BIOCHEMICAL-CHARACTERIZATION OF THE AMILORIDE-SENSITIVE NA+/H+ ANTIPORT IN CHINESE-HAMSTER LUNG FIBROBLASTS

Abstract

Net H+ fluxes across the plasma membrane of Chinese hamster lung fibroblasts (CC139) were monitored by pH-stat titration. Na+-depleted cells release H+ on addition of Na+. Na+- or Li+-loaded cells take up H+ from the medium when shifted to a Na+,Li+-free medium. This reversible Na+ (or Li+)-dependent H+ flux is inhibited by amiloride and does not occur in digitonin-permeabilized cells. A similar Na+/H+ exchanger was identified in vascular smooth muscle cells, corneal and aortic endothelial cells, lens epithelial cells of bovine origin and human platelets. Kinetic studies carried out with CCl39 cells indicate that half-saturation of the system is observed at pH = 7.8, in the absence of Na+, external Na+ stimulates H+ release and inhibits H+ uptake in a competitive manner (Ki [inhibition constant] = 2-3 mM), amiloride is a competitive inhibitor for Na+ (Ki .simeq. 1 .mu.M) and a noncompetitive inhibitor for H+ and a coupling ratio of 1.3 .+-. 0.3 for the H+/Li+ exchange suggests a stoichiometry of 1:1. Evidently, CCl39 cells possess in their plasma membrane a reversible, electroneutral and amiloride-sensitive Na+/H+ antiporter, with 2 distinct and mutually exclusive binding sites for Na+ and H+. The rapid stimulation of the Na+/H+ antiporter in G0/G1-arrested CCl39 cells on addition of growth factors, together with the fact that intracellular H+ concentration is, under physiological conditions, around the apparent K0.5 of the system, strongly suggests a key role of this antiport in pHi regulation and mitogen action.