Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100191, China.
Cardiovasc Res. 2011 Jun 1;90(3):565-72. doi: 10.1093/cvr/cvr016. Epub 2011 Jan 19.
Soluble guanylyl cyclase (sGC) is a heterodimer. The dimerization of the enzyme is obligatory for its function in mediating actions caused by agents that elevate cyclic guanosine monophosphate (cGMP). The present study aimed to determine whether sGC dimerization is modulated by thiol-reducing agents and whether its dimerization influences relaxations in response to nitric oxide (NO).
The dimers and monomers of sGC and cGMP-dependent protein kinase (PKG) were analysed by western blotting. The intracellular cGMP content was measured by enzyme-linked immunosorbent assay. Changes in isometric tension were determined in organ chambers. In isolated porcine coronary arteries, the protein levels of sGC dimer were decreased by the thiol reductants dithiothreitol, l-cysteine, reduced l-glutathione and tris(2-carboxyethyl) phosphine. The effect was associated with reduced cGMP elevation and attenuated relaxations in response to nitric oxide donors. The dimerization of sGC and activation of the enzyme were also decreased by dihydrolipoic acid, an endogenous thiol antioxidant. Dithiothreitol at concentrations markedly affecting the dimerization of sGC had no significant effect on the dimerization of PKG or relaxation in response to 8-Br-cGMP. Relaxation of the coronary artery in response to a NO donor was potentiated by hypoxia when sGC was partly inhibited, coincident with an increase in sGC dimer and enhanced cGMP production. These effects were prevented by dithiothreitol and tris(2-carboxyethyl) phosphine.
These results demonstrate that the dimerization of sGC is exquisitely sensitive to thiol reductants compared with that of PKG, which may provide a novel mechanism for thiol-dependent modulation of NO-mediated vasodilatation in conditions such as hypoxia.
可溶性鸟苷酸环化酶（sGC）是一种异二聚体。酶的二聚化对于其介导升高环鸟苷酸（cGMP）的试剂引起的作用是必需的。本研究旨在确定 sGC 二聚化是否受巯基还原剂调节，以及其二聚化是否影响对一氧化氮（NO）的反应性松弛。
通过 Western blot 分析 sGC 和 cGMP 依赖性蛋白激酶（PKG）的二聚体和单体。通过酶联免疫吸附测定法测量细胞内 cGMP 含量。在器官室中测定等长张力变化。在分离的猪冠状动脉中，巯基还原剂二硫苏糖醇、半胱氨酸、还原型 l-谷胱甘肽和三（2-羧乙基）膦降低 sGC 二聚体的蛋白水平。这种作用与 cGMP 升高减少和对 NO 供体的反应性松弛减弱有关。二氢硫辛酸，一种内源性巯基抗氧化剂，也降低 sGC 的二聚化和酶的激活。二硫苏糖醇在显著影响 sGC 二聚化的浓度下对 PKG 的二聚化或对 8-Br-cGMP 的反应性松弛没有显著影响。当 sGC 部分抑制时，NO 供体对冠状动脉的松弛作用被缺氧增强，同时 sGC 二聚体增加和 cGMP 产生增强。这些作用被二硫苏糖醇和三（2-羧乙基）膦所阻止。
与 PKG 相比，sGC 的二聚化对巯基还原剂极为敏感，这可能为缺氧等条件下巯基依赖的 NO 介导的血管舒张的调节提供了一种新的机制。
