Characterization of a Na

Ca exchange protein in cultured human retinal pigment epithelial cells

Brian Kennedy, L. Haugh-Scheidt, R. K. Getz, N. J. Mangini

Research output: Contribution to journalArticle

Abstract

Purpose. Changes in free [Ca2+]i in either intracellular or extracellular compartments, have dramatic physiological effects. Even though the retinal pigment epithelium (RPE) has beer implicated in retinal Ca2+ regulation, little work has been done to characterize plasma membrane Ca2+ transport in the RPE. This work identifies and characterizes the Na:Ca exchange protein in cultured human RPE (HRPE). This transporter, along with the Ca-ATPase, is known to regulate [Ca2+]i in many excitable and non-excitable cell types. Methods. Experiments employed adult HRPE maintained in cell culture. Western blotting of SDS-PAGE separations of whole cell lysates of HRPE and light microscopic immunocytochemistry in fixed HRPE monolayers were used to identify the Na:Ca exchanger. Experiments used polyclonal (π) and monoclonal (C2C12) antibodies. Results. Western blotting with the polyclonal antibody detected three major bands in HRPE lysates. A high molecular weight band (128 ± 2 kD, mean ± SEM) and two lower molecular weight bands at 70 ± 1 and 57 ± 2 kD. In other preparations, the Na:Ca exchanger is detected at ∼120 kD (assumed to be the full size protein) and at ∼70 kD (a proteolytic fragment). The polyclonal antibody also gives prominent staining, by immunohistochemistry, in intact HRPE monolayers. Though the monoclonal antibody was not found to react in Western blots, reaction product was detected immunohistochemically. This discrepancy could be due to the fact that this antibody reportedly recognizes a protein hinge epitope that is strongly conformation dependent. Conclusions. The present work provides evidence for the presence of Na:Ca exchange protein in cultured HRPE.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

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Retinal Pigments
Epithelial Cells
Retinal Pigment Epithelium
Proteins
Western Blotting
Antibodies
Molecular Weight
Immunohistochemistry
Monoclonal Antibodies
Cell Separation
Adenosine Triphosphatases
Epitopes
Polyacrylamide Gel Electrophoresis
Cell Culture Techniques
Cell Membrane
Staining and Labeling
Light

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Characterization of a Na : Ca exchange protein in cultured human retinal pigment epithelial cells. / Kennedy, Brian; Haugh-Scheidt, L.; Getz, R. K.; Mangini, N. J.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

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