A study of H+ transport in gastric microsomal vesicles using fluorescent probes

Abstract

Fluorescent amines, 9-aminoacridine, acridine orange and quinacrine, were used as probes for a pH gradient (ΔpH) across gastric microsomal vesicles. Analysis of probe uptake data indicates that 9-aminoacridine distributes across the membrane as a weak base in accordance with the ΔpH. On the other hand, acridine orange and quinacrine show characteristics of binding to membrane sites in addition to accumulation in response to ΔpH. A discussion of the advantages and limitations of the probes is presented. Application of these probes to pig gastric microsomal vesicles indicates that the K +-stimulated ATPase is responsible for the transport of H + into the vesicles and thus develops a ΔpH across the membrane. The ΔpH generated by the K +-ATPase has a definite requirement for internal K +. The proton gradient can be discharged slowly after ATP depletion or rapidly either by detergent disruption of the vesicles or by increasing their leakiness using both H + and K + ionophores. On the other hand, the sole use of the K + ionophore, valinomycin, stimulates the ATP-induced formation of ΔpH by increasing the availability of K + to internal sites. This stimulation by valinomycin requires the presence of permeable anions like Cl -. Analysis of the Cl - requirement indicates that in the presence of valinomycin the net effect is the accumulation of HCl inside the gastric vesicles. With an external pH of 7.0, the ATP-generated ΔpH was calculated to be from 4 to 4.5 pH units. The results are consistent with the hypothesis that the K +-stimulated ATPase drives a K +/H + exchange across the gastric vesicles. Since other lines of evidence suggest that these gastric microsomes are derived from the tubulovesicular system of the oxyntic cell, the participation of the ATP-driven transport processes in gastric HCl secretion is of interest.