Byron K L, Babnigg G, Villereal M L
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637.
J Biol Chem. 1992 Jan 5;267(1):108-18.
Lys-Bradykinin (BK), a mitogen for human foreskin fibroblasts (HSWP cells) (Owen, N. E., and Villereal, M. L. (1983) Cell 32, 979-985), elicits a rapid, transient elevation of intracellular free Ca2+ concentration ([Ca2+]i) in these cells. We have used image analysis of fura-2-loaded HSWP cells to examine the BK-induced [Ca2+]i changes in individual cells. BK-stimulated Ca2+ entry and release of intracellular Ca2+ stores can be distinguished by stimulating cells in the presence or absence of extracellular Ca2+, or by inhibiting Ca2+ entry with 5 mM NiCl2. BK-sensitive intracellular Ca2+ stores can be depleted by exposure of the cells to BK in Ca(2+)-free medium; refilling of the stores requires extracellular Ca2+. A component of BK-stimulated Ca2+ entry persists after removal of agonist, but inactivates with a t1/2 of approximately 5 min. Although previous studies have attributed the Ca2+ entry which persists after agonist removal to a "capacitative Ca2+ entry" pathway activated by the depletion of the intracellular Ca2+ stores, we find that a large component of this BK-stimulated Ca2+ entry is not due to capacitative Ca2+ entry since (1) ionomycin can deplete the BK-sensitive intracellular Ca2+ stores without appreciably stimulating Ca2+ entry and without inhibiting the BK-stimulated Ca2+ entry and (2) this Ca2+ entry pathway inactivates at a time when the Ca2+ pools are still empty and a capacitance entry pathway should still be open. On the other hand, refilling of the intracellular Ca2+ stores can occur after the noncapacitative Ca2+ entry component has inactivated or when it is inhibited by Ni2+; in these cases refilling occurs without a detectable elevation of [Ca2+]i suggesting that refilling of internal Ca2+ pools might occur by a capacitative route.
赖氨酰缓激肽（BK）是一种人类包皮成纤维细胞（HSWP细胞）的促有丝分裂原（欧文，N.E.，和维勒雷亚尔，M.L.（1983年）《细胞》32卷，979 - 985页），它能使这些细胞内的游离钙离子浓度（[Ca2+]i）迅速短暂升高。我们利用负载fura - 2的HSWP细胞的图像分析来检测BK诱导的单个细胞内[Ca2+]i的变化。BK刺激的钙离子内流和细胞内钙离子储存的释放可以通过在有或无细胞外钙离子存在的情况下刺激细胞，或者用5 mM氯化镍抑制钙离子内流来区分。BK敏感的细胞内钙离子储存可以通过将细胞暴露于无钙培养基中的BK而耗尽；储存的重新填充需要细胞外钙离子。BK刺激的钙离子内流的一个成分在去除激动剂后仍然持续存在，但以约5分钟的半衰期失活。尽管先前的研究将激动剂去除后持续存在的钙离子内流归因于由细胞内钙离子储存耗尽激活的“容量性钙离子内流”途径，但我们发现，这种BK刺激的钙离子内流的很大一部分并非由于容量性钙离子内流，因为（1）离子霉素可以耗尽BK敏感的细胞内钙离子储存，而不会明显刺激钙离子内流，也不会抑制BK刺激的钙离子内流，并且（2）这种钙离子内流途径在钙离子池仍然为空且容量性内流途径应该仍然开放的时候失活。另一方面，在非容量性钙离子内流成分失活后或当它被镍离子抑制时，细胞内钙离子储存可以重新填充；在这些情况下，重新填充发生时[Ca2+]i没有可检测到的升高，这表明内部钙离子池的重新填充可能通过容量性途径发生。
