Activation of BKCa Channels in Rat Cerebrovascular Smooth Muscle Cells and Vasodilation Induced by Neurogenic H2S and Its Relationship with VEGFR2.

To explore the mechanism of action of CBS-derived H2S in inducing cerebral vasodilation and activating BKCa channels. Sprague-Dawley (SD) rat middle cerebral arteries (MCA) were isolated from rat brains, and a pressure myography system was used to measure the effects of different concentrations of L-cysteine (L-Cys, 1 × 10-5.5 to 1 × 10-3.5 mol/L), a substrate for cystathionine-β-synthase (CBS)-a hydrogen sulfide (H2S)-producing enzyme. Additionally, the effects of pretreatment with the CBS inhibitor amino-oxoacetate (AOAA, 1 mmol/L), the vascular endothelial growth factor receptor 2 inhibitor semaxanib (SU5416, 10 μmol/L), and the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin (IBTX, 100 nmol/L) were investigated to determine their impacts on CBS-derived H2S-induced vasodilation. Acute digestion of rat vascular smooth muscle cells (VSMCs) was performed, and whole-cell patch-clamp techniques were used to measure current changes in neurons or astrocytes (ASTs), as well as acutely digested VSMCs, in the presence of L-Cys, AOAA (1 mmol/L), SU5416 (10 μmol/L), and IBTX (100 nmol/L). Additionally, neurons or ASTs were co-cultured with VSMCs to determine CBS-derived H2S levels. Neurons or ASTs co-incubated with blood vessels and then treated with L-Cys produced H2S, which exhibited a concentration-dependent dilatory effect on middle cerebral artery occlusion (MCA) pre-contracted with 100 nmol/L U46619 (p < 0.01). However, the addition of AOAA significantly attenuated this dilatory effect (p < 0.01). SU5416 and IBTX significantly inhibited cerebral vascular dilation (p < 0.01). H2S produced by adding L-Cys after co-incubation of neurons or ASTs with VSMCs significantly increased BKCa channel current (p < 0.01). However, this effect was significantly attenuated after adding AOAA (p < 0.01). SU5416 and IBTX significantly inhibited the activation of BKCa channels (p < 0.01). Wild-type rat neurons or astrocytes (ASTs) were co-cultured with CSE(Cystathionine γ-lyase)-knockout vascular smooth muscle cells (VSMCs-CSE KO); the addition of L-Cys significantly increased hydrogen sulfide (H2S) levels in the co-culture system (p < 0.01), while the addition of AOAA reduced H2S production (p < 0.01). However, the addition of SU5416 had no statistical significance. Neurogenic H2S, the H2S produced by neurons and ASTs, could induce cerebral vasodilation in rats via VEGFR2(Vascular Endothelial Growth Factor Receptor 2)-mediated activation of BKCa channels in the smooth muscle cells.