Effects of divalent cations and sulfhydryl reagents on the p-aminohippurate (PAH) transporter of renal basal-lateral membranes.

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.