INVOLVEMENT OF TYROSINE KINASE IN THE INDUCTION OF CYCLOOXYGENASE AND NITRIC-OXIDE SYNTHASE BY ENDOTOXIN IN CULTURED-CELLS

1 Cyclo-oxygenase (COX) and nitric oxide synthase (NOS) are two enzymes which have distinct cytokine-inducible isoforms (COX-2 and iNOS). Many cytokine receptors have an intracellular tyrosine kinase domain. Here we have used the tyrosine kinase inhibitors, erbstatin and genistein, to investigate the potential role of tyrosine kinase activation in the induction on COX-2 and iNOS caused by endotoxin (lipopolysaccharide; LPS) in bovine aortic endothelial cells (BAEC) and J774.2 macrophages. 2 The main COX metabolites, 6-oxo-prostaglandin F-1 alpha (6-oxo-PGF(1 alpha)) (for BAEC) and PGF(2 alpha) (for 774.2 macrophages) were measured by radioimmunossay: (i) accumulation of COX metabolites from endogenous arachidonic acid was measured at 24 h after addition of LPS (1 mu g ml(-1)); (ii) in experiments designed to measure 'COX activity', COX metabolites generated by BAEC or J774.2 macrophages activated with LPS were assayed (at 12 h after LPS administration) after incubation of the washed cells with exogenous arachidonic acid (30 mu M for 15 min). Western blot analysis with a specific antibody to COX-2 was used to determine the expression of COX-2 protein caused by LPS in cell extracts. Accumulation of nitrite (measured by the Griess reaction) was used as an indicator of NO formation and, hence, iNOS activity. 3 Erbstatin (0.05 to 5 mu g ml(-1)) or genistein (0.5 to 50 mu g ml(-1)) caused a dose-dependent inhibition of the accumulation of COX metabolites in the supernatant of BAEC or J774.2 macrophages activated with LPS. Erbstatin or genistein also caused a dose-dependent inhibition of 'COX activity' in both cell types. Western blot analysis showed that erbstatin (5 mu g ml(-1)) or genistein (50 mu g ml(-1)) inhibited the expression of COX-2 protein in BAEC and J774.2 macrophages activated with LPS (1 mu g ml(-1) for 24 h). 4 Erbstatin or genistein also caused a dose-dependent inhibition of nitrite accumulation in J774.2 macrophages activated with LPS (1 mu g ml(-1) for 24h). In contrast to J774.2 macrophages, BAEC stimulated with LPS (1 mu g ml(-1) for 24 h) did not produce detectable amounts (<1 mu M) of nitrite. 5 These results suggest that tyrosine phosphorylation is part of the signal transduction mechanism that mediates (i) the induction of COX-2 and iNOS elicited by LPS in J774.2 macrophages, and (ii) the induction of COX-2 by LPS in BAEC.