Abstract 5251: H2O2-induced Protein Kinase G Dimerization and Vasodilation in Human Coronary Arteriole
Flow-induced dilation of human coronary arterioles (HCA) is mediated by a unique mechanism involving the release of H2O2 from the mitochondria of endothelial cells and subsequent smooth muscle relaxation via K+-channel-dependent membrane hyperpolarization. The precise mechanisms by which H2O2 induces smooth muscle hyperpolarization remain largely undefined. An important mechanism of action of H2O2 involves the oxidation of key cysteine residues in its target proteins, including protein kinase G 1-alpha (PKG-1α). Here we hypothesize that H2O2 dilates HCA through direct oxidation and activation of PKG-1α leading to the subsequent opening of large-conductance Ca2+-activated K+ channels (BKCa) in smooth muscle cells. In isolated HCA, H2O2 (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 BKCa 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 H2O2-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-1α and BKCa channels in HCA smooth muscles were confirmed by immunohistochemistry and Western blot analysis. In cultured human coronary arterial smooth muscle cells, H2O2 induced dose-dependent dimerization of PKG-1α, 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-1α via H2O2-induced protein dimerization and subsequent opening of BKCa channels serves as a novel mechanism of flow-induced H2O2-mediated relaxations in HCA. This signaling pathway may have implications for the pathogenesis and therapy of coronary disease.