Jones Bertina F, Boyles Rebecca R, Hwang Sung-Yong, Bird Gary S, Putney James W
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences-NIH, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
Hepatology. 2008 Oct;48(4):1273-81. doi: 10.1002/hep.22461.
The process of capacitative or store-operated Ca(2+) entry has been extensively investigated, and recently two major molecular players in this process have been described. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca(2+) stored in the endoplasmic reticulum, and Orai proteins constitute pore-forming subunits of the store-operated channels. Store-operated Ca(2+) entry is readily demonstrated with protocols that provide extensive Ca(2+) store depletion; however, the role of store-operated entry with modest and more physiological cell stimuli is less certain. Recent studies have addressed this question in cell lines; however, the role of store-operated entry during physiological activation of primary cells has not been extensively investigated, and there is little or no information on the roles of STIM and Orai proteins in primary cells. Also, the nature of the Ca(2+) influx mechanism with hormone activation of hepatocytes is controversial. Hepatocytes respond to physiological levels of glycogenolytic hormones with well-characterized intracellular Ca(2+) oscillations. In the current study, we have used both pharmacological tools and RNA interference (RNAi)-based techniques to investigate the role of store-operated channels in the maintenance of hormone-induced Ca(2+) oscillations in rat hepatocytes. Pharmacological inhibitors of store-operated channels blocked thapsigargin-induced Ca(2+) entry but only partially reduced the frequency of Ca(2+) oscillations. Similarly, RNAi knockdown of STIM1 or Orai1 substantially reduced thapsigargin-induced calcium entry, and more modestly diminished the frequency of vasopressin-induced oscillations.
Our findings establish that store-operated Ca(2+) entry plays a role in the maintenance of agonist-induced oscillations in primary rat hepatocytes but indicate that other agonist-induced entry mechanisms must be involved to a significant extent.
钙池调控性或储存-操作性Ca(2+)内流过程已得到广泛研究，最近已描述了该过程中的两个主要分子参与者。基质相互作用分子(STIM)1作为内质网中储存的Ca(2+)水平的传感器，而Orai蛋白构成储存-操作性通道的孔形成亚基。通过能使钙池大量排空的实验方案很容易证明储存-操作性Ca(2+)内流；然而，适度且更具生理性的细胞刺激下储存-操作性内流的作用尚不确定。最近的研究已在细胞系中探讨了这个问题；然而，在原代细胞的生理性激活过程中储存-操作性内流的作用尚未得到广泛研究，并且关于STIM和Orai蛋白在原代细胞中的作用几乎没有信息。此外，肝细胞激素激活时Ca(2+)内流机制的性质存在争议。肝细胞对糖原分解激素的生理水平会产生特征明确的细胞内Ca(2+)振荡反应。在本研究中，我们使用药理学工具和基于RNA干扰(RNAi)的技术来研究储存-操作性通道在维持大鼠肝细胞激素诱导的Ca(2+)振荡中的作用。储存-操作性通道的药理学抑制剂阻断了毒胡萝卜素诱导的Ca(2+)内流，但仅部分降低了Ca(2+)振荡的频率。同样，RNAi敲低STIM1或Orai1可显著降低毒胡萝卜素诱导的钙内流，并更适度地降低血管加压素诱导的振荡频率。
我们的研究结果表明，储存-操作性Ca(2+)内流在维持原代大鼠肝细胞激动剂诱导的振荡中起作用，但表明其他激动剂诱导的内流机制也必须在很大程度上参与其中。
