Investigation of calcium sensitizing mechanisms and enzymes responsible for synthesis of hydrogen sulfide in the relaxant effect of bovine retinal artery

Hydrogen sulfide (H2S) is a gasotransmitter expressed in ocular tissues and involved in regulation of ocular vascular tone. NaHS, a H2S donor, induces vasodilation in bovine retinal artery partially via Kv and Kir channel activation. In this study we aimed to investigate the role of extracellular Ca2+, Ca2+ sensitization and the possible mechanisms and also to determine the levels of the enzymes responsible for the synthesis of H2S (cystathionine beta synthase-CBS, cystathionine gamma lyase-CSE, 3-mercapturuvate sulphidetransferase-3-MST) and distributions (CBS, CSE) in retina and retinal artery. The role of extracellular Ca2+ was assessed at CaCl2 or PGF2α precontracted arteries in the absence of Ca2+, the role of Ca2+ sensitization pathways were investigated in the presence of specific inhibitors. The protein levels of RhoA / Rho kinase, myosin phosphatase (MP) enzymes and protein-type 1 phosphatase inhibitor protein CPI-17,which have role in Ca2+ sensitization mechanisms were investigated by Western blot. ELISA and immunofluorescence microscopic analyzes were performed to determine the presence, amount and distrubution of the enzymes that synthesized H2S in the retina and retinal artery. As a result, it is shown that extracellular Ca2+, myosin light chain kinase, protein kinase C, tyrosine kinase, RhoA/Rho kinase and CPI-17 did not play a role in the vasodilator effect of NaHS. Additionally, incubation with MP inhibitor significantly reduced the relaxation effect of NaHS. Incubation of the arteries with PGF2α + NaHS showed that the MYPT1 subunit of MP phosphorylation levels reduced significantly compared to the control and only PGF2α incubated arteries. Besides, the presence of all three enzymes, particularly CSE, were found in retina and retinal artery and they were found to be higher in retinal artery than the retina. Moreover, CBS was shown to be present in the endothelial and smooth muscle layer of the retinal artery whereas, CSE in the endothelium. In terms of distribution, in the retina, CBS and CSE were found in cone-basil cells and retinal ganglion cells, besides, CSE radiating in bipolar cells. Our findings suggest that the mechanism responsible for the relaxation effect of NaHS is mainly MP activation and that H2S is synthesized endogenously in the retina and retinal artery. Our study, showing the presence of H2S in retinal artery and its contribution to vascular tone proposes that H2S may have protective or therapeutic effects in retinal vascular diseases. Key Words: Hydrogen sulfide, Ca2+ sensitization, retinal artery, retina, vasodilation. This study was funded by Scientific Research Projects Coordination Unit of Istanbul University and TUBITAK. BAP Project Number: TDK-2018-29789 and TUBITAK Project Number: 115S196.