Amiloride-sensitive Na+-H+ exchange has been identified in basolateral membrane vesicles from rat liver, but little is currently known about its regulation or its role in maintenance of resting intracellular pH (pH(i)) in intact hepatocytes. We have assessed Na+-H+ exchange activity in isolated or cultured rat hepatocytes in nominally HCO3--free solution under basal conditions and after intracellular acidification by an NH4Cl pulse by measuring 1) pH(i), using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxy fluorescein, 2) net H+ efflux by pH-stat titration, and 3) amiloride-inhibitable 22Na uptake. Under resting conditions, Na+-H+ exchange did not contribute measurably to Na+ uptake and accounted for <20% of net H+ efflux. Hepatocyte pH(i) averaged 7.07 ± 0.03, significantly above H+ electrochemical equilibrium (6.92 ± 0.08) determined using an electrogenic proton ionophore. Transient removal of extracellular Na+ or exposure to amiloride reversibly lowered pH(i) by 0.09 ± 0.01 and 0.12 ± 0.03 pH units, respectively, within 5-10 min. After intracellular acidification by an NH4Cl pulse, Na+ uptake rate increased about twofold, the increase being entirely amiloride inhibitable. Net H+ efflux increased about threefold, and 70% of the increase was amiloride inhibitable. Recovery of pH(i) after an NH4Cl pulse was reversibly blocked by exposure to amiloride or removal of Na+. Na+-H+ exchange activity (calculated from the rate of change in pH(i) and intracellular buffering capacity) was inversely related to pH(i) and was estimated to approach zero at pH(i) 7.25-7.50. Thus under resting conditions in HCO3--free medium, Na+-H+ exchange helps maintain hepatocyte pH(i) above electrochemical equilibrium but contributes minimally to total Na+ influx or net H+ efflux. After intracellular acidification, Na+-H+ exchange activity increases markedly, accounts for up to 50-70% of total Na+ uptake or net H+ efflux, and appears entirely responsible for rapid recovery of pH(i). Analysis of the relationship between Na+-H+ exchange activity and pH(i) suggests an estimated setpoint above resting pH(i).
Na+-H+ exchange activity in rat hepatocytes: Role in regulation of intracellular pH
PERSICO, Marcello;
1989-01-01
Abstract
Amiloride-sensitive Na+-H+ exchange has been identified in basolateral membrane vesicles from rat liver, but little is currently known about its regulation or its role in maintenance of resting intracellular pH (pH(i)) in intact hepatocytes. We have assessed Na+-H+ exchange activity in isolated or cultured rat hepatocytes in nominally HCO3--free solution under basal conditions and after intracellular acidification by an NH4Cl pulse by measuring 1) pH(i), using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxy fluorescein, 2) net H+ efflux by pH-stat titration, and 3) amiloride-inhibitable 22Na uptake. Under resting conditions, Na+-H+ exchange did not contribute measurably to Na+ uptake and accounted for <20% of net H+ efflux. Hepatocyte pH(i) averaged 7.07 ± 0.03, significantly above H+ electrochemical equilibrium (6.92 ± 0.08) determined using an electrogenic proton ionophore. Transient removal of extracellular Na+ or exposure to amiloride reversibly lowered pH(i) by 0.09 ± 0.01 and 0.12 ± 0.03 pH units, respectively, within 5-10 min. After intracellular acidification by an NH4Cl pulse, Na+ uptake rate increased about twofold, the increase being entirely amiloride inhibitable. Net H+ efflux increased about threefold, and 70% of the increase was amiloride inhibitable. Recovery of pH(i) after an NH4Cl pulse was reversibly blocked by exposure to amiloride or removal of Na+. Na+-H+ exchange activity (calculated from the rate of change in pH(i) and intracellular buffering capacity) was inversely related to pH(i) and was estimated to approach zero at pH(i) 7.25-7.50. Thus under resting conditions in HCO3--free medium, Na+-H+ exchange helps maintain hepatocyte pH(i) above electrochemical equilibrium but contributes minimally to total Na+ influx or net H+ efflux. After intracellular acidification, Na+-H+ exchange activity increases markedly, accounts for up to 50-70% of total Na+ uptake or net H+ efflux, and appears entirely responsible for rapid recovery of pH(i). Analysis of the relationship between Na+-H+ exchange activity and pH(i) suggests an estimated setpoint above resting pH(i).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
