Hydrogen sulfide-mediated vasodilation requires heme oxygenase-derived carbon monoxide

<jats:p> Background: Hydrogen sulfide (H <jats:sub>2</jats:sub> S) is an important endothelial-derived vasodilator, but the signaling mechanism remains incompletely understood. We previously demonstrated that H <jats:sub>2</jats:sub> S-mediated vasodilation requires transient receptor potential vanilloid type 4 (TRPV4) channels. Because H <jats:sub>2</jats:sub> S has been reported to enhance heme oxygenase (HO) activity and HO-derived carbon monoxide (CO) regulates endothelial signaling, we hypothesized that H <jats:sub>2</jats:sub> S-mediated vasodilation requires HO-2-derived CO. Methods: Pressure myography was performed in isolated rat mesenteric arteries to determine the contribution of HO, TRPV4, eBK, and SK/IK channels to H <jats:sub>2</jats:sub> S-mediated vasodilation. HO-2 sulfhydration was assessed using a maleimide assay, and spatial association among HO-2 and TRPV4 was examined using proximity ligation assays in human aortic endothelial cells. Results: H <jats:sub>2</jats:sub> S elicited concentration-dependent vasodilation that was abolished by HO inhibition. Repletion of CO restored H <jats:sub>2</jats:sub> S-mediated vasodilation in the presence of HO inhibition. CO-mediated vasodilation was abolished by TRPV4 and SK/IK inhibition but was unaffected by eBK inhibition. H <jats:sub>2</jats:sub> S increased HO-2 sulfhydration and enhanced HO activity. In endothelial cells, HO-2 and TRPV4 exhibited close spatial association. Conclusions: These findings support a model in which H <jats:sub>2</jats:sub> S stimulates HO-2-derived CO production, leading to TRPV4-dependent endothelial signaling, SK/IK activation, and vasodilation. Together, the data support the existence of an endothelial HO-2/TRPV4/SK/IK signaling domain that contributes to H <jats:sub>2</jats:sub> S-mediated vascular reactivity. </jats:p>