Department of Physiology, Shandong University School of Medicine, Jinan, China.
J Sex Med. 2012 Nov;9(11):2806-13. doi: 10.1111/j.1743-6109.2012.02879.x. Epub 2012 Aug 20.
Hydrogen sulfide (H(2) S) is generated in mammalian cells mainly by one of the two pyridoxal-5'-phosphate-dependent enzymes, cystathione-γ-lyase (CSE), and cystathione-β-synthase (CBS) using L-cysteine as the main substrate. In previous studies, we found that CBS and CSE were functionally expressed in vas deferens (VD) and H(2) S-mediated VD smooth muscle relaxation. However, the detail mechanisms that H(2) S-relaxed VD smooth muscle were unknown so far.
The aim of this study is to explore the molecular target sites of H(2) S in VD smooth muscle.
Isolated rat VD smooth muscle strips were used for tension recording in vitro. Double immunofluorescence staining was used to identify the localization of large conductance Ca(2+) -activated K(+) (BK(Ca)) channels.
Changes in tonic contraction of isolated rat VD smooth muscle strip were measured after the treatment of drugs. The expression of BKca channels in rat VD smooth muscle cells was also assessed.
The results showed that L-NG-nitroarginine methyl ester (a nitric oxide synthase inhibitor) did not affect the response of VD to sodium hydrosulphide (NaHS), suggesting that nitric oxide pathway was not involved. Further studies revealed that transient receptor potential (TRP) channels did not contribute to the NaHS-induced relaxant effect. Glibenclamide, an ATP-sensitive K channel blocker, did the same thing, whereas BK(Ca) channel blockers iberiotoxin or tetraethylammonium largely reversed the relaxant effect, suggesting that H(2) S may target BK(Ca) channels. We also confirmed that BK(Ca) channels were localized in VD smooth muscle cells. Then, studies revealed that NaHS-induced VD smooth muscle relaxation was abolished by N-ethylmaleimide, which was widely used as a sulfhydryl alkylation compound protecting thiols from oxidation, whereas DL-Dithiothreitol, a strong reducing agent, did not affect the response of VD to NaHS.
We concluded that H(2) S relaxed the VD smooth muscle by targeting BK(Ca) channels via redox-mediated mechanism.
在哺乳动物细胞中，硫化氢（H₂S）主要由两种吡哆醛-5'-磷酸依赖性酶之一胱硫醚-γ-裂合酶（CSE）和胱硫醚-β-合酶（CBS）生成，以 L-半胱氨酸为主要底物。在之前的研究中，我们发现 CBS 和 CSE 在输精管（VD）中具有功能表达，并且 H₂S 介导的 VD 平滑肌松弛。然而，迄今为止，H₂S 松弛 VD 平滑肌的详细机制尚不清楚。
本研究旨在探讨 VD 平滑肌中 H₂S 的分子靶位。
采用离体大鼠 VD 平滑肌条进行张力记录。采用双重免疫荧光染色法鉴定大电导钙激活钾（BK（Ca））通道的定位。
药物处理后，测量离体大鼠 VD 平滑肌条的紧张性收缩变化。还评估了大鼠 VD 平滑肌细胞中 BKca 通道的表达。
结果表明，L-NG-硝基精氨酸甲酯（一氧化氮合酶抑制剂）对 VD 对硫氢化钠（NaHS）的反应没有影响，提示一氧化氮途径不参与其中。进一步的研究表明，瞬时受体电位（TRP）通道对 NaHS 诱导的松弛作用没有贡献。ATP 敏感性钾通道阻滞剂格列本脲也有同样的作用，而 BK（Ca）通道阻滞剂 Iberiotoxin 或四乙铵则大大逆转了松弛作用，提示 H₂S 可能靶向 BK（Ca）通道。我们还证实 BK（Ca）通道定位于 VD 平滑肌细胞中。然后，研究表明，NaHS 诱导的 VD 平滑肌松弛被 N-乙基马来酰亚胺消除，N-乙基马来酰亚胺被广泛用作保护巯基不被氧化的巯基烷化化合物，而强还原剂 DL-二硫苏糖醇则不影响 VD 对 NaHS 的反应。
我们的结论是，H₂S 通过氧化还原介导的机制靶向 BK（Ca）通道来松弛 VD 平滑肌。
