Pfliigers Arch (1993) 422:546-551
Journal of Physiology 9 Springer-Verlag1993
K + recirculation in A6 cells at increased N a + transport rates M, Granitzer, W. Nagel*, J. Crabb~ D6partement de Physiologic, Universit6 Catholique de Louvain, Tour Harvey ENDO 5530, Av. Hippocrate 55, B-1200 Bruxelles, Belgium Received July 13, 1992/Received after revision September 25, 1992/Accepted October 19, 1992
Abstract. Homocellular regulation of K + at increased transcellular N a § transport implies an increase in K § exit to m a t c h the intracellular K + load. Increased K § conductance, gi(, was suggested to account for this gain. We tested whether such a mechanism is operational in A6 monolayers. N a § transport was increased f r o m 5.1 _+ 1.0 ~A/cm 2 to 20.7 + 1.3 laA/cm 2 by preincubation with 0.1 gmol/1 dexamethasone for 24 h. Basolateral K + conductances were derived f r o m transference numbers of K +, tK, and basolateral m e m b r a n e conductances, gb, using conventional microelectrodes and circuit analysis with application of amiloride. Activation o f N a + transp o r t induced an increase in gb f r o m 0.333 + 0.067 mS/ cmz to 1.160_+ 0.196 m S / c m 2 and tK was reduced to 0.22 +_ 0.01 f r o m a value of 0.70 +__0.05 in untreated control tissues. As a result, gK remained virtually unchanged at increased N a + transport rates. The increase in gb after dexamethasone was due to activation of a conductive leak p a t h w a y presumably for C1-. Increased K + effiux, IK, was a consequence o f the larger driving force for K + exit due to depolarization at an elevated N a + transport rate. The relationship between calculated K + fluxes and N a + transport rate, measured as the Is~, is described by the linear function IK = 0.624 x INa--0.079, which conforms with a stoichiometry 2: 3 for the fluxes of K + and N a + in the N a + / K + - A T P a s e pathway. Our data show that homocellular regulation of K + in A6 cells is not due to up-regulation of gK. Key words: Mieroelectrodes - Basolateral K + conductance - K + currents -- Homocellular regulation
Introduction
body homeostasis necessitates particular mechanisms to maintain their own cellular homeostasis. In N a + - r e a b sorbing epithelia, for example, an increase in apical N a § entry accelerates the turnover rate of the N a + / K +ATPase and could result in overload of the cells with K § unless gross disturbances of the cellular K § content are prevented by associated increases in K § efflux f r o m the cells. The m a j o r fraction of this efflux occurs via K § channels in the basolateral membrane. Accordingly, the magnitude o f K § exit is determined by basolateral K + conductance and effective electrochemical force for K +, but the relative contribution of these determinators is not obvious a priori. In a popular hypothesis, Schultz considered a cooperative link between the activity of the Na § § and basolateral K § permeability as the main process accounting for the "homocellular regulation of K § [19]. We tested whether this mechanism is operational in the cultured N a § A6 cells. Spontaneous fluctuations in transcellular N a + transport in this tight epithelium are paralleled by, or result from, changes in apical m e m b r a n e N a § conductance. The conductance of the basolateral m e m b r a n e , on the other hand, seems to be unrelated to the rate of transport [2]. This lack in balance would be at variance with the above hypothesis regarding homocellular regulation o f K + [19]. Since the rate of spontaneous N a + transport in A6 cells is low, an actually existing cross-talk might have been overlooked. In the present study, the analysis was extended by including tissues with larger transepithelial N a + transport, which was induced by preincubation with dexamethasone for 24 h. Furthermore, the contribution of K + to basolateral m e m b r a n e conductance and the relationship between the m e m b r a n e currents of N a + and K + was assessed in the individual preparations.
The large variation in transcellular ion transport accomplished by epithelial tissues in order to maintain total
Materials and methods
*Permanent address." Physiologisches Institut der Universitfit Mfinchen, Pettenkoferstr. 12, W-8000 Mfinchen, Germany Correspondence to: M. Granitzer
A6 cells (American Type Culture Collection, Bethesda, USA), cultured from the distal tubule of the Xenopus laevis kidney were grown to confluent epithelia on microporous filter membranes (Millicell-
547 HA: 0.45 I~m pore diameter, Millipore PIHA 030050; Millipore, Bedford, Mass., USA) at 28~ in a humidified incubator gassed with 2% CO2 in air [2]. Both sides were supplied with 2 ml fresh medium three times a week. Cell growth medium contained 35% Leibovitz medium L-15 (Gibco, Grand Island, N. Y., USA) and 35% Ham's medium F-12 (Gibco) diluted with 20% H20 and supplemented with 10% fetal bovine serum (Gibco). Glutamine and NaHCO3 were added at concentrations of 2 mmol/l and 8 mmol/1 respectively. Osmolarity amounted to 247 mosmol/1 and pH was 7.4. Fifteen days after plating, the monolayers with the permeable filter membrane were mounted horizontally on a steel grid in a modified Ussing-type chamber, allowing access for microelectrodes as described previously [2]. The apical and basolateral sides of the tissue were continuously superfused at a rate of 5 - 8 ml/min and 2 - 3 ml/min, respectively, with a solution containing (in mmol/1) NaC1 70, KC1 2.5, CaC12 1, MgC12 1, KH2PO4 1, NaHCOa 18 and HEPES 5; pH was maintained at 7.4 with 5% C Q . The osmolarity of this solution was 170 mosmol/1. In basolateral high-K + solutions, 20 mmol/1 KC1 was substituted for equal amounts of NaC1. The speed of change in basolateral K + concentration was limtied by the presence of the supporting steel grid (150 ~tm thickness) in addition to the filter membrane of t 50 Ixm. Time constants for exchange are in the order of 2 rain [5]. Transepithelial and intracellular parameters were recorded with a voltage-clamp/microelectrode amplifier system with stimulation unit and logic control circuitry for sample-andhold circuits (Frankenberger, Germering, FRG). The latter provided analog on-line determination of tissue conductance (G0, fractional resistance of the apical membrane (fR,) and microelectrode input resistance. Tissues were continuously short-circuited except for brief perturbations of transepithelial voltage (Vs (20 mV, 400 ms every 2 s) necessary for the determination of Gt and fR, from the change in transepithelial current (It) and apical membrane potential (V,) using the relationships Gt A[t/AVt and fR~ = A Va/AV,, respectively. Transepithelial current and intracellular potential at short-circuit are indicated by I~ and V~. Cells were impaled across the apical surface with a stepping-motor micromanipulator (Frankenberger, Germering, FRG). Microelectrodes were prepared from microflbre borosilicate tubes (Hilgenberg, Malsfeld, FRG) and had an input resistance greater than 50 MQ after filling with 1 mol/1 KC1. Impalements were evaluated according to previously described criteria [2]. I~o, Gt, V~o and fR, were monitored on a multichannel strip-chart recorded. In addition, they were collected every 2 s on a PC-compatible computer using a 14-bit analog/digital converter for reproduction. Experiments were performed at an ambient temperature of 20--22~ Apical and basolateral membrane conductances, g, and gb, were obtained from standard equivalent-circuit analysis using the relationships =
g. = (a~-a~)/(f_e~ • fR~)
O)
gb = (Gt-- Gi)/(fR;-fRa)
(2)
Values with ' reflect measurement in the presence of I0 I-tmol/l amiloride at the apical side. The apparent transference number for K +, tK, of the basolateral membrane was obtained after blocking the apical membrane with 10 gmol/1 amiloride, comparing the voltage change in response to a short-term increase in basolateral K + concentration from 3.5 retool/1 (c1) to 20 retool/1 (c2) with the theoretical value predicted by the Nernst equation: tK = A ~ c / ( R T / z F x In c2/el) = A V'c/43.9 mV
(3)
Determination of tK in the presence of amiloride is possible in A6 cells since the magnitude ofgb is negligibly affected by the inhibition of Na + transport [4, 8], and inactivation of leak conductance requires much longer periods of time than those employed (5]. Basolateral K + conductance was derived from tK and gb using the relationship
gK ---- tK X gb
(4)
The K + current, I~ across the basolateral membrane, was estimated from & = g~(Vsc- v~)
(5)
where VKis the chemical driving force for K + across the basolateral membrane. Na + transport was stimulated by preincubation with 0.1 tool/1 dexamethasone in the culture medium for 24 h. Dexamethasone was purchased from Sigma (St. Louis, Mo., USA). Amiloride hydrochloride was a generous gift of Dr. G. Fanelli (Merck, Sharp and Dohme; West Point, Pa.). Values are means __ SEM. Significance of differences is calculated using Student's t-test.
Results Figure 1 shows typical recordings f r o m two different A6 m o n o l a y e r s either with n o r m a l (A) or increased (B) N a + t r a n s p o r t rate, in which the effect o f a sudden increase in basolateral K + c o n c e n t r a t i o n was tested. Is~ o f the control tissue was 4.1 g A / c m 2 and increased after dexam e t h a s o n e to 16 t,tA/cm 2. I n the dexamethasone-stimulated tissue, Vsc was considerable lower than u n d e r control conditions and Gt was m u c h higher. The apical fractional resistance, fRa, was a b o u t similar in b o t h preparations. Application o f 10 gmol/1 amiloride to the apical side led to p r o m p t changes o f the electrical p a r a m eters characteristic for inhibition o f apical N a + entry. Note, however, that the m a g n i t u d e o f Vsc in the presence o f amiloride, V~, was considerably lower in the dexamethasone-stimulated tissue than u n d e r control conditions. Basolateral m e m b r a n e conductance, gb, as calculated f r o m the amiloride-sensitive Gt and fRa using Eq.(1) was 486 ~tS/cm 2 for the c o n t r o l tissue, whereas a significantly higher value o f 1316 g S / c m 2 was o b t a i n e d for the stimulated monolayer. I n order to discriminate between different ions involved in basolateral m e m b r a n e conductance, the chemical gradient for K + across this m e m b r a n e was altered by a short-term increase o f the basolateral K + c o n c e n t r a t i o n f r o m 3.5 retool/1 to 20 retool/1. I n contrast to the control tissue, electrical parameters o f the stimulated tissue were little affected by this challenge, b o t h in the N a + - t r a n s p o r t i n g state a n d during exposure to amiloride. F r o m the decrease in V'so after elevation o f basolateral K + (Eq. 3), a tK o f only 0.18 is calculated for the stimulated tissue c o m p a r e d with 0.76 in the unstimulated A 6 monolayer. These data, in c o n j u n c t i o n with the respective values o f gb, reveal that basolateral m e m b r a n e K + conductance, gK, was even lower after d e x a m e t h a s o n e (237 ~S/cm 2) t h a n in the control tissue (369 ~S/cm2), despite the e n o r m o u s increase in gb. The correlation between a p p a r e n t transference n u m bers for K +, tK and V~ is s h o w n in Fig. 2. It reveals that depolarization was associated with a strong decrease in tK. Accordingly, dexamethasone-treated tissues have the lowest values o f tK, since increase in the N a + t r a n s p o r t rate is paralleled with depolarization o f the cells. O n the average, tK was 0.70 _+ 0.05 (n = 6) a n d 0.22 + 0.01 (n = 8) for control and dexamethasone-treated tissues respectively.
548
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V,c (mY) Fig. 2. Correlation between apparent transference numbers for K +, tK a n d cell potential, Vso, for A6 at s p o n t a n e o u s ( 9 a n d stimulated ( V ) t r a n s p o r t rate (y = - 0 . 0 0 9 x + 0.013: r = - 0 . 9 6 )
Figure 3 illustrates the influence of the rate of Na § transport, measured as the Iso, on the values gb and gK for A6 tissues under control conditions and after stimulation of Na § transport with dexamethasone. It appears that gb increased with Isc for the combined data (gb =
A
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Fig. 1. D e t e r m i n a t i o n of tK in control (A) and after d e x a m e t h a s o n e (B). The time course of response in electrical p a r a m e t e r s after change of basolateral K + c o n c e n t r a t i o n reflects limited exchange across the inner c h a m b e r surface. Apical superfusion with 10 gmol/1 amiloride is indicated by the shaded areas. Arrows depict the change in
0.058 x Isc -0.002; r = 0.83). Basolateral conductance was significantly larger after dexamethasone than in control tissues. Mean values were 0.333 _+ 0.067 mS/cm 2 and 1.160_+ 0.196 mS/cm 2 for control (5.1 +__1.0 gA/cm 2) and hormone-treated (20.7 _+ 1.3 gA/cm 2) tissues respectively. In contrast, gK was not different between tissues with normal (0.242_+0.061 mS/cm 2) and stimulated (0.235 _+ 0.024 mS/cm 2) Na § transport rates and there was no correlation at all between g~ and Isc. Implicit in these latter findings is the conclusion that the increase in gb after dexamethasone was due to conductive pathways for ions other than K +. Equivalent-circuit analysis was done in a number of experiments under control (n = 30) conditions and after 24 h dexamethasone (n = 21) without determination of trc in the individual tissues. In these cases, tic was estimated from the relationship between tic and Vs~ (Fig. 2). The data of these experiments are summarized in Table 1. They demonstrate that all transport parameters aside from g~: were significantly different from the control after dexamethasone. The increase in Na § transport, resulting from the activation of apical Na + conductance by dexamethasone, depolarized the cells by more than 30 mV with considerable reduction of the driving force
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Fig. 3. Correlation between [sr a n d basolateral gb (A) and gK (B) in control tissues ( 9 a n d after 24 h dexam e t h a s o n e ( V ) . -.., T h e regression line for the pooled data. The slope between Isc a n d gK is n o t different f r o m zero (r = 0.17)
Table 1. Transport parameters in polarized A6 monolayes under control conditions and after stimulation with 0.1 gmol/1 dexamethasone a
Parameter
Control
Dexamethasone
I~o(i.tA/cm2) V~o(mV) fR, g , ( g S / c m 2) gb (~tS/cm2) gK (gS/cm 2)
5.0 -62 0.70 69 345 237
22.5 + 1.2 --28 + 2 0.77_+ 0.02 399 + 38 1143 -]- 141 216 __ 29*
• 0.3 __ 4 • 0.01 • 4 __ 25 • 17
Values are means • SEM from 30 control and 21 stimulated tissues. Differences between control and dexamethasone are significant at a level of 2P < 0.01, except where indicated by *
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549
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Fig. 5. Correlation between Vsc and leak conductance, gl~k, of the basolateral membrane. ( V = Control; 9 = Dexamethasone) 30-
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linear for the pooled data from control and dexamethasone-treated tissues, can be described by the relationship Ii~ = 0.624 x Isc-0.079; r = 0.92. The slope of 0.62 is not significantly different from a ratio of 2:3 between fluxes of K § and Na + and would thus agree with the stoichiometry of the Na +/K +-ATPase. Figure 5 shows a plot of basolateral leak conductance, gleak, determined as the difference between gb and gK, versus the basolateral membrane potential for the whole range of Na § transport rates. It appears that gl,,k was quiescent below - 4 0 mV and started to increase after further depolarization. These data suggest that the secondary increase in gb is provoked by depolarization resulting from the gain in Na § transport after dexamethasone. Discussion
for Na § entry. Although total basolateral conductance increased remarkably, the gb/ga ratio decreased from 5.0 in control tissues to 2.9 in stimulated monolayers. This underscores the fact that the steroid was predominantly effective at the apical membrane. It should be particularly emphasized that g~: was virtually not affected by the more than fourfold increase in Na § transport. Thus, a concerted response of apical Na § and basolateral K § conductance is evidently absent. To account for the necessary balance between Na § entry into and passive K + exit from the cytosol, one might question whether it results from alteration of the driving force. The depolarization after increase in Na + entry will raise the driving force for K § exit. Accordingly, we calculated the magnitude of passive basolateral K § currents from the determined values of gK and the effective driving force for K 4, A IxK/F = Vs~- VK. To obtain V~:, we assumed that V~ after amiloride estimates the Nernst potential for K + in control tissues. The mean value of VL ( - 77 mV) was assumed for dexamethasonetreated tissues since direct determination of V~o gave unreasonably low values (see below). The correlation of K § fluxes, thus calculated, across the basolateral border with the simultaneously obtained I~ is shown in Fig. 4. The relationship between I~c and IK, which is evidently
Our data demonstrate that homocellular regulation of K § in the A6 cell line does not involve up-regulation of basolateral K § conductance to enhance the recirculation of K § at increased Na § transport. Similar results have been reported for isolated frog proximal tubular cells [9], where stimulation of apical Na § entry by alanine depolarizes the cells only transiently; a subsequent, almost complete repolarization was believed to reflect increase in basolateral K § conductance [J 5]. Using the patch-clamp technique, it was shown, however, that the basolateral membrane of the proximal tubule contains a single population of K § channels with inward rectification, which did not increase in activity [9]. For other tissues such as Necturnus urinary bladder [7], toad skin [17] or alveolar epithelia [11], increase in g~: was reported and could point to variation in the response pattern between different target epithelia and/or stimulating agents. The lack in up-regulation ofgK in A6 cells after increae in Na § transport might not be too surprising. We have previously shown for unstimulated tissues that gic decreases upon depolarization [4]. Since the increase in Na + transport after dexamethasone depolarized apical and basolateral membrane potentials (in the short-circuited tissue) by more than 30 mV, reduction of gK to about
550 60% of the value in unstimulated tissues would be expected. Yet, g~cwas not significantly different at the spontaneous or increased N a + transport rate. This could indicate that the basolateral m e m b r a n e contains additional populations o f K + channels with independent regulation, which overlay the inactivation o f inwardly rectifying K + channels. The nature of these putative K + channels can at present only be speculated. In view o f virtually unchanged basolateral K + conductance at an increased N a + transport rate, despite considerable elevation o f gb, one must question which ionic species accounts for this gain. Notably, this gain was not due to specific interference of the steroid on basolateral m e m b r a n e conductance, but occurred secondary to the stimulation of transcellular N a + flux [3]. It appears that the leak conductance can be attributed essentially to an increase in the conductance of the basolateral m e m b r a n e to C I - , as demonstrated by its sensitivity to the C1- channel blocker, 5-nitro-2-(3phenylpropyl-amino)benzoate (NPPB) and to substitution of basolateral C1- with the i m p e r m e a n t anion, gluconate [5]. Furthermore, evidence for strong voltage sensitivity of these C1- channels was obtained [5]. In the present study, we observed a strong correlation between Vsc and gle,k, calculated as the difference between gb and gK- Although not explicitly proven, the identity between gleak and gcl appears highly likely. The role of the increase in gc~ is not clear. It cannot elicit the observed depolarization because it is secondary to and induced by the depolarization. In other epithelial cells including those of renal origin, C1- channels in the basolateral m e m b r a n e p r o b a b l y serve to maintain cell volume [14]. Since basolateral m e m b r a n e K + conductance seems essentially unchanged at an increased N a + transport rate, it must be questioned how homocellular regulation of K + can be achieved. Activation of K +-selective channels in the apical m e m b r a n e , as observed in proximal tubules of amphibia [1, 10, 12] and primary cultured m a m m a l i a n proximal tubule cells [13, 16, 20, 21] under conditions where efflux of K + across the basolateral m e m b r a n e is impeded, can be excluded for A6 cells, since K +-selective channels are absent f r o m the apical m e m b r a n e of the differentiated m o n o l a y e r [22]. Amiloride-insensitive, non-selective cation channels have been discovered recently in the apical m e m b r a n e [6], but we have never observed negative Iso after suppression of the apical N a + conductance, which could indicate K + secretion. Thus, we must consider another i m p o r t a n t determinant of K + efflux. K + exit occurs mainly through basolateral K + channels and is therefore determined by both conductance and electrochemical gradient for this ion across the basolateral membrane. The electrochemical gradient for K + exit is markedly increased by cell depolarization due to increased apical N a + entry. Plotting the passive K + current versus the whole range of N a + transport rates shows that recirculation of the increased p u m p e d K + can easily be accomplished by alteration o f the driving force. The m o s t i m p o r t a n t source of error in the calculation of IK results f r o m the uncertainty implicit in the use o f V~ as VK. Equation (5) applies only if basolateral m e m b r a n e conductance for ions other than K + is negligible. This assumption m a y be valid for control tissues with tK
greater than 0.7, but is certainly unreasonable for dexamethasone-treated tissues with tic below 0.2. Therefore VK for the stimulated tissues was assumed to be - 7 7 mV, which is the m e a n value of V~o in A6 tissues at the spontaneous transport rate. At this VK, the slope that represents the N a + / K + ratio is 0.62. The real VK is p r o b a b l y somewhat higher since some other conductive pathway, presumably for C I - , is in parallel with that of K + . F r o m measurements of intracellular K + in A6 cells [18], we can extrapolate a value of 91 mmol/1 for intracellular K + at 170 m o s m o l / k g medium osmolality. This corresponds to a value of - 8 2 m V for VK and is only slightly larger than the values used. In conclusion: following stimulation of N a + transport by dexamethasone, cellular K + balance in A6 cells is not achieved by an increase in basolateral m e m b r a n e K + conductance, but exclusively by an increase in the driving force favouring K + exit. Thus, homocellular regulation of K + does not necessarily have to involve change in K + conductance per se. Acknowledgements. We gratefully acknowledge the help of Dr. M.
Hunter for his valuable suggestions. The work was financially supported by research grants fi'om F. N. R. S. (1.5.090.90F), Actions de Recherche Concert6es (89-95/135) and the Deutsche Forschungsgemeinschaft.
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