Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles |
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Authors: | Jonathan M Wilkes Barry H Hirst |
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Institution: | (1) Department of Physiological Sciences, University of Newcastle upon Tyne, Medical School, NE2 4HH Newcastle upon Tyne, England |
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Abstract: | Summary 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 (k
H
+
was determined by addition of vesicles (pH
i
= 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 × 10–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 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 k
H
+
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 (pH
i
< pH
o
) 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
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. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K+-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na+/H+ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA). |
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Keywords: | amiloride brush-border membranes intestine Na+/H+ antiport parietal cell proton conductance |
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