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.
Eason
S
S
Clinical Research Initiative, University of Glasgow, United Kingdom.
Martin
W
W
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D007371:Interferon-gamma / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins
D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C