Kinetics of sodium succinate cotransport across renal brush-border membranes.

The kinetics of Na/succinate cotransport across renal brush-borders was studied using membrane vesicles. Initial rates of succinate uptake (Js) were estimated from 1-s uptakes measured under voltage-clamped conditions. Lowering the external (cis) sodium concentration reduced the succinate Kt without affecting the Jsmax. Increasing the intravesicular (trans) sodium concentration reduced both Jsmax and Kt. This trans inhibition by Na was relieved by trans-succinate. Varying the membrane potential from -60 to +60 reduced succinate transport in an exponential manner, which was reflected by a Kt effect, i.e. an increase in the Kt with no change in Jsmax. The membrane potential results suggest that the succinate/sodium-carrier complex bears a net positive charge. On the basis of these and earlier observations, we propose that Na/succinate cotransport across renal brush-borders is described by an Iso Ordered Quad Quad reaction kinetics in which three Na ions first bind to the carrier to increase its affinity for succinate.

Specificity of the Na+-dependent monocarboxylic acid transport pathway in rabbit renal brush border membranes.

The substrate specificity of a Na+-dependent transport pathway for L-lactate was studied in rabbit renal brush border membrane vesicles. Jmax for L-lactate transport was unaffected by the presence of a fixed concentration of two different short-chain monocarboxylic acids, while the apparent Kt(Ka) for L-lactate increased, and this is compatible with competitive inhibition. The inhibitor constants ("Ki"'s) for the transport pathway for the two solutes examined closely corresponded to the respective "Ki"'s derived from a Dixon plot. A broad range of compounds were then tested as potential inhibitors of L-lactate transport, and the "Ki"'s thereby derived yielded specific information regarding optimal substrate recognition by the carrier. A single carboxyl group is an absolute requirement for recognition, and preference is given to 3 to 6 C chain molecules. Addition of ketone, hydroxyl and, particularly, amine groups at any carbon position, diminishes substrate-carrier interaction. Intramolecular forces, notably the inductive effects of halogens, may play a role in enhancing substrate-carrier interaction; however, no correlation was found between pKa and "Ki" for the substrates examined. We conclude that a separate monocarboxylic acid transport pathway, discrete from either the D-glucose, alpha or beta neutral amino-acid, or dicarboxylic acid carriers, exists in the renal brush border, and this handles a broad range of monocarboxylates.

Interactions between lithium and renal transport of Krebs cycle intermediates.

The effect of lithium on the renal transport of Krebs cycle intermediates was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The di- and tricarboxylic acids are avidly transported across renal brush border membranes by a sodium cotransport system. Lithium acted as a potent, specific, competitive inhibitor (Ki = 1.2 mM) of succinate/sodium cotransport when added to the uptake medium. Similar effects were observed for citrate but not D-glucose, L-phenylalanine, L-proline, L-alanine, or L-lactate transport. Intravesicular lithium behaved as a noncompetitive inhibitor of succinate transport in the absence of sodium. These results account for the observation that therapeutic doses of lithium increase the renal excretion of Krebs cycle intermediates. The existence of a transport system for alpha-ketoglutarate in synaptosomes suggests a possible target for lithium in the central nervous system.

Pathways for carboxylic acid transport by rabbit renal brush border membrane vesicles.

Brush border membrane vesicles were purified from rabbit renal cortex using a calcium-precipitation procedure, and the uptake of carboxylic acids was determined by a rapid-filtration method. L-Lactate, pyruvate (monocarboxylic acids), and succinate (dicarboxylic acid) demonstrated features of Na+ cotransport: enhanced initial rate (1 s) of uptake with an inward Na+ gradient compared with the Na+ -free control condition and transient accumulation of substrate within the vesicles. Kinetic parameters derived for L-lactate and succinate show that each substrate is transported via single pathway and that the two substrates exhibit marginal cross-inhibition. A range of monocarboxylic acids including pyruvate and ketone bodies appear to interact with the monocarboxylic acid carrier. The kinetics of Nat-dependent pyruvate uptake suggest at least two transport pathways-namely, that this monocarboxylate shares both the mono- and dicarboxylic acid carriers. We conclude that isolated rabbit renal microvillus membranes possess independent transport systems for mono- and polycarboxylic acids.

Stoichiometry of Na+-succinate cotransport in renal brush-border membranes.

The coupled transport of Na+ with succinate and citrate into rabbit renal brush-border membrane vesicles was studied. Initial rates of transport (J) were estimated from 1-s uptakes under well defined transmembrane conditions (membrane potential, pH, ionic composition). A Na+ gradient increased J up to 700 times compared to Na+ replacement with sorbitol. J was a sigmoid function of increasing Na+ concentration; the kinetic interaction of Na+ with the succinate-citrate transport system was described by the equation: J = Jmax/(1 + (Ks/[Na]))3. Assuming that the Na+ interaction involves equivalent, noninteracting binding sites, these results indicate that 3 Na+ ions are translocated/molecule of organic substrate. Consistent with the latter observation was the direct demonstration of a stoichiometric coupling between simultaneously determined fluxes of 22Na+ and 14C-labeled substrates. In excess of 2 Na+ ions were transported/molecule of organic substrate. These results explain the electrogenicity of the transport of succinate and citrate as evidenced by the response of J to K+ diffusion potentials and the effects of transport on the fluorescence of a potential-sensitive cyanine dye.

Organic and inorganic solute transport in renal and intestinal membrane vesicles preserved in liquid nitrogen.

The transport of organic solutes (sugars, amino acids, and metabolic intermediates) and inorganic solutes (Na+/H+ exchange and Na+-SO = 4 cotransport) in renal brush border and in intestinal brush border and basal lateral membrane vesicles is preserved when the vesicles are stored in liquid nitrogen. The preservation allows comparisons among transport systems of renal and intestinal cells obtained from the same animal.

Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles.

We have confirmed previous demonstrations of sodium gradient-stimulated transport of L-alanine, phenylalanine, proline, and beta-alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these 14C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability of D-glucose to inhibit tracer uptake. L-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the 14C-L-alanine uptake could be inhibited by proline and beta-alanine, while lysine and MeAIB were no more effective than D-glucose. Roughly 50% of the 14C-phenylalanine uptake could be inhibited by proline and beta-alanine; lysine was as effective as proline and beta-alanine, and the effects of pairs of these amino acids at 50 mM each were not cumulative. MeAIB was no more effective than D-glucose. We conclude that three pathways mediate the uptake of neutral L, alpha-amino acids. One system is inaccessible to lysine, proline, and beta-alanine. The second system carries a major fraction of the L-alanine flux; it is sensitive to proline and beta-alanine, but not to lysine. The third system carries half the 14C-phenylalanine flux, and it is sensitive to proline, lysine, and beta-alanine. Since the neutral, L, alpha-amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classical A system, and since all of the L-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classical ASC system. L-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective than D-glucose in inhibiting lysine uptake, while proline and beta-alanine appear to inhibit a component of the lysine flux. We conclude that the 14C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, beta-alanine, and L-alanine, and one which is inhibited by L-alanine and phenylalanine but inaccessible to proline, beta-alanine, and MeAIB. Fluxes of 14C-proline and 14C-MeAIB are completely inhibited by L-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit 14C-beta-alanine uptake. However, beta-alanine inhibits only 38% of the 14C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports beta-alanine.

Effect of pH on the transport of Krebs cycle intermediates in renal brush border membranes.

Lowering extravesicular pH stimulated Na+-dependent citrate transport in renal brush border membrane vesicles: e.g., at pHout = 5.5, the initial rate of citrate uptake was increased 10-fold compared to parallel control experiments at pH 7.5. The same experimental conditions had little effect on succinate uptake. The influence of pH on citrate transport is a product of the extravesicular H+ concentration; pH gradients did not potentiate the effects nor were proton gradients capable of driving transport in the absence of Na+. The effect of pH is adequately explained if only the mono- and divalent species of citrate (Cit1-, Cit2-) are considered acceptable substrates for transport. The stimulatory influence of pH on transport correlated quite well with pH-related increases in the concentrations of Cit1- and Cit2-, and over the same pH range [Cit3-] was inversely related to citrate uptake. A model of the Na+-dependent dicarboxylate transport system is discussed in which three sodium ions are translocated per molecule of dicarboxylic acid.

Na+-dependent transport of tricarboxylic acid cycle intermediates by renal brush border membranes. Effects on fluorescence of a potential-sensitive cyanine dye.

The effect of the transport of tricarboxylic acid cycle intermediates on the membrane potential of renal brush border vesicles was studied using fluorescence of the cyanine dye, 3,3'-dipropylthiadicarbocyanine iodide. The behavior of the dye in the preparation was established with valinomycin-induced K+-diffusion potentials; increases in fluorescence were associated with depolarizing conditions. Addition of 1 mm succinate or citrate to membrane/dye suspensions produced transient increases in fluorescence, indicative of a depolarizing event(s) associated with the transport of these substrates. The transient response in fluorescence was Na+ dependent, of greater magnitude under Na+-gradient as compared to Na+-equilibrium conditions, and was a saturable function of substrate concentration. The specificity of the fluorescence response was identical to that obtained from studies of the competitive inhibition of succinate transport by tricarboxylic acid cycle intermediates and analogs We conclude that the major tricarboxylic acid cycle intermediates are transported via a common Na+-dependent transport system in renal brush border membranes.

Specificity of the transport system for tricarboxylic acid cycle intermediates in renal brush borders.

Uptake studies employing renal brush border membranes were used to examine the structural specificity of the TCA cycle intermediate transport system. The kinetics of reciprocal inhibition between succinate and citrate revealed these compounds to be transported by a common mechanism. The Michaelis constant for succinate (0.11 mM) was significantly lower than that of citrate (0.28 mM), indicating that the system has a higher affinity for succinate than for citrate. The specificity of the transport system was determined from the relative inhibitory constants of 40 organic acids on the transport of succinate. The results established that the system is highly specific for 4-carbon, terminal dicarboxylic acids in the trans-configuration, including the major intermediates of the TCA cycle. The system is comparatively insensitive to monocarboxylates. Substitution of one of several polar, non-charged residues on the alpha-carbon of succinate permitted interaction of the substrate with the transport system, but substitutions on both the alpha and beta-carbons did not. The structural specificity of the system is fundamentally different from that of the dicarboxylate and tricarboxylate exchange systems of mitochondria. The role of this transport system in the reabsorption of TCA cycle intermediates from the proximal tubule is discussed.

Effects of metabolic intermediates on sugar and amino acid uptake in rabbit renal tubules and brush border membranes.

1. The effects of tricarboxylic acid cycle intermediates on the renal transport of alpha-methyl-D-glucoside and alpha-amino-isobutyric acid were examined using separated renal tubules of the rabbit. 2. The effect of citrate on alpha-methyl-D-glucoside and alpha-amino-isobutyric acid uptake was markedly biphasic with maximum stimulation of transport occurring at a citrate concentration of 0.64 mM. Biphasic effects were also apparent for L-malate, succinate, fumarate, alpha-ketoglutarate and oxaloacetate. 3. The route of uptake of alpha-methyl-D-glucoside into separated renal tubules is primarily across the brush border (luminal) membrane. 4. Tricarboxylic acid cycle intermediates produced significant stimulation of renal O2 consumption; however, the effects on O2 consumption were not biphasic suggesting that reduced stimulation of transport at high substrate concentration was not caused by a reduction in the supply of metabolic energy. 5. In purified renal cortical brush border membrane vesicles, citrate and alpha-ketoglutarate inhibited the uptake of alpha-methyl-D-glucoside and alpha-amino-isobutyric acid indicating that inhibition of their transport in respiring renal tubules by high concentrations of tricarboxylic acid cycle intermediates occurs via an effect at the membrane level.

Effects of dibutyryl cyclic AMP on the transport of alpha-methyl-D-glucoside and alpha-aminoisobutyric acid in separated tubules and brush border membranes from rabbit kidney.

The effect of dibutyryl cyclic AMP on the transport of alpha-methyl-D-glucoside and alpha-aminoisobutyric acid in separated tubules and purified brush border membranes from rabbit kidney was investigated using a rapid filtration procedure. Dibutyryl cyclic AMP stimulated the uptake of alpha-methyl-D-glucoside and alpha-aminoisobutyric acid by separated renal tubules in agreement iwth prior studies utilizing renal slices (Rea, C. and Segal, S. (1973) Biochim. Biophys. Acta 311, 615--624; Weiss, I.W., Morgan, K. and Phang, J.M. (1972) J. Biol. Chem. 247, 760--764). However, in contrast to previous reports, no preincubation of the tissue with dibutyryl cyclic AMP was required for stimulation of transport to be manifest. Dibutyryl cyclic AMP stimulated oxygen consumption by separated tubules suggesting that stimulation of transport may occur by a linkage with renal oxidative metabolism. Dibutyryl cyclic AMP increased the uptake of alpha-aminoisobutyric acid into purified renal brush border membranes. However the uptakes of alpha-methyl-D-glucoside, proline, leucine and phosphate into brush border membranes were significantly inhibited.

Transport of p-aminohippuric acid, uric acid and glucose in highly purified rabbit renal brush border membranes.

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na+ + K+)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%. Transport of D-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (EA = 11.3--37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 muM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.

Transport of tricarboxylic acid cycle intermediates by membrane vesicles from renal brush border.

The uptake of citrate and alpha-ketoglutarate by membrane vesicles from rabbit renal brush border was studied by a rapid filtration technique. Both compounds exhibited transport characteristics similar to those seen for the sodium-dependent cotransport systems previously described for sugars and amino acids in brush border membranes. The estimated sodium-dependent Vmax and Km were 17 nmol per mg of protein per min and 0.18 mM for citrate and 17 nmol per mg of protein per min and 1.0 mM for alpha-ketoglutarate. The initial rate of citrate transport was 5 times that of sugars and amino acids under comparable conditions. Uptake rates of 0.1 mM citrate and alpha-ketoglutarate were inhibited by greater than 90% by 10 mM succinate, malate, fumarate, or oxaloacetate, indicating the presence in the brush border membrane of a transport system highly specialized for the renal conservation of intermediates of the tricarboxylic acid cycle.

Effects of renal fuels on uptake of PAH and uric acid by separated renal tubules of the rabbit.

A rapid-filtration procedure was used to examine the effects of a wide variety of renal fuels on the uptake of p-aminohippuric acid (PAH) and uric acid (UA) by separated rabbit renal tubules. PAH and UA uptakes in 15 min over a range of substrate concentrations of 0.01-10.2 mM were determined. All tricarboxylic acid cycle intermediates and pyruvate showed biphasic stimulation of PAH and UA uptake. alpha-Ketoglutarate produced a 320 +/- 54% increase in PAH uptake and a 192 +/- 60% increase in UA uptake at 0.16 mM, the concentration at which uptake was maximal, while causing 20 +/- 3 (PAH) and 35 +/- 7% (UA) inhibition at 10.2 mM. Citrate produced a 373 +/- 19% increase in PAH uptake and a 246 +/- 41% increase in UA uptake at 0.64 mM. PAH and UA uptake were also stimulated by acetate, glucose frutose, phosphoenolypyruvate and L-glutamic acid. The data indicate a direct relationship between stimulation of PAH and uric acid transport and stimulation of renal cortical oxidative metabolism.

Uptake of uric acid by separated renal tubules of the rabbit. II. Effects of drugs.

The effect of various drugs on the rate of uptake of uric acid by separated renal tubules of the rabbit was investigated. Determinations were made of 150 values and slopes of the inhibition curves. The most potent inhibitor of uric acid uptake was sulfinpyrazone, with an 150 of 0.02 mM. Calcium ipodate and benzbromarone were also potent inhibitors. Uricosuric drugs, with the exception of benzbromarone, had shallow inhibition slopes. Diuretic drugs had steep inhibition slopes. More than one mechanism of action is probably involved in the inhibition of uric acid uptake in separated renal tubules by drugs.

Uptake of uric acid by separated renal tubules of the rabbit. I. Characteristics of transport.

A rapid filtration procedure was used to determine rates of uric acid uptake by a preparation of separated renal cortical tubules of the rabbit. The rate of uric acid uptake was temperature dependent and showed saturation kinetics with a K of 3.2 mM. The uptake was 50% lower when measured under nitrogen as compared with uptake under oxygen. The uptake rate increased with increasing sodium concentration and decreased with increasing potassium concentration. Uptake was stimulated by citrate, succinate and pyruvate, and inhibited by alpha-ketoglutarate. Parathyroid hormone and 10(-4) M adenosine 3':5'-monophosphate increased the uric acid uptake rate when preincubated with the tubules for 130 minutes before the addition of uric acid. Uric acid uptake in this preparation appears to occur by some form of carrier-mediated active transport.