Article ID:
Nitric oxide (NO) modulates activity of an inwardly rectifying K+ channel in cultured human proximal tubule cells. In this study, we investigated which NO synthase (NOS) isoform(s) were 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 non-selective NOS inhibitor, Nω-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 L-arginine. 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 cases out of 40. In 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, 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 activity of the inwardly rectifying K+ channel in cultured human proximal tubule cells.
