Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

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Abstract
Kinetic properties of the Na+-H+ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pHi) and Na+ activity (aNai) with the aid of double-barreled H+- and Na+-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pHi was 7.12±0.01 and aNai was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH 4 + solution followed by an Na+-free N-methyl-d-glucamine (NMDG+) solution. Intracellular Na+ and H+ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na+ solution rapidly increased pHi. This Na+-induced increase in pHi was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNai. The rate of pHi recovery induced by the standard Na+ solution increased in a saturable manner as pHi decreased, and was negligible at pHi 7.2–7.3, indicating an inactivation of the Na+-H+ antiport. The apparent K m for intracellular H+ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNai to less than 1 mmol/l by prolonged exposure to NMDG+ solution significantly increased the rate of Na+-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na+ concentration increased following Michaelis-Menten kinetics (V max was 0.55 pH/min and the apparent K m was 75 mmol/l at pHi 6.88). The results clearly showed that the Na+-H+ antiport activity is dependent on the chemical potential gradient of both Na+ and H+ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na+ and H+ concentrations.