The secretory transport of tetraethylammonium (TEA) was investigated in perfused and nonperfused isolated S1, S2, and S3 segments of proximal tubules from rabbit kidneys. In the perfused tubules the transepithelial net secretory flux and in nonperfused tubules the TEA cellular uptake were saturable (Km = 67 microM, Vmax = 2,480 fmol X min-1 X mm-1 in perfused S2 segments), energy dependent, and inhibited by mepiperphenidol. The net secretory flux of TEA (J b leads to j TEA) at a bath TEA concentration of 40 microM differed for the three segments and decreased in the order S1 greater than S2 greater than S3. The concentration of TEA in the perfusate leaving the tubule was approximately twice as great and the intracellular TEA concentration approximately 40 times as great as that in the bath. In nonperfused segments (40 microM TEA in the incubation medium) the TEA tissue water-to-medium ratio reached 100. In the three segments the ability to accumulate TEA across the peritubular membrane, thus, was similar, but the transepithelial secretory flux differed significantly. The differences in secretory rate between the three segments presumably result from differences in the luminal membrane permeability.
Proteins from rabbit kidney brush border membranes were solubilized with 1% Nonidet P-40 (crude membrane proteins) and fractionated according to their isoelectric points (pI) by chromatofocusing. The eluate was pooled into three fractions according to the pI of the samples (1, greater than 6.8; 2, 6.8-5.4; 3, 5.4-4.0). The crude membrane proteins as well as the three fractions were reconstituted into liposomes and transport of Pi was measured by a rapid filtration technique in the presence of an inwardly directed K+ or Na+ gradient. Arsenate-inhibitable Na+-dependent transport of Pi was reconstituted into an osmotically active intravesicular space from both the crude membrane proteins and Fraction 1. In contrast, Fractions 2 and 3 were inactive. Treatment of the crude membrane proteins and the three fractions with the method for extracting phosphorin (a Pi-binding proteolipid found in brush border membranes) yielded Mn2+-dependent binding of Pi characteristic of phosphorin only in the extracts from crude membrane proteins and Fraction 1, the same fractions in which Na+-dependent transport of Pi was found in the reconstituted system. When reconstituted into liposomes, phosphorin was, however, unable to yield Na+-dependent transport of Pi. Moreover, we cannot eliminate the possibility that Na+-Pi transport can occur in the absence of phosphorin, since complete recovery of Na+-Pi transport was not achieved. However, the present data showing localization of the recovered binding and transport systems for Pi in the same protein fraction lend support to the hypothesis that phosphorin might be a constituent of the renal Pi transport system. Whether the presence of phosphorin is necessary or accessory for Na+-dependent Pi transport in intact brush border membrane vesicles or in liposomes reconstituted with crude or purified membrane proteins requires further investigation.
Segments of rabbit proximal tubules (S1, S2, and S3) were incubated (30 min, 37 degrees C, pH 7.4) in Ringer solution (under O2) containing [14C]urate or [3H]PAH and probenecid, pyrazinoic acid (PZA), urate, or PAH, all at 1 mM. The ratio [14C]urate (counts/min per nanoliter of tubular cell water)/(counts/min per nanoliter of incubation medium) (T/M) reached 2.92 +/- 0.31 (mean +/- SE; n = 15), 6.08 +/- 0.42 (n = 42), and 1.72 +/- 0.31 (n = 14) for S1, S2, and S3 segments, respectively. In these segments the uptake of [3H]PAH was 22.7 +/- 4.6 (n = 19), 133 +/- 18.9 (n = 24), and 66.2 +/- 15.3 (n =0). The accumulation of [14C]urate was saturated at 2 x 10(-4) M in the medium, and that of [3H]PAH at 7 x 10(-5) M in the medium. Secretory transport was saturated at the same bath PAH concentration in perfused tubules. Urate and PAH transports inhibited each other, thus suggesting a common transport mechanism mainly located in the S2 segment. Probenecid was a strong and PZA a weak inhibitor of this transport system.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.