Rammelkamp Center for Education and Research, MetroHealth Medical Center, Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 2500 MetroHealth Dr., Cleveland, OH 44109, USA.
Am J Physiol Heart Circ Physiol. 2011 Feb;300(2):H493-506. doi: 10.1152/ajpheart.01073.2010. Epub 2010 Dec 10.
Diamide is a membrane-permeable, thiol-oxidizing agent that rapidly and reversibly oxidizes glutathione to GSSG and promotes formation of protein-glutathione mixed disulfides. In the present study, the acute effect of diamide on free cytosolic Ca2+ concentration ([Ca2+]i) was examined in fura-2-loaded bovine aortic endothelial cells. At low concentrations (50, 100 μM), diamide reversibly increased spontaneous, asynchronous Ca2+ oscillations, whereas, at higher concentrations (250, 500 μM), diamide caused an immediate synchronized Ca2+ oscillation in essentially all cells of the monolayer, followed by a time-dependent rise in basal [Ca2+]i. The effects of diamide on [Ca2+]i dynamics were independent of extracellular Ca2+. Inhibition of phospholipase C by U-73122 prevented the observed changes in [Ca2+]i. Additionally, the diamide-induced oscillations, but not the rise in basal [Ca2+]i, were blocked by inhibition of the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) by 2-aminoethyl diphenyl borate. However, diamide failed to alter the plasmalemmal distribution of a green fluorescent protein-tagged phosphatidylinositol-4,5-bisphosphate binding protein, demonstrating that diamide does not activate phospholipase C. Inhibition of glutathione reductase by N,N'-bis(2-chloroethyl)-N-nitrosourea or depletion of glutathione by l-buthionine-sulfoximine enhanced the effects of diamide, which, under these conditions, could only be reversed by addition of dithiothreitol to the wash buffer. Biochemical assays showed that both the IP3R and the plasmalemmal Ca2+-ATPase pump could be reversibly glutathionylated in response to diamide. These results demonstrate that diamide promotes Ca2+ release from IP3-sensitive internal Ca2+ stores and elevates basal [Ca2+]i in the absence of extracellular Ca2+, effects that may be related to a diamide-induced glutathionylation of the IP3R and the plasmalemmal Ca2+-ATPase Ca2+ pump, respectively.
二酰胺是一种膜通透性的硫醇氧化剂，可快速可逆地将谷胱甘肽氧化为 GSSG，并促进蛋白-谷胱甘肽混合二硫键的形成。在本研究中，我们用 fura-2 负载的牛主动脉内皮细胞检测了二酰胺对胞浆游离 Ca2+浓度([Ca2+]i)的急性影响。在低浓度（50、100 μM）时，二酰胺可逆地增加自发的、异步的 Ca2+振荡，而在较高浓度（250、500 μM）时，二酰胺引起单层中几乎所有细胞的同步 Ca2+振荡，随后基础[Ca2+]i 呈时间依赖性升高。二酰胺对[Ca2+]i 动力学的影响与细胞外 Ca2+无关。用 U-73122 抑制磷脂酶 C 可阻止观察到的[Ca2+]i 变化。此外，二酰胺诱导的振荡，但不是基础[Ca2+]i 的升高，可被 2-氨基乙基二苯基硼酸盐抑制肌醇-1,4,5-三磷酸（IP3）受体（IP3R）而阻断。然而，二酰胺未能改变绿色荧光蛋白标记的磷脂酰肌醇-4,5-二磷酸结合蛋白的质膜分布，表明二酰胺不会激活磷脂酶 C。用 N,N'-双（2-氯乙基）-N-亚硝脲抑制谷胱甘肽还原酶或用 L-丁硫氨酸亚砜亚胺耗尽谷胱甘肽增强了二酰胺的作用，在这些条件下，只有在洗涤缓冲液中添加二硫苏糖醇才能逆转二酰胺的作用。生化分析表明，IP3R 和质膜 Ca2+-ATP 酶泵都可被二酰胺可逆地谷胱甘肽化。这些结果表明，二酰胺促进 IP3 敏感的内部 Ca2+库释放 Ca2+，并在不存在细胞外 Ca2+的情况下升高基础[Ca2+]i，这些作用可能分别与二酰胺诱导的 IP3R 和质膜 Ca2+-ATP 酶泵的谷胱甘肽化有关。
