H2O2-Induced Dilation in Human Coronary Arterioles: Role of Protein Kinase G Dimerization and Large-Conductance Ca2+-Activated K+ Channel Activation

Rationale: Hydrogen peroxide (H2O2) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H2O2 elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H2O2 involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-I alpha, thereby modulating their activities. Objective: Here we hypothesize that H2O2 dilates HCAs through direct oxidation and activation of PKG-I alpha leading to the opening of the large-conductance Ca2+-activated K+ (BKCa) channel and subsequent smooth muscle hyperpolarization. Methods and Results: Flow and H2O2 induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BKCa channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H2O2-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H2O2 elicited dose-dependent, reversible dimerization of PKG-I alpha, and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K+ current with a unitary conductance of 246-pS in freshly isolated coronary SMCs. Addition of H2O2 into the bath solution significantly increased the probability of BKCa single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-I alpha inhibitor DT-2. H2O2 exhibited an attenuated stimulatory effect on BKCa channel open probability in inside-out membrane patches. Conclusions: H2O2 dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-I alpha and subsequent opening of smooth muscle BKCa channels. (Circ Res. 2012;110:471-480.)