Mangini N J, Haugh-Scheidt L, Valle J E, Cragoe E J, Ripps H, Kennedy B G
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine 60612, USA.
Exp Eye Res. 1997 Dec;65(6):821-34. doi: 10.1006/exer.1997.0390.
Regulation of intracellular free Ca2+ concentration ([Ca2+]i) by an Na+/Ca2+ exchanger was studied in cultures of human retinal pigment epithelial cells using Ca(2+)-indicator dyes (fura-2 and fluo-3) and digital fluorescence imaging. Mean resting [Ca2+]i of cultured RPE in a control Ringer solution was 189 +/- 16 nM. Replacing extracellular Na+ with N-methyl-D-glucamine elicited a two-fold rise in [Ca2+]i; the magnitude of the [Na+]o-free-induced rise in [Ca2+]i varied as a function of extracellular [Ca2+]. The [Na+]o-free response was not significantly affected by the Ca2+ channel blocker nifedipine, or by pretreatment with thapsigargin which depletes intracellular Ca2+ stores. By contrast, the [Na+]o-free-induced rise in [Ca2+]i was significantly reduced by CBDMB, an amiloride derivative that is highly selective for Na+/Ca2+ exchange inhibition. These findings indicate that removal of extracellular Na+ promotes net [Ca2+]i gain via Na+/Ca2+ exchange. Western and Northern blot analyses, respectively, confirmed the presence of Na+/Ca2+ exchanger protein and mRNA in cultures of human RPE. Specifically, Western blot analysis of whole cell lysates of cultured RPE using a polyclonal antibody made against the canine cardiac exchanger identified a major band at approximately 126 kD. Northern blot analysis of total human RPE RNA using a restriction fragment cRNA probe coding for the canine cardiac Na+/Ca2+ exchanger showed that the major exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-type Na+/Ca2+ exchanger in cultures of human RPE.
利用钙指示剂染料（fura-2和fluo-3）及数字荧光成像技术，在人视网膜色素上皮细胞培养物中研究了钠钙交换体对细胞内游离钙离子浓度（[Ca2+]i）的调节作用。在对照林格氏液中培养的视网膜色素上皮细胞的平均静息[Ca2+]i为189±16 nM。用N-甲基-D-葡糖胺替代细胞外钠离子可使[Ca2+]i升高两倍；无细胞外钠离子诱导的[Ca2+]i升高幅度随细胞外[Ca2+]的变化而变化。无细胞外钠离子反应不受钙通道阻滞剂硝苯地平或用毒胡萝卜素预处理（耗尽细胞内钙储存）的显著影响。相比之下，高度选择性抑制钠钙交换的氨氯地平衍生物CBDMB可显著降低无细胞外钠离子诱导的[Ca2+]i升高。这些发现表明，去除细胞外钠离子可通过钠钙交换促进[Ca2+]i的净增加。蛋白质免疫印迹和Northern印迹分析分别证实了人视网膜色素上皮细胞培养物中存在钠钙交换体蛋白和mRNA。具体而言，用针对犬心脏交换体制备的多克隆抗体对培养的视网膜色素上皮细胞全细胞裂解物进行蛋白质免疫印迹分析，在约126 kD处鉴定出一条主要条带。用编码犬心脏钠钙交换体的限制性片段cRNA探针对人视网膜色素上皮细胞总RNA进行Northern印迹分析，结果显示主要的交换体相关转录本约为6.8 kb。总之，我们的发现证明了人视网膜色素上皮细胞培养物中存在一种心脏型钠钙交换体。
