PGE2 inhibits basolateral 50pS potassium channels in the thick ascending limb of the rat kidney
2008; Elsevier BV; Volume: 74; Issue: 4 Linguagem: Inglês
10.1038/ki.2008.198
ISSN1523-1755
AutoresRuimin Gu, Yan Jin, Yuanyuan Zhai, Lei Yang, Chengbiao Zhang, Wennan Li, Lijun Wang, Shumin Kong, Baofeng Yang, Wen‐Hui Wang, Yunhong Zhang,
Tópico(s)Ion Transport and Channel Regulation
ResumoTo study the inhibition of the inwardly rectifying basolateral 50 pS potassium channels by PGE2 we performed patch-clamp studies on the basolateral membrane of the rat kidney thick ascending limb. PGE2's effect was mimicked by the selective EP1- and EP3-receptor agonist, sulprostone, but was prevented by inhibiting protein kinase-C with calphostin-C. The mitogen-activated protein kinase inhibitor PD98059 (ERK) or SB203580 (p38) increased basal channel activity; however, while neither alone prevented the inhibitory effect of PGE2, but using both of them together completely abolished PGE2's effect on channel activity. Treatment with PGE2 stimulated phosphorylation of both p38 and ERK in primary cultures of medullary thick ascending limb cells. The PGE2-mediated mitogen-activated protein kinase activation was not affected by indomethacin, but was completely blocked by calphostin-C. These studies show that inhibition of basolateral 50 pS potassium channels by PGE2 is mediated by protein kinase-C, which in turn stimulates mitogen-activated protein kinases in the thick ascending limb of the rat kidney. To study the inhibition of the inwardly rectifying basolateral 50 pS potassium channels by PGE2 we performed patch-clamp studies on the basolateral membrane of the rat kidney thick ascending limb. PGE2's effect was mimicked by the selective EP1- and EP3-receptor agonist, sulprostone, but was prevented by inhibiting protein kinase-C with calphostin-C. The mitogen-activated protein kinase inhibitor PD98059 (ERK) or SB203580 (p38) increased basal channel activity; however, while neither alone prevented the inhibitory effect of PGE2, but using both of them together completely abolished PGE2's effect on channel activity. Treatment with PGE2 stimulated phosphorylation of both p38 and ERK in primary cultures of medullary thick ascending limb cells. The PGE2-mediated mitogen-activated protein kinase activation was not affected by indomethacin, but was completely blocked by calphostin-C. These studies show that inhibition of basolateral 50 pS potassium channels by PGE2 is mediated by protein kinase-C, which in turn stimulates mitogen-activated protein kinases in the thick ascending limb of the rat kidney. The thick ascending limb (TAL) is responsible for absorption of 20–25% of the filtered NaCl load and plays a key role in the ability of the kidney to concentrate urine.1.Greger R. Ion transport mechanisms in thick ascending limb of Henle’s loop of mammalian nephron.Physiol Rev. 1985; 65: 760-797Crossref PubMed Scopus (527) Google Scholar, 2.Giebisch G. Renal potassium transport:mechanisms and regulation.Am J Physiol. 1998; 274: F817-F833PubMed Google Scholar, 3.Hebert S.C. Roles of Na–K–2Cl and Na–Cl cotransporters and ROMK potassium channels in urinary concentrating mechanism.Am J Physiol. 1998; 275: F325-F327PubMed Google Scholar Potassium (K) channels in the TAL are an important component for maintaining the function of TAL: apical K channels are essential for K recycling across the luminal membrane,4.Simon D.B. Karet F.E. Rodriguez-Soriano J. et al.Genetic heterogeneity of Bartter’s syndrome revealed by mutations in the K+ channel, ROMK.Nat Genet. 1996; 14: 152-156Crossref PubMed Scopus (677) Google Scholar while basolateral K channels play a key role in generating the cell membrane potential in the TAL.5.Hebert S.C. Desir G. Giebisch G. et al.Molecular diversity and regulation of renal potassium channels.Physiol Rev. 2005; 85: 319-371Crossref PubMed Scopus (249) Google Scholar Several types of basolateral K channels have been identified with patch-clamp experiments.6.Gu R.M. Wang W.H. Arachidonic acid inhibits K channels in basolateral membrane of the thick ascending limb.Am J Physiol Renal Physiol. 2002; 283: F407-F414Crossref PubMed Scopus (22) Google Scholar,7.Paulais M. Lourdel S. Teulon J. Properties of an inwardly rectifying K+ channel in the basolateral membrane of mouse TAL.Am J Physiol Renal Physiol. 2002; 282: F866-F876Crossref PubMed Scopus (25) Google Scholar We and others have confirmed that a 50 pS inwardly rectifying K channel was highly active in the basolateral membrane. However, the regulatory mechanism of the 50 pS K channels is not completely understood. We have demonstrated previously that arachidonic acid inhibited basolateral 50 pS K channels through a cytochrome P450-dependent pathway,6.Gu R.M. Wang W.H. Arachidonic acid inhibits K channels in basolateral membrane of the thick ascending limb.Am J Physiol Renal Physiol. 2002; 283: F407-F414Crossref PubMed Scopus (22) Google Scholar and that cAMP stimulated the 50 pS K channels.8.Gu R. Wang J. Zhang Y. et al.Adenosine stimulates the basolateral 50 pS K channels in the thick ascending limb of the rat kidney.Am J Physiol Renal Physiol. 2007; 293: F299-F305Crossref PubMed Scopus (14) Google Scholar Prostaglandins such as prostaglandin-E2 (PGE2) have been shown to inhibit Cl transport by antagonizing vasopressin's effect in the TAL.9.Tago K. Schuster V.L. Stokes J.B. Regulation of chloride self exchange by cAMP in cortical collecting tubule.Am J Physiol. 1986; 251: 40-48PubMed Google Scholar,10.Culpepper R.M. Andreoli T.E. PGE2, forskolin, and cholera toxin interactions modulating NaCl transport in mouse mTALH.Am J Physiol Renal Fluid Electrolyte Physiol. 1984; 247-16: F784-F792Google Scholar Cl transport in the TAL is a two-step process: Cl enters the cells through an Na/K/Cl co-transporter and leaves the cells across the basolateral membrane via Cl channels.1.Greger R. Ion transport mechanisms in thick ascending limb of Henle’s loop of mammalian nephron.Physiol Rev. 1985; 65: 760-797Crossref PubMed Scopus (527) Google Scholar,11.Hebert S.C. Reeves W.B. Molony D.A. et al.The medullary thick limb: function and modulation of the single-effect multiplier.Kidney Int. 1987; 31: 580-588Abstract Full Text PDF PubMed Scopus (45) Google Scholar As Cl exit across the basolateral membrane is electrogenic, alteration of basolateral K channel activity is expected to affect the Cl exit and indirectly the activity of the Na/Cl/K co-transporter in the apical membrane. Thus, it is likely that the PGE2-induced inhibition of Cl transport in the TAL is partially mediated by regulation of the basolateral K channels. The main goal of this study is to examine whether PGE2 regulates the basolateral 50 pS K channels in the TAL and to investigate the pathway by which PGE2 regulates the K channels. We first examined the effect of PGE2 on the 50 pS K channels in the basolateral membrane of the TAL. Figure 1 is a channel recording showing that application of 10 μM PGE2 inhibited the 50 pS K channels in the basolateral membrane of the TAL and reduced NPo from 0.30±0.05 to 0.04±0.03 (n=5, P<0.01). As prostaglandin E receptor 3 (EP3) has been shown to be a major type of PGE2 receptor expressed in the TAL,12.Breyer M.D. Breyer R.M. Prostaglandin E receptors and the kidney.Am J Physiol Renal Physiol. 2000; 279: F12-F23PubMed Google Scholar we next examined the effect of sulprostone (a specific EP1- and EP3-receptor agonist)13.van Baal J. de Jong M.D. Zijlstra F.J. et al.Endogenously produced prostanoids stimulate calcium reabsorption in the rabbit cortical collecting system.J Physiol. 1996; 497: 229-239Crossref PubMed Scopus (20) Google Scholar on the 50 pS K channels in the basolateral membrane of the TAL. Figure 2 is a recording demonstrating that sulprostone (10 μM) mimicked the effect of PGE2 and decreased NPo from 0.32±0.06 to 0.05±0.03 (n=5, P<0.01). This suggests that the inhibitory effect of PGE2 on the 50 pS K channels was mediated by stimulating either EP1 or EP3 receptors in the TAL. Stimulation of EP3 receptor has been shown to activate pertussis toxin-sensitive G-protein and decrease cAMP generation.14.Breyer M.D. Breyer R.M. G protein-coupled prostanoid receptors and the kidney.Annu Rev Physiol. 2001; 63: 579-605Crossref PubMed Scopus (185) Google Scholar We have demonstrated previously that stimulation of protein kinase-A (PKA) with the membrane-permeable cAMP analog increased the activity of the basolateral 50 pS K channels.8.Gu R. Wang J. Zhang Y. et al.Adenosine stimulates the basolateral 50 pS K channels in the thick ascending limb of the rat kidney.Am J Physiol Renal Physiol. 2007; 293: F299-F305Crossref PubMed Scopus (14) Google Scholar If the inhibitory effect of PGE2 on the basolateral 50 pS K channels was due to decrease of cAMP and inhibition of PKA, inhibiting PKA should mimic the effect of PGE2. Thus, we examined the effect of H8 (inhibitor of PKA) on the 50 pS K channels in the TAL. We confirmed the previous finding that inhibition of PKA had no significant effect on the basolateral 50 pS K channels in the TAL (data not shown),8.Gu R. Wang J. Zhang Y. et al.Adenosine stimulates the basolateral 50 pS K channels in the thick ascending limb of the rat kidney.Am J Physiol Renal Physiol. 2007; 293: F299-F305Crossref PubMed Scopus (14) Google Scholar suggesting that PKA did not play a role in determining the basal activity of the basolateral K channels. In addition to decreasing cAMP, stimulation of EP3 receptor has also been reported to activate PKC and Rho-kinase.15.Claudino R.F. Kassuya C.A.L. Ferreira J. et al.Pharmacological and molecular characterization of the mechanisms involved in prostaglandin E2-induced mouse paw edema.J Pharmacol Exp Ther. 2006; 318: 611-618Crossref PubMed Scopus (44) Google Scholar,16.Shum W.W.C. Le Gy Jones R.L. et al.Involvement of Rho-kinase in contraction of guinea-pig aorta induced by prostanoid EP3 receptor agonists.Br J Pharmacol. 2003; 139: 1449-1461Crossref PubMed Scopus (30) Google Scholar Thus, we examined whether PKC was responsible for mediating the inhibitory effect of PGE2 on the basolateral 50 pS K channels in the medullary thick ascending limb (mTAL). We first tested the effect of 300 nM calphostin-C on the 50 pS K channel in the absence of PGE2. Figure 3 is a representative recording showing that application of calphostin-C had no significant effect on the basal level of channel activity (control, 0.24±0.06 and calphostin-C, 0.26±0.06) (n=3), suggesting that PKC is not involved in determining the basal level of the 50 pS K channel activity. However, blocking PKC with 300 nM calphostin-C was restored the channel activity inhibited by PGE2 (Figure 4). Data summarized in Figure 5 demonstrate that inhibition of PKC with 300 nM calphostin-C restored channel activity to 0.26±0.03 (n=4), a value which was not significantly different from that in the control. This strongly suggests that the inhibitory effect of PGE2 on the basolateral 50 pS K channel was mediated by stimulation of PKC in the TAL. Figure 5 also shows that stimulation of PKC with 5 μM phorbol 12-myristate 13-acetate significantly decreased activity of the 50 pS K channels to 0.06±0.02 (n=4, P<0.01).Figure 2A channel recording showing the effect of 10 μM sulprostone on the basolateral 50 pS K channels. The experiment was performed on a cell-attached patch and the holding potential was 0 mV. The channel close level is indicated by a dotted line and C. Two parts of the record indicated by numbers are extended to show the fast time resolution.View Large Image Figure ViewerDownload (PPT)Figure 3A channel recording showing the effect of calphostin-C on the 50 pS K channels in the TAL. The experiment was performed on a cell-attached patch and the holding potential was −20 mV (depolarization). The channel close level is indicated by a dotted line and C. Two parts of the record indicated by numbers are extended to show the fast time resolution.View Large Image Figure ViewerDownload (PPT)Figure 4A channel recording demonstrating the effect of 10 μM PGE2 on the basolateral 50 pS K channels in the absence and presence of 300 nM calphostin-C. The experiment was performed on a cell-attached patch and the holding potential was 0 mV. The channel close level is indicated by a dotted line and C. Three parts of the record indicated by numbers are extended to show the fast time resolution.View Large Image Figure ViewerDownload (PPT)Figure 5Effect of PGE2 (10 μM), PGE2+calphostin-C (cal.c) (300 nM), and phorbol 12-myristate 13-acetate (5 μM) on the basolateral 50 pS K channels. The experiments were carried out on cell-attached patches.View Large Image Figure ViewerDownload (PPT) Stimulation of PKC has been shown to activate mitogen-activated protein kinase (MAPK) such as p38 and extracellular signal-regulated kinase (ERK) in the colleting duct.17.Jin Y. Wang Z. Zhang Y. et al.PGE2 inhibits apical K channels in the CCD through activation of the MAPK pathway.Am J Physiol Renal Physiol. 2007; 293: F1299-F1307Crossref PubMed Scopus (15) Google Scholar To test whether p38 and ERK are also involved in mediating the effect of PGE2 on the basolateral 50 pS K channels in the TAL, we examined the effect of PD98059, an inhibitor of ERK,18.Alessi D.R. Cuenda A. Cohen P. et al.PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.J Biol Chem. 1995; 270: 27489-27494Crossref PubMed Scopus (3123) Google Scholar on the basolateral 50 pS K channels in the TAL. Figure 6 is a recording demonstrating that inhibiting ERK with 50 μM PD98059 increased the 50 pS K channel activity from 0.18±0.04 to 0.59±0.09 (n=4, P<0.01). This suggests that ERK is involved in determining the basal activity of the basolateral 50 pS K channels in the TAL. Alternatively, basolateral 50 pS K channels were inhibited by ERK under control conditions and inhibition of ERK reactivated the 50 pS K channels. We next examined the effect of PGE2 on the basolateral 50 pS K channels in the TAL treated with 50 μMPD98059. Although inhibition of ERK increased the activity of the basolateral 50 pS K channels, treatment of the TAL with PD98059 did not abolish the 10 μM PGE2-induced inhibition of the 50 pS K channels and decreased NPo from 0.59±0.09 to 0.3±0.07 (n=4, P<0.01) (Figure 6). However, upon examination of the time course of the experiments, it is apparent that the onset time of PGE2 effect on the 50 pS K channels was also increased. Thus, inhibition of ERK could not completely abolish the effect of PGE2 on the basolateral 50 pS K channels in the TAL. We then examined the effect of inhibiting p38 on the basolateral 50 pS K channels in the TAL. Similar to PD98059, inhibiting p38 with 5 μM SB20358019.Cuenda A. Rouse J. Doza Y.N. et al.SB203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1.FEBS Lett. 1995; 364: 229-233Abstract Full Text PDF PubMed Scopus (1947) Google Scholar also increased basolateral 50 pS K channel activity from 0.21±0.06 to 0.49±0.08 (Figure 7) (n=4, P<0.01). This suggests that p38 MAPK inhibited the 50 pS K channels under control conditions. Moreover, in the presence of SB203580, PGE2 was still able to inhibit the 50 pS K channels in the TAL and decreased NPo from 0.49±0.08 to 0.29±0.08 (n=4, P<0.01). Thus, similar to PD98059, inhibition of p38 alone was not sufficient to abolish the PGE2-induced inhibition of the basolateral 50 pS K channels. We then examined the possibility that the effect of PGE2 was the result of stimulating both ERK and p38 MAPKs. Data summarized in Figure 7 demonstrate that inhibition of both p38 and ERK not only significantly increased the activity of the basolateral 50 pS K channels from 0.18±0.05 to 0.70±0.09 (n=4, P<0.001), but also abolished the PGE2-induced inhibition of the basolateral 50 pS K channels completely. Thus, it is suggested that PGE2 inhibits basolateral 50 pS K channels in the TAL through activation of PKC–MAPK pathways.Figure 7Effect of 10 μM PGE2 on the basolateral 50 pS K channels in the TAL treated with PD98059, SB203580 or both agents. The experiments were performed on cell-attached patches. The asterisk indicates the significant difference between the MAPK inhibitor and the corresponding control. # Indicates a significant difference between the PD group and PD+PGE2 group or between the SB and SB+PGE2 group.View Large Image Figure ViewerDownload (PPT) We next used the mTAL cell line, which has been shown to possess the properties of the native mTAL,20.Eng B. Mukhopadhyay S. Vio C.P. et al.Characterization of a long-term rat mTAL cell line.Am J Physiol Renal Physiol. 2007; 293: F1413-F1422Crossref PubMed Scopus (20) Google Scholar to study the effect of PGE2 on ERK and p38 phosphorylation. We treated mTAL cell with 10 μM PGE2 for 15 min and examined the phosphorylation of p38 and ERK. Figure 8 is a western blot showing that application of 10 μM PGE2 stimulates phosphorylation of both p38 and ERK by 85±10% (n=4, P<0.05) and 50±8% (n=4), respectively. Inhibition of cyclooxygenase with 10 μM indomethacin did not significantly affect the basal level of MAPK phosphorylation, suggesting that endogenous PGE2 was not involved in regulating p38 and ERK phosphorylation. Moreover, in the presence of indomethacin, application of PGE2 still stimulates p38 and ERK phosphorylation by 70±10% (n=4, P<0.05) and 50±8% (n=4, P<0.05), respectively. Thus, our results indicate that PGE2 stimulates ERK and p38 MAPK. As the inhibitory effect of PGE2 on the 50 pS K channels was abolished by blockade of PKC, we examined whether calphostin-C could abolish the effect of PGE2 on the MAPK phosphorylation. We treated mTAL cells with 300 nM calphostin-C or the vehicle for 20 min, followed by addition of 10 μM PGE2 for 5 min in the presence or absence of calphostin-C. As shown in Figure 9, inhibiting PKC did not significantly affect the basal level of MAPK phosphorylation, but it abolished the effect of PGE2 on both ERK and p38 phosphorylation. This suggests that PKC is involved in mediating the effect of PGE2 on MAPK phosphorylation.Figure 9Effect of PGE2 on the phosphorylation of p38 and ERK in the presence of PKC inhibitor. Western blots show the effect of PGE2 on the phosphorylation of p38 (a) and ERK (b) in the presence or absence of 300 nM calphostin-C. Data are summarized in a bar graph (bottom panel). The asterisk indicates a significant difference in comparison with the control.View Large Image Figure ViewerDownload (PPT) In this study, we have shown that PGE2 inhibited the basolateral 50 pS K channels in the TAL and that inhibiting PKC abolished the effect of PGE2 on the 50 pS K channel activity. As the effect of PGE2 on the basolateral K channels was mimicked by sulprostone, results strongly suggest that the effect of PGE2 is mediated by stimulation of either EP1 or EP3 receptor. Both EP1 and EP3 receptors are expressed in the kidney.12.Breyer M.D. Breyer R.M. Prostaglandin E receptors and the kidney.Am J Physiol Renal Physiol. 2000; 279: F12-F23PubMed Google Scholar,21.Breyer M.D. Zhang Y. Guan Y. et al.Regulation of renal function by prostaglandin E receptors.Kidney Int. 1998; 54: 88-94Abstract Full Text Full Text PDF Scopus (79) Google Scholar A large body of evidence indicates that EP1 receptors are mainly located in the distal nephron segment such as the connecting tubule and collecting duct,22.Breyer M.D. Davis L. Jacobson H.R. et al.Differential localization of prostaglandin E receptor subtypes in human kidney.Am J Physiol. 1996; 270: F912-F918PubMed Google Scholar,23.Guan Y. Zhang Y. Breyer R.M. et al.Prostaglandin E2 inhibits renal collecting duct Na+ absorption by activating the EP1 receptor.J Clin Invest. 1998; 102: 194-201Crossref PubMed Scopus (117) Google Scholar whereas EP3 receptor is a major PG receptor in the TAL.12.Breyer M.D. Breyer R.M. Prostaglandin E receptors and the kidney.Am J Physiol Renal Physiol. 2000; 279: F12-F23PubMed Google Scholar It is well demonstrated that EP3 receptor is a G-protein-coupled receptor and that stimulating EP3 receptor inhibits adenylate cyclase and decreases cAMP generation. However, the observation that inhibiting PKA had no significant effect on the basolateral 50 pS K channel, suggests that PKA is not involved in determining the basal activity of the 50 pS K channels. Thus, it is unlikely that the inhibitory effect of PGE2 on the 50 pS K channels was the result of inhibition of PKA, although activation of PKA stimulated the basolateral 50 pS K channel activity in the TAL.8.Gu R. Wang J. Zhang Y. et al.Adenosine stimulates the basolateral 50 pS K channels in the thick ascending limb of the rat kidney.Am J Physiol Renal Physiol. 2007; 293: F299-F305Crossref PubMed Scopus (14) Google Scholar In addition to decreasing camp generation, stimulation of EP3 receptor has also been reported to activate PKC, MAPK, and Rho-kinase,15.Claudino R.F. Kassuya C.A.L. Ferreira J. et al.Pharmacological and molecular characterization of the mechanisms involved in prostaglandin E2-induced mouse paw edema.J Pharmacol Exp Ther. 2006; 318: 611-618Crossref PubMed Scopus (44) Google Scholar,16.Shum W.W.C. Le Gy Jones R.L. et al.Involvement of Rho-kinase in contraction of guinea-pig aorta induced by prostanoid EP3 receptor agonists.Br J Pharmacol. 2003; 139: 1449-1461Crossref PubMed Scopus (30) Google Scholar and to increase inositol triphosphate/Ca2+ release.24.Sugimoto Y. Narumiya S. Prostaglandin E receptors.J Biol Chem. 2007; 282: 11613-11617Crossref PubMed Scopus (861) Google Scholar The finding that inhibiting PKC restored the channel activity inhibited by PGE2, strongly suggests that PKC was responsible for mediating the effect of PGE2 on the basolateral K channels. The role of PKC in inhibiting the basolateral 50 pS K channels is also suggested by the observation that stimulation of PKC with phorbol 12-myristate 13-acetate inhibited the basolateral K channels. Relevant to our observation is that PGE2 inhibited vasopressin-induced stimulation of bicarbonate transport in the mTAL by activating PKC-δ.25.Aristimuno P. Good D.W. PKC isoforms in rat medullary thick ascending limb: selective activation of the d-isoform by PGE2.Am J Physiol. 1997; 272: F624-F631PubMed Google Scholar,26.Good D.W. PGE2 reverses AVP inhibition of HCO3- absorption in rat mTAL by activation of protein kinase C.Am J Physiol. 1996; 270: F978-F985PubMed Google Scholar Also, we have demonstrated that PGE2 inhibits the apical 70 pS K channels by a PKC-dependent signaling mechanism in the TAL.27.Liu H.J. Wei Y. Ferreri N. et al.Vasopressin and PGE2 regulate the apical 70 pS K channel in the thick ascending limb of rat kidney.Am J Physiol (Cell Physiol). 2000; 278: C905-C913PubMed Google Scholar However, we could not completely exclude the possibility that EP1 receptor is responsible for the inhibitory effect of PGE2 on the basolateral K channels in the TAL. This study has also demonstrated that p38 and ERK were involved in mediating the effect of PGE2 on the basolateral 50 pS K channels in the TAL. This view is supported by two lines of evidence: (1) PGE2 stimulated phosphorylation of p38 and ERK in the mTAL cells and (2) inhibition of p38 and ERK abolished the inhibitory effect of PGE2 on the 50 pS K channels. Because inhibition of PKC abolished the effect of PGE2 on the phosphorylation of both p38 and ERK, MAPK may be a downstream signaling molecule, which mediates the effect of PGE2 on the basolateral K channels. MAPK plays an important role in the regulation of renal K channels. We have demonstrated previously that p38 and ERK inhibited the big conductance K channels and renal outer-modullary K channel (Kirl.1) in the CCD.28.Li D.M. Wang Z.J. Sun P. et al.Inhibition of mitogen-activated protein kinase stimulates the Ca2+-dependent big conductance K channels (BK) in cortical collecting duct.Proc Natl Acad Sci USA. 2006; 103: 19569-19574Crossref PubMed Scopus (58) Google Scholar,29.Babilonia E. Li D. Wang Z.J. et al.Mitogen-activated protein kinase (MAPK) inhibits ROMK-like small conductance K channels in the CCD of K-restricted rats.J Am Soc Nephrol. 2006; 17: 2687-2696Crossref PubMed Scopus (39) Google Scholar The observation that inhibiting p38 or ERK stimulated the basolateral 50 pS K channel activity suggests that MAPKs were involved in determining the basal activity of the basolateral 50 pS K channels. This is in contrast to PKC, which is not involved in determining the basal activity of the 50 pS K channels because inhibition of PKC did not significantly alter basolateral K channel activity. We speculate that MAPK is activated under control conditions (without PGE2) and application of PGE2 causes further stimulation of MAPK by a PKC-dependent mechanism. PKC has been shown to stimulate MAPK in different types of cells.30.Baines C.P. Zhang J. Wang G.W. et al.Mitochondrial PKCepsilon and MAPK form signaling modules in the murine heart: enhanced mitochondrial PKCepsilon–MAPK interactions and differential MAPK activation in PKCepsilon-induced cardioprotection.Circ Res. 2002; 90: 390-397Crossref PubMed Scopus (362) Google Scholar,31.Ghayor C. Rey A. Caverzasio J. Prostaglandin-dependent activation of ERK mediates cell proliferation induced by transforming growth factor beta in mouse osteoblastic cells.Bone. 2005; 36: 93-100Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar The mechanism by which PKC stimulates MAPK in response to PGE2 is not clear. PKC has been shown to stimulate NADPH oxidase32.Bey E.A. Xu B. Bhattacharjee A. et al.Protein kinase C delta is required for p47phox phosphorylation and translocation in activated human monocytes.J Immunol. 2004; 173: 5730-5738Crossref PubMed Scopus (152) Google Scholar and stimulation of NADPH oxidase increases superoxide production, which is a potent stimulator for p38 and ERK in a variety of tissues, including kidney.33.Babilonia E. Lin D. Zhang Y. et al.Role of gp91phox-containing NADPH oxidase in mediating the effect of K restriction on ROMK channels and renal K excretion.J Am Soc Nephrol. 2007; 18: 2037-2045Crossref PubMed Scopus (28) Google Scholar The physiological significance of this study is to illustrate the mechanism by which PGE2 inhibits Cl absorption in the TAL through inhibiting basolateral K channels. Figure 10 is a cell model illustrating the way PGE2 inhibits basolateral K channels in the TAL. Stimulation of EP1 or EP3 receptor activates PKC, which further increases p38 and ERK activity. As a consequence, PGE2 inhibits basolateral K channels. PGE2-induced inhibition of the basolateral K channel activity is expected to depolarize the basolateral cell membrane potential and decrease the driving force for Cl exit across the basolateral membrane. Decreasing Cl exit would raise intracellular Cl concentrations, which leads to inhibition of the Na/K/Cl co-transporter.34.Gamba G. Molecular physiology and pathophysiology of electroneutral cation–chloride cotransporters.Physiol Rev. 2005; 85: 423-493Crossref PubMed Scopus (569) Google Scholar A large body of evidence indicates that PGE2 inhibits Cl absorption in the TAL.10.Culpepper R.M. Andreoli T.E. PGE2, forskolin, and cholera toxin interactions modulating NaCl transport in mouse mTALH.Am J Physiol Renal Fluid Electrolyte Physiol. 1984; 247-16: F784-F792Google Scholar, 35.Stokes J.B. Effect of prostaglandin E2 on chloride transport across the rabbit thick ascending limb of Henle. Selective inhibition of the medullary portion.J Clin Invest. 1979; 64: 495-502Crossref PubMed Scopus (267) Google Scholar, 36.Peterson L.N. McKay A.J. Borzecki J.S. Endogenous prostaglandin E2 mediates inhibition of rat thick ascending limb Cl reabsorption in chronic hypercalcemia.J Clin Invest. 1993; 91: 2399-2407Crossref PubMed Scopus (25) Google Scholar The inhibitory effect of PGE2 on Cl transport may be partially mediated by inhibiting the Na/K/Cl co-transporter.36.Peterson L.N. McKay A.J. Borzecki J.S. Endogenous prostaglandin E2 mediates inhibition of rat thick ascending limb Cl reabsorption in chronic hypercalcemia.J Clin Invest. 1993; 91: 2399-2407Crossref PubMed Scopus (25) Google Scholar,37.Kaji D.M. Chase Jr, H.S. Eng J.P. et al.Prostaglandin E2 inhibits Na–K–2Cl cotransport in medullary thick ascending limb cells.Am J Physiol. 1996; 271: F354-F361PubMed Google Scholar However, it has been reported that PGE2 had no effect on the basal level of Cl transport in the absence of vasopressin, although it abolished vasopressin-induced stimulation of Cl transport in the mouse medullary TAL.10.Culpepper R.M. Andreoli T.E. PGE2, forskolin, and cholera toxin interactions modulating NaCl transport in mouse mTALH.Am J Physiol Renal Fluid Electrolyte Physiol. 1984; 247-16: F784-F792Google Scholar We have now demonstrated that PGE2 may inhibit Cl absorption by inhibiting basolateral K channel activity in the absence of vasopressin. The discrepancy may be due to the different concentrations of PGE2 and different part of TAL because we used high concentrations of PGE2 and cortical TAL in our experiments. The finding that PGE2 at high concentrations may affect the activity of epithelial transport in the TAL, has also been reported in our previous study.27.Liu H.J. Wei Y. Ferreri N. et al.Vasopressin and PGE2 regulate the apical 70 pS K channel in the thick ascending limb of rat kidney.Am J Physiol (Cell Physiol). 2000; 278: C905-C913PubMed Google Scholar We have shown that PGE2 at low concentrations (<1 μM) abolished the stimulatory effect of vasopressin on the apical K channels in the TAL, while 5–10 μM PGE2 inhibited apical K channels by PKC. PGE2-induced inhibition of basolateral K channels may be partially responsible for the inhibition of salt transport in the TAL during K-restriction, which is known to cause impairment of the epithelial transport in the Henle's loop. Because K-restriction increases cyclooxygenase-2 expression and PGE2 generation in the kidney,17.Jin Y. Wang Z. Zhang Y. et al.PGE2 inhibits apical K channels in the CCD through activation of the MAPK pathway.Am J Physiol Renal Physiol. 2007; 293: F1299-F1307Crossref PubMed Scopus (15) Google Scholar increasing PGE2 production could suppress the salt transport by inhibiting apical K channels and the Na/K/Cl co-transporter and basolateral K channels. We conclude that PGE2 inhibits the basolateral 50 pS K channels and that the effect of PGE2 is mediated by stimulating the PKC–MAPK-dependent pathway. Pathogen-free Sprague–Dawley rats of either sex (40–50 g) were purchased from the animal facility of the Second Affiliated Hospital of Harbin Medical University. The rats were fed normal rat chow and had free access to water for 7 days before experiments. Rats were killed by cervical dislocation (the weight of rats was less than 90 g) and kidneys were removed immediately. Thin coronal sections were cut with a razor blade and kidney slices were incubated in a HEPES buffer solution containing collagenase type-1A (1 mg/ml; Sigma, St Louis, MO, USA) at 37 °C for 45–60 min. After collagenase treatment, the kidney slices were rinsed with a solution containing (in mM) 140 NaCl, 5 KCl, 1.5 MgCl2, 1.8 CaCl2, and 10 HEPES (pH=7.4) at 4 °C. The cortical TAL was dissected and the isolated tubule was transferred onto a 5 × 5-mm cover glass coated with polylysine (Sigma) to immobilize the tubule. The cover glass was placed in a chamber mounted on an inverted microscope (Nikon, Melville, NY, USA). The TAL tubules were superfused with HEPES-buffered NaCl solution plus 5 mM glucose. The basolateral membrane of cells, which had no sign of swelling and a clear board to their neighbor cells, was then patched. The animal protocol was approved by the animal use committee of Harbin Medical University. The patch pipette was pulled with Narishege PP-81 electrode puller and the pipette solution contained (in mM) 140 KCl, 1.8 MgCl2, and 10HEPES (pH=7.4). We used an Axon 200A patch-clamp amplifier to record channel current. The current was low-pass filtered at 0.5 kHz and digitized with an Axon interface (Digidata 1200). Data were collected with an IBM-compatible Pentium computer at a rate of 2 kHz and analyzed using pClamp software system 7.0 (Axon Instruments, Burlingame, CA, USA). Channel activities were defined as NPo, a product of channel open probability (Po) and channel number (N). NPo was calculated from data samples of 60-s duration in the steady state as follows:NPo=∑(1t1+2t2+…iti) where ti is the fractional open time spent at each of the observed current levels. We selected a 60-s-long trace at the steady state after the onset of PGE2 effect to calculate the channel activity. The channel conductance was determined by measuring K current at several different holding potentials. The method used to culture mTAL cell has been described previously.20.Eng B. Mukhopadhyay S. Vio C.P. et al.Characterization of a long-term rat mTAL cell line.Am J Physiol Renal Physiol. 2007; 293: F1413-F1422Crossref PubMed Scopus (20) Google Scholar Briefly, mTAL cells were grown in Dulbecco's modified Eagle's medium/F12(1:1) medium with 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate, and 1.2 g/l sodium bicarbonate supplemented with 0.005 mM dexamethasone and 5% fetal bovine serum. mTAL cells were treated with PGE2 for 5 min. After treatment with PGE2, cells were washed with ice-cold phosphate-buffered saline twice and incubated for 30 min in radio-immunoprecipitation assay lysis buffer (1 × phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride, 10 μl/ml Cocktail, 100 mM sodium orthovanadate). Proteins homogenized from mTAL cells were separated by electrophoresis on 8–10% SDS–polyacrylamide gels and transferred to Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with Odyssey blocking buffer for fluorescent western blotting (Rockland, Gilbertsville, PA, USA) and incubated with the primary antibody at 4 °C for 12 h. The membrane was washed four times (5 min for each wash) with phosphate-buffered saline containing 0.1% Tween-20, followed by incubation with the secondary antibody for an additional 30 min. The membrane was then washed three times (10 min for each wash) with phosphate-buffered saline and scanned by Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA) at wavelength of 680 or 800 nm. Phorbol 12-myristate 13-acetate, PGE2, sulprostone, calphostin-C, SB203580, and PD98059 were purchased from Biomol (Plymouth Meeting, PA, USA) and dissolved in either ethanol or dimethylsulfoxide solution. The final concentration of ethanol or dimethylsulfoxide was less than 0.1% and had no effect on channel activity. Data are shown as mean±s.e.m. We used either Student's t-test or one-way analysis of variance to determine the significance between the two groups. P<0.05 was considered statistically significant. This work was supported by National Nature Science Foundation of China #30570673 (RMG), #30770800 (RMG), the Research Fund of High Education for Doctoral Program #20040226007 (RMG), Nature Science Foundation of HeilongJiang province #D0319 (RMG), National Basic Research Program of China #2007CB512000 (BFY), and NIH HL34300 (WHW). We thank Ms Melody Steinberg for editing the paper, Dr MM Carroll for stimulating discussions during the course of the study and Dr N Ferreri for the cultured mTAL cells.
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