Anion inhibition of the proton pump in rat liver multivesicular bodies.
Rat liver multivesicular bodies (MVB), as well as other hepatic subcellular organelles, are acidified by an electrogenic ATP-dependent proton pump that requires Cl- for maximal acidification (Van Dyke, R. W., Hornick, C. A., Belcher, J., Scharschmidt, B. F., and Havel, R.J. (1985) J. Biol. Chem. 260, 11021-11026), suggesting that Cl- serves as a permeable charge-compensating anion. However, we have observed that NO3- is unable to substitute for Cl-. This study was undertaken therefore to examine more closely the effects of Cl- on MVB acidification and to determine whether NO3- and other anions interact with the proton pump. ATP-dependent vesicle acidification and membrane potential (psi) were measured using the fluorescent dyes acridine orange and Oxonol V (bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol), respectively. Cl- both stimulated acidification (Km = 23.2 +/- 4.2 mM) and decreased psi (IC50 = 3.4 +/- 0.6 mM) in a concentration-dependent, nonlinear fashion. In the presence of saturating Cl- (100 mM), however, NO3- (shown to be more permeable than Cl-) and the impermeant anions SO4(2-) and PO4(2-), inhibited both ATP-dependent acidification and psi in a concentration-dependent manner. Other anions, including gluconate and HCO3-, had no effect. The inhibitory effect of NO3- was reversible. Neither SO4(2-) nor PO4(2-) appeared to block Cl- movement across the vesicle membrane as assessed by the ability of Cl- to decrease an established psi. In additional experiments, the effects of anions on relaxation of a previously established pH gradient were measured. Compared to Cl- or gluconate, NO3- had no significant effect on pH gradient relaxation, even when MVB were preloaded with NO3-, indicating that rapid cycling of NO3-/HNO3 across the MVB membrane does not occur. The organic nitrate, isosorbide dinitrate, also inhibited both acidification and psi and, similar to NO3-, had no effect on pH gradient relaxation. By contrast, NO2- potently inhibited both MVB acidification and psi but also rapidly relaxed a pre-established pH gradient, suggesting that NO2- increases MVB membrane proton permeability. Finally, MVB exhibited N-ethylmaleimide-sensitive ATPase activity that was inhibited 23.9% by NO3- (100 mM). In conclusion, although MVB are permeable to a variety of anions (Cl-, Br-, NO3-, NO2-), only Cl- and Br- support maximal rates of acidification by the proton pump.(ABSTRACT TRUNCATED AT 400 WORDS)
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000032:analysis; D000818:Animals; D000838:Anions / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D002712:Chlorides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007548:Isosorbide Dinitrate / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009566:Nitrates / Q000494:pharmacology; D011522:Protons; D051381:Rats; D011919:Rats, Inbred Strains
D000838:Anions; D002712:Chlorides; D009566:Nitrates; D011522:Protons; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000251:Adenosine Triphosphatases; D007548:Isosorbide Dinitrate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid