Altamirano J, Brodwick M S, Alvarez-Leefmans F J
Departamento de Neurobiología, Instituto Mexicano de Psiquiatría, México 14370, D.F. México.
J Gen Physiol. 1998 Aug;112(2):145-60. doi: 10.1085/jgp.112.2.145.
The possible role of Ca2+ as a second messenger mediating regulatory volume decrease (RVD) in osmotically swollen cells was investigated in murine neural cell lines (N1E-115 and NG108-15) by means of novel microspectrofluorimetric techniques that allow simultaneous measurement of changes in cell water volume and [Ca2+]i in single cells loaded with fura-2. [Ca2+]i was measured ratiometrically, whereas the volume change was determined at the intracellular isosbestic wavelength (358 nm). Independent volume measurements were done using calcein, a fluorescent probe insensitive to intracellular ions. When challenged with approximately 40% hyposmotic solutions, the cells expanded osmometrically and then underwent RVD. Concomitant with the volume response, there was a transient increase in [Ca2+]i, whose onset preceded RVD. For hyposmotic solutions (up to approximately -40%), [Ca2+]i increased steeply with the reciprocal of the external osmotic pressure and with the cell volume. Chelation of external and internal Ca2+, with EGTA and 1,2-bis-(o -aminophenoxy) ethane-N,N,N ',N '-tetraacetic acid (BAPTA), respectively, attenuated but did not prevent RVD. This Ca2+-independent RVD proceeded even when there was a concomitant decrease in [Ca2+]i below resting levels. Similar results were obtained in cells loaded with calcein. For cells not treated with BAPTA, restoration of external Ca2+ during the relaxation of RVD elicited by Ca2+-free hyposmotic solutions produced an increase in [Ca2+]i without affecting the rate or extent of the responses. RVD and the increase in [Ca2+]i were blocked or attenuated upon the second of two approximately 40% hyposmotic challenges applied at an interval of 30-60 min. The inactivation persisted in Ca2+-free solutions. Hence, our simultaneous measurements of intracellular Ca2+ and volume in single neuroblastoma cells directly demonstrate that an increase in intracellular Ca2+ is not necessary for triggering RVD or its inactivation. The attenuation of RVD after Ca2+ chelation could occur through secondary effects or could indicate that Ca2+ is required for optimal RVD responses.
利用新型显微分光荧光技术，在小鼠神经细胞系（N1E - 115和NG108 - 15）中研究了Ca2 +作为第二信使介导渗透肿胀细胞调节性容积减小（RVD）的可能作用。该技术能够同时测量负载fura - 2的单个细胞中细胞水容积和[Ca2 +]i的变化。[Ca2 +]i采用比率法测量，而容积变化则在细胞内等吸收波长（358 nm）处测定。使用对细胞内离子不敏感的荧光探针钙黄绿素进行独立的容积测量。当用约40%的低渗溶液刺激时，细胞发生渗透性膨胀，然后经历RVD。伴随容积反应，[Ca2 +]i出现短暂升高，其起始先于RVD。对于低渗溶液（高达约 - 40%），[Ca2 +]i随外部渗透压的倒数和细胞容积急剧增加。分别用乙二醇双四乙酸（EGTA）和1,2 - 双（邻氨基苯氧基）乙烷 - N，N，N'，N' - 四乙酸（BAPTA）螯合外部和内部的Ca2 +，可减弱但不能阻止RVD。即使[Ca2 +]i伴随性降低至静息水平以下，这种不依赖Ca2 +的RVD仍会发生。在负载钙黄绿素的细胞中也获得了类似结果。对于未用BAPTA处理的细胞，在无Ca2 +的低渗溶液引起的RVD松弛过程中恢复外部Ca2 +，会使[Ca2 +]i增加，而不影响反应的速率或程度。在间隔30 - 60分钟施加的两次约40%低渗刺激中的第二次刺激后，RVD和[Ca2 +]i的增加被阻断或减弱。失活在无Ca2 +的溶液中持续存在。因此，我们对单个神经母细胞瘤细胞内Ca2 +和容积的同时测量直接表明，细胞内Ca2 +的增加对于触发RVD或其失活并非必要。Ca2 +螯合后RVD的减弱可能通过继发效应发生，或者可能表明Ca2 +对于最佳RVD反应是必需的。
