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 language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Feb 15 1996|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience