Involvement of tyrosine kinase and protein kinase C in the induction of nitric oxide synthase by lipopolysaccharide and interferon-gamma in J774 macrophages.

The combination of lipopolysaccharide (LPS; 100 ng/ml) and interferon-gamma (IFN-gamma; 10 IU/ml) synergistically stimulated induction of nitric 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 combination, stimulated accumulation of nitrite. They were also unable to substitute 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 concentration-dependent inhibition of nitrite accumulation when added prior to stimulation with LPS and IFN-gamma, but enhanced nitrite accumulation when added 12 hours following stimulation with LPS and IFN-gamma. 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 powerful, concentration-dependent inhibition of nitrite accumulation when added 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 only a slight inhibition of nitrite accumulation when added prior to stimulation with LPS and IFN-gamma, but slightly enhanced nitrite accumulation when added 12 hours following stimulation with LPS and IFN-gamma. The tyrosine kinase inhibitors, genistein (10(-7) - 10(-4) M) and herbimycin A (5.2 x 10(-9) - 1.74 x 10(-6) M), produced a powerful concentration-dependent inhibition of nitrite accumulation when added prior to stimulation with LPS and IFN-gamma. In contrast, herbimycin A had only a slight inhibitory effect when added 12 hours following stimulation with LPS and IFN-gamma, and genistein had no effect. When used in combination 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 inhibitory effects on nitrite accumulation, but herbimycin A, together with Ro 31-8220 (3 x 10(-6) M) or chelerythrine chloride (10(-5) M), produced no further effects. These results provide strong evidence for the involvement of tyrosine kinases in the induction of nitric oxide synthase by LPS and IFN-gamma in J774 macrophages. They also suggest a role for protein kinase C, but elucidation of the precise mechanisms by which this pathway interacts with tyrosine kinase to regulate the expression of nitric oxide synthase requires further investigation.