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2Cl− Cotransporter via a PTK-Dependent Pathway in Renal Epithelium
Biochemical and Biophysical Research Communications 285, 880 – 884 (2001) doi:10.1006/bbrc.2001.5251, available online at http://www.idealibrary.com on
Forskolin Activation of Apical Cl ⫺ Channel and Na ⫹/K ⫹/2Cl ⫺ Cotransporter via a PTK-Dependent Pathway in Renal Epithelium Naomi Niisato and Yoshinori Marunaka 1 Department of Cellular and Molecular Physiology, Kyoto Prefectural University of Medicine, Kyoto 602-0841, Japan
Received May 29, 2001
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Forskolin induced the transepithelial Cl transport (secretion) by activating the apical Cl ⴚ channel and basolateral Na ⴙ/K ⴙ/2Cl ⴚ cotransporter in renal epithelial A6 cells via an increase in cytosolic cAMP concentration. The cAMP activation of apical Cl ⴚ channel and Na ⴙ/K ⴙ/2Cl ⴚ cotransporter was partially mediated through a protein kinase A (PKA)-dependent pathway, but a PKA-independent pathway was also suggested to be involved in the cAMP activation. Therefore, we assessed a possibility of involvement of protein tyrosine kinase (PTK)-dependent pathway as a PKA-independent pathway in the cAMP activation by applying a PTK inhibitor, tyrphostin A23 (AG18). Tyrphostin A23 abolished the forskolin-induced transepithelial Cl ⴚ secretion by partially diminishing the activity of the Cl ⴚ channel and completely inhibiting the Na ⴙ/K ⴙ/2Cl ⴚ cotransporter. Further, forskolin increased phosphorylation of protein tyrosine, suggesting that cAMP activates PTK. These observations suggest that cAMP activates the Cl ⴚ channel and the Na ⴙ/K ⴙ/2Cl ⴚ cotransporter by activating PTK. © 2001 Academic Press Key Words: Cl ⴚ secretion; bumetanide; NPPB; ouabain; cAMP; tyrphostin A23; protein kinase A; protein tyrosine kinase.
induced Cl ⫺ secretion. Generally, Cl ⫺ secretion is composed of two steps; i.e., Cl ⫺ uptake mediated by basolateral Na ⫹/K ⫹/2Cl ⫺ cotransporter and Cl ⫺ release through apical Cl ⫺ channels. In the cAMP-induced Cl ⫺ secretion of renal epithelial A6 cells, Cl ⫺ is taken up by bumetanide-sensitive Na ⫹/K ⫹/2Cl ⫺ cotransporter in the basolateral membrane and is released through Cl ⫺ channels in the apical membrane. A6 cell has at least two types of Cl ⫺ channels, 3-pS Cl ⫺ channel and 8-pS Cl ⫺ channel (2), in the apical membrane which contribute to transepithelial Cl ⫺ transport (secretion). The 3-pS Cl ⫺ channel is activated by cytosolic Ca 2⫹, while the 8-pS Cl ⫺ channel, which is thought to be a CFTR, is regulated by cAMP in PKA-dependent and -independent pathways (2, 9, 10). However, the regulatory mechanism of cAMP-induced Cl ⫺ secretion is still unclear. The purpose of the present study is to clarify the regulatory mechanism of the Cl ⫺ secretion in renal epithelial A6 cells. The present study indicates that cAMP (forskolin)-induced Cl ⫺ transport (secretion) is regulated by a protein tyrosine kinase (PTK)-dependent pathway which is involved in activation of the basolateral Na ⫹/K ⫹/2Cl ⫺ cotransporter and the apical Cl ⫺ channel. MATERIALS AND METHODS
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Cl transport is regulated by various hormones and factors through Ca 2⫹- and cAMP-dependent pathways. In renal epithelium, vasopressin, forskolin and 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase), which increase cytosolic cAMP concentration, induce Cl ⫺ transport (secretion) (1) via cAMPdependent pathways (2–5). Most of previous studies indicate that cAMP-induced Cl ⫺ secretion is regulated via PKA-dependent pathways (6 – 8). However, the PKA-dependent regulatory mechanism would not be complete resolution to understandings of cAMP1
To whom correspondence should be addressed. Fax: 075-2510295. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Solutions. The bathing solution (255 mOsm/kg H 2O) contained following ion concentrations (in mM); 120 NaCl, 3.5 KCl, 1 CaCl 2, 1 MgCl 2, 5 glucose, 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes). The pH of solution used in the present study was adjusted to 7.4 by NaOH. The bathing solution was stirred with air. Materials. Medium NCTC-109 and fetal bovine serum were purchased from GIBCO (Grand Island, NY). Benzamil and 5-nitro-2-(3phenylpropylamino)benzoic acid (NPPB, a Cl ⫺ channel blocker) were purchased from Sigma (St. Louis, MO). Forskolin and tyrphostin A23 were obtained from Calbiochem (San Diego, CA). Tissue culturetreated Transwell was purchased from Costar Corporation (Cambridge, MA). Nunc filters were obtained from Nunc (Roskilde, Denmark). Cell culture. A6 cells were purchased from American Type Culture Collection (ATCC). A6 cells (passage 72– 84) were grown on plastic flasks in NCTC-109 medium modified for amphibian cells supplemented with 10% fetal bovine serum (osmolality ⫽ 255
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mOsm/kg H 2O) (5, 11). The flasks were kept in a humidified incubator at 27°C with 2.0% CO 2 in air. Cells were seeded onto tissue culture-treated Transwell filters for electrophysiological measurements or onto Nunc filters for Western blotting at density of 5 ⫻ 10 4 cells/well and were cultured for 9 –13 days. Electrophysiological measurements: Short-circuit current (Isc) and Cl ⫺ conductance. For measurements of Isc and conductance monolayers grown on tissue culture-treated Transwell filters were rinsed with the same solution as the experimental solution, and were transferred to a modified Ussing chamber (Jim’s Instrument, Iowa City, IA) designed to hold the filter cup (12). Isc and conductance were measured with an amplifier VCC-600 (Physiologic Instrument, San Diego, CA) (12, 13). Transepithelial voltage was measured with a pair of calomel electrodes that were immersed in a saturated KCl solution and bridged to the modified Ussing chamber by a pair of polyethylene tubes filled with a solution of 2% agarose in 2 M KCl. The NPPB-sensitive Isc and conductance were represented as transepithelial Cl ⫺ transport and Cl ⫺ conductance, respectively (14). A positive current represents a net flow of cation from the apical to the basolateral solution (cation absorption) or a net flow of anion from the basolateral to the apical solution (anion secretion). Therefore, the Cl ⫺ secretion is represented as a positive current (Isc) in the present study. The experiments were performed at 24 –25°C. Western blotting. We were employed in western blotting using the same method previously reported (14). Cells were incubated for 30 min in the bathing solution with or without 10 M forskolin which was applied to the basolateral solution at 24 –25°C. The cells with and without forskolin application were lysed by lysis buffer (50 mM Hepes, 150 mM NaCl, 1.5 mM MgCl 2, 1 mM EGTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 100 mM NaF, 10 mM pyrophosphate, 200 M Na-orthovanadate, 250 g/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 100 kallikrein inactivator units/ml aprotinin, pH 7.4) on ice. We homogenized cells by sonication and centrifuged at 12,000g for 10 min at 4°C to remove insoluble debris. The cell lysate containing 25 g protein was boiled in SDS sample buffer (60 mM Tris–HCl, 2% (w/v) SDS, 5% (v/v) glycerol, pH 6.8) and then subjected to 10% (w/v) SDS–polyacrylamide gel electrophoresis (SDS– PAGE). After electrophoresis, we transferred proteins to nitrocellulose membranes. Nonspecific binding was blocked by incubation in 5% (w/v) bovine serum albumin for 60 min. Membranes were immunoblotted with a monoclonal anti-phosphotyrosine antibody, PY99 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). After overnight incubation at 4°C, the membrane was washed with Tris-buffered saline (TBS) and incubated for 60 min at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG. After washing, blots were developed with an enhanced chemiluminescence (ECL) detection kit from Amersham (Oakville, Ontario, Canada). Data presentation. All data shown in the present study are represented as means ⫾ SE. Where SE bars are not visible, they are smaller than the symbol. The unpaired Student’s t test, ANOVA and Duncan’s multiple range comparison test were used for statistical analysis as appropriate and a P value ⬍0.05 was considered significant.
RESULTS AND DISCUSSION We have reported that cAMP induces the transepithelial Cl ⫺ transport (secretion) and that the cAMPinduced Cl ⫺ secretion is partially mediated through a protein kinase A (PKA)-dependent pathway in renal epithelium (15). However, the regulatory mechanism of cAMP-induced Cl ⫺ secretion is not fully understood. The aim of this study is to clarify the regulatory mechanism of cAMP-induced Cl ⫺ secretion in renal epithelial A6 cells.
FIG. 1. Effect of PTK inhibitor, tyrphostin A23, on time course of forskolin-stimulated Isc. Forskolin of 10 M was applied to the basolateral side with (closed circles) or without (open circles) 100 M tyrphostin A23 which was added 60 min before application of forskolin. NPPB (100 M) was applied to the apical side 30 min after addition of forskolin. Benzamil (10 M) was applied to the apical solution 70 min before addition of forskolin. Data are presented as mean ⫾ SE (n ⫽ 8).
Our previous study (15) has reported contribution of PKA-dependent pathways to the cAMP-induced Cl ⫺ transport (secretion), but a PKA-independent pathway is also suggested to be involved in the cAMP-induced Cl ⫺ secretion (10). Therefore, we have to consider a PKA-independent pathway to fully understand the regulatory mechanism of cAMP-induced Cl ⫺ secretion. As a possible PKA-independent pathway, protein tyrosine kinase (PTK) is considered, since PTK has been reported to regulate epithelial ion transport (12). Based upon these observations, we studied a possible contribution of PTK-dependent pathways in the cAMPinduced Cl ⫺ secretion. To study a possible role of PTK in the cAMP-induced Cl ⫺ secretion, we assessed effects of tyrphostin A23 (AG18), a PTK inhibitor, on the Cl ⫺ secretion induced by forskolin which increases cytosolic cAMP concentration. In the present study, we treated monolayered A6 cells with 10 M benzamil, a specific blocker of epithelial Na ⫹ channel, before starting the experiments to focus our present study on the regulation of forskolin-induced Cl ⫺ secretion by abolishing Na ⫹ transport. Forskolin of 10 M was applied to the basolateral solution for elevation of cytosolic cAMP concentration, and we measured the Cl ⫺ secre-
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FIG. 2. Effect of PTK inhibitor, tyrphostin A23 (TY), on forskolin-induced NPPB-sensitive Isc. The NPPB-sensitive Isc was measured by addition of 100 M NPPB to the apical solution at 30 min after application of 10 M forskolin to the basolateral solution. Tyrphostin A23 or H89 (5 M) was added to the bilateral sides 60 min before application of forskolin. Benzamil (10 M) was applied to the apical solution 70 min before addition of forskolin. Data are presented as mean ⫾ SE (n ⫽ 8).
tion by adding 100 M NPPB, a Cl ⫺ channel blocker, to the apical solution 30 min after application of forskolin. The basal Isc was negligibly small compared with Isc after application of 10 M forskolin to the basolateral solution (Fig. 1). Forskolin biphasically increased Isc; a transient increase in Isc with its peak around 3 min after application of forskolin followed by a sustained increase in Isc (open circles in Fig. 1). Most of the forskolin-induced Isc was sensitive to NPPB (100 M) which was applied to the apical solution, suggesting that forskolin induces Cl ⫺ secretion. Next, we tested the effect of tyrphostin A23 on the Isc; tyrphostin A23 was bilaterally applied at ⫺60 min (closed circles) in Fig. 1. Tyrphostin A23 had no effects on the basal Isc, but abolished the forskolin-induced Isc (closed circles in Fig. 1), suggesting that a PTK-dependent pathway may be involved in the regulation of the cAMP-induced Cl ⫺ secretion. On the other hand, H89 (5 M, a super maximal concentration), an inhibitor of protein kinase A (PKA), partially inhibited the NPPB-sensitive Isc unlike tyrphostin A23 (Fig. 2). The Cl ⫺ secretion is mediated through two steps: (1) the releasing step of Cl ⫺ through the apical Cl ⫺ channel, and (2) the uptake step of Cl ⫺ through the basolateral Na ⫹/K ⫹/2Cl ⫺ cotransporter. Therefore, based upon the observation that tyrphostin A23 abolished the cAMP-induced Cl ⫺ secretion, it is suggested that tyrphostin A23 completely inhibits at least one of the releasing and uptake steps of Cl ⫺. Namely, we considered two possibilities: (1) tyrphostin A23 completely
inhibits the apical Cl ⫺ channel, and/or (2) tyrphostin A23 abolishes the Na ⫹/K ⫹/2Cl ⫺ cotransporter (15, 16). To assess activity of the apical Cl ⫺ channel, we measured the NPPB-sensitive conductance. Interestingly, tyrphostin A23 partially reduced the NPPB-sensitive conductance, but did not abolish it (Fig. 3). H89 also partially decreased the NPPB-sensitive conductance (Fig. 3). It is notable that the NPPB-sensitive conductance in tyrphostin A23-treated cells was not significantly different from that in H89-treated cells (Fig. 3). These observations indicate that the inhibitory action of tyrphostin A23 on the apical Cl ⫺ channel would be identical to that of H89, nevertheless tyrphostin A23 abolished the NPPB-sensitive Isc unlike H89. In other words, the tyrphostin A23-caused diminution of the Cl ⫺ conductance is a partial factor of the tyrphostin A23-caused abolishment of the transepithelial Cl ⫺ transport, but the inhibitory effect of tyrphostin A23 on the apical Cl ⫺ channel is not fully accountable for the abolishment of the transepithelial Cl ⫺ transport (NPPB-sensitive Isc). This indicates that tyrphostin A23 abolishes the Cl ⫺ transport by completely blocking the uptake step of the Cl ⫺ transport; i.e., the Na ⫹/K ⫹/ 2Cl ⫺ cotransporter was completely blocked by tyrphostin A23. Further, we have to consider a possibility that tyrphostin A23 abolishes activity of the Na ⫹/K ⫹/2Cl ⫺ cotransporter by inhibiting the Na ⫹/K ⫹ ATPase in the basolateral membrane which generates the Na ⫹ gradi-
FIG. 3. Effect of PTK inhibitor, tyrphostin A23 (TY), on forskolin-induced NPPB-sensitive conductance. The NPPB-sensitive conductance was measured by addition of 100 M NPPB to the apical solution at 30 min after application of 10 M forskolin to the basolateral solution. Tyrphostin A23 or H89 was added to the bilateral sides 60 min before application of forskolin. Benzamil (10 M) was applied 70 min to the apical solution before addition of forskolin. Data are presented as mean ⫾ SE (n ⫽ 10). NS represents no significance.
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on the Cl ⫺ secretion is partially explained to be mediated through the inhibition of the Na ⫹/K ⫹ ATPase, it is not fully explained. Namely, tyrphostin A23 would partially diminish the forskolin-induced Cl ⫺ secretion by decreasing the driving force, the Na ⫹ gradient, for the Na ⫹/K ⫹/2Cl ⫺ cotransporter, however within the time period of experiments in the present study (i.e., within about 2 h) the tyrphostin-caused diminution of the Na ⫹ gradient is not large enough to abolish the forskolininduced Cl ⫺ secretion. Finally, to clarify whether forskolin affects PTK activity, we studied effects of forskolin on phosphorylation of protein tyrosine. Forskolin increased phosphorylation of protein tyrosine as shown in Fig. 6. These observations suggest that forskolin increases phosphorylation of protein tyrosine by activating PTK. Although effects of cAMP on the Na ⫹/K ⫹/2Cl ⫺ cotransporter are reported, we still do not fully understand the regulatory mechanism of the Na ⫹/K ⫹/2Cl ⫺ cotransporter by cAMP. It is reported that cAMP activates the Na ⫹/K ⫹/2Cl ⫺ cotransporter in Ehrlich cells and renal epithelium (17, 18). On the other hand, a study (19) indicates that the Na ⫹/K ⫹/2Cl ⫺ cotransFIG. 4. Effect of ouabain on time course of forskolin-stimulated Isc. Forskolin of 10 M was applied to the basolateral solution in the presence of 1 mM ouabain (basolateral solution, closed squares) or 100 M tyrphostin A23 (TY, apical and basolateral solutions, closed triangles) which was added 60 min before application of forskolin. NPPB (100 M) was applied to the apical solution 30 min after addition of forskolin. Data are presented as mean ⫾ SE (n ⫽ 4).
ent for the driving force to function the Na ⫹/K ⫹/2Cl ⫺ cotransporter. Indeed, we have reported that tyrphostin A23 abolishes the Na ⫹/K ⫹ ATPase activity in A6 cells (14). To examine whether tyrphostin A23 abolishes the Cl ⫺ secretion by inhibiting the Na ⫹/K ⫹ ATPase, we studied the effect of ouabain (an inhibitor of the Na ⫹/K ⫹ ATPase) on the Cl ⫺ secretion. Before testing the inhibitory effect of ouabain on the Cl ⫺ secretion, we compared the inhibitory effect of ouabain on the Na ⫹/K ⫹ ATPase with that of tyrphostin A23 in their extent and time course. Ouabain of 1 mM showed identical effects on the Na ⫹/K ⫹ ATPase in the magnitude and time course to tyrphostin A23 of 100 M (data not shown; c.f. (14)). Based upon this observation, we applied 1 mM ouabain to the cell 60 min before adding forskolin. As shown in Fig. 4, in the presence of 1 mM ouabain, forskolin showed a smaller increase in Isc compared with control (in the absence of ouabain). However, the inhibitory action of ouabain was much smaller than that of tyrphostin A23 (Fig. 4). We also measured the NPPB-sensitive Isc. As shown in Fig. 5, ouabain diminished the NPPB-sensitive Isc, but ouabain did not diminish it unlike tyrphostin A23 which abolished it (Fig. 5). These observations mean that although the abolishing action of tyrphostin A23
FIG. 5. Effects of ouabain and tyrphostin A23 (TY) on forskolininduced NPPB-sensitive Isc. The NPPB-sensitive Isc was measured by addition of 100 M NPPB to the apical solution 30 min after application of 10 M forskolin to the basolateral solution. Ouabain (1 mM) was added to the basolateral solution 60 min before application of forskolin. Tyrphostin A23 (100 M) was added to the apical and basolateral solutions 60 min before application of forskolin. Data are presented as mean ⫾ SE (control, n ⫽ 8; TY, n ⫽ 8; ouabain, n ⫽ 4).
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FIG. 6. Effects of forskolin on phosphorylation of protein tyrosine. Forskolin increased phosphorylation of protein tyrosine in a time-dependent manner. This Western blotting result is a typical one. Three more similar results were also obtained.
porter in erythrocytes might be regulated by PTK. However, the relationship between regulation of the Na ⫹/K ⫹/2Cl ⫺ cotransporter by cAMP (or PKA) and PTK is unknown. The present study leads us to a conclusion that cAMP induces renal Cl ⫺ secretion by stimulating the basolateral Na ⫹/K ⫹/2Cl ⫺ cotransporter and the apical Cl ⫺ channel through activation of PTK. ACKNOWLEDGMENTS This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (13670046), and the Salt Science Research Foundation (0143).
REFERENCES 1. Chalfant, M. L., Coupaye-Gerard, B., and Kleyman, T. R. (1993) Am. J. Physiol. 264, C1480 –C1488.
2. Marunaka, Y., and Eaton, D. C. (1990) Am. J. Physiol. 258, C352–C368. 3. Marunaka, Y. (1993) Jpn. J. Physiol. 43, 553–560. 4. Marunaka, Y., and Tohda, H. (1993) Biochim. Biophys. Acta 1153, 105–110. 5. Doi, Y., and Marunaka, Y. (1995) Am. J. Physiol. 268, C762– C770. 6. Hume, J. R., Duan, D., Collier, M. L., Yamazaki, J., and Horowitz, B. (2000) Physiol. Rev. 80, 31– 81. 7. McCann, J. D., and Welsh, M. J. (1990) Annu. Rev. Physiol. 52, 115–135. 8. Morris, A. P. (1999) Adv. Pharmacol. 46, 209 –251. 9. Marunaka, Y., and Eaton, D. C. (1990) J. Gen. Physiol. 95, 773–789. 10. Shintani, Y., and Marunaka, Y. (1996) Biochem. Biophys. Res. Commun. 223, 234 –239. 11. Nakahari, T., and Marunaka, Y. (1995) Am. J. Physiol. 268, F64 –F72. 12. Niisato, N., Liu, M., Van Driessche, W., and Marunaka, Y. (2000) J. Membr. Biol. 175, 63–77. 13. Marunaka, Y., and Niisato, N. (2001) J. Membr. Biol. 180, 91– 99. 14. Niisato, N., and Marunaka, Y. (1999) J. Physiol. (London) 518, 417– 432. 15. Niisato, N., and Marunaka, Y. (1997) Pflug. Arch.–Eur. J. Physiol. 434, 227–233. 16. Niisato, N., and Marunaka, Y. (1997) J. Membr. Biol. 157, 53– 61. 17. Krarup, T., Jakobsen, L. D., Jensen, B. S., and Hoffmann, E. K. (1998) Am. J. Physiol. Cell Physiol. 275, C239 –C250. 18. Aoki, Y., Albrecht, F. E., Bergman, K. R., and Jose, P. A. (1996) Am. J. Physiol. Regul. Integr. Comp. Physiol. 271, R1561–R1567. 19. Flatman, P. W., and Creanor, J. (1999) J. Physiol. (London) 517, 699 –708.
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Forskolin Activation of Apical Cl ⫺ Channel and Na ⫹/K ⫹/2Cl ⫺ Cotransporter via a PTK-Dependent Pathway in Renal Epithelium Naomi Niisato and Yoshinori Marunaka 1 Department of Cellular and Molecular Physiology, Kyoto Prefectural University of Medicine, Kyoto 602-0841, Japan
Received May 29, 2001
ⴚ
Forskolin induced the transepithelial Cl transport (secretion) by activating the apical Cl ⴚ channel and basolateral Na ⴙ/K ⴙ/2Cl ⴚ cotransporter in renal epithelial A6 cells via an increase in cytosolic cAMP concentration. The cAMP activation of apical Cl ⴚ channel and Na ⴙ/K ⴙ/2Cl ⴚ cotransporter was partially mediated through a protein kinase A (PKA)-dependent pathway, but a PKA-independent pathway was also suggested to be involved in the cAMP activation. Therefore, we assessed a possibility of involvement of protein tyrosine kinase (PTK)-dependent pathway as a PKA-independent pathway in the cAMP activation by applying a PTK inhibitor, tyrphostin A23 (AG18). Tyrphostin A23 abolished the forskolin-induced transepithelial Cl ⴚ secretion by partially diminishing the activity of the Cl ⴚ channel and completely inhibiting the Na ⴙ/K ⴙ/2Cl ⴚ cotransporter. Further, forskolin increased phosphorylation of protein tyrosine, suggesting that cAMP activates PTK. These observations suggest that cAMP activates the Cl ⴚ channel and the Na ⴙ/K ⴙ/2Cl ⴚ cotransporter by activating PTK. © 2001 Academic Press Key Words: Cl ⴚ secretion; bumetanide; NPPB; ouabain; cAMP; tyrphostin A23; protein kinase A; protein tyrosine kinase.
induced Cl ⫺ secretion. Generally, Cl ⫺ secretion is composed of two steps; i.e., Cl ⫺ uptake mediated by basolateral Na ⫹/K ⫹/2Cl ⫺ cotransporter and Cl ⫺ release through apical Cl ⫺ channels. In the cAMP-induced Cl ⫺ secretion of renal epithelial A6 cells, Cl ⫺ is taken up by bumetanide-sensitive Na ⫹/K ⫹/2Cl ⫺ cotransporter in the basolateral membrane and is released through Cl ⫺ channels in the apical membrane. A6 cell has at least two types of Cl ⫺ channels, 3-pS Cl ⫺ channel and 8-pS Cl ⫺ channel (2), in the apical membrane which contribute to transepithelial Cl ⫺ transport (secretion). The 3-pS Cl ⫺ channel is activated by cytosolic Ca 2⫹, while the 8-pS Cl ⫺ channel, which is thought to be a CFTR, is regulated by cAMP in PKA-dependent and -independent pathways (2, 9, 10). However, the regulatory mechanism of cAMP-induced Cl ⫺ secretion is still unclear. The purpose of the present study is to clarify the regulatory mechanism of the Cl ⫺ secretion in renal epithelial A6 cells. The present study indicates that cAMP (forskolin)-induced Cl ⫺ transport (secretion) is regulated by a protein tyrosine kinase (PTK)-dependent pathway which is involved in activation of the basolateral Na ⫹/K ⫹/2Cl ⫺ cotransporter and the apical Cl ⫺ channel. MATERIALS AND METHODS
⫺
Cl transport is regulated by various hormones and factors through Ca 2⫹- and cAMP-dependent pathways. In renal epithelium, vasopressin, forskolin and 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase), which increase cytosolic cAMP concentration, induce Cl ⫺ transport (secretion) (1) via cAMPdependent pathways (2–5). Most of previous studies indicate that cAMP-induced Cl ⫺ secretion is regulated via PKA-dependent pathways (6 – 8). However, the PKA-dependent regulatory mechanism would not be complete resolution to understandings of cAMP1
To whom correspondence should be addressed. Fax: 075-2510295. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Solutions. The bathing solution (255 mOsm/kg H 2O) contained following ion concentrations (in mM); 120 NaCl, 3.5 KCl, 1 CaCl 2, 1 MgCl 2, 5 glucose, 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes). The pH of solution used in the present study was adjusted to 7.4 by NaOH. The bathing solution was stirred with air. Materials. Medium NCTC-109 and fetal bovine serum were purchased from GIBCO (Grand Island, NY). Benzamil and 5-nitro-2-(3phenylpropylamino)benzoic acid (NPPB, a Cl ⫺ channel blocker) were purchased from Sigma (St. Louis, MO). Forskolin and tyrphostin A23 were obtained from Calbiochem (San Diego, CA). Tissue culturetreated Transwell was purchased from Costar Corporation (Cambridge, MA). Nunc filters were obtained from Nunc (Roskilde, Denmark). Cell culture. A6 cells were purchased from American Type Culture Collection (ATCC). A6 cells (passage 72– 84) were grown on plastic flasks in NCTC-109 medium modified for amphibian cells supplemented with 10% fetal bovine serum (osmolality ⫽ 255
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mOsm/kg H 2O) (5, 11). The flasks were kept in a humidified incubator at 27°C with 2.0% CO 2 in air. Cells were seeded onto tissue culture-treated Transwell filters for electrophysiological measurements or onto Nunc filters for Western blotting at density of 5 ⫻ 10 4 cells/well and were cultured for 9 –13 days. Electrophysiological measurements: Short-circuit current (Isc) and Cl ⫺ conductance. For measurements of Isc and conductance monolayers grown on tissue culture-treated Transwell filters were rinsed with the same solution as the experimental solution, and were transferred to a modified Ussing chamber (Jim’s Instrument, Iowa City, IA) designed to hold the filter cup (12). Isc and conductance were measured with an amplifier VCC-600 (Physiologic Instrument, San Diego, CA) (12, 13). Transepithelial voltage was measured with a pair of calomel electrodes that were immersed in a saturated KCl solution and bridged to the modified Ussing chamber by a pair of polyethylene tubes filled with a solution of 2% agarose in 2 M KCl. The NPPB-sensitive Isc and conductance were represented as transepithelial Cl ⫺ transport and Cl ⫺ conductance, respectively (14). A positive current represents a net flow of cation from the apical to the basolateral solution (cation absorption) or a net flow of anion from the basolateral to the apical solution (anion secretion). Therefore, the Cl ⫺ secretion is represented as a positive current (Isc) in the present study. The experiments were performed at 24 –25°C. Western blotting. We were employed in western blotting using the same method previously reported (14). Cells were incubated for 30 min in the bathing solution with or without 10 M forskolin which was applied to the basolateral solution at 24 –25°C. The cells with and without forskolin application were lysed by lysis buffer (50 mM Hepes, 150 mM NaCl, 1.5 mM MgCl 2, 1 mM EGTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 100 mM NaF, 10 mM pyrophosphate, 200 M Na-orthovanadate, 250 g/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 100 kallikrein inactivator units/ml aprotinin, pH 7.4) on ice. We homogenized cells by sonication and centrifuged at 12,000g for 10 min at 4°C to remove insoluble debris. The cell lysate containing 25 g protein was boiled in SDS sample buffer (60 mM Tris–HCl, 2% (w/v) SDS, 5% (v/v) glycerol, pH 6.8) and then subjected to 10% (w/v) SDS–polyacrylamide gel electrophoresis (SDS– PAGE). After electrophoresis, we transferred proteins to nitrocellulose membranes. Nonspecific binding was blocked by incubation in 5% (w/v) bovine serum albumin for 60 min. Membranes were immunoblotted with a monoclonal anti-phosphotyrosine antibody, PY99 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). After overnight incubation at 4°C, the membrane was washed with Tris-buffered saline (TBS) and incubated for 60 min at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG. After washing, blots were developed with an enhanced chemiluminescence (ECL) detection kit from Amersham (Oakville, Ontario, Canada). Data presentation. All data shown in the present study are represented as means ⫾ SE. Where SE bars are not visible, they are smaller than the symbol. The unpaired Student’s t test, ANOVA and Duncan’s multiple range comparison test were used for statistical analysis as appropriate and a P value ⬍0.05 was considered significant.
RESULTS AND DISCUSSION We have reported that cAMP induces the transepithelial Cl ⫺ transport (secretion) and that the cAMPinduced Cl ⫺ secretion is partially mediated through a protein kinase A (PKA)-dependent pathway in renal epithelium (15). However, the regulatory mechanism of cAMP-induced Cl ⫺ secretion is not fully understood. The aim of this study is to clarify the regulatory mechanism of cAMP-induced Cl ⫺ secretion in renal epithelial A6 cells.
FIG. 1. Effect of PTK inhibitor, tyrphostin A23, on time course of forskolin-stimulated Isc. Forskolin of 10 M was applied to the basolateral side with (closed circles) or without (open circles) 100 M tyrphostin A23 which was added 60 min before application of forskolin. NPPB (100 M) was applied to the apical side 30 min after addition of forskolin. Benzamil (10 M) was applied to the apical solution 70 min before addition of forskolin. Data are presented as mean ⫾ SE (n ⫽ 8).
Our previous study (15) has reported contribution of PKA-dependent pathways to the cAMP-induced Cl ⫺ transport (secretion), but a PKA-independent pathway is also suggested to be involved in the cAMP-induced Cl ⫺ secretion (10). Therefore, we have to consider a PKA-independent pathway to fully understand the regulatory mechanism of cAMP-induced Cl ⫺ secretion. As a possible PKA-independent pathway, protein tyrosine kinase (PTK) is considered, since PTK has been reported to regulate epithelial ion transport (12). Based upon these observations, we studied a possible contribution of PTK-dependent pathways in the cAMPinduced Cl ⫺ secretion. To study a possible role of PTK in the cAMP-induced Cl ⫺ secretion, we assessed effects of tyrphostin A23 (AG18), a PTK inhibitor, on the Cl ⫺ secretion induced by forskolin which increases cytosolic cAMP concentration. In the present study, we treated monolayered A6 cells with 10 M benzamil, a specific blocker of epithelial Na ⫹ channel, before starting the experiments to focus our present study on the regulation of forskolin-induced Cl ⫺ secretion by abolishing Na ⫹ transport. Forskolin of 10 M was applied to the basolateral solution for elevation of cytosolic cAMP concentration, and we measured the Cl ⫺ secre-
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FIG. 2. Effect of PTK inhibitor, tyrphostin A23 (TY), on forskolin-induced NPPB-sensitive Isc. The NPPB-sensitive Isc was measured by addition of 100 M NPPB to the apical solution at 30 min after application of 10 M forskolin to the basolateral solution. Tyrphostin A23 or H89 (5 M) was added to the bilateral sides 60 min before application of forskolin. Benzamil (10 M) was applied to the apical solution 70 min before addition of forskolin. Data are presented as mean ⫾ SE (n ⫽ 8).
tion by adding 100 M NPPB, a Cl ⫺ channel blocker, to the apical solution 30 min after application of forskolin. The basal Isc was negligibly small compared with Isc after application of 10 M forskolin to the basolateral solution (Fig. 1). Forskolin biphasically increased Isc; a transient increase in Isc with its peak around 3 min after application of forskolin followed by a sustained increase in Isc (open circles in Fig. 1). Most of the forskolin-induced Isc was sensitive to NPPB (100 M) which was applied to the apical solution, suggesting that forskolin induces Cl ⫺ secretion. Next, we tested the effect of tyrphostin A23 on the Isc; tyrphostin A23 was bilaterally applied at ⫺60 min (closed circles) in Fig. 1. Tyrphostin A23 had no effects on the basal Isc, but abolished the forskolin-induced Isc (closed circles in Fig. 1), suggesting that a PTK-dependent pathway may be involved in the regulation of the cAMP-induced Cl ⫺ secretion. On the other hand, H89 (5 M, a super maximal concentration), an inhibitor of protein kinase A (PKA), partially inhibited the NPPB-sensitive Isc unlike tyrphostin A23 (Fig. 2). The Cl ⫺ secretion is mediated through two steps: (1) the releasing step of Cl ⫺ through the apical Cl ⫺ channel, and (2) the uptake step of Cl ⫺ through the basolateral Na ⫹/K ⫹/2Cl ⫺ cotransporter. Therefore, based upon the observation that tyrphostin A23 abolished the cAMP-induced Cl ⫺ secretion, it is suggested that tyrphostin A23 completely inhibits at least one of the releasing and uptake steps of Cl ⫺. Namely, we considered two possibilities: (1) tyrphostin A23 completely
inhibits the apical Cl ⫺ channel, and/or (2) tyrphostin A23 abolishes the Na ⫹/K ⫹/2Cl ⫺ cotransporter (15, 16). To assess activity of the apical Cl ⫺ channel, we measured the NPPB-sensitive conductance. Interestingly, tyrphostin A23 partially reduced the NPPB-sensitive conductance, but did not abolish it (Fig. 3). H89 also partially decreased the NPPB-sensitive conductance (Fig. 3). It is notable that the NPPB-sensitive conductance in tyrphostin A23-treated cells was not significantly different from that in H89-treated cells (Fig. 3). These observations indicate that the inhibitory action of tyrphostin A23 on the apical Cl ⫺ channel would be identical to that of H89, nevertheless tyrphostin A23 abolished the NPPB-sensitive Isc unlike H89. In other words, the tyrphostin A23-caused diminution of the Cl ⫺ conductance is a partial factor of the tyrphostin A23-caused abolishment of the transepithelial Cl ⫺ transport, but the inhibitory effect of tyrphostin A23 on the apical Cl ⫺ channel is not fully accountable for the abolishment of the transepithelial Cl ⫺ transport (NPPB-sensitive Isc). This indicates that tyrphostin A23 abolishes the Cl ⫺ transport by completely blocking the uptake step of the Cl ⫺ transport; i.e., the Na ⫹/K ⫹/ 2Cl ⫺ cotransporter was completely blocked by tyrphostin A23. Further, we have to consider a possibility that tyrphostin A23 abolishes activity of the Na ⫹/K ⫹/2Cl ⫺ cotransporter by inhibiting the Na ⫹/K ⫹ ATPase in the basolateral membrane which generates the Na ⫹ gradi-
FIG. 3. Effect of PTK inhibitor, tyrphostin A23 (TY), on forskolin-induced NPPB-sensitive conductance. The NPPB-sensitive conductance was measured by addition of 100 M NPPB to the apical solution at 30 min after application of 10 M forskolin to the basolateral solution. Tyrphostin A23 or H89 was added to the bilateral sides 60 min before application of forskolin. Benzamil (10 M) was applied 70 min to the apical solution before addition of forskolin. Data are presented as mean ⫾ SE (n ⫽ 10). NS represents no significance.
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on the Cl ⫺ secretion is partially explained to be mediated through the inhibition of the Na ⫹/K ⫹ ATPase, it is not fully explained. Namely, tyrphostin A23 would partially diminish the forskolin-induced Cl ⫺ secretion by decreasing the driving force, the Na ⫹ gradient, for the Na ⫹/K ⫹/2Cl ⫺ cotransporter, however within the time period of experiments in the present study (i.e., within about 2 h) the tyrphostin-caused diminution of the Na ⫹ gradient is not large enough to abolish the forskolininduced Cl ⫺ secretion. Finally, to clarify whether forskolin affects PTK activity, we studied effects of forskolin on phosphorylation of protein tyrosine. Forskolin increased phosphorylation of protein tyrosine as shown in Fig. 6. These observations suggest that forskolin increases phosphorylation of protein tyrosine by activating PTK. Although effects of cAMP on the Na ⫹/K ⫹/2Cl ⫺ cotransporter are reported, we still do not fully understand the regulatory mechanism of the Na ⫹/K ⫹/2Cl ⫺ cotransporter by cAMP. It is reported that cAMP activates the Na ⫹/K ⫹/2Cl ⫺ cotransporter in Ehrlich cells and renal epithelium (17, 18). On the other hand, a study (19) indicates that the Na ⫹/K ⫹/2Cl ⫺ cotransFIG. 4. Effect of ouabain on time course of forskolin-stimulated Isc. Forskolin of 10 M was applied to the basolateral solution in the presence of 1 mM ouabain (basolateral solution, closed squares) or 100 M tyrphostin A23 (TY, apical and basolateral solutions, closed triangles) which was added 60 min before application of forskolin. NPPB (100 M) was applied to the apical solution 30 min after addition of forskolin. Data are presented as mean ⫾ SE (n ⫽ 4).
ent for the driving force to function the Na ⫹/K ⫹/2Cl ⫺ cotransporter. Indeed, we have reported that tyrphostin A23 abolishes the Na ⫹/K ⫹ ATPase activity in A6 cells (14). To examine whether tyrphostin A23 abolishes the Cl ⫺ secretion by inhibiting the Na ⫹/K ⫹ ATPase, we studied the effect of ouabain (an inhibitor of the Na ⫹/K ⫹ ATPase) on the Cl ⫺ secretion. Before testing the inhibitory effect of ouabain on the Cl ⫺ secretion, we compared the inhibitory effect of ouabain on the Na ⫹/K ⫹ ATPase with that of tyrphostin A23 in their extent and time course. Ouabain of 1 mM showed identical effects on the Na ⫹/K ⫹ ATPase in the magnitude and time course to tyrphostin A23 of 100 M (data not shown; c.f. (14)). Based upon this observation, we applied 1 mM ouabain to the cell 60 min before adding forskolin. As shown in Fig. 4, in the presence of 1 mM ouabain, forskolin showed a smaller increase in Isc compared with control (in the absence of ouabain). However, the inhibitory action of ouabain was much smaller than that of tyrphostin A23 (Fig. 4). We also measured the NPPB-sensitive Isc. As shown in Fig. 5, ouabain diminished the NPPB-sensitive Isc, but ouabain did not diminish it unlike tyrphostin A23 which abolished it (Fig. 5). These observations mean that although the abolishing action of tyrphostin A23
FIG. 5. Effects of ouabain and tyrphostin A23 (TY) on forskolininduced NPPB-sensitive Isc. The NPPB-sensitive Isc was measured by addition of 100 M NPPB to the apical solution 30 min after application of 10 M forskolin to the basolateral solution. Ouabain (1 mM) was added to the basolateral solution 60 min before application of forskolin. Tyrphostin A23 (100 M) was added to the apical and basolateral solutions 60 min before application of forskolin. Data are presented as mean ⫾ SE (control, n ⫽ 8; TY, n ⫽ 8; ouabain, n ⫽ 4).
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FIG. 6. Effects of forskolin on phosphorylation of protein tyrosine. Forskolin increased phosphorylation of protein tyrosine in a time-dependent manner. This Western blotting result is a typical one. Three more similar results were also obtained.
porter in erythrocytes might be regulated by PTK. However, the relationship between regulation of the Na ⫹/K ⫹/2Cl ⫺ cotransporter by cAMP (or PKA) and PTK is unknown. The present study leads us to a conclusion that cAMP induces renal Cl ⫺ secretion by stimulating the basolateral Na ⫹/K ⫹/2Cl ⫺ cotransporter and the apical Cl ⫺ channel through activation of PTK. ACKNOWLEDGMENTS This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (13670046), and the Salt Science Research Foundation (0143).
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