Department of Physiology, College of Medicine, Konyang University, Daejeon 302-718, Korea.
Korean J Physiol Pharmacol. 2011 Dec;15(6):431-6. doi: 10.4196/kjpp.2011.15.6.431. Epub 2011 Dec 27.
Vascular smooth muscle cells can obtain a proliferative function in environments such as atherosclerosis in vivo or primary culture in vitro. Proliferation of vascular smooth muscle cells is accompanied by changes in ryanodine receptors (RyRs). In several studies, the cytosolic Ca(2+) response to caffeine is decreased during smooth muscle cell culture. Although caffeine is commonly used to investigate RyR function because it is difficult to measure Ca(2+) release from the sarcoplasmic reticulum (SR) directly, caffeine has additional off-target effects, including blocking inositol trisphosphate receptors and store-operated Ca(2+) entry. Using freshly dissociated rat aortic smooth muscle cells (RASMCs) and cultured RASMCs, we sought to provide direct evidence for the operation of RyRs through the Ca(2+)- induced Ca(2+)-release pathway by directly measuring Ca(2+) release from SR in permeabilized cells. An additional goal was to elucidate alterations of RyRs that occurred during culture. Perfusion of permeabilized, freshly dissociated RASMCs with Ca(2+) stimulated Ca(2+) release from the SR. Caffeine and ryanodine also induced Ca(2+) release from the SR in dissociated RASMCs. In contrast, ryanodine, caffeine and Ca(2+) failed to trigger Ca(2+) release in cultured RASMCs. These results are consistent with results obtained by immunocytochemistry, which showed that RyRs were expressed in dissociated RASMCs, but not in cultured RASMCs. This study is the first to demonstrate Ca(2+) release from the SR by cytosolic Ca(2+) elevation in vascular smooth muscle cells, and also supports previous studies on the alterations of RyRs in vascular smooth muscle cells associated with culture.
血管平滑肌细胞在体内动脉粥样硬化等环境或原代体外培养中可获得增殖功能。血管平滑肌细胞的增殖伴随着兰尼碱受体（RyRs）的变化。在几项研究中，在平滑肌细胞培养过程中，细胞溶质 Ca(2+)对咖啡因的反应降低。虽然咖啡因通常用于研究 RyR 功能，因为直接测量肌浆网（SR）中的 Ca(2+)释放很困难，但咖啡因具有其他非靶向作用，包括阻断肌醇三磷酸受体和储存操作的 Ca(2+)内流。使用新鲜分离的大鼠主动脉平滑肌细胞（RASMCs）和培养的 RASMCs，我们试图通过直接测量透化细胞中 SR 中的 Ca(2+)释放，为 RyRs 通过 Ca(2+)-诱导的 Ca(2+)释放途径的操作提供直接证据。另一个目标是阐明培养过程中 RyRs 发生的变化。透化的新鲜分离的 RASMCs 用 Ca(2+)灌注可刺激 SR 中的 Ca(2+)释放。咖啡因和兰尼碱也可诱导分离的 RASMCs 中的 Ca(2+)从 SR 中释放。相比之下，兰尼碱、咖啡因和 Ca(2+)不能触发培养的 RASMCs 中的 Ca(2+)释放。这些结果与免疫细胞化学结果一致，该结果表明 RyRs 在分离的 RASMCs 中表达，但在培养的 RASMCs 中不表达。这项研究首次证明了血管平滑肌细胞中通过细胞溶质 Ca(2+)升高从 SR 释放 Ca(2+)，并支持了先前关于与培养相关的血管平滑肌细胞中 RyRs 变化的研究。
