Lipopolysaccharide plus hypoxia and reoxygenation synergistically reduce electrical coupling between microvascular endothelial cells by dephosphorylating connexin40

Abstract

We showed that lipopolysaccharide (LPS) or hypoxia and reoxygenation (H/R) decreases electrical coupling between microvascular endothelial cells by targeting the gap junction protein connexin40 (Cx40), tyrosine kinase-, ERK1/2-, and PKA-dependently. Since LPS can compromise microvascular blood flow, resulting in micro-regional H/R, the concurrent LPS + H/R could reduce coupling to a much greater extent than LPS or H/R alone. We examined this possibility in a model of cultured microvascular endothelial cells (mouse skeletal muscle origin) in terms of electrical coupling and the phosphorylation status of Cx40. To assess coupling, we measured the spread of electrical current injected into the cell monolayer and computed the intercellular resistance as an inversed measure of coupling. In wild type cells, but not in Cx40 null cells, concurrent LPS + H/R synergistically increased resistance by approximately 270%, well above the level observed for LPS or H/R alone. Cx37 and Cx43 protein expression did not differ between Cx40 null and wild type cells. LPS + H/R increased resistance PKA- and PKC-dependently. By immunoprecipitating Cx40, we found that LPS + H/R reduced serine phosphorylation to a much greater degree than that observed for LPS or H/R alone. Further, PKA-specific, but not PKC-specific serine phosphorylation of Cx40 was also significantly reduced following LPS + H/R. This reduction was prevented by tyrosine kinase and MEK1/2 inhibition, by PKA activation, and mimicked in control cells by PKA inhibition. We conclude that LPS + H/R initiates tyrosine kinase- and ERK1/2-sensitive signaling that synergistically reduces inter-endothelial electrical coupling by dephosphorylating PKA-specific serine residues of Cx40.