Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

• Published in: The Journal of membrane biology Vol. 126; no. 2; p. 115
• Main Authors: Wilkes, J M, Hirst, B H
• Format: Journal Article
• Language: English
• Published: United States 01.03.1992
• Subjects: Amiloride - pharmacology; Animals; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Membrane Permeability - drug effects; Diffusion; Electric Conductivity; Gastric Mucosa - drug effects; Gastric Mucosa - metabolism; Hydrogen-Ion Concentration; Intestinal Mucosa - drug effects; Intestinal Mucosa - metabolism; Kinetics; Membrane Potentials; Protons; Rabbits; Sodium - metabolism
• ISSN: 0022-2631
• DOI: 10.1007/BF00231910

Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (kH+) was determined by addition of vesicles (pHi = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 x 10(-3) (gastric parietal cell stimulation-associated vesicles; SAV) and 5 x 10(-3) (duodenal brush-border membrane vesicles; dBBMV) to 11 x 10(-3) sec-1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mM, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in kH+ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na+/H+ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pHi less than pHo) 22Na+ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K(+)-diffusion potential ([K+]i much much greater than [K+]o). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K(+)-ionophore valinomycin, indicating that the native K+ permeability is rate limiting. In the presence of valinomycin, the K(+)-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K+ permeability. Valinomycin was without effect, but the K+/H+ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes.