Possible role of protein kinase C-ε isoenzyme in inhibition of interleukin 1β induction of nitric oxide synthase in rat renal mesangial cells

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Abstract
In cultured glomerular mesangial cells, interleukin 1 beta (IL-1 beta) has been shown to induce a dose- and time-dependent accumulation of nitrite, a stable metabolite of nitric oxide (NO). In parallel, increased levels of mRNA of an inducible macrophage-type of nitric oxide synthase (iNOS) were observed after incubating mesangial cells with IL-1 beta. Here we report that addition of the biologically active phorbol esters, phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), dose-dependently inhibited the IL-1 beta-stimulated increase in iNOS mRNA levels and nitrite production. In contrast, the biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate, had no effect on cytokine induction of iNOS and nitrite formation. Incubation of mesangial cells with PMA or PDBu alone, in the absence of IL-1 beta, did not trigger any iNOS expression. Time-course studies indicated that phorbol ester needs to be added for only 1 h in order to maximally inhibit cytokine-induced nitrite production. Down-regulation of protein kinase C (PKC)-alpha and -delta isoenzymes by 8 h PMA or PDBu treatment before stimulation with IL-1 beta still resulted in full inhibition of iNOS induction. In contrast, a 24 h treatment of mesangial cells with PMA or PDBu, a regimen that also causes depletion of PKC-epsilon, abolished inhibition of IL-1 beta-induced iNOS expression and nitrite production. In addition, the selective PKC inhibitor calphostin C potentiated IL-1 beta induction of iNOS activity. In summary these data suggest that IL-1 beta induction of iNOS expression is tonically suppressed by PKC and the epsilon-isoenzyme is the most likely candidate mediating this effect.

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