Schilling W P, Cabello O A, Rajan L
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030.
Biochem J. 1992 Jun 1;284 ( Pt 2)(Pt 2):521-30. doi: 10.1042/bj2840521.
Previous studies in non-excitable cells have suggested that depletion of internal Ca2+ stores activates Ca2+ influx from the extracellular space via a mechanism that does not require stimulation of phosphoinositide hydrolysis. To test this hypothesis in vascular endothelial cells, the effect of the Ca(2+)-ATPase/pump inhibitor 2,5-di-t-butylhydroquinone (BHQ) on cytosolic free Ca2+ concentration ([Ca2+]i) was examined. BHQ produced a dose-dependent increase in [Ca2+]i, which remained elevated over basal values for several minutes and was substantially inhibited in the absence of extracellular Ca2+. Application of bradykinin after BHQ demonstrated that the BHQ-sensitive compartment partially overlapped the bradykinin-sensitive store. Similar results were obtained with thapsigargin and cyclopiazonic acid, two other Ca(2+)-ATPase inhibitors. Although BHQ had no effect on phosphoinositide hydrolysis, both 45Ca2+ influx and efflux were stimulated by this agent. These results suggest that depletion of the agonist-sensitive Ca2+ store is sufficient for activation of Ca2+ influx. Several characteristics of the Ca(2+)-influx pathway activated by internal store depletion were compared with those of the agonist-activated pathway. Bradykinin-stimulated Ca2+ influx was increased at alkaline extracellular pH (pHo), and was inhibited by extracellular La3+, by depolarization of the membrane, and by the novel Ca(2+)-influx blocker 1-(beta-[3-(4-methoxyphenyl)propoxy]-4- methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365). Additionally, bradykinin stimulated influx of both 45Ca2+ and 133Ba2+, consistent with the hypothesis that the agonist-activated influx pathway is permeable to both of these bivalent cations. Likewise, activation of Ca2+ influx by BHQ, thapsigargin and cyclopiazonic acid was blocked by La3+, membrane depolarization and SKF 96365, but was unaffected by nitrendipine or BAY K 8644. Furthermore, Ca2+ influx stimulated by BHQ was increased at alkaline pHo and BHQ stimulated the influx of both 45Ca2+ and 133Ba2+ to the same extent. These results demonstrate that the agonist-activated Ca(2+)-influx pathway and the pathway activated by depletion of the agonist-sensitive internal Ca2+ store are indistinguishable.
以往在非兴奋性细胞中的研究表明，细胞内钙库的耗竭会通过一种不需要刺激磷酸肌醇水解的机制激活细胞外空间的钙内流。为了在血管内皮细胞中验证这一假说，研究了钙 - 腺苷三磷酸酶/泵抑制剂2,5 - 二叔丁基对苯二酚（BHQ）对细胞内游离钙浓度（[Ca2+]i）的影响。BHQ使[Ca2+]i呈剂量依赖性增加，在基础值之上持续升高数分钟，且在无细胞外钙的情况下显著受到抑制。在BHQ作用后应用缓激肽表明，BHQ敏感区部分与缓激肽敏感储存区重叠。使用另外两种钙 - 腺苷三磷酸酶抑制剂毒胡萝卜素和环匹阿尼酸也得到了类似结果。尽管BHQ对磷酸肌醇水解没有影响，但该试剂刺激了45Ca2+的内流和外流。这些结果表明，激动剂敏感钙库的耗竭足以激活钙内流。将由细胞内储存耗竭激活的钙内流途径的几个特征与激动剂激活途径的特征进行了比较。缓激肽刺激的钙内流在碱性细胞外pH（pHo）下增加，并受到细胞外La3+、膜去极化以及新型钙内流阻滞剂1 - （β - [3 - （4 - 甲氧基苯基）丙氧基] - 4 - 甲氧基苯乙基） - 1H - 咪唑盐酸盐（SKF 96365）的抑制。此外，缓激肽刺激了45Ca2+和133Ba2+的内流，这与激动剂激活的内流途径对这两种二价阳离子均通透的假说一致。同样，BHQ、毒胡萝卜素和环匹阿尼酸激活的钙内流受到La3+、膜去极化和SKF 96365的阻断，但不受尼群地平或BAY K 8644的影响。此外，BHQ刺激的钙内流在碱性pHo下增加，且BHQ刺激45Ca2+和133Ba2+内流的程度相同。这些结果表明，激动剂激活的钙内流途径与由激动剂敏感细胞内钙库耗竭激活的途径无法区分。
