Effect of substituted benzoylglycines (hippurates) and phenylacetylglycines on p-aminohippurate transport in dog renal membrane vesicles.

The effect of substituted benzoylglycines (hippurates) and phenylacetylglycines on the transport of p-aminohippurate (PAH) was studied in basolateral (BLMV) and brush border membrane vesicles (BBMV) isolated from dog kidney cortex. The probenecid-sensitive part of 100 microM [3H]PAH uptake into BLMV and BBMV was measured in the presence and absence of 5 mM glycine conjugate. The benzoyl- and phenylacetylglycines studied were substituted in the 2-, 3-, or 4-position with an H, CH3, OCH3 or OH group. Benzoylglycines were stronger inhibitors of PAH transport than phenylacetylglycines and the inhibitory potency of the conjugates was in general lower against the transporter in BBMV than BLMV. The specificities of the transporters in both membranes appear to be very similar. The inhibitory potency of the benzoylglycines, expressed as the apparent inhibition constant (logKi), did not show a linear relationship with their lipophilicity as determined by reversed phase HPLC. Deviation from linearity was caused mainly by the 3-OH and 4-OH analogs, which showed a greater inhibitory potency than expected from their lipophilicity. Phenylacetylglycines only showed a small variation in logKi values, indicating that insertion of a CH2 group between the ring and the carbonyl practically abolishes the influence of the ring and its substituents. In conclusion, both hydrophobic and electronic properties are important determinants of affinity for the PAH transport system. An additional partially negative hydroxyl group in the ring, located preferably at the 3- or 4-position, increases the interaction with the transport system.

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

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.