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Our study found the uptake of [14C]succinate into bovine adrenocortical cells to be sodium-dependent, inhibited by lithium, and to have an apparent K m of 146 µmol/l. Succinate uptake was inhibited by glutarate, fumarate, α-ketoglutarate, and maleate but not by 2,3-dimethylsuccinate or cis-aconitate, specific inhibitors of the basolateral Na+-dicarboxylate transporter of renal proximal tubule cells. Succinate uptake was highest at pH 6.0 and decreased with increasing pH. Transport of succinate was not significantly inhibited by citrate at pH 7.4 whereas at pH 6.0 inhibition of succinate uptake by citrate was small but significant. The affinity of the adrenal dicarboxylate transporter towards succinate ranges in between the low affinity of the renal luminal dicarboxylate transporter and the high affinity of the respective basolateral transporter. The pH dependency of succinate uptake and the missing inhibition by citrate at pH 7.4 differ from both the luminal and from the basolateral dicarboxylate transporters in kidney, liver, intestine, and placenta. These functional characteristics provide evidence for the existence of a Na+-dicarboxylate cotransporter in adrenocortical cells which may supply cholesterol metabolism with reducing substrates. [ABSTRACT FROM AUTHOR]

Several earlier studies suggested that the uptake of phallotoxins by liver cells is a carrier mediated process using a transport system normally handling bile acids (see Frimmer 1982). In this study we have shown whether ileal cells, well known to transport bile acids too, are able to take up phallotoxins. Isolated epithelial cells prepared from guinea pig ileum accumulated [C]-cholate, whereas [H]-demethylphalloin ([H]-DMP) was not taken up. The same observation was made with isolated jejunal cells but the uptake of [C]-cholate was much slower. [H]-DMP, however, was partly bound to intestinal cells. This process was not inhibited by cholate, iodipamide, oligomycin and carbonylcyano-chlorophenylhydrazone (CCCP), compounds known to decrease the uptake of phallotoxins into liver cells. Substituting Na for choline and also Cl for SCN did not influence the binding of [H]-DMP. Frozen intestinal cells from the guinea pig bound two times more [H]-DMP after thawing compared with intact cells. Supplementary uptake experiments on isolated brush border membrane vesicles from rat ileum revealed that phalloidin does not inhibit taurocholate uptake and that taurocholate does not interfer with [H]-DMP binding. The results suggest that [H]-demethylphalloin is not recognized by the bile acid carrier of the guinea pig and the rat ileum. It is concluded that the transport system for bile acids present in ileal cells is different from that of liver cells. [ABSTRACT FROM AUTHOR]

Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H+ uptake as visualized by the delta pH indicator, acridine orange. The reoriented H(+)-pump is electrogenic because permeant extravesicular anions or intravesicular K+ plus valinomycin enhance H+ transport. ATP stimulates H+ uptake with an apparent Km of 93 microM. Support of H+ uptake and Pi liberation by ATP greater than GTP approximately ITP greater than UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H+ uptake (Ki, 24 microM), Mg2+ and Mn2+ support ATP-driven H+ uptake, but Ca2+, Ba2+, and Zn2+ do not, 1 mM Zn2+ inhibits MgATP-driven H+ transport completely. NEM-sensitive Pi liberation is stimulated by Mg2+ and Mg2+ and, unlike H+ uptake, also by Ca2+ suggesting Ca2(+)-dependent ATP hydrolysis unrelated to H+ transport. The inside-out oriented H(+)-pump is relatively insensitive toward oligomycin, azide, N,N'-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparent Ki, 0.77 microM), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparent Ki, 0.39 microM). Taken together, the H(+)-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of "vacuolar type" (V-type) H(+)-ATPases. Hence, V-type H(+)-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas. [ABSTRACT FROM AUTHOR]

Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H+ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl- channels with a single-channel conductance of 73 +/- 2 pS in symmetrical 140 mM KCl solution (n = 25). The current-voltage relationship was linear in the range of -60 to +80 mV, but channel kinetic properties depended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10-15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2-6 msec. Clamp voltages more negative than -50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl- = Br- = I- greater than SO4(2-) approximately F-. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 1.0 mM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1 mM) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl(-)-flux measurements and studies on the H+ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H+ uptake into the endosomes. [ABSTRACT FROM AUTHOR]

In the presence of inhibitors for mitochondrial H+-ATPase, (Na+ + K+)- and Ca2+-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane ("ecto-ATPases"). These ATPases accept several nucleotides, are stimulated by Ca2+ and Mg2+, and are inhibited by N.N'-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brush-border and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg2+ and Mn2+, but not by Ca2+, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator. ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H+ secretion is electrogenic and requires either exit of a permeant anion (Cl-) or entry of a cation, e.g., Na+ via electrogenic Na+/D-glucose and Na+/L-phenylalanine uptake. In the presence of Na+, ATP-driven H+ efflux is stimulated by blocking the Na+/H+ exchanger with amiloride. These data prove the coexistence of Na+-coupled substrate transporters, Na+/H+ exchanger, and an ATP-driven H+ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H+ pumps with (a part of) the DCCD- and NEM- sensitive ATPases at the cytosolic side of the brush-border membrane. [ABSTRACT FROM AUTHOR]

Dicyclohexylcarbodiimide (DCCD) and the 5-ethyl-isopropyl-6-bromo-derivative of amiloride (Br-EIPA) have been used as affinity and photoaffinity labels of the Na+/H+ exchanger in rat renal brush-border membranes. Intravesicular acidification by the Na+/H+ exchanger was irreversibly inhibited after incubation of vesicles for 30 min with DCCD. The substrate of the antiporter, Na+, and the competitive inhibitor, amiloride, protected from irreversible inhibition. The Na+-dependent transport systems for sulfate, dicarboxylates, and neutral, acidic, and basic amino acids were inhibited by DCCD, but not protected by amiloride. An irreversible inhibition of Na+/H+ exchange was also observed when brush-border membrane vesicles were irradiated in the presence of Br-EIPA. Na+ and Li+ protected. [14C]-DCCD was mostly incorporated into three brush-border membrane polypeptides with apparent molecular weights of 88,000, 65,000 and 51,000. Na+ did not protect but rather enhanced labeling. In contrast, amiloride effectively decreased the labeling of the 65,000 molecular weight polypeptide. In basolateral membrane vesicles one band was highly labeled by [14C]DCCD that was identified as the alpha-subunit of the Na+,K+-ATPase. [14C]-Br-EIPA was mainly incorporated into a brush-border membrane polypeptide with apparent molecular weight of 65,000. Na+ decreased the labeling of this protein. Similar to the Na+/H+ exchanger this Na+-protectable band was absent in basolateral membrane vesicles. We conclude that a membrane protein with an apparent molecular weight of 65,000 is involved in rat renal Na+/H+ exchange. [ABSTRACT FROM AUTHOR]

Rat small intestinal brush-border membrane (BBM) vesicles were solubilized to test for the presence of an intravesicular H+-ATPase. Several detergents that in rat renal BBM vesicles revealed a bafilomycin-sensitive H+-ATPase failed to expose a similar ATPase in small intestinal vesicles. Antibodies against the 31-kDa and 70-kDa H+-ATPase subunits labelled respective antigens in the BBM of renal proximal tubules, but not in the BBM of enterocytes. The results demonstrate that a bafilomycin-sensitive, vacuolar-type H+-ATPase is absent from the BBM of enterocytes and, hence, cannot contribute to H+ secretion. [ABSTRACT FROM AUTHOR]