Sodium-calcium exchanger in cultured human retinal pigment epithelium

Nancy J. Mangini, Laura Haugh-Scheidt, Jason E. Valle, Edward J. Cragoe, Harris Ripps, Brian G. Kennedy

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)821-834
Number of pages14
JournalExperimental eye research
Volume65
Issue number6
DOIs
StatePublished - Dec 1997

Keywords

  • Calcium
  • Fluo-3
  • Fura-2
  • Retinal pigment epithelium
  • Sodium/calcium exchanger
  • Transport

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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  • Cite this

    Mangini, N. J., Haugh-Scheidt, L., Valle, J. E., Cragoe, E. J., Ripps, H., & Kennedy, B. G. (1997). Sodium-calcium exchanger in cultured human retinal pigment epithelium. Experimental eye research, 65(6), 821-834. https://doi.org/10.1006/exer.1997.0390