Lipopolysaccharide-induced reductions in cellular coupling correlate with tyrosine phosphorylation of connexin 43

We have previously shown in cultured rat microvascular endothelial cells (RMEC) that lipopolysaccharide (LPS) stimulates a protein tyrosine kinase (PTK)-dependent reduction in cellular coupling. We hypothesized that connexin 43 (Cx43) becomes phosphorylated following exposure to LPS. Cx43 was immunoprecipitated from control and LPS-treated RMEC monolayers. Tyrosine phosphorylation of Cx43, detected by immunoblot, was found only in the LPS treatment. To verify these results, Cx43 was radiolabeled with [P-32]-orthophosphate. Radiolabeled Cx43 exhibited a slight increase in phosphorylation in response to LPS; phosphoamino acid analysis displayed equivalent amounts of phosphoserine in control and LPS treatments, but detected phosphotyrosine only in the LPS treatment. The PTK inhibitors PP-2 (10 nM) and geldanamycin (200 nM) were found to block the response to LPS in terms of Cx43 tyrosine phosphorylation and cellular coupling. The phosphatase inhibitor BpV (1 muM) accentuated the effect of LPS, while the putative phosphatase activator C-6-ceramide prevented it. When measuring cell communication, phosphatase inhibition also blocked the reversal of the LPS response following LPS washout. We conclude that Cx43 is tyrosine phosphorylated following exposure to LPS and suggest that the LPS-induced increase in intercellular resistance maybe mediated by tyrosine phosphorylation of this con nexin. Altering tyrosine kinase and phosphatase activities can modulate the LPS-induced tyrosine phosphorylation of Cx43 and reductions in cellular coupling.