Study of the Ca2+/Na+ exchange mechanism in vesicles isolated from apical membranes of lens epithelium of spiny dogfish (Squalus acanthias) and bovine eye

Abstract

Apical membrane vesicles of dogfish and bovine lens epithelium were prepared by differential centrifugation and Mg2+ precipitation. Enrichment of the apical enzyme markers, for Na+,K(+)-ATPase was achieved. Na+,K(+)-ATPase was enriched 25.9-fold and 23.6-fold for dogfish and bovine lens epithelia, respectively, and acid phosphatase was enriched 10.4-fold and 12.6-fold, respectively. There was no enrichment of the cytoplasmic marker, NADH reductase. The majority of the apical membrane vesicles isolated from both dogfish and bovine lens epithelia were oriented right-side out. Electron micrographs of the purified vesicles show the presence of circular sealed vesicles with an average size of approximately 0.2-0.5 microns. Incubation of the vesicles with either the acetoxymethyl ester of SBFI, a new indicator for sodium, or with Fura-2, the calcium-specific indicator, leads to a large accumulation of the de-esterified SBFI or Fura-2 in the lens epithelial apical vesicles as determined by fluorescence measurements. When an outwardly direct Ca2+ gradient is formed across the vesicular membranes, the influx of Na+ is stimulated 77.8 and 63.0% for dogfish and bovine lens epithelia, respectively. When an outwardly directed Na+ gradient is formed across the vesicular membranes, the Ca2+ influx is also greatly enhanced. Both cases indicate that there is a bidirectional Ca2+/Na+ exchanger present in the apical side of the lens epithelial cell. The exchanger is inhibited by 50 microM bepridil or by 200 microM La3+. The stimulatory effects are also observed in membrane vesicles that are 'short-circuited' with valinomycin and high concentrations of K+.(ABSTRACT TRUNCATED AT 250 WORDS)