SYNERGISTIC COOPERATION BETWEEN PHORBOL ESTER AND IFN-GAMMA FOR INDUCTION OF NITRIC-OXIDE SYNTHESIS IN MURINE PERITONEAL-MACROPHAGES

The role of protein kinase C (PKC) in the induction of nitric oxide (N O) synthesis in murine peritoneal macrophages was examined. Phorbol es ter, a PKC activator, had no effect on NO synthesis by itself, whereas IFN-gamma alone had modest activity. When phorbol ester was used in c ombination with IFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesi s was reflected as increased amount of inducible NO synthase (iNOS) mR NA, as determined by Northern blotting. The optimal effect of phorbol ester was shown at 6 h after treatment with IFN-gamma. Phorbol ester a lso induced the release of NO to the incubation medium by bacillus Cal mette-Guerin-infected peritoneal macrophages. Prolonged incubation of cells with phorbol ester, which down-regulates PKC activity, abolished the synergistic cooperative effect on NO production with IFN-gamma. I n addition, such PKC inhibitors as staurosporin or polymyxin B reduced NO production induced by lFN-gamma plus phorbol ester. When the cells were treated with both actinomycin D and phorbol ester after IFN-gamm a stimulation, more NO was produced and more iNOS mRNA was expressed t han in the cells treated with actinomycin D alone. On the basis of the se observations, we conclude that PKC might not be directly involved i n the expression of NO synthase, but, instead, might be involved in th e stabilization of the iNOS mRNA already expressed by the treatment of IFN-gamma.