Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

Abstract

The developmental maturation of Na(+)-H+ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na+ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside = 5.2/7.5) significantly stimulated Na+ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na+ uptake which increased with advancing age. Na+ uptake represented an electroneutral process. The amiloride sensitivity of the pH-stimulated Na+ uptake was investigated using [amiloride] 10(-2)-10(-5) M. At 10(-3) M amiloride concentration, Na+ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed a Km for amiloride-sensitive Na+ uptake of 21.8 +/- 6.4, 24.9 +/- 10.9 and 11.8 +/- 4.17 mM and Vmax of 8.76 +/- 1.21, 5.38 +/- 1.16 and 1.99 +/- 0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na(+)-H+ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.