Regulation of intracellular free Ca2+ concentration ([Ca2+](i)) by an Na+Ca2+ exchanger was studied in cultures of human retinal pigment epithelial cells using Ca2+-indicator dyes (furs-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 ~ 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 ~ 6.8 kb. In sum, our findings demonstrate the presence of a cardiac type Na+/Ca2+ exchanger in cultures of human RPE.
- Retinal pigment epithelium
- Sodium/calcium exchanger
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience