Lo T M, Thayer S A
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA.
Brain Res. 1995 Dec 15;704(1):10-8. doi: 10.1016/0006-8993(95)01099-8.
Following mobilization with the inositol 1,4,5-trisphosphate (IP3)-generating agonist bradykinin, Ca2+ stores in neuroblastoma x glioma hybrid, NG108-15 cells require extracellular Ca2+ to refill. The process by which this store refills with Ca2+ was characterized by recording bradykinin-induced intracellular free Ca2+ concentration transients as an index of the degree of refilling of the store. Cyclopiazonic acid, a microsomal Ca2+ ATPase inhibitor, reversibly depleted intracellular Ca2+ stores in these cells, but did not recruit detectable Ca2+ influx, suggesting that these cells lack substantial capacitative Ca2+ entry. The paucity of voltage-sensitive Ca2+ channels in undifferentiated NG108-15 cells, suggested that a channel analogous to that proposed to mediate capacitative Ca2+ entry in nonexcitable cells might assist refilling IP3-sensitive Ca2+ stores in these cells. The possibility that compounds shown previously to inhibit capacitative Ca2+ entry in nonexcitable cells might inhibit the refilling of the IP3-sensitive store in NG108-15 cells was explored. The IP3-sensitive store was depleted by exposure to bradykinin, allowed to refill briefly in the presence of the test compound and then challenged again with bradykinin to evaluate the degree of refilling of the store. The imidazole derivatives, econazole (10 microM), L-651582 (10 microM) and SKF 96365 (20 microM), all completely blocked the bradykinin-induced Ca2+ response. Calmodulin antagonists, W-7 (100 microM) and trifluoperazine (10 microM), were also effective, although at concentrations well above those required to inhibit calmodulin. Because of the high concentrations required to inhibit bradykinin responses, the possibility that these agents might have additional effects was explored. Compounds were tested in a paradigm in which the store was preloaded with Ca2+ before treatment. All of these agents depleted, at least partially, the preloaded store. Econazole was the least effective of the compounds tested for releasing stores, although it was comparable to the other compounds for inhibition of refilling. Although NG108-15 cells refill intracellular Ca2+ stores by a plasmalemmal Ca2+ leak, this leak shares a pharmacology similar to the capacitative Ca2+ entry pathway described for nonexcitable cells.
用能产生肌醇 1,4,5 - 三磷酸（IP3）的激动剂缓激肽进行动员后，神经母细胞瘤×胶质瘤杂交细胞 NG108 - 15 中的 Ca2 + 储存库需要细胞外 Ca2 + 来重新填充。通过记录缓激肽诱导的细胞内游离 Ca2 + 浓度瞬变来表征该储存库用 Ca2 + 重新填充的过程，以此作为储存库重新填充程度的指标。环匹阿尼酸是一种微粒体 Ca2 + ATP 酶抑制剂，它能可逆地耗尽这些细胞内的 Ca2 + 储存库，但不会引发可检测到的 Ca2 + 内流，这表明这些细胞缺乏大量的容量性 Ca2 + 内流。未分化的 NG108 - 15 细胞中电压敏感性 Ca2 + 通道较少，这表明类似于在非兴奋性细胞中介导容量性 Ca2 + 内流的通道可能有助于这些细胞中 IP3 敏感的 Ca2 + 储存库的重新填充。研究了先前显示能抑制非兴奋性细胞中容量性 Ca2 + 内流的化合物是否可能抑制 NG108 - 15 细胞中 IP3 敏感储存库的重新填充。通过暴露于缓激肽使 IP3 敏感储存库耗尽，在存在测试化合物的情况下短暂重新填充，然后再次用缓激肽刺激以评估储存库的重新填充程度。咪唑衍生物酮康唑（10 μM）、L - 651582（10 μM）和 SKF 96365（20 μM）均完全阻断了缓激肽诱导的 Ca2 + 反应。钙调蛋白拮抗剂 W - 7（100 μM）和三氟拉嗪（10 μM）也有效，尽管其浓度远高于抑制钙调蛋白所需的浓度。由于抑制缓激肽反应需要高浓度，因此研究了这些药物可能具有其他作用的可能性。在一种模式下测试化合物，即在处理前将储存库预加载 Ca2 +。所有这些药物至少部分耗尽了预加载的储存库。酮康唑是测试的释放储存库的化合物中效果最差的，尽管它在抑制重新填充方面与其他化合物相当。尽管 NG108 - 15 细胞通过质膜 Ca2 + 泄漏来重新填充细胞内 Ca2 + 储存库，但这种泄漏与描述的非兴奋性细胞的容量性 Ca2 + 内流途径具有相似的药理学特性。
