Sodium-calcium exchanger in cultured human retinal pigment epithelium

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.