. (Ea) equals EL. Calculated values for Eamil and EL are presented in Table 1. Ea (EaamiI +E. TABLE 1. Membrane parameters used to calculate the Na+-K+ selective permeability of the amiloride pathway in the apical membrane for animals on control diets as compared to low-na+ diets Control Diet Method 1 Ea(mV) EAL(mV) -17+± Eamil (mv) Ebl (mv) Ga (#S//SF) OaL(,S/#F) Gaamil (ts/#f) PNa/PK Method 2 P1mi (/ua/luf) Oaamil (ps/1tf) Eami, + Vse (mv) PaC (mv) Elamil (mv) PNa/PK 2-95± PNa/PK was calculated for the amiloride-sensitive pathway using the constant-field equation and Na+ and K+ activity values previously published by Wills & Lewis (1980). Using the same data we can estimate (indirectly) the e.m.f. ofthe amiloride-sensitive and insensitive pathway in the apical membrane using a second method. Given that the short-circuit current (Isc) is equal to the sum of the amiloride-sensitive current (Iamil) plus a leak current (IL), the magnitude of these individual currents is equal to the product of their conductance and net driving force (the net electrochemical gradient). This latter term is the sum of the e.m.f. and the apical membrane potential under short-circuited conditions (Vas), i.e.: Iamil = Oamii( Vay + Eamii) and similarly IL = GaL( VaS + EL), where Ga is the apical membrane conductance before amiloride, Gaamil is that after amiloride and GaL represents the amiloride insensitive apical membrane conductance. Since Iamil, IL, VSc, Gami1, GL are easily determined (see Methods) one can solve for Eamil and EL. Table 1 summarizes the net electrochemical gradient for the amiloride pathway, as well as the short circuit apical membrane potential (Vsa). Taking the difference 173

6 174 S. A. LEWIS AND N. K. WILLS between the net electrochemical gradient and the VsC yields the equivalent driving force through the amiloride-sensitive channels. As is obvious from Table 1 this value is less than the equivalent Na+ Nernst potential. This indicates that the amiloridesensitive pathway is permeable to ions other than Na+. In addition the selectivity of the amiloride channel is different between control and low-na+ diet conditions. Using the constant-field equation it is possible to calculate the selectivity of the amiloride-sensitive pathway. The competing ion is likely to be K+, rather than Cl-, since Lewis & Diamond (1976) showed that Cl- permeability was very low through the urinary bladder. Thus by imposing the constant-field conditions, we calculated the selectivity of the amiloride pathway to both Na+ and K+. These selectivities are shown in Table 1 for both methods. It is obvious that the PNa/PK (Na+ to K+ selectivity) is greater for the low Na+ diet condition than the control condition. The reason for such a change will be offered in the discussion. Kinetic properties of the Na+ pump Returning to our previous finding that ada+ does not differ between control and low-na+ diet animals (Wills & Lewis, 1980), we next investigated the in situ Na+ and K+ activation of the electrogenic Na+-K+-ATPase. Na+ activation Two methods were used to determine the dependence of pump rate on intracellular Na+ activity. The first is a non-steady-state method described by Wills & Lewis (1980) and the second is the steady-state method of Lewis, Wills & Eaton (1978). Non-steady-state pump properties. In these experiments the pump was reversibly inhibited by removal of serosal K+. Due to this inhibition, ai a+ increased while ai + and Vbl decreased. After min, 7 mm-k+ was added to the serosal solution and ai a+, aij+ and Vbl were monitored as a function of time. As previously reported (Wills & Lewis, 1980) a rapid hyperpolarization of the basolateral membrane occurred in the absence of changes in aka+ or ak+. After 60 sec ada+ decreased and ak+ increased. From the ion activities and measured permeability of the basolateral membrane we can calculate the 'passive' component of the basolateral membrane potential at any time after serosal K+ addition (i.e. ignoring the contribution of the electrogenic pump). The difference between the measured and calculated basolateral membrane potential represents the 'electrogenic' potential, i.e. the Na+-pump current times the membrane resistance at known intracellular Na+ activities. The basolateral membrane resistance was calculated using the method of Lewis & Wills (1982). In brief, the ratio of the change in the basolateral membrane potential to the transepithelial potential after pump stimulation yields the ratio of apical membrane to junctional resistance (Ra/Rj). Simultaneous measurement of RT and a (see Methods) allows one to calculate Ra Rbl and R;. Dividing Rbl into the potential generated by the pump yields the Na+-pump current (Ip). A plot of Ip against a a+ is shown in Fig. 1. The curve is sigmoidal and can be fitted by the equation for a model of highly co-operative binding (Nelson & Blaustein, 1980): Jmax 4P = 1 + (Km/a a+)n (3)

7 Na+ PERMEABILITY AND PUMP KINETICS The best fit of the model to the data using a non-linear curve-fitting routine (Brown & Dennis, 1972) gave the following parameters: n = 2-9, Km = 19 mm and ImNax = 18tA/cm2. Steady-state pump properties. The previous method assumes that the lateral space K+ activity is equal to the bulk solution K+ activity (a +). Small changes in this restricted space K+ activity can change the shape of the curve. To avoid this problem we used a gradient-free condition as described by Lewis, Wills & Eaton (1978). Mucosal NaCl Ringer solution was replaced with an isosmotic K2SO4 Ringer solution, ana+. (mm) Fig. 1. Determination of the Na+-pump current as a function of intracellular Na+ activity (ai +). Method for calculating the pump current is described in the text. Tesot uv is a best fit of the data by a model of highly co-operative binding (eqn. 1). The best fit paramterswere Km o 19 M;aI of 18,sA/m and a Hill coefficient (n) of 2-9. and then the polyene antibiotic nystatin (120 units/ml) was added to the mucosal solution so as to reduce the apical membrane resistance to a very low value ( K2 cm2). Next, to eliminate the possibility of K+-diffusion potential developing across the basolateral membrane, the serosal solution was changed to a K2S04 Ringer solution. This caused the VT and Vbl to decrease to zero. Lastly NaCl was added to first the serosal and then mucosal solutions in defined final concentrations. Only when the NaCl was added to the mucosal solution was there a hyperpolarization of VT and VbW. The VT change was biphasic, consisting of a rapid, near step increase followed by a slower sigmoidal hyperpolarization (see Fig. 7 of Lewis, Wills & Eaton, 1978). Monitoring Vbj demonstrated that the rapid phase of VT was restricted to the apical membrane and consisted of a rapid step followed by an exponential-like decrease to a value greater than the initial potential. Vbj then increased in a sigmoidal fashion to a steady-state value within 2 to 3 min after mucosal NaCl addition. Since the rapid hyperpolarization across the apical membrane reflects a NaCl diffusion potential

8 176 S. A. LEWIS AND N. K. WILLS through the nystatin channels, we can use this potential change to calculate the intracellular Na+ activity associated with the prior steady-state Vbl hyperpolarization i.e. the previous pump current. Calculation of aia+ was determined using the following equation: AVT = V'- VT RF1 (ak++a% +'+xao- ai++aoa++xa ) K+ +nia+ xci- ak +Na+ +xciwhere a +, ai + and ai + are as previously described, ai I- in intracellular chloride activity, aoa+ and at,- are control extracellular Na+ and Cl- activities and a a+' and aol' are extracellular Na+ and Cl- activities after addition of an aliquot of NaCl to < 15/ ala+ (mm) Fig. 2. Similar to Fig. 1 except that Na+ entry across the apical membrane was greatly enhanced by reducing apical resistance using nystatin. To eliminate the possibility that the hyperpolarization was caused by a K+ gradient across the basolateral membrane both apical and basolateral surfaces were bathed in a high K+ Ringer solution. Ip and ai a+ were calculated as described in the text. Using a model of highly co-operative binding (eqn. 1) the best fit parameters were: Km of 14 mm; INa of 27-3,A/cm2 and an n of 2-8. Vertical bars are s.e. of the mean. the mucosal and serosal chambers. x is the selective permeability of the nystatin channels for Cl/Na (see Lewis, Eaton, Clausen & Diamond, 1977). The above equation has two unknowns, ai a+ and as -. In order to solve for aka+ we assume (for electroneutrality) that ai a+ = ai I- and aj(+ = as+. Fig. 2 is a plot of the calculated ai a+ as a function of the electrogenic current across the basolateral membrane of control animals. Again the current is calculated from the basolateral resistance and the basolateral potential generated by increasing a' a+. Using eqn. (3) the following values were determined to best fit the data, Km of 14 mm, n = 2-8 and a maximum pump rate (IpaX) = 27x5 #ta/cm2, for control animals. Identical experiments were performed on low-na+ diet animals. The values for Kin, n and Iax were not significantly different from control (Km = 15-2 mnt n = 2-9 and IifX = 31-7 jua/cm2). These data indicate that neither the density nor the Na+-binding properties of the pump are altered by endogenous aldosterone.

9 Na+ PERMEABILITY AND PUMP KINETICS K+ activation In the previous experiment we were unable to demonstrate any influence of aldosterone on IWaX, Km or the number of Na+ ions which must bind to activate the system. However, these experiments were conducted under conditions of high serosal K+. In this section we will investigate the effect of serosal K+ activity on the current generating ability of the pump. The experimental protocol for studying the K+ activation of the pump was to increase the apical membrane conductance for Na+ but not to Cl-. Since Cl- entry into the cell will result in cell swelling, we were not able to use nystatin to increase apical membrane Na+ conductance. However, Wills (1981) and Lewis & Wills (1982) found that gramicidin D can increase the apical conductance of rabbit urinary bladder to cations but not anions. After gramicidin-d action was complete a K+-free Ringer solution was placed in the serosal chamber and Na+ allowed to enter the cell. After a 5 min incubation in K+-free Ringer solution, K+ was added back to final concentrations of 0 7, 1-4, 2-1, 3-5, 5-6 and 7 mm. Each K+ addition was bracketed by a 5 min incubation in a K+-free Ringer solution. Simultaneous monitoring of VT, Vb1, RT and Rbl (see Lewis & Wills, 1982) showed that K+ addition to the serosal solution caused a rapid hyperpolarization of VT and Vbl without significant changes in RT and Rbl. This hyperpolarization was confined to the basolateral membrane and plateaued within 90 sec after K+ addition. From the magnitude of the hyperpolarization and Rbl we calculated a pump current at each serosal K+ activity (Fig. 3). Again using eqn. (1) the parameters which gave the best fit of the model to the data were for control animals: Imp" = 18-7 #sa/cm2, Km = 2-3 mm and n = These experiments were also performed on low-na+ diet animals, with best fit values of IWaX 19-8 #sa/cm2, Km = 2-7 mm and an n = The two groups were not significantly different. Ouabain inhibition Lewis, Wills & Eaton (1978) demonstrated that serosal addition of 10-4 M-ouabain completely inhibits the Na+ pump. The K+-activation experiments offer a convenient method for determining the dose-response curve of the pump ta ouabain. After gramicidin D had full effect on the apical membrane, a K+-free solution is placed in the serosal chamber and then an aliquot of ouabain added (10-8, 10-7, 5 x 10-7, 10-6, 5 x 10-6, 10-5 or 10-4 M). 5 min after ouabain addition 7 mm-k+ was added to the serosal solution and the basolateral membrane hyperpolarization and resistance measured. The serosal solution was then replaced with a K+-free Ringer solution, the next higher dose of ouabain added, and K+ added 5 min later. From this data we can determine the % inhibition of the current as a function of ouabain concentration. Since ouabain is a non-competitive inhibitor, a model of highly co-operative binding was fitted to the data. We chose a multi-site model since it is well established that the ouabain-pump interaction is a first order kinetic reaction. If our experimental conditions offer significant ionic build-up in the lateral intercellular spaces, this simple model will fail. Fig. 4 shows that the best-fit parameters to a multi-site model yields first order kinetics where n = 1-03 and 50 % inhibition occurs at a concentration of approximately 1 /M. Thus one ouabain molecule is required to inhibit one pump. Again these ouabain kinetic studies were identical whether the experiments were studied on control or low-na+ diet animals. 177

10 178 S. A. LEWIS AND N. K. WILLS L : a'+ (mm) Fig. 3. Dependence of the pump current on serosal K+ activity. Experimental protocol is as described in the text. The smooth curve is best fit, to eqn. 1. Derived parameter values are Imax of 18-4 /sa/cm2; Km of 2-3 mm and an n of 1-8. Note that at normal serosal K+ activity (5 3 mm) pump is at 82 % of its maximum rate. Vertical bars are S.E. of the mean C A T 1 Un _ Log ouabain (M) Fig. 4. Dose-response curve for ouabain inhibition of the pump current. Smooth curve was best fitted by eqn. 1. Half maximal inhibition (Ki) is 1 x 10-6 M, with an n of This indicates simple Michaelis-Menten binding kinetics. Note abscissa is in logarithmic units. Vertical bars are S.E. of the mean.

11 Na+ PERMEABILITY AND PUMP KINETICS 179 DISCUSSION That aldosterone apparently induces apical membrane selectivity changes without affecting aia+ or the Na+-pump kinetics holds important implications for the mechanism of Na+ transport in the urinary bladder. We shall discuss in turn the apical membrane selectivity changes, the constancy of aa+ as a function of increased Na+ transport and lastly the stoichiometry of the Na+ pump. Apical selectivity In a number of epithelia, aldosterone has been reported to increase the apical membrane Na+ permeability (e.g. in toad urinary bladder, Palmer, Li, Lindemann & Edelman 1982; hen coprodaeum, Christensen & Bindslev, 1982; rabbit descending colon, Thompson, Suzuki & Schultz, 1982; Lewis, Eaton & Diamond, 1976). More recent evidence from current fluctuation analysis, suggests that the increase in Na+ transport is a consequence of the addition or activation of Na+ channels (Christensen & Bindslev, 1982; Palmer et al. 1982; Zeiske, Wills & Van Driessche, 1982; Loo, Lewis & Diamond, 1982). With the ability to measure intracellular Na+ and K+ activities and membrane conductances we now have been able to determine the selective permeability of the apical membrane in the presence and absence of amiloride, and before and after aldosterone stimulation. In control conditions we found that there was a significant permeability of the amiloride-sensitive pathway to K+ (PNa/PK = 2-6). After stimulation of endogenous aldosterone levels, the selectivity of the amiloride-sensitive pathway increased to approximately 8-6. The following are possible explanations for the increased selectivity of the amiloride inhibited channel: (i) there are two intrinsically different sets of amiloride-sensitive pathways. The first set acts as a poorly-selective filter. The second is produced in response to increased, endogenous aldosterone levels and is highly selective for Na+ over K+; (ii) the amiloride-sensitive pathways age or cycle. Initially these channels may be highly Na+-selective with pronounced amiloride sensitivity. As they age (or are degraded by the composition of the urine) their selectivity to Na+ over K+ is reduced and finally the amiloride sensitivity is lost. Aldosterone might then promote synthesis ofnew pathways, and in addition, might increase the turnover (degradation or loss) of these pathways; (iii) a combination of the above two suggestions. At present we have no evidence for or against the first hypothesis. There are three lines of evidence for the second hypothesis. First, the K+ conductance of the amiloride-sensitive channel decreases with respect to the Na+ conductance after aldosterone stimulation. Although one may be tempted to speculate that the selectivity of a single channel is increasing, to prove such a hypothesis, the single channel conductances for the two conditions must be measured. In this respect we note that (Lewis, 1975) after in vitro aldosterone addition, I., (an index of Na+ transport) increases, before any measurable increase in apical conductance. This result indicates an increased turnover of amiloride-sensitive channels or alternately an alteration in the selectivity of pre-existing channels by aldosterone. Again in the absence of channel density and single-channel conductances we cannot differentiate among the three possibilities. The last and perhaps the strongest line of evidence involves the physiological

12 180 S. A. LEWIS AND N. K. WILLS function and structure of the mammalian urinary bladder. During expansion cycles the bladder accommodates an increase in volume by first eliminating membrane infolding and, during extended stretch an incorporation of cytoplasmic vesicles (Lewis & Diamond, 1976; Minsky & Chlapowski, 1978). Recent measurements indicate that the selectivity of the amiloride-sensitive channels in the cytoplasmic vesicles after apical fusion were not different between control and diet animals, however, the selectivity of these channels was at least, 30: 1 (PNa: PK) and was not dependent upon dietary conditions (Lewis & de Moura, 1982). This suggests that the channels are degraded as a function of time during exposure to the luminal contents of the bladder. Thus the selectivity differences are most likely caused by channel degradation and not a result of two populations of amiloride-sensitive pathways. If the apical membrane channels are K+-selective after an exposure time to urine, this implies that the bladder secretes K+. Consequently, the measured I., does not equal the net Na+ flux under short circuit conditions. At first one might think that if the PNa/PK is 2 5/1 then the net Na+ flux compared to the I., will yield a ratio of 1P67, a value greater than if the Na+ channel was only Na+ selective. However, Lewis & Diamond (1976) measured a flux of This dilemma can be accounted for by considering the net driving forces for these ions. The net driving force for K+ exit across the apical membrane under short-circuit conditions is the difference between the chemical driving force and the short-circuit potential across the apical membrane thus: /tkrt K K=Vasc RF~~In K O (2) F a F ni-(2 This yields a value near -20 mv, the driving force favouring K+ exit from cell to lumen. The Na+ driving force is described by an equation similar to that above, however the apical potential and chemical gradient are additive thus the driving force favouring Na+ entry is some 130 mv. Because of the finite permeability to K+ and a net driving force out of the cell, the I., will equal the difference between the inward Na+ current and the outward K+ current i.e. ISc will be an underestimate of net Na+ flux. The extent of this underestimate is easily calculated using the constant-field current equation: 'Na PNa. aka+ - ala+ev/irt -K PK a+a+evf/rt () Where Iasa and IK are the Na+ and K+ currents crossing the apical membrane, V is the apical membrane potential under short-circuit conditions referenced to the mucosal solution (-52 mv), subscripts i and o refer to the cell and mucosal solution activities respectively, for Na+ and K+, and the usual constants, R, T and F. For control diet animals the PNa/PK used was 2-6. The ratio of Na+ to K+ currents is then -37: 1 (the negative sign resulting from the opposing flow of Na+ to K+ current). Since In = INa+Ias, the amount INa will over-estimate I., is 3%, a value in remarkable agreement with the ratio of 1-03 reported by Lewis & Diamond (1976). Higher selectivity values will reduce the error between I.c and INa* It is interesting to note that Clausen, Lewis & Diamond (1979) using impedance analysis generated a relationship between apical conductance (Ga) and I, The slope

13 Na+ PERMEABILITY AND PUMP KINETICS of this curve will equal the net electrochemical driving force for this channel or pathway. The slope is 72 mv. The average basolateral membrane potential was approximately 55 mv. Thus the channel had a driving force of ca. 17 mv yielding a selectivity of the apical channel of - 2:1 Na+ to K+ in agreement with our present results. Pump capacity and a'na+ The finding of apical membrane selectivity changes does not explain why ai a+ remains essentially constant after Na+ transport is stimulated by placing animals on low-na+ diets. Moreover we have been unable to demonstrate an effect of aldosterone on the kinetic properties of the Na+ pump. How can the apparent constancy of ai a+ be explained by our measurements? There are two possible explanations for such a constant aka+. First the kinetics of the pump may be so steep that a small increment in aka+ will cause a large change in the Isc or net Na+-transport rate. The second possibility is that the pump is extremely efficient at eliminating Nat from the cell and is limited in its efficacy only by the energy available to it. Thus the rate of Na+ transport is not limited per se by the kinetic qualities of the pump but rather by the energy supply to the pump. Unfortunately we cannot differentiate between these two possibilities since we do not know the maximum energy available for the splitting of an ATP molecule in the cell cytoplasm nor do we know the 'efficiency' of coupling between ATP hydrolysis and Na+-K+ exchange (not electroneutral). While our data indicates that for certain conditions there might be an energy limitation for the pump, results from other conditions suggested that there is a kinetic limitation of the pump. From our kinetic data derived from electrical measurements of the pump activity one can predict whether the ai a+ should change over the measured range of Na+ transport. To do this one must consider the total amount of Na+ that the pump must accommodate at any time. One cannot simply use the Isc or the Na+ that is entering across the apical membrane under short-circuit conditions to estimate the Na+ load to the pump since Lewis, Wills & Eaton (1978) demonstrated that the basolateral membrane has a significant Na+ permeability. The Na+ current which enters across the basolateral membrane is in the order of 3.5 /ZA/cm2 (130 mv driving force and 37,000 Q cm2 resistance). Thus even if the Na+ transport across the epithelium is doubled (2-1 to 4 Q A/cm2), the amount of Na+ that the pump must handle is only increased by 33 %. Combining our kinetic data from the electrical measurements and assuming that the pump current represents only one-third of the total Na+ transported (i.e. a Na+ to K+ stoichiometry of 3:2), we estimate that the ana+ will change from a calculated value of 5-6 to 6-3 mm a value well within the measuring error of our experimental equipment. Consequently the lack of change in aka+ after an increase in Na+ transport is a property of the steep kinetics of the pump rather than an alteration of the pump kinetics. Kinetic properties of the Na+ pump and apical membrane negative feed-back A problem of great importance and increasing interest is the consideration of the apical and basolateral membranes not as separate entities but rather as a single inter-active or co-operative system. Recently Wills & Lewis (1980) demonstrated that 181

14 182 S. A. LEWIS AND N. K. WILLS at an intracellular Na+ activity of approximately 25 mm the apical permeability to Na+ was significantly decreased. This phenomenon was termed negative feed-back by Lewis, Eaton & Diamond (1976) for the rabbit urinary bladder. According to our calculations, at this value of Na+ activity, the Na+ pump is within 15 % of the maximal pump rate. Thus inhibition of Na+ entry occurs at an activity close to that for saturation of the Na+ pump in this preparation. Whether Na+ is the direct effector for a decrease in apical membrane Na+ permeability or another mediator such as Ca remains to be resolved (cf. Taylor & Windhager (1979)). Pump stoichiometry It is tempting to speculate that the Hill coefficients (see Results) represent the number of Na+ or K+ ions translocated per pump cycle. However this would be an over interpretation of the Hill coefficient, as this coefficient simply reflects ions bound per cycle, not necessarily ions transported. An estimate of the stoichiometry can be performed in the following manner: for steady-state conditions (when aia+ is not changing in the cell), the amount of Na+ being transported by the pump (INa) must be equal to the net rate of Na+ entry across both the apical and basolateral membranes. The apical entry rate is equal to the Ir, and the basolateral entry rate is calculated using the constant-field current equation and measured values for PNa, basolateral membrane potential (short-circuit case), intracellular and extracellular Na+ activities (see above). This calculation yielded a basolateral current of 3 5,tA/cm2. For control conditions the net Na+ pump current (sum of I., and Na+ entering across the basolateral membrane) was 5-6 #ia/cm2 and for diet animals was 7 5 #ta/cm2. We can now compute the apparent electrogenic current given the measured ai a+ for control and diet case using eqn. 3 and the best fit values for Ki, Imax and n. The ratio of the electrogenic pump current to total Na+ current (Ip/Ipa = Ija - ji/ipa) will equal the fraction of Na+ that is being transported in an electrogenic manner. For the control and diet animals this ratio was 0-25 and 0-38 respectively. This indicates that for every 3 Na+ ions transported there are two K+ ions transported in the opposite direction or that the coupling ratio is 3 Na+: 2 K+. It must be emphasized that this is only a rough approximation since the influence of electroneutral Na+ movement into or out of the cell has not been considered. Such a stoichiometry is not unusual and has been reported in other epithelia using radiotracer and electrical methodology (Nielsen, 1979; Zeuthen & Wright, 1981; Kirk, Halm & Dawson, 1980; Eaton, 1981; Hviid Larsen, Fuchs & Lindemann, 1979). The work of Eaton and co-workers (Eaton, 1981, Eaton, Frace & Silverthorn, 1982) indicates a 3:2 stoichiometry for the basolateral pump of the rabbit urinary bladder. We find this value is in good agreement with our electrical measurements. Our data disagree with the maximum current of that reported by these authors but agrees with that reported by Lewis, Wills & Eaton (1978). The reason for such a difference is not immediately clear. Energetics Since the coupling ratio seems to be approximately 3 Na+:2 K+ we can now calculate the minimum energy available to the pump from ATP using the equation

15 Na+ PERMEABILITY AND PUMP KINETICS developed by Chapman & Johnsen (1978), which is shown below. Em - RT (x n a+a++l ak++aj) (4) EM F(x-y) xi i yi a0j + RT'4 where A is the energy from ATP hydrolysis, x and y are the number of Na+ and K+ translocated per pump cycle, R, T and F have there usual meaning, and the activities are as described in the text. Pooling the data for control and diet animals we find that the minimum available energy is in the range of 10-2 kcal/mole ATP. It has been known for a number of years that in addition to increasing apical membrane permeability aldosterone also increases the metabolic machinery of the cell. Such an increase in metabolism might result in an increase in the ATP levels of the cell. If an increase in ATP levels does occur this then will result in an increase in the energy released during ATP hydrolysis, i.e. the phosphate potential. This increase in energy will then shift the equilibrium potential. A simple re-arrangement of eqn. (4), (shown below) 183 ana+ = ana+ exp (EmF(xY)/RT+Y ln ai +/a ++A/RT) can be used to demonstrate that for our conditions of Na+ and K+ activities and membrane potential that an increase in energy from -10 kcal/mole to -11 kcal/mole will result in ai a+ decreasing from 5 9 mm to 3-4 mm if the pump is at equilibrium. Whether the pump is working near equilibrium or not will require accurate measurements of the 'phosphate' potentials of this epithelium or perhaps the voltage intercept of the I- V relationship before and after pump stimulation. This intercept can then be readily converted into an energy term. We wish to thank William Alles for perfect technical assistance, and Dr C. Clausen for his help in computer programming. This work was supported by NIH Grants no. AM (to S. A. Lewis) and AM (to N. K. Wills). REFERENCES BROWN, K. M. & DENNIS, J. E., Jr. (1972). Derivative free analogues of the Levenberg-Marquardt and Gauss algorithms for non-linear least squares approximation. Numer. Math. 18, CHAPMAN, J. B. & JOHNSON, E. A. (1978). The reversal potential for an electrogenic sodium pump: a method for determining the free energy of ATP breakdown? J. gen. Phy8iol. 72, CHRISTENSEN, 0. & BINDSLEE, N. (1982). Fluctuation analysis of short-circuit current in a warm-blooded sodium retaining epithelium: site, current density and interaction with Triamterene. J. Membrane Biol. 65, CLAUSEN, C., LEWIS, S. A. & DIAMOND, J. M. (1979). Impedance analysis of a tight epithelium using a distributed resistance model. Biophys. J. 26, EATON, D. C. (1981). Intracellular sodium ion activity and sodium transport in rabbit urinary bladder. J. Physiol. 316, EATON, D. C., FRACE, A. M. & SILVERTHORN, S. U. (1982). Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder. J. Membrane Biol. 67, HVIID LARSEN, E., FUCHS, W. & LINDEMANN, B. (1979). Dependence of Na-pump flux on intracellular Na-activity in frog skin epithelium (R. esculenta). Pflfigers Arch. 382, R13. KIRK, K. L., HALM, D. R. & DAWSON, D. C. (1980). Active sodium transport by turtle colon via an electrogenic Na-K exchange pump. Nature, Lond. 287, KOEFOED-JOHNSEN, V. & USSING, H. H. (1958). The nature of the frog skin potential. Acta physiol. scand. 43,

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