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Pflfigers Arch (1988) 412: 80- 85

9 Springer-Verlag1988

Kinetic properties of Na +]H + exchange in cultured bovine pigmented ciliary epithelial cells Horst Helbig, Christoph Korbmacher, Susanne Berweck, Dagmar Kiihner, and Michael Wiederholt Institut fiir Klinische Physiologic, Klinikum Steglitz der Freien Universitgt Berlin, Hindenburgdamm 30, D-t000 Berlin 45 Abstract. Uptake studies with 22Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na + transport. A large part of Na + uptake was sensitive to amiloride, quinidine and harmaline. Na + uptake was stimulated by intracellular acidification (using the NH~- prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparent KM for Na* from 38 to 86 mM without considerable changes in Vmax. In the presence of 5 mM Na + half maximal inhibition of amiloride sensitive Na + uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na + the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H + seemed to be competitive with a Ki of 2 0 - 4 0 nM. 10 gM amiloride increased the apparent Kra for Na + from 33 mM to 107 mM, while V~x remained nearly unchanged. ICso for amiloride was 6 gM at 5 mM Na + and 36 gM in the presence of 150 mM Na +. Thus, amiloride behaves as a competitive inhibitor with a Ki of about 5 ~M. The affinities of Na § to the transport site (KM ~ 16 raM), to the inhibitory site for protons (KM m 21 mM), and to the inhibitory site for amiloride (KM ~ 26 raM) were in the same order of magnitude. In summary, we have presented evidence for the presence of a Na+/H + exchanger in cultured bovine pigmented ciliary epithelial cells. The kinetic data suggest the presence of only one common extracellular binding site for Na § H § and amiloride. Key words: Ciliary epithelium - Tissue culture - Na+/H + exchange - Amiloride

Introduction The intraocular pressure (IOP) is determined by the interaction between formation of the aqueous humor in the ciliary body and its effiux through the trabecular meshwork. Glaucoma is a common disease, in which in most cases the regulatory mechanisms for IOP fail to maintain the intraocular pressure in the normal range resulting in an increased IOP. The high pressure is believed to damage the optic nerve leading to progressive loss of visual function. The basic pathophysiologieal disorder underlying glaucoma and the Offprint requests to: H. Helbig

exact mechanism of action of many drugs used in glaucoma therapy are unknown. Moreover, there is a lack of knowledge about the physiological mechanisms of aqueous humor formation as well as aqueous outflow, and their regulation. With our experiments in cultured ciliary epithelial cells we tried to characterize ion transport mechanisms possibly involved in aqueous humor formation. It is now generally accepted that active cellular ion transport across the ciliary epithelium is crucial for aqueous humor formation and that the maintenance of IOP cannot be solely explained by ultrafiltration across the ciliary capillaries [8, 44]. The investigation of ion transport properties is complicated by the complex anatomy of the ciliary body with fragile processes and their unique epithelial organization. The ciliary epithelium consists of two different epithelial layers. The pigmented epithelium (PE) is located at the blood side, while the non-pigmented layer (NPE) faces the aqueous humor. Both layers exhibit properties of actively transporting epithelia [19]. Many attempts have been made to investigate the mechanisms of ion transport in the ciliary epithelium in situ [25, 29, 39, 42, 43]. Nevertheless, besides technical difficulties in the application of the methods of epithelial and membrane physiology, the double layered anatomy renders the interpretation of data difficult. Therefore, we have tried to develope techniques to grow both layers separately in tissue culture. Recently we have characterized in cultured bovine PE mechanisms of transmembranal chloride transport [20] and electrical membrane properties [I 7]. Furthermore, in a virus transformed cell clone derived from bovine pigmented ciliary epithelium we found a Na+/H + antiport participating in the maintenance of intracellular pH [18]. The present study was designed to investigate the mechanisms of Na + uptake in cultured PE. We found that the majority of Na + uptake occurs via a Na+/H + exchange. There are conflicting reports in the literature about the kinetic properties of Na+/H + exchange with regard to the interaction between Na +, protons and amiloride, and some authors have postulated the presence of distinct carriers mediating Na+/H + exchange [31]. Therefore, we have also investigated the interaction of extracellular Na +, H + and amiloride in cultured PE. Our data are in agreement with competition between Na +, protons and amiloride for one single common extracellular binding site.

Materials and methods Cell culture. Primary cultures of bovine pigmented ciliary epithelial cells were established as described previously [17,

8~ 20]. In brief, bovine eyes were enucleated, the ciliary processes were cut off and incubated in trypsin/EDTA solution, until single epithelial cells were released. This single cell suspension, containing mainly PE and NPE, was seeded in plastic tissue culture flasks (Nunc, Roskilde, Denmark) with Dulbecco's modification of minimal essential medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ ml penicillin, 100 gg/ml streptomycin and 2.5 ~g/ml amphotericin B. The cultures were gassed with a 5% CO2/95% air mixture and incubated at 37~ The medium was changed twice a week. Amphotericin B was no longer included in the culture medium after the first medium exchange. Only PE were able to grow under these conditions [17, 20]. NPE loosely attached to the substrate, but never flattened or grew. The same growth behavior has been described by others [9, 10, 28]. PE formed a confluent monolayer within I week. Subcultures were performed weekly with a split ratio of I : 2. Confluent monolayers in the first and second subpassage with a cell density of 1 - 3 x 10 s cells/cm 2 were used for our experiments. 22Na uptake measurements. 2ZNa uptake studies were performed essentially as described previously [20, 23]. In short, confluent monolayers of cultured bovine pigmented ciliary epithelial cells grown on 25 cm:" tissue culture flasks were preincubated for 35 min at 37~ in a Na+-free solution (isoosmotically replaced by N M D G , see below) containing 20 m M N H + . This preincubation was choosen in order to stimulate N a + / H + exchange by intracellular acidification to obtain higher uptake values. It has been shown that intracellular acidification had no influence on the affinity of the extracellular binding site for Na + [2, 15]. In one kinetic experiment without preincubation with N H + we found a KM of 21 m M for Na +, which is in the same range as the KM value of 33 mM observed under stimulated conditions. However, Vm,x was about 3-fold increased by the preincubation procedure. Immediately before addition of the 2~Na containing solution, cells were rinsed twice with a solution containing the same ionic concentrations and inhibitors as the uptake saline (but no label). Subsequently cells were incubated with the uptake saline containing 1 0 30 kBq/ml 22Na and 0.1 mM ouabain (to prevent active extrusion of Na + by the Na+/K+-ATPase) in a shaking bath at 37 ~C. Uptake was terminated by three rapid washes with ice-cold isotonic MgC12 solution (buffered with HEPES/Tris to pH 7.4). Cells were dissolved in 0.2 M N a O H and the radioactivity in the lysate measured in a gammacounter. Uptake was referred to the surface area and expressed as Na + uptake per surface of a confluent monolayer. All experiments have been performed 2 to 4 times with qualitatively similar results. For the kinetic experiments uptake time was 3 min, and each point represents mean of five determinations. Solutions and source of chemicals. All solutions were buffered with 10 m M HEPES to pH 7.4, if not indicated otherwise. Control-Ringer solution contained the following ionic concentrations (in raM): 151 NaCI, 4 KC1, 1.7 CaC12, I KHzPO4, 0.9 MgSO4. In solutions containing a lower Na + concentration, Na + was replaced by N-methyl-Dglucamin (NMDG), if not indicated otherwise. Glucose concentration was 5 mM. (ZZNa)-NaCl was purchased from New England Nuclear (Boston, MA, USA). Amiloride, quinine and quinidine were from Sigma (St. Louis, MO,

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USA). Ouabain (g-strophanthin) was obtained from Merck (Darmstadt, FRG). Harmaline (harmaline-HC1) was from Fluka (Buchs, Switzerland). Celt culture media and supplements were purchased from Biochrom K G (Berlin). Results

The time course of Na uptake in the presence of 10 m M Na + in the uptake saline (after preincubation with 20 mM N H +) is illustrated in Fig. 1 a. A small overshoot was observed, which was probably caused by the transient gradient for N H 2 . In the presence of I mM amiloride the time course of Na + uptake was markedly slowed. Amiloride sensitive Na + uptake was nearly linear for the first 3 - 4 rain. The results of experiments showing the effect of various drugs on Na + uptake are depicted in Fig. 1 b. Na + uptake was markedly inhibited by substances, which are known inhibitors of the plasma membrane N a + / H + exchange, such as amiloride [6, 24], quinine, quinidine [30], harmaline [1, 3] and Li + [30]. The interaction between extracellular Na + and protons was investigated in experiments like the one shown in Fig. 2. With decreasing pH, N a + uptake was reduced. The concentration of H + necessary for half maximal inhibition of

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[H§ (nM) Fig. 2A, g. Effect of extracellular pH on amiloride sensitive Na § uptake9 A Na + uptake was measured at varying extracellular pH values with 5 and 50 mM Na § in the uptake saline in the presence and absence of i mM amiloride. Amiloride (1 mM) insensitive Na § uptake was subtracted. Control means uptake at pH 89 which is believed not to inhibit Na + uptake. B Dixon plot of the data from (A). Vmeans t mM amiloride sensitive Na + uptake in nmol - cm -2 9min -1, r 2 for the regression lines was 09 (5 mM Na +) and 0.99 (50 mM Na+), respectively

amiloride sensitive N a + u p t a k e was dependent on the N a + concentration (Fig. 2a). A t 5 m M extracellular N a § IC5o for p r o t o n s was 50 n M (pH 7.30), while in the presence o f 50 m M N a + 139 n M H + (pH 6.86) were necessary for 50% inhibition o f amiloride sensitive sodium uptake. Dixon plot o f the same d a t a is depicted in Fig. 2 b. The inhibitory constant Ki for protons can be calculated from the point o f intersection o f the two straight lines. The d a t a reveal a Ki for H § o f 43 n M (pH 7.36). W i t h increasing N a + concentration N a + uptake exhibits saturation kinetics and approaches a maximal value for N a + uptake. We have performed these experiments at different p H values (Fig. 3 a). Reciprocal plot o f the d a t a (Lineweaver-Burk, Fig. 3b) reveals straight lines with an intersection near the Y axis, indicating that the maximal velocity o f amiloride sensitive N a + uptake is not m a r k e d l y influenced by changes in extracellular pH. On the other hand, the a p p a r e n t KM values increased with increasing proton concentration. A t p H 7.0 KM~pv was 86 m M N a +, while at p H 7.5 only 38 m M N a § were necessary to achieve half maximal amiloride sensitive N a + uptake. The calculated Ki1 for H + from these d a t a is 22 n M (pH 7.66). The KM for N a + in the absence o f inhibiting extracellular protons can be calculated to be 16 raM.

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The results o f experiments designed to determine the type o f inhibition o f amiloride are shown in Figs. 4 and 5. Dose-response curves for amiloride at two fixed N a + concentrations are depicted in Fig. 4a. The Dixon plot from these d a t a (Fig. 4b) reveals the ICso values for amiloride. A t 5 m M N a + 6 g M amiloride inhibited half o f total amiloride sensitive N a § uptake, while at 1 5 0 r a M N a § ICso for amiloride was 36 gM. The intersection o f the two regression lines reveals a Ki o f 5.2 ~tM for amiloride. Figure 5a illustrates the effect of varying the N a § concentration at a fixed concentration o f amiloride on sodium uptake. The kinetic analysis (Eadie-Scatchard) o f the data is depicted in Fig. 5 b. The maximal velocity in the EadieScatchard plot is given by the intersection o f the regression line with the abscissa. The straight lines calculated by the method o f least squares intersect near the X axis, suggesting that Vmax is not dependent on the presence o f amiloride. However, the N a § concentration, which gives half maximal N a § uptake, was 33 m M in the absence o f amiloride, but was increased to 1 0 7 m M in the presence o f 1 0 ~ M amiloride. Ki for amiloride calculated from these d a t a was 4.5 g M I 1 K~avp = K~4x [I]/Ki + KM (according to [40]). KMavp means the apparent KM in the presence of an inhibitor concentration [I]. KM means the Michaelis-Menthen constant for the substrate in the absence of the inhibitor I

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Discussion A large p a r t o f N a + uptake into cultured pigmented ciliary epithelial cells was sensitive to the diuretic drug amiloride. Two different ion t r a n s p o r t systems have been reported to be inhibitable by amiloride: an amiloride sensitive N a + channel and a N a + / H + antiport. Both transporters can be differentiated. (i) The K~ for inhibition with amiloride is < i g M for the channel, whereas K~ for amiloride inhibition of N a + / H + exchange is ~ 10 IxM [6]. (ii) N a + uptake via N a + / H + exchange is trans-stimulated and cis-inhibited by H + [4, 5, 33]. These arguments were tested in our study. We found evidence for N a + / H + exchange operating in the p l a s m a m e m b r a n e of cultured pigmented ciliary epithelia. In leaky epithelia grown on plastic in tissue culture the label has not only access to the apical membrane, but m a y also diffuse to the basolateral membrane. Thus, with the technique used in the present study it is not possible to localize the carrier to the apical or the basolateral m e m b r a n e [22]. Some other methodical problems should be discussed. We used the p a r t o f N a + uptake, which was sensitive to 1 m M amiloride for kinetic evaluations. This procedure requires that N a + / t t + exchange is totally inhibited by 1 m M amiloride. In some epithelia the sensitivity o f N a + / H +

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Fig. 5A, B. Na + dependence of Na + uptake in the presence and absence of 10 gM amiloride. A Na + uptake was measured with increasing Na + concentrations in the presence of 10-3 M, 10-s M and in the absence of amiloride. Na + uptake in the presence of 1 mM amiloride was thought to be unspecific and subtracted. B Eadie Scatchard plot of the data from (A). V means amiloride (t mM) sensitive Na + uptake in nmol 9 cm -2 min -1. r 2 for the regression line was 0.90 (0 amiloride) and 0.94 (10-5 M amiloride), respectively 9

exchange to amiloride is low with a K~ near 100 g M [26, 32]. In that case only a b o u t 6 0 - 7 0 % of N a + / H + exchange were inhibited by 1 m M amiloride at physiological N a + concentration. However, in cultured PE the N a + / H + exchange was more sensitive to amiloride. The shape o f the dose-response curve for amiloride indicates that even at high extracellular N a + concentrations N a + / H + exchange was almost totally inhibited. We can calculate that with a Ki of 5 g M 97% of the N a + / H + exchange is inhibited by 1 m M amiloride at 1 5 0 r a M extracellular N a +. Therefore, we think it is a p p r o p r i a t e to use the N a + u p t a k e sensitive to 1 m M amiloride for kinetic evaluations. Furthermore, due to the limited accuracy of the method, smaller non-competitive components o f inhibition of amiloride or protons cannot be excluded. There are conflicting results concerning the interaction between N a + and H + at the extracellular binding site(s) of the N a + / H + exchanger. Some authors found pure competitive inhibition o f N a + uptake by extracelhilar p r o t o n s [5, 38]. F r o m these d a t a a kinetic model for N a + / H + exchange was postulated, which includes only one c o m m o n extracellular binding site for N a + and H + [2]. Other studies describe noncompetitive inhibition or mixed type inhibition

84 o f N a + uptake by extracellular protons [7, 14, 31, 41]. Kinetic models have been postulated including two distinct binding sites for N a + and H + [35] or an additional extracellular regulatory site for H + [36]. Our own d a t a show a decreased affinity for N a + to the N a + / H + exchange and no change in V~ax, when extracellular p H was lowered. Moreover, the degree o f inhibition o f N a + uptake by extracellular p r o t o n s depended on the sodium concentration present in the medium. The Ki for p r o t o n s in our study o f a b o u t 2 0 - 4 0 n M is in the same order o f magnitude as the affinity constants for H + between 16 and 40 n M found by others [5, 7, 14, 35]. The affinity constant for N a + to the inhibitory binding site for H + was 21 m M 2. This value is in the same order o f magnitude as the KM for transport o f N a +, which was calculated to be 16 m M in the absence o f inhibiting protons. These data indicate that protons inhibit N a + uptake by binding to the N a + transport site. Thus, the results from these experiments favor a competition between extracellular N a + and H + for the N a + transport site, suggesting a kinetic model with only one extracellular binding site. There is not only disagreement a b o u t the m o d e o f inhibition o f N a + uptake via N a +/H + exchange by extracellular protons, but also the inhibitory mode o f amiloride is controversially discussed. In cultured PE we observed a decreased affinity for N a + and only m i n o r changes in Vm,x, when amiloride was present in the medium. Ki for amiloride was a b o u t 5 gM. The affinities o f amiloride to the N a + / H + exchanger (expressed as K~ values) reported in the literature vary by over two orders o f magnitude from 0.5 g M [34] to 230 g M [26]. However, most K~ values in the literature were near 10 p M [6]. The dose-response curve in our study for amiloride was shifted by different N a + concentrations. The binding constant for N a + to this inhibitory binding site was 26 m M 2, which is very similar to the K~ o f 33 m M for N a + uptake at p H 7.5. Thus, it is likely that the transp o r t site and the amiloride binding site are identical and N a + and amiloride bind mutually exclusive to the same binding site. Such a competitive model for amiloride inhibition has been described by others [7, 16, 24, 30, 34]. However, mixed type inhibition [21] and non-competitive inhibition [31, 32] have been also reported. Moreover, there are other hints for the presence of an additional distinct binding site for amiloride (see [11, 13]). However, our d a t a are best explained by a simple competition between N a + and amiloride for one c o m m o n extracellular binding site. N a + / H + exchange has been reported to be present in the plasma membranes o f m a n y different m a m m a l i a n epithelial and non-epithelial cells. It has been shown to subserve a variety o f different physiological functions, such as intracellular p H and volume regulation, mediation o f responses to hormones and growth factors, and is responsible for N a + uptake in some sodium transporting epithelia [2, 12]. In a recent study with a virus-transformed cell clone derived from bovine PE we found involvement o f N a + / H + exchange in intracellular p H regulation during steady state and after an acid load [18]. In addition, N a + / H + exchange working in parallel with a C I - / H C O ; - antiport, which we 2 IC5o = K i x ( l + [S]/KM) (according to [40]). concentration of an inhibitor, which gives 50% presence of the substrate concentration [S]. Ki = for the inhibitor. KM = affinity constant of S to site

ICso means the inhibition in the affinity constant the same binding

have characterized in PE [20], could also mediate uptake o f NaC1 into the ciliary epithelium as described in other epithelia [27, 37]. In summary, we have characterized a N a + / H + exchange in cultured bovine pigmented ciliary epithelial cells. N a + u p t a k e via N a + / H + exchange was competitively inhibited by extracellular protons and amiloride, suggesting a kinetic model with only one c o m m o n extracellular binding site for N a +, H + and amiloride.

Acknowledgements. The expert technical assistance of A. Krolik and M. Koch is gratefully acknowledged. The present work was supported by the Deutsche Forschungsgemeinschaft grant D F G Wi-328 (1 i-2) and the Alcon Research Institute.

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Received January 19/Received after revision March 1/ Accepted March 7, 1988