title: 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)
authors.affiliation: Department of Physiology and Biophysics, New York University Medical Center, NY 10016.
authors.forename: J
authors.initials: J
authors.lastname: Ye
authors.forename: J A
authors.initials: JA
authors.lastname: Zadunaisky
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000648:ultrastructure; D004284:Dogfish / Q000378:metabolism*; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007474:Ion Exchange; D007904:Lens Cortex, Crystalline / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D009245:NADH Dehydrogenase / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014617:Vacuoles / Q000648:ultrastructure
chemical_list: D005456:Fluorescent Dyes; D012964:Sodium; D006861:Hydrogen Peroxide; D009245:NADH Dehydrogenase; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium