Mechanism of vascular smooth muscle cells activation by hydrogen peroxide: role of phospholipase C gamma

Background. Hydrogen peroxide (H2O2) formation is a critical factor in processes involving ischaemia/ reperfusion. However, the precise mechanism by which reactive oxygen species (ROS) induce vascular damage are insufficiently known. Specifically, activation of phospholipase C gamma (PLCγ) is a probable candidate pathway involved in vascular smooth muscle cells (VSMC) activation by H2O2. Methods. The activation of human venous VSMC was measured as cytosolic free calcium mobilization, shape change and protein phosphorylation, focusing on the role of tyrosine phosphorylation‐activated PLCγ. Results. The exposure of VSMC to exogenous H2O2 caused a rapid increase in cytosolic free calcium concentration ([Ca2+]i), and induced a significant VSMC shape change. Both effects were dependent on a tyrosine kinase‐mediated mechanism, as determined by the blockade of short‐term treatment of VSMC with the protein tyrosine kinase inhibitor, genistein. Giving further support to the putative role of phospholipase C (PLC)‐dependent pathways, the [Ca2+]i and VSMC shape change response were equally inhibited by the specific PLC blocker, 1‐(6‐((17‐beta‐methoxyestra‐1,3,5(10)trien‐17‐yl)amino)hexyl)‐1H‐pyrrole‐2,5‐dione (U73122). In addition, U73122 had a protective effect against the deleterious action (24 h) of H2O2 on non‐confluent VSMC. As a further clarification of the specific pathway involved, the exposure to H2O2 significantly stimulated the tyrosine phosphorylation of PLCγ with a concentration‐ and time‐profile similar to that of [Ca2+]i mobilization. Conclusions. The present study reveals that H2O2 activates PLCγ on VSMC through tyrosine phosphorylation and that this activation has a major role in rapid [Ca2+]i mobilization, shape‐changing actions and damage by H2O2 in this type of cells. These findings have direct implications for understanding the mechanisms of the vascular actions of H2O2 and may help to design pharmacologically protective strategies.