H 2 O 2 -Induced Dilation in Human Coronary Arterioles: Role of Protein Kinase G Dimerization and Large-Conductance Ca 2+ -Activated K + Channel Activation

Abstract

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