Lipopolysaccharide mediated increase in intercellular resistance 13 tyrosine phosphorylation dependent

Introduction: We have previously shown that lipopolysaccharide (LPS, E.coli055:B5) mediates a 50% increase in intercellular resistance (Ri) in isolated rat microvascular endothelial cell(RMEC). We are testing the hypothesis that a tyrosine kinase pathway is involved in the LPS mediated regulation of gap junction coupling. Methods: RMECs were isolated from adult male Wistar rats by immunoisolation using magnetic beads. RMECs in passage 5-15 were treated ± LPS (10 μg/ml) and various inhibitors for 2 hours. Intercellular resistance (a measure of cellular coupling) was measured by the degree of spatial decay in a cell monolayer of a current injected into one cell. Results: Inhibitors of receptor tyrosine kinase (Tyrphostin A9; Tyr), cytosolic tyrosine kinase (PP2) inhibited the LPS mediated increase in Ri. The tyrosine phosphatase activator (Ceramide; Cer) also inhibited this response, but the tyrosine phosphatases inhibitor BpV enhanced the LPS response. None of these agents affected intercellular resistance on their own. Conclurions: RMEC activation by LPS is dependent on a tyrosine kinase pathway where an increased phosphorylation results in higher Ri. This suggest a possible role for gap junctional phosphorylation. Treatment Concentration RI(MΩ) n Control 3.2 ± 0.1 64 LPS 5.3 ± 0.1*64 LPS + Tyr 1.5μM 3.3 ± 0.2 12 LPS + PP2 10μM 3.5 ± 0.1 12 LPS + Cer 10μM 3.9 ± 0.1 12 LPS + BpV 1μM 7.3 ± 0.2**12 (*p<0.05 compared to control;**p<0.05 compared to IPS alone; all values are means ± SEM).