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

Endothelial cells synthesize numerous vasoactive compounds, including carbon monoxide (CO), nitric oxide (NO), and hydrogen sulfide (H2S). The mechanism of H2S-mediated vasodilation, as well as potential interactions with NO and CO, has not been established. Our previous work demonstrated that endothelium-dependent dilation by H2S requires activation of both transient receptor potential vanilloid type 4 channels (TRPV4) and endothelial large-conductance calcium-sensitive potassium channels (eBK). However, the mechanism by which H2S activates TRPV4/eBK channels is unclear. We also demonstrated that both endothelial TRPV4 and heme oxygenase (HO)-generated CO regulate eBK activity. In addition, H2S has been shown to regulate HO activity by reacting with a ferric verdoheme intermediate of HO, thereby increasing HO activity. Thus, we hypothesized that H2S-mediated vasodilation requires HO-2 derived CO. Methods: Pressure myography was conducted using isolated mesenteric arteries (160 mm – 200 mm) from male Sprague-Dawley rats (Envigo 150 - 300g, N=5 animals/group) to assess the effect of HO inhibition (Chromium mesophorphin, 1 uM) on H2S-mediated dilation (vehicle control). CO levels were repleted with a sub-vasoactive concentration of the CORM-3 (100 nM). The order of experimental treatments was alternated. Sulfhydration of HO-2 was detected using the maleimide assay (N=5 animals/group). Protein-protein co-localization (TRPV4, HO-2, eBK) was qualitatively determined using proximity ligation assays in human aortic endothelial cells. Data are presented as Mean ± SD. Results: In pressurized, U46619-constricted mesenteric arteries, H2S elicited a dose-dependent vasodilation blocked by inhibition of HO. Repletion of CO with the CO-donor, CORM-3, in the presence of HO-inhibition restored the H2S-mediated vasodilation. Treatment with H2S increased HO-2 sulfhydration. Using a proximity ligation assay, we observed co-localization between HO-2, TRPV4, and eBK channels in human aortic endothelial cells. Conclusions: Taken together, our results support the postulate that a HO-2/TRPV4/eBK signaling domain exists in endothelial cells. H2S-induced CO production may be the link between H2S and TRPV4/eBK activation, leading to subsequent vasodilation. Funding: R01 HL160606-04 (J.S.N), UNM Comprehensive Cancer Center Fluorescence Microscopy Shared Resource Facility, NCI P30CA118100. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.