Modulation of intracellular pH by secretagogues and the Na+/H+ antiporter in cultured bovine chromaffin cells

Abstract

The possible physiological role of cytosolic pH changes in adrenal medullary chromaffin cell secretion was examined by investigating the effects of catecholamine secretagogues on cytosolic pH, which was monitored using the intracellular fluorescent indicator 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Anti-fluorescein antibodies were used to reduce background fluorescence from extracellular 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Stimulation with both cholinergic agonists (acetylcholine, nicotine) and a depolarizing agent (high K+) transiently acidified the cytosol of the chromaffin cell. This acidification was antagonized by reducing extracellular Ca2+ concentration and by Ca2+ antagonists (Co2+, verapamil), indicating that it occurred secondarily to Ca2+ influx, possibly as a result of exchange of Ca2+ ions for protons across organelle membranes. Taken together with previously published data [Kuijpers G.A. J. et al. (1989) J. biol. Chem. 264, 698-705] showing no effect of cytosolic acidification on nicotine-induced catecholamine secretion, these results indicate that secretagogue-induced cytosolic pH changes do not represent a causal step in stimulus-secretion coupling of the chromaffin cell. The cytosolic pH recovery to pre-stimulatory cytosolic pH levels was delayed by amiloride and by 5-(N,N-dimethyl)amiloride, at concentrations that otherwise substantially inhibited cytosolic pH recovery from the rebound acidification induced by a 40-fold sudden dilution of NH4Cl. This latter type of recovery results from activation of an Na+/H+ exchange mechanism. Therefore, the data suggest that Na+/H+ exchange is actively involved in the dissipation of the small acid loads generated by secretagogues.