McCarty N A, O'Neil R G
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77030.
J Membr Biol. 1991 Aug;123(2):161-70. doi: 10.1007/BF01998086.
The Ca2+ entry pathways in the basolateral plasma membrane of the isolated, nonperfused proximal straight tubule (PST) of rabbit kidney were investigated using fura-2 fluorescence microscopy. Under isotonic conditions, reduction of bath [Ca2+] from 1 mM to 1 microM caused intracellular free calcium concentration ([Ca2+]i) to fall close to zero. Treatment with 10 microM verapamil, a calcium channel blocker, had a similar effect. Treatment with verapamil or low Ca2+ also induced fluctuations in cell volume. However, isotonic treatment with 10 microM nifedipine, a dihydropyridine (DHP)-type calcium channel blocker, did not affect [Ca2+]i or cell volume, indicating that the endogenous Ca2+ entry pathway is verapamil-sensitive but DHP-insensitive. When cells were exposed to hypotonic solutions in the presence of 1 mM Ca2+, they swelled and underwent normal RVD while [Ca2+]i increased transiently to a peak before decreasing to a late phase plateau level above the baseline level (see McCarty, N.A., O'Neil, R.G. 1991. J. Membrane Biol. 123:149-160). When cells were swollen in the presence of verapamil or low bath [Ca2+], RVD was abolished and [Ca2+]i fell well below the baseline during the late phase response. In contrast, when cells were swollen in the presence of nifedipine, RVD and the late phase rise in [Ca2+]i were abolished, but [Ca2+]i did not fall below the baseline level in the late phase, indicating that nifedipine inhibited the swelling-induced Ca2+ entry but that Ca2+ entry by another pathway was undisturbed. It was concluded that PST cells are characterized by two Ca2+ permeability pathways in the basolateral membrane. Under both isotonic and hypotonic conditions, Ca2+ entry occurs at a slow rate via a verapamil-sensitive, DHP-insensitive "baseline" Ca2+ entry pathway. Cell swelling activates a separate DHP-sensitive, verapamil-sensitive Ca2+ entry pathway, which is responsible for the supply of Ca ions to the Ca(2+)-dependent mechanism by which cell volume regulation is achieved.
利用fura-2荧光显微镜研究了兔肾分离的、非灌注近端直管（PST）基底外侧质膜中的Ca2+进入途径。在等渗条件下，将浴液中的[Ca2+]从1 mM降至1 microM会导致细胞内游离钙浓度（[Ca2+]i）降至接近零。用10 microM维拉帕米（一种钙通道阻滞剂）处理也有类似效果。用维拉帕米或低钙处理还会引起细胞体积波动。然而，用10 microM硝苯地平（一种二氢吡啶（DHP）型钙通道阻滞剂）进行等渗处理对[Ca2+]i或细胞体积没有影响，这表明内源性Ca2+进入途径对维拉帕米敏感但对DHP不敏感。当细胞在1 mM Ca2+存在下暴露于低渗溶液时，它们会肿胀并经历正常的调节性容积减小（RVD），而[Ca2+]i会先短暂升高至峰值，然后降至高于基线水平的晚期平台水平（见McCarty, N.A., O'Neil, R.G. 1991. J. Membrane Biol. 123:149 - 160）。当细胞在维拉帕米或低浴液[Ca2+]存在下肿胀时，RVD被消除，且在晚期反应期间[Ca2+]i降至基线以下。相反，当细胞在硝苯地平存在下肿胀时，RVD和[Ca2+]i的晚期升高被消除，但[Ca2+]i在晚期不会降至基线水平以下，这表明硝苯地平抑制了肿胀诱导的Ca2+进入，但另一条途径的Ca2+进入未受干扰。得出的结论是，PST细胞的特征是基底外侧膜中有两种Ca2+通透途径。在等渗和低渗条件下，Ca2+通过一条对维拉帕米敏感、对DHP不敏感的“基线”Ca2+进入途径以缓慢速率进入。细胞肿胀会激活一条独立的对DHP敏感、对维拉帕米敏感的Ca2+进入途径，该途径负责为实现细胞体积调节的Ca(2+)依赖性机制供应Ca离子。
