Na+ and H+ gradient-dependent transport of p-aminohippurate in membrane vesicles from dog kidney cortex

Abstract

The transport of p-aminohippurate (PAH) was studied in basolateral (BLMV) and brush border membrane vesicles (BBMV) isolated from dog kidney cortex. Imposition of an inwardly directed 100 mN Na+ gradient stimulated the uptake of 50 microM [3H]PAH into BLMV, whereas a pH gradient (pHout = 6.0, pHin = 7.4) only slightly enhanced uptake. The Na+ gradient-dependent uptake of PAH was electroneutral, saturable and sensitive to inhibition by probenecid and several anionic drugs, with (apparent) Km = 0.79 +/- 0.16 mM, Vmax = 0.80 +/- 0.05 nmol/mg protein, 15 sec and Ki for probenecid = 0.08 +/- 0.01 mM. Simultaneous imposition of the pH gradient (outward OH- gradient) and inward Na+ gradient stimulated PAH uptake significantly over that with an Na+ gradient alone. These results are consistent with an Na+ gradient-stimulated PAH/OH- exchange mechanism in the basolateral membrane. In BBMV, PAH uptake could be stimulated by an outwardly directed OH- gradient as well as an inward Na+ gradient. Both gradients could drive PAH transport via a mediated probenecid-sensitive pathway. Na+ gradient-stimulated uptake was electrogenic with a (apparent) Km = 4.93 +/- 0.57 mM, Vmax = 6.71 +/- 0.36 nmol/mg protein, 15 sec and Ki,prob = 0.13 +/- 0.01 mM. The kinetic parameters for PAH/OH- exchange were virtually the same, (apparent) Km = 5.72 +/- 0.49 mM, Vmax = 7.87 +/- 0.33 nmol/mg protein, 15 sec and Ki,prob = 0.16 +/- 0.02 mM. When both the Na+ and pH (outward OH-) gradient were simultaneously imposed an almost twofold stimulation in uptake was observed over that with either an Na+ or pH gradient alone. These results suggested that both gradients stimulate PAH transport in BBMV via the same pathway. However, inhibition experiments with various organic anions showed that the specificities of Na+ and pH gradient-stimulated PAH uptake do not entirely overlap. Thus, our results support a simple transport in BBMV, but it cannot be excluded that two separate pathways are involved.