1. Interleukin-1ß reduces K+ channel activity through phosphorylation processes mediated by protein kinase C in human kidney proximal tubule cells.
- Author
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Nakamura, K., Komagiri, Y., and Kubokawa, M.
- Subjects
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CYTOKINES , *CELLS , *KIDNEYS , *WOUNDS & injuries - Abstract
It is well known that proinflammatory cytokines, such as interleukin-1ß (IL-1ß), interferon-γ (IFN-γ), tumor necrosis factor-α, induce cell injury in various organs, including kidney. Some investigators reported that changes in K+ channel activity contributed to the renal tubular cell injury during ischemia or endotoxemia (1, 2). Thus, it is possible that the proinflammatory cytokines may cause cell injury partly by modulating K+ channel activity in the kidney. However, little information was available regarding the effects of cytokines on activity of renal K+ channels. In cultured human kidney proximal tubule cells purchased from Lonza (Walkersville, MD, USA) which isolated these cells under informed consent, an inwardly rectifying K+ channel with an inward conductance of 40 pS was most frequently observed under the control condition (3). We have reported that IFN-γ possessed an acute stimulatory effect and a delayed suppressive effect on activity of this 40 pS K+ channel (4). In this study, we investigated the effect of IL-1ß on activity of the 40 pS K+ channel in cultured human proximal tubule cells, using the patch-clamp technique and Fura-2 Ca2+ imaging. Values are means±S.E.M., compared by ANOVA. In cell-attached patches, IL-1ß (15 pg/ml) significantly reduced channel activity to 33±9 % of the control (n=10, p<0.01) in a few minutes. This acute suppressive effect was blocked by an IL-1 receptor antagonist (IL-1ra, 20 ng/ml), suggesting that the effect of IL-1ß was mediated by its specific receptor. An inhibitor of protein kinase C (PKC), GF109203X (500 nM), also blocked the acute suppressive effect of IL-1ß. In inside-out patches, effects of phorbol 12-myristate 13-acetate (PMA) and PKC were examined in the presence of ATP (1 mM) and Ca2+ (1 µM). While PMA (500 nM) alone had no appreciable effect, the subsequent addition of PKC (1 U/ml) reduced channel activity to 21±6 % of the control (n=6, p<0.01). Since it has been reported that phospholipase C (PLC) plays important role in activation of PKC by providing diacyl glycerol and inositol 1,4,5-triphosphate which in turn causes Ca2+ release from the intracellular store (5), we next examined the effect of a PLC inhibitor, neomycin, on the acute suppressive effect of IL-1ß in cell-attached patches. Neomycin (300 µM) completely blocked the suppressive effect of IL-1ß. Finally, we examined whether IL-1ß would actually increase intracellular Ca2+ concentration ([Ca2+]i). The Fura-2 Ca2+ imaging revealed that IL-1ß transiently caused a 1.5±0.2-fold increase in [Ca2+]i (n=9, p<0.01). In accordance with the patch-clamp experiments, this increase in [Ca2+]i was blocked by IL-1ra or neomycin. These results suggested that the acute suppressive effect of IL-1ß on activity of the 40 pS K+ channel in cultured human kidney proximal tubule cells would be dependent, at least in part, on the PKC-mediated phosphorylation processes. [ABSTRACT FROM AUTHOR]
- Published
- 2013