| Under the physiological conditions, the vascular endothelium play a important role in the maintenance of vascular homeostasis by producing and releasing endothelium-derived vasoactive substances, such as NO, PGI2 and endothelium-derived hyperpolarizing factor (EDHF). Together with thromboxane and endothelin-A2 (TXA2) and other vascular contraction factors, they regulate vascular tension and local blood flow. The nature and mechanism of NO and PGI2 have been widely and in-depth studied, but the study of EDHF, particularly the study about role of EDHF in cerebral blood vessel was few.Hydrogen sulfide (H2S) has long been seen as a harmful gas molecules to health. Recent studies showed that H2S is an important member of the gasotransmitter family, and together with NO, carbon monoxide (CO) known as the "triumvirate." In mammalian cells, the mainly enzymes synthesis of H2S were cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). It is generally considered that CSE is the only enzyme which produced H2S in cardiovascular system. In the blood vessels, CSE not only expressed in vascular smooth muscle cells, but also expressed in vascular endothelial cells. The present study was to gain insight into whether H2S mediated the EDHF dilation in rat middle cerebral artery (MCA) or not. Purpose:1. To study whether H2S mediated the EDHF dilation in rat MCA or not.2. To explore the alteration of the EDHF-mediated dilation during cerebral ischemia/reperfusion in rat MCA.Methods:1. The relaxant effects of ACh, NaHS, L-Cys on isolated and perfused MCAs were detected by vasomotoricity experiment in vitro. The effects of Non-NO-non-PGI2 relaxation was induced by ACh in the presence of Nω-nitro-L–arginine-methyl-ester (L-NAME, 3×10-5mol/L) and indomethacin(Indo, 10-5mol/L). The effects of tetraethylammonium(TEA, 1×10-3mol/L) or DL-propargylglycine(PPG, 1×10-4 mol/L) on the non-NO-non-PGI2 relaxation were also observed.2. The rat MCAs was digested to get a mixture cell solution by using collagenaseⅡ, and the endothelial cells of MCAs were purified from the mixture cell solution by using magnetic cell separation system. RT-PCR method was used to detect the expression of CSEmRNA that is the synthase of endogenous H2S in the endothelial cells of rat MCAs.3. Focal cerebral ischemia/reperfusion injury was caused by middle cerebral artery occlusion (MCAO). The neurological functional disturbance score was observed, cerebral infart volume and H2S level in the cerebrum were detected, and the alteration of EDHF-mediated dilation during cerebral ischemia/reperfusion were also observed. Then used RT-PCR method to detect the effect of cerebral ischemia/reperfusion on the expression of CSEmRNA in the endothelial cells of rat MCAs. Results:1. In rat MCA preconstricted by 30mmol/L KCl, ACh(10-710-4.5 mol/L) produced concentration-dependent relaxation effect in the presense and absence of L-NAME(3×10-5 mol/L) and Indo(10-5 mol/L). ACh-induced vasorelaxation in the presense of L-NAME and Indo was markly inhibited by TEA (an inhibitor of Ca2+-sensitive K+ channel at 1×10-3mol/L). ACh-induced vasorelaxation in the presense of L-NAME and Indo was also significantly inhibited by 10-4 mol/L PPG (an inhibitor of CSE). NaHS, a donor of H2S had the concentration-dependent relaxation at the range of 10-5～10-2.5mol/L, and the vasorelaxation was obviously abolished by 1×10-3mol/L TEA. The addition of L-cysteine led to a concentration-dependent relaxation that was prevented by the CSE inhibitors PPG or the removal of endothelium.2. In the isolated and purified endothelial cells of rat MCAs, the band of CSEmRNA was detected by the RT-PCR method .3. During cerebral ischemia/reperfusion, EDHF-mediated dilation in rat MCA was potentialized obviously, the expression of CSEmRNA in rat MCAs endothelial cells is upregulated, and the H2S level in the cerebrum increased significantly.Conclusions:1. In the rat MCA, ACh-induced non-NO- non-PGI2 relaxation, the so-called EDHF dilation might be mediated by H2S.2. In the cerebral ischemia/reperfusion rat MCA, the EDHF-mediated dilation potentialized obviously.
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