Activity of an inwardly rectifying K(+) channel with inward conductance of about 40 pS in cultured human renal proximal tubule epithelial cells (RPTECs) is regulated at least in part by protein phosphorylation and dephosphorylation. In this study, we examined involvement of calcineurin (CaN), a Ca(2+)/calmodulin (CaM)-dependent phosphatase, in modulating K(+) channel activity. In cell-attached mode of the patch-clamp technique, application of a CaN inhibitor, cyclosporin A (CsA, 5 microM) or FK520 (5 microM), significantly suppressed channel activity. Intracellular Ca(2+) concentration ([Ca(2+)]( i )) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca(2+)]( i ), we speculated that inhibiting CaN enhances Ca(2+)-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca(2+)/CaM-dependent protein kinase-II (CaMKII) on CsA-induced channel suppression. Although the PKC inhibitor GF109203X (500 nM) did not influence the CsA-induced channel suppression, the CaMKII inhibitor KN62 (20 microM) prevented channel suppression, suggesting that the channel suppression resulted from CaMKII-dependent processes. Indeed, Western blot analysis showed that CsA increased phospho-CaMKII (Thr286), an activated CaMKII in inside-out patches, application of CaM (0.6 microM) and CaMKII (0.15 U/ml) to the bath at 10(-6) M Ca(2+) significantly suppressed channel activity, which was reactivated by subsequent application of CaN (800 U/ml). These results suggest that CaN plays an important role in supporting K(+) channel activity in RPTECs by preventing CaMKII-dependent phosphorylation.
ϩ channel in cultured human renal proximal tubule cells (RPTECs) is stimulated and inhibited by nitric oxide (NO) at low and high concentrations, respectively. In this study, we investigated the effects of IFN-␥, one of the cytokines which affect the expression of inducible NO synthase (iNOS), on intracellular NO and channel activity of RPTECs, using RT-PCR, NO imaging, and the cellattached mode of the patch-clamp technique. Prolonged incubation (24 h) of cells with IFN-␥ (20 ng/ml) enhanced iNOS mRNA expression and NO production. In these cells, a NOS inhibitor, N -nitro-L-arginine methyl ester (L-NAME; 100 M), elevated channel activity, suggesting that NO production was so high as to suppress the channel. This indicated that IFN-␥ would chronically suppress channel activity by enhancing NO production. Acute effects of IFN-␥ was also examined in control cells. Simple addition of IFN-␥ (20 ng/ml) to the bath acutely stimulated channel activity, which was abolished by inhibitors of IFN-␥ receptor-associated Janus-activated kinase [P6 (1 M) and AG490 (10 M)]. However, L-NAME did not block the acute effect of IFN-␥. Indeed, IFN-␥ did not acutely affect NO production. Moreover, the acute effect was not blocked by inhibition of PKA, PKG, and phosphatidylinositol 3-kinase (PI3K). We conclude that IFN-␥ exerted a delayed suppressive effect on K ϩ channel activity by enhancing iNOS expression and an acute stimulatory effect, which was independent of either NO pathways or phosphorylation processes mediated by PKA, PKG, and PI3K in RPTECs.
Nitric oxide (NO) modulates the activity of an inwardly rectifying K + channel in cultured human proximal tubule cells. In this study, we investigated which NO synthase (NOS) isoform(s) was involved in the endogenous production of NO and hence the regulation of channel activity. The patch-clamp experiments using the cell-attached mode showed that a nonselective NOS inhibitor, N ω -nitro-L-arginine methyl ester (L-NAME; 100 µM), suppressed channel activity, whereas a NOS substrate, L-arginine (500 µM), stimulated it. A neuronal NOS (nNOS)/inducible NOS (iNOS)-selective inhibitor, 1-(α,α,α-trifluoro-o-tolyl)-imidazole (TRIM; 100 µM), suppressed channel activity to the same extent as L-NAME. TRIM also blocked the stimulatory effect of Larginine. In contrast, an NO donor, sodium nitroprusside (10 µM) or 8-bromoguanosine 3',5'-cyclic monophosphate (100 µM) stimulated channel activity even in the presence of TRIM. RT-PCR revealed that iNOS mRNA alone was expressed in most of the cultures, i.e., 34 out of 40. In the other 6 cases, endothelial NOS (eNOS) and iNOS mRNA were simultaneously expressed. This finding was confirmed at the protein level by Western blotting. Indeed, in the patch-clamp experiments TRIM sometimes failed to suppress the channel activity, but the following addition of L-NAME suppressed it. However, since the suppressive effect of TRIM was usually similar to that of L-NAME, the involvement of eNOS in K + channel regulation would be relatively low. These results suggest that iNOS plays a pivotal role in the endogenous production of NO under the basal condition, which is involved in the activity of the inwardly rectifying K + channel in cultured human proximal tubule cells.
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