Abstract MP06: The Protein Tyrosine Phosphatase Inhibitors Induce The Vasoconstriction Via Src/epidermal growth factor/rho-kinase Cascade

Abstract

Orthovanadate (OVA), a protein tyrosine phosphatase (PTPase) inhibitor, exerts contractile effects on smooth muscle in a Rho-kinase-dependent manner, but the precise mechanisms are not elucidated. The aim of this study was to determine the potential roles of Src and epidermal growth factor receptor (EGFR) in the OVA-induced contraction of rat aortas and the phosphorylation of myosin phosphatase target subunit 1 (MYPT1; an index of Rho-kinase activity) in vascular smooth muscle cells (VSMCs). The concentration-response curves for OVA (8–1000μM) were constructed in endothelium-denuded rings using stepwise increases in the concentration of OVA, and the maximal force was observed at concentrations of >500 μM (16 ± 2 in % of 60 mmol/L KCl). OVA (500 μM) evoked rapid contraction of endothelium-denuded aortas, presenting as a hyperbolic rise in tension development within 10 min followed by a slow linear rise. Aortic contraction by OVA was significantly blocked not only by Rho kinase inhibitors Y-27632 and hydroxyfasudil, but also by Src inhibitors PP2 and Src inhibitor No. 5 and the EGFR inhibitors AG1478 and EGFR inhibitor 1. OVA induced rapid increases in the phosphorylation of MYPT1 (Thr-853; 180 ± 24 % in OVA vs 98 ± 22 in vehicle), Src (Tyr-416; 225 ± 34 % in OVA vs 102 ± 30 in vehicle), and EGFR (Tyr-1173; 173 ± 31 % in OVA vs 101 ± 18 in vehicle) in VSMCs, and Src inhibitors abolished these effects. OVA-induced Src phosphorylation was abrogated by Src inhibitors, but not affected by inhibitors of EGFR and Rho-kinase. Inhibitors of Src and EGFR, but not Rho-kinase, also blocked OVA-induced EGFR phosphorylation. Furthermore, a metalloproteinase inhibitor TAPI-0 and an inhibitor of heparin-binding EGF not only abrogated the OVA-induced aortic contraction, but also OVA-induced EGFR and MYPT1 phosphorylation, suggesting the involvement of EGFR transactivation. OVA also induced EGFR phosphorylation at Tyr-845, one of residues phosphorylated by Src. These results suggest that OVA-induced vasocontraction is mediated by the Rho-kinase-dependent inactivation of myosin light chain phosphatase via signaling downstream of Src-induced transactivation of EGFR.