Vascular smooth muscle Na+-H+ exchanger kinetics and its activation by angiotensin II

Abstract

We have studied the kinetic properties of basal and angiotensin II (ANG II)-stimulated Na+-H+ exchange in cultured rat aortic smooth muscle cells. Initial rates of 22Na+ influx were measured in the presence of ouabain (1 mM) and bumetanide (0.1 mM) with and without amiloride after intracellular acidification by preincubation in Na-free media. The kinetics of amiloride (100 microM)-sensitive Na+ influx were studied under the following conditions: 1) constant intracellular pH (pHi; 6.8) and varying external Na+ (Na+o), which gave a Km of 23.6 +/- 2.0 (SD, n = 3) mM and a maximum velocity (Vmax) of 25 nmol.mg protein-1.min-1 (varying the amiloride concentration gave a Ki of 22 microM for inhibition under these conditions); and 2) constant Na+o (100 mM) and varying pHi (from 7.4 to 6.2), which indicated that amiloride-sensitive Na+ influx was stimulated by cell acidification when an outward H+ gradient was imposed. ANG II-stimulated amiloride-sensitive Na+ influx for up to 30 min with a half-maximal activation 10(-8) M. The pHi dependence from cell pH (pHi 7.2-6.2) of amiloride-sensitive Na+ influx stimulated by ANG II was similar to that of the basal values, a finding indicating that ANG II did not change the affinity of Na+-H+ exchange for intracellular H+. However, at pHi 6.8, ANG II increased the Vmax of amiloride-sensitive Na+ influx from 25 to 33 nmol.mg protein-1.min-1 and markedly decreased the Km for Na+o from 23.6 +/- 7.4 to 3.7 (SD, n = 4; P less than 0.005) mM.(ABSTRACT TRUNCATED AT 250 WORDS)