Mediation of phorbol ester-induced oxidant generation in murine epidermal cells by protein kinase C

Murine epidermal cells have previously been shown to produce an oxidant response to the mouse skin tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA); however, the cellular source of these oxidants has not been well characterized. The demonstration that phospholipase C also elicits this oxidant response suggests that protein kinase C is the common mediator. In order to pursue this hypothesis, studies using protein kinase C inhibitors were carried out; both the induced oxidant response and the induction of ornithine decarboxylase (ODC), a known protein kinase C mediated event, were studied. At 100 μm-palmitoylcarnitine inhibited TPA-induced ODC by 50% and phospholipase C-induced ODC by 95%. At the same dose level, the negative analogue acetylcarnitine had no inhibitory effect on ODC with either inducer. The protein kinase C inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H-7) and its negative analogue N-(2-guanidinoethyl)-5-isoquinolinesulphonamide hydrochloride (HA-1004) were also used. At 100 μm, H-7 completely inhibited both TPA and phospholipase C-induced ODC; at the same dose HA-1004 had no effect. When these agents were included in the chemiluminescence assay for oxidant generation, similar results were seen: at 100 μm-palmitoylcarnitine and H-7, but not acetylcarnitine or HA-1004, suppressed the response by nearly 100%. These results suggest that the oxidant response to TPA in epidermal cells is mediated at least in part by protein kinase C.