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A specific system of transport for p-aminohippurate (PAH) is demonstrated in rabbit renal basal-lateral membrane vesicles. The PAH uptake into an intravesicular space is inhibited by probenecid in concentrations above 0.2 mM. The transport is saturable and is also temperature-dependent with an optimum between 37 and 45 degrees C. Divalent cations are able to enhance the uptake 2- to 3-fold. The stimulatory effect of the divalent cations diminishes in the following order: Mg++ = Mn++, Ba++, Ca++ and Sr++. Maximum stimulation occurs between 2.5 and 5 mM Mg++. The divalent cation stimulatory effect is not the result of changes in the size of the vesicles, in the degree of vesiculation, in the net charge of the membrane or of a transient potential difference across the membrane. Several inhibitors, more inhibitory than probenecid, were found. These are: lithium diiodosalicylate; 4-acetamido-4'-isothiocyano 2,2'-disulfonic acid stilbene; the mercurials, mersalyl acid, p-chloromercuriphenyl sulfonate and Hg++; and 5,5'-dithiobis(nitrobenzoate). Among these, mersalyl acid is the most potent inhibitor for PAH uptake. Its inhibitory effect is probably a combination of its reactivity toward sulfhydryl groups and its anionic character. The results with sulfhydryl reagents indicate that the PAH transport system contains sulfhydryl groups which are essential for the uptake activity. These sulfhydryl groups are probably buried in a hydrophobic region within the lipoprotein matrix of the basal-lateral membrane.

In basal-lateral membranes of the renal cortex, thiol substituted analogs of salicylate inhibited the uptake of p-aminohippurate (PAH). The mercury-containing analogs, thimerosal and mercaptide V (formed from 2 mol of thiosalicylate and 1 mol of Hg++), were highly inhibitory. Compared with probenecid, thimerosal and mercaptide V yielded dose-response curves of steeper slope and higher maximal effect. The dose-response curves of thimerosal and mercaptide V were similar in shape, although mercaptide V was more potent. The inhibition by thimerosal, mercaptide V and mersalyl acid, an anionic sulfhydryl reagent which also inhibits uptake of PAH ( Tse et al., J. Pharmacol. Exp. Ther. 226: 19-26, 1983), was found to be competitive with Ki values of 0.39 +/- 0.05, 0.12 +/- 0.06 and 0.05 +/- 0.02 mM, respectively. The inhibitory effects of thimerosal and mercaptide V were only partially reversible. Thimerosal and mercaptide V reacted 35 and 62%, respectively, with membrane sulfhydryl groups which may explain the nonreversibility of the inhibitor. The nonreversibility is probably not due to irreversible destruction of the vesicles, as the "glucose space" of the vesicles was not affected by these two compounds. That derivatives of salicylates capable of reacting with sulfhydryl groups were more inhibitory than thiosalicylate is consistent with a hypothesis that the PAH transporter contains a sulfhydryl group(s) essential for uptake. Based on the degree of inhibition, the degree of reversibility, the degree of membrane sulfhydryl group oxidation and the computer-generated three dimensional models of thimerosal, mercaptide V and mersalyl acid, a model of the PAH transporter is proposed.