Lo T M, Thayer S A
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
Am J Physiol. 1993 Mar;264(3 Pt 1):C641-53. doi: 10.1152/ajpcell.1993.264.3.C641.
Bradykinin-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single NG108-15 cells with indo-1-based dual-emission microfluorimetry (50% effective concentration, 16 nM). A 1-min exposure to 30 nM bradykinin completely depleted the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store; refilling the store required extracellular Ca2+ (half time, 2 min). Refilling the IP3-sensitive store was completely blocked by 1 microM La3+ and 10 microM nitrendipine, but not 10 microM verapamil, 10 microM flunarizine, 1 microM nitrendipine, or 0.1 microM La3+. Thapsigargin irreversibly depleted the Ca2+ store and prevented its refilling (half-maximal inhibitory concentration, 3 nM). Influx of Ca2+ across the plasma membrane did not increase after depletion of the IP3-sensitive store by exposure to bradykinin, although maintained presence of the agonist produced significant Ca2+ influx. Similarly, Mn2+ and Ba2+ influx, as measured by indo-1 quenching and spectral shifts, did not increase following depletion of IP3-sensitive store. In contrast to depletion of the IP3-sensitive Ca2+ store by bradykinin, thapsigargin (10 nM) treatment produced Ca2+ and Ba2+ influx. We conclude that after Ca2+ mobilization, the IP3-sensitive Ca2+ store in NG108-15 cells is refilled with cytoplasmic Ca2+ via a thapsigargin-sensitive Ca(2+)-Mg(2+)-ATPase. Cytoplasmic Ca2+ is replenished by a persistent leak of Ca2+ across the plasma membrane. This leak is not modulated by the status of the intracellular Ca2+ store. In NG108-15 cells, agonist and thapsigargin-evoked Ca2+ entry are mediated by activation of plasmalemmal Ca2+ channels independent of the status of the IP3-sensitive intracellular Ca2+ store.
用基于indo-1的双发射显微荧光测定法（半数有效浓度为16 nM）在单个NG108 - 15细胞中记录缓激肽诱导的细胞内游离钙离子浓度（[Ca2+]i）的增加。用30 nM缓激肽处理1分钟可完全耗尽肌醇1,4,5 - 三磷酸（IP3）敏感的钙离子储存；重新填充该储存需要细胞外钙离子（半衰期为2分钟）。1 microM La3+和10 microM尼群地平可完全阻断IP3敏感储存的重新填充，但10 microM维拉帕米、10 microM氟桂利嗪、1 microM尼群地平或0.1 microM La3+则不能。毒胡萝卜素不可逆地耗尽钙离子储存并阻止其重新填充（半数最大抑制浓度为3 nM）。暴露于缓激肽使IP3敏感储存耗尽后，跨质膜的钙离子内流并未增加，尽管激动剂的持续存在会产生显著的钙离子内流。同样，通过indo-1淬灭和光谱位移测量的Mn2+和Ba2+内流，在IP3敏感储存耗尽后也未增加。与缓激肽耗尽IP3敏感钙离子储存相反，毒胡萝卜素（10 nM）处理会产生Ca2+和Ba2+内流。我们得出结论，钙离子动员后，NG108 - 15细胞中IP3敏感的钙离子储存通过毒胡萝卜素敏感的Ca(2+)-Mg(2+)-ATP酶由细胞质钙离子重新填充。细胞质钙离子通过钙离子持续跨质膜泄漏得到补充。这种泄漏不受细胞内钙离子储存状态的调节。在NG108 - 15细胞中，激动剂和毒胡萝卜素诱发的钙离子内流是由质膜钙离子通道的激活介导的，与IP3敏感的细胞内钙离子储存状态无关。
