Delayed and acute effects of interferon-γ on activity of an inwardly rectifying K⁺ channel in cultured human proximal tubule cells

Abstract

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.

Effects of cytokines on potassium channels in renal tubular epithelia

“…channels in distal nephron segments, those in the proximal tubule cells have recently been reported to be affected by cytokines [26,27]. In cultured human renal proximal tubule epithelial cells (RPTECs) isolated from the normal kidney, an inwardly rectifying K ?…”

“…channel [26,27]. The IFN-c-induced suppression and stimulation of the channel are highly likely dependent on the IFN-c receptor and its closely related janus kinase (JAK), since the JAK inhibitors blocked both effects [27]. The delayed suppression of channel activity was mediated, at least in part, by nitric oxide (NO) [26,27].…”

“…channel; a protein kinase G (PKG)-dependent stimulatory effect at micromolar concentrations and a peroxynitrite-dependent suppressive effect at higher millimolar concentrations [29]. Incubation of RPTECs with IFN-c for 24 h augments the expression of inducible NO synthase (iNOS), which results in production of a large amount of NO [26,27]. The excessive NO then reacts with superoxide to form peroxynitrite, which in turn impairs K ?…”

“…The excessive NO then reacts with superoxide to form peroxynitrite, which in turn impairs K ? channel activity through oxidation or nitrosylation of the channel and/or its related proteins [27]. On the other hand, the mechanisms involved in the acute stimulatory effect of IFN-c are still unknown.…”

“…The activity of the 40 pS K ? channel is stimulated by protein kinase A (PKA) [28] and phosphatidylinositol-3-kinase [27], as well as PKG [30]. However, IFN-c seems to circumvent these pathways to stimulate the channel activity [27].…”