INVOLVEMENT OF TYROSINE KINASE AND PROTEIN-KINASE-C IN THE INDUCTION OF NITRIC-OXIDE SYNTHASE BY LIPOPOLYSACCHARIDE AND INTERFERON-GAMMA INJ774 MACROPHAGES

The combination of lipopolysaccharide (LPS; 100 ng/ml) and interferon- gamma (IFN-gamma; 10 IU/ml) synergistically stimulated induction of ni tric oxide synthase activity in J774 macrophages, measured by nitrite accumulation during an overnight incubation. Neither the phorbol ester , phorbol 12-myristate 13-acetate (PMA; 10(-9) - 3 x 10(-6) M) nor the calcium ionophore, A23187 (10(-7) - 10(-4) M), alone or in combinatio n, stimulated accumulation of nitrite. They were also unable to substi tute for IFN-gamma in priming J774 macrophages to stimulation with LPS . Phorbol 12-myristate 13-acetate (10(-9) - 3 x 10(-6) M) produced a c oncentration-dependent inhibition of nitrite accumulation when added p rior to stimulation with LPS and IFN-gamma, but enhanced nitrite accum ulation when added 12 hours following stimulation with LPS and IFN-gam ma. Of the protein kinase C inhibitors tested, staurosporine (10(-9) - 3 x 10(-6) M) and Ro 31-8220 (3 x 10(-9) - 10(-5) M) produced a power ful, concentration-dependent inhibition of nitrite accumulation when a dded prior to stimulation with LPS and IFN-gamma, but had only slight inhibitory effects when added 12 hours after stimulation with LPS and IFN-gamma. Chelerythrine chloride (10(-8) - 3 x 10(-5) M) produced onl y a slight inhibition of nitrite accumulation when added prior to stim ulation with LPS and IFN-gamma, but slightly enhanced nitrite accumula tion when added 12 hours following stimulation with LPS and IFN-gamma. The tyrosine kinase inhibitors, genistein (10(-7) - 10(-4) M) and her bimycin A (5.2 x 10(-9) - 1.74 x 10(-6) M), produced a powerful concen tration-dependent inhibition of nitrite accumulation when added prior to stimulation with LPS and IFN-gamma. In contrast, herbimycin A had o nly a slight inhibitory effect when added 12 hours following stimulati on with LPS and IFN-gamma, and genistein had no effect. When used in c ombination prior to stimulation with LPS and IFN-gamma, herbimycin A ( 1.7 x 10(-7) M) and staurosporine (3 x 10(-8) M) produced additive inh ibitory effects on nitrite accumulation, but herbimycin A, together wi th Ro 31-8220 (3 x 10(-6) M) or chelerythrine chloride (10(-5) M), pro duced no further effects. These results provide strong evidence for th e involvement of tyrosine kinase in the induction of nitric oxide synt hase by LPS and IFN-gamma in J774 macrophages. They also suggest a rol e for protein kinase C, but elucidation of the precise mechanisms by w hich this pathway interacts with tyrosine kinase to regulate the expre ssion of nitric oxide synthase requires further investigation.