H 2 O 2 -Induced Dilation in Human Coronary Arterioles: Role of Protein Kinase G Dimerization and Large-Conductance Ca 2 (cid:1) -Activated K (cid:1) Channel Activation

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)-I (cid:2) , thereby modulating their activities. Objective: Here we hypothesize that H 2 O 2 dilates HCAs through direct oxidation and activation of PKG-I (cid:2) leading to the opening of the large-conductance Ca 2 (cid:1) -activated K (cid:1) (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-I (cid:2) , and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K (cid:1) 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-I (cid:2) 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-I (cid:2) and subsequent opening of smooth muscle BK Ca channels. ( Circ Res . 2012;110:471-480.) Student–Newman–Keuls multiple- comparisons test. To compare concentration-response between treatment groups, we used a 2-way repeated-measures ANOVA. Probability values of P (cid:7) 0.05 were considered statistically significant. -channel activity, we demonstrate that H 2 O 2 -induced dilation of human coronary arterioles depends on increased opening of BK Ca channels. In particular, our patch-clamp studies showed that H 2 O 2 activated BK Ca currents recorded from the cell-attached patches (intracel-lular components present) but not from inside-out patches (intracellular components absent) and that the stimulatory effect of H 2 O 2 on BK Ca activity was markedly reduced by the PKG-I (cid:1) inhibitor DT-2. Taken together, these data provided strong evidence that H 2 O 2 -induced BK Ca channel opening requires intracellular signaling, ie, the activation of PKG-I (cid:1) in smooth muscle cells. 2 relaxes coronary arterioles remain unclear. In this study, we found that exogenous H 2 O 2 dilates human coronary arterioles by opening smooth muscle BK Ca channels through dimerization and transloca- tion of PKG-I (cid:1) . Flow or shear stress, which releases endothelium-derived H 2 O 2 , dilates coronary arterioles by activating a signaling pathway similar to that induced by exogenous H 2 O 2 . Our findings provide further support for the proposed role of H 2 O 2 as a diffusible endothelium-derived hyperpolarizing factor in both animals and humans and have implications for antioxidant treatment strategies in patients with coronary disease.