CYTOSOLIC ACIDIFICATION STIMULATES A CALCIUM INFLUX THAT ACTIVATES NA+-H+ EXCHANGE IN LLC-PK1
CYTOSOLIC ACIDIFICATION STIMULATES A CALCIUM INFLUX THAT ACTIVATES NA+-H+ EXCHANGE IN LLC-PK1
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DOI：
10.1152/ajprenal.1991.261.4.f617
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发表时间：
1991-10-01
影响因子：
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通讯作者：
HARRIS, RC
中科院分区：
文献类型：
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作者：
BURNS, KD;HOMMA, T;HARRIS, RC
我们之前已经证明，LLC-PK1 细胞中胞浆酸化刺激的 Na+-H+ 交换被钙调蛋白拮抗剂抑制。 为了进一步研究 Ca2+-钙调蛋白依赖性过程在这些细胞的细胞内 pH (pH(i)) 调节中的作用，我们研究了 pH(i) 变化对胞质 Ca2+ 浓度 ([Ca2+]i) 的影响。 在含有 1.8 mM CaCl2 的等渗无 Na+ 缓冲液中维持的 fura-2/乙酰氧基甲酯 (fura-2/AM) 负载细胞中，[Ca2+]i 为 168 +/- 59 nM (n = 5)。 NH4Cl 诱导碱化后，[Ca2+]i 降低至 83 +/- 28 nM，并部分恢复至 126 +/- 42 nM。 NH4Cl 冲洗后，胞质酸化导致 [Ca2+]i 增加至 481 +/- 166 nM (P < 0.05；n = 5)，这取决于细胞外 Ca2+。 在用 KCl-尼日利亚霉素酸化的细胞中还观察到 [Ca2+]i 增加，随着细胞碱化，[Ca2+]i 返回到基线。 Ca-45(2+) 通量没有增加与初始 NH4Cl 诱导的 [Ca2+]i 减少相关，表明碱化过程中 Ca2+ 流入细胞内储存。 膜去极化不会改变[Ca2+]i。 与维拉帕米或钙调蛋白拮抗剂 N-(6-氨基己基)-5-氯-1-萘磺酰胺 (W-7) 预孵育可抑制酸化诱导的 [Ca2+]i 增加。 在缺乏细胞外 Ca2+ 的情况下，负载 2,'7'-双（羧乙基）-5(6)-羧基荧光素乙酰氧基甲基酯 (BCECF/AM) 的 LLC-PK1 细胞中 Na+ 依赖性 pH(i) 恢复和 LLC-PK1/CL4 细胞中胞质酸化刺激的基底外侧 Na+-H+ 交换活性均减弱。 结果表明，细胞质酸化激活 LLC-PK1 细胞中细胞外 Ca2+ 的流入。 此外，在没有 Ca2+ 流入的情况下，Na+-H+ 交换受到抑制。 我们假设这些细胞中存在 pH(i) 敏感的 Ca2+ 流入途径。
We have previously shown that cytosolic acidification-stimulated Na+-H+ exchange in LLC-PK1 cells is inhibited by calmodulin antagonists. To investigate further the role of Ca2+-calmodulin-dependent processes in intracellular pH (pH(i)) regulation in these cells, we studied the effects of pH(i) changes on cytosolic Ca2+ concentration ([Ca2+]i). In fura-2/acetoxymethylester (fura-2/AM)-loaded cells maintained in isotonic Na+-free buffer containing 1.8 mM CaCl2, [Ca2+]i was 168 +/- 59 nM (n = 5). After NH4Cl-induced alkalinization, [Ca2+]i decreased to 83 +/- 28 nM and partially recovered to 126 +/- 42 nM. Cytosolic acidification, after NH4Cl washout, caused an increase in [Ca2+]i to 481 +/- 166 nM (P < 0.05; n = 5) that was dependent on extracellular Ca2+. An increase in [Ca2+]i was also observed in cells acidified with KCl-nigericin, with a return of [Ca2+]i to baseline with cell alkalinization. No increase in Ca-45(2+) flux occurred in association with initial NH4Cl-induced [Ca2+]i decrease, suggesting Ca2+ flux into an intracellular store during alkalinization. Membrane depolarization did not alter [Ca2+]i. The acidification-induced [Ca2+]i increase was inhibited by preincubation with verapamil or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Na+-dependent pH(i) recovery in 2,'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded LLC-PK1 cells and cytosolic acidification-stimulated basolateral Na+-H+ exchange activity in LLC-PK1/CL4 cells were both attenuated in the absence of extracellular Ca2+. The results indicate that cytosolic acidification activates an influx of extracellular Ca2+ in LLC-PK1 cells. Furthermore, in the absence of Ca2+ influx, Na+-H+ exchange is inhibited. We postulate that a pH(i)-sensitive Ca2+ influx pathway exists in these cells.