Na+-H+ exchange in gastric glands as measured with a cytoplasmic-trapped, fluorescent pH indicator

Abstract

We have used the pH-sensitive, fluorescent, cytoplasmic-trapped dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+-H+ exchange in gastric glands isolated from rabbit stomachs by high-pressure perfusion and collagenase digestion. The fluorescence of BCECF-loaded glands was calibrated in terms of cytosolic pH (pHc) by permeabilizing the cell membranes and titrating the extracellular solution to different pH values. In one set of experiments in Cl--free solutions, glands were treated with 0.1 mM ouabain for 45 min to increase cellular cytosolic molar sodium ion concentration [( Na+]c) to high levels. Subsequent suspension of these cells in a Na+-free Ringer's solution (to generate [Na+]c greater than [Na+]o) caused cells to acidify rapidly (t1/2 approximately equal to 60 sec) from pHc approximately equal to 7.15 to pHc approximately equal to 6.55. Subsequent addition of 100 mM Na+ or Li+, but not K+, caused cells rapidly to increase pHc (t1/2 approximately equal to 30 sec) toward the control value. These changes of pHc were blocked when ouabain-treated glands had been preequilibrated for 10 min with 1 mM amiloride, and this block was overcome by adding 10 microM monensin (an ionophore that artificially exchanges Na+ for H+). In another set of experiments in Cl--containing Ringer's solution, glands were acid-loaded by treatment with 30 mM NH4Cl for 4 min, followed by washing the NH4Cl from the solutions. Under these conditions, pHc decreased from 7.02 to approximately equal to 6.5; subsequent alkalinization of cells back to control pHc was stimulated by Na+ (t1/2 approximately equal to 60 sec), but not K+, and was inhibited by 1 mM amiloride. This amiloride block also was overcome by further addition of 10 microM monensin. We conclude that gastric glands contain a Na+-H+ exchanger that appears independent of Cl-, not activated by K+, and blocked by 1 mM amiloride. This exchanger is likely localized to the serosal membrane of gland cells. Na+-H+ exchange may play an important role in regulation of pHc in oxyntic and chief cells exposed to high luminal acidity, where back diffusion of H+ into cells may occur at rapid rates.