Abstract 5251: H 2 O 2 -induced Protein Kinase G Dimerization and Vasodilation in Human Coronary Arteriole

Abstract

Flow-induced dilation of human coronary arterioles (HCA) is mediated by a unique mechanism involving the release of H 2 O 2 from the mitochondria of endothelial cells and subsequent smooth muscle relaxation via K + -channel-dependent membrane hyperpolarization. The precise mechanisms by which H 2 O 2 induces smooth muscle hyperpolarization remain largely undefined. An important mechanism of action of H 2 O 2 involves the oxidation of key cysteine residues in its target proteins, including protein kinase G 1-alpha (PKG-1 α ). Here we hypothesize that H 2 O 2 dilates HCA through direct oxidation and activation of PKG-1 α leading to the subsequent opening of large-conductance Ca 2+ -activated K + channels (BK Ca ) in smooth muscle cells. In isolated HCA, H 2 O 2 (10 −6 −3×10 −4 M) induced dose-dependent dilations in both endothelium-intact and -denuded vessels (relaxations at 10 −4 M of 83.5±3.7% and 85.1±8.4%, respectively; n=4 – 6). The relaxations were largely abolished by iberiotoxin, a BK Ca blocker (3.4±2.1% and 19.0±10.2% in intact and denuded vessels, respectively). The PKG inhibitor Rp-8-Br-cGMP also markedly inhibited H 2 O 2 -induced dilations (4.5±6.0%; n=6), whereas the guanylyl cyclase inhibitor ODQ has no significant effects (75.6±8.4%; n=3). The expression of PKG-1 α and BK Ca channels in HCA smooth muscles were confirmed by immunohistochemistry and Western blot analysis. In cultured human coronary arterial smooth muscle cells, H 2 O 2 induced dose-dependent dimerization of PKG-1 α , which was subsequently reduced to the monomer forms by the reducing agent β -mecaptomethanol. We also examined flow-mediated dilation in HCA pretreated with iberiotoxin or Rp-8-Br-cGMP. Both agents markedly inhibited relaxation responses as compared to control (maximal relaxations of 36.0±3.8% vs. 69.2±6.8% in controls, and 30.1±4.8% vs. 57.8±3.0% in controls, respectively; n=4 –5). It is concluded that the activation of PKG-1 α via H 2 O 2 -induced protein dimerization and subsequent opening of BK Ca channels serves as a novel mechanism of flow-induced H 2 O 2 -mediated relaxations in HCA. This signaling pathway may have implications for the pathogenesis and therapy of coronary disease.