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V Ganapathy

Publications and source records attributed to V Ganapathy.

At least 217 records · Page 12Linked to original sources
PubMed1988

Na+ + Cl- -gradient-driven, high-affinity, uphill transport of taurine in human placental brush-border membrane vesicles.

Uptake of taurine in human placental brush-border membrane vesicles was greatly stimulated in the presence of an inwardly-directed Na+ + Cl- -gradient and uphill transport of taurine could be demonstrated under these conditions. Na+ as well as Cl- were obligatory for this uptake and both ion gradients could energize the uphill transport. This Na+ + Cl- -gradient-dependent taurine uptake was stimulated by an inside-negative membrane potential, demonstrating the electrogenicity of the process. The uptake system was highly specific for beta-amino acids and the Km of the system for taurine was 6.5 +/- 0.4 microM.

Biological Transport↗
PubMed1988

Evidence for an organic cation-proton antiport system in brush-border membranes isolated from the human term placenta.

Uptake of guanidine, an endogenous organic cation, into brush-border membrane vesicles isolated from human term placentas was investigated. Initial uptake rates were manyfold greater in the presence of an outward-directed H+ gradient ([pH]o greater than [pH]i) than in the absence of a H+ gradient ([pH]o = [pH]i). Guanidine was transiently accumulated inside the vesicles against a concentration gradient in the presence of the H+ gradient. The H+ gradient-dependent stimulation of guanidine uptake was not due to a H+-diffusion potential because an ionophore (valinomycin or carbonylcyanide p-trifluoromethoxyphenylhydrazone)-induced inside-negative membrane potential failed to stimulate the uptake. In addition, uphill transport of guanidine could be demonstrated even in voltage-clamped membrane vesicles. The H+ gradient-dependent uptake of guanidine was inhibited by many exogenous as well as endogenous organic cations (cis-inhibition) but not by cationic amino acids. The presence of unlabeled guanidine inside the vesicles stimulated the uptake of labeled guanidine (trans-stimulation). These data provide evidence for the presence of an organic cation-proton antiporter in human placental brush-border membranes. Kinetic analysis of guanidine uptake demonstrated that the uptake occurred via two saturable, carrier-mediated transport systems, one being a high affinity, low capacity type and the other a low affinity, high capacity type. Studies on the effects of various cations on the organic cation-proton antiporter and the Na+-H+ exchanger revealed that these two transport systems are distinct.

Carrier Proteins↗
PubMed1988

Fatty acid-induced alterations in transport systems of the small intestinal brush-border membrane.

We have investigated the effects of fatty acids on the Na+-H+ exchanger and other carrier-mediated transport systems in intestinal brush-border membrane vesicles. The Na+-H+ exchanger (i.e. H+ gradient-dependent, dimethylamiloride-sensitive Na+ uptake) was strongly inhibited by fatty acids and the inhibition was concentration dependent. Unsaturated fatty acids showed more inhibition than saturated fatty acids. Among unsaturated fatty acids, ricinoleic acid was found to be the most potent inhibitor. Inhibition of the Na+-H+ exchanger by oleic acid was partially reversible, and the nature of the inhibition was found to be non-competitive with respect to Na+. The dimethylamiloride-sensitive Na+ uptake measured in the absence of an H+ gradient was also inhibited by oleic acid, suggesting that the inhibition of the Na+-H+ exchanger by fatty acids was not due to the accelerated dissipation of the H+ gradient. Treatment of the membrane vesicles with oleic acid also inhibited other carrier-mediated transport systems as well, such as the H+ gradient-driven transport of glycylsarcosine and the Na+ gradient-driven transport of D-glucose and L-alanine, whereas it did not affect the permeability of L-glucose, a non-carrier-mediated process. However, the inhibitory effects of oleic acid on the transport of D-glucose and L-alanine appeared to be related to the enhanced collapse of the Na+ gradient rather than a direct effect on the carrier systems because transport of these solutes when measured in the absence of a Na+ gradient ([Na+]i = [Na+]o) was not affected by oleic acid. These data demonstrate that fatty acids bring about significant alterations in the activities of various transport systems of the small intestinal brush-border membrane, either by directly interacting with the transport protein or by abolishing the energy source that is necessary for the transport process.

Animals↗
PubMed1988

Sodium-gradient-driven, high-affinity, uphill transport of succinate in human placental brush-border membrane vesicles.

Brush-border membrane vesicles isolated from normal human term placentas were shown to accumulate succinate transiently against a concentration gradient, when an inward-directed Na+ gradient was imposed across the membrane. This uptake was almost totally due to transport into intravesicular space, non-specific binding to the membranes being negligible. The dependence of the initial uptake rate of succinate on Na+ concentration exhibited sigmoidal kinetics, indicating interaction of more than one Na+ ion with the carrier system. The Hill coefficient for this ion was calculated to be 2.7. The Na+-dependent uptake of succinate was electrogenic, resulting in the transfer of positive charge across the membrane. Kinetic analysis showed that succinate uptake in these vesicles occurred via a single transport system, with an apparent affinity constant of 4.8 +/- 0.2 microM and a maximal velocity of 274 +/- 4 pmol/20 s per mg of protein. Uptake of succinate was strongly inhibited by various C4 or C5 dicarboxylic acids, whereas monocarboxylic acids, amino acids and glucose showed little or no effect. Li+ and K+ could not substitute for Na+ in the uptake process. Instead, Li+ was found to have a significant inhibitory effect on the Na+-dependent uptake of succinate.

Biological Transport, Active↗
PubMed1988

Distribution and properties of the glycylsarcosine-transport system in rabbit renal proximal tubule. Studies with isolated brush-border-membrane vesicles.

The distribution and properties of the peptide-transport system in rabbit renal proximal tubule was examined with glycylsarcosine as the substrate and using brush-border-membrane vesicles derived from pars convoluta (outer cortex) and pars recta (outer medulla). The dipeptide was transported into these vesicles against a concentration gradient in the presence of an inward-directed H+ gradient, demonstrating the presence of a H+-coupled peptide-transport system in outer-cortical as well as outer-medullary brush-border membranes. Even though the transport was electrogenic and was energized by a H+ gradient in both membranes, the system was more active in outer medullary membranes than in outer cortical membranes. Kinetic analysis showed that, although the affinity of the transport system for glycylsarcosine was similar in both membrane preparations, the capacity of the system was significantly greater in outer medulla than in outer cortex. In addition, the pH profiles of the peptide-transport systems in these membrane preparations also showed dissimilarities. The greater dipeptide uptake in one membrane vis-à-vis the other may probably be due to the difference in the affinity of the transport system for H+ and/or the difference in peptide/H+ stoichiometry.

Animals↗
PubMed1988

Transport of guanidine in rabbit intestinal brush-border membrane vesicles.

The characteristics of guanidine uptake were studied in brush-border membrane vesicles isolated from the rabbit proximal intestine. Guanidine uptake was manyfold greater in the presence of an outward-directed H+ gradient (intracellular pH = 5.5; extracellular pH = 7.2) than in the absence of a H+ gradient (intracellular and extracellular pH = 7.2). The time course of guanidine uptake exhibited an overshoot phenomenon in the presence of the H+ gradient, indicating occurrence of uphill transport. This H+ gradient-stimulated guanidine uptake was not due to an inside-negative H+-diffusion potential because carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, a protonophore, failed to have any effect on guanidine uptake. Moreover, the transient uphill transport of guanidine was observed even in voltage-clamped membrane vesicles. However, under the conditions that effectively dissipated the H+ gradient, there was no active transport of guanidine. This H+ gradient-dependent transport mechanism for guanidine is distinct from the Na+-H+ exchanger, because amiloride did not inhibit guanidine uptake even at a concentration as high as 100 microM. These data provide evidence for the presence of a guanidine-H+ antiport system in the rabbit intestinal brush-border membrane. The presence of a carrier for guanidine in these membranes is further substantiated by the trans-stimulation of the uptake of radiolabeled guanidine by unlabeled guanidine and by the inhibition of guanidine uptake by imipramine under equilibrium exchange conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Evidence for active dipeptide transport in isolated proximal straight tubules.

Transport of the dipeptide glycylsarcosine (Gly-Sar) was examined in isolated proximal straight tubules of the rabbit kidney by an in vitro microperfusion technique to determine whether it can be actively transported intact. The unidirectional lumen-to-bath flux of Gly-Sar was measured by two separate methods, namely its appearance rate (JA) in the bathing fluid and its disappearance rate (JD) from the luminal fluid. In addition, the cell Gly-Sar concentration was measured immediately after the last flux period. Mean luminal fluid Gly-Sar concentration was 0.22 mM. Transepithelial Gly-Sar flux (260.0 fmol.min-1.mm-1) was greater than could be accounted for by passive leakage, whereas cellular Gly-Sar accumulation (2.72 mM) was greater than could be attributed to passive equilibration across the luminal membrane. High-pressure liquid chromatographic analysis of cellular extract indicated that 63% of the transported Gly-Sar was hydrolyzed within the cell. Analysis of the bath solution revealed that 47% of the radioactivity that crossed the tubule cell was in the form of intact dipeptide, whereas the remainder of the radioactivity was in the form of hydrolytic and metabolic products of Gly-Sar. This indicates that the dipeptide Gly-Sar is actively transported intact at the luminal membrane into the cytosol of proximal straight tubule cells with subsequent hydrolysis. It then exits across the basolateral membrane as intact Gly-Sar and its hydrolytic and metabolic products.

Algorithms↗
PubMed1988

Effects of phenothiazines on the Na+-H+ exchanger of the brush-border membrane from the proximal small intestine of the rabbit.

We investigated the effects of various phenothiazines on the Na+-H+ exchanger in brush-border membrane vesicles isolated from the proximal small intestine of the rabbit. The Na+-H+ exchanger activity was assayed by measuring the dimethylamiloride-sensitive Na+ uptake into these vesicles in the presence of an outward-directed H+ gradient ([pH]o greater than [pH]i). All phenothiazines that were tested inhibited the Na+-H+ exchanger and chlorpromazine sulfoxide (CPZ-SO) was the most potent inhibitor among them. CPZ-SO inhibited the dimethylamiloride-sensitive Na+ uptake in a dose-dependent manner and IC50 for this process was 90 microM. Even though CPZ-SO drastically reduced the initial rates of Na+ uptake, the equilibrium uptake of Na+ into the vesicles was not affected, indicating that the integrity of membrane vesicles was not altered by the drug. Furthermore, the inhibition of the exchanger activity by phenothiazines was not due to nonspecific dissipation of the H+ gradient, because CPZ-SO inhibited the dimethylamiloride-sensitive Na+ uptake even in the absence of a H+ gradient. The inhibition of the exchanger by CPZ-SO was freely and completely reversible. Kinetic analyses of the Na+-H+ exchanger carried out in the presence and absence of CPZ-SO revealed that the nature of the inhibition depended on the composition of the buffers used in the assay of the exchanger because the inhibition was of a mixed type in the mannitol medium, whereas it was of a competitive type in the KCl medium.

Amiloride↗
PubMed1987

Evidence for histidyl and carboxy groups at the active site of the human placental Na+-H+ exchanger.

The Na+-H+ exchanger of the human placental brush-border membrane was inhibited by pretreatment of the membrane vesicles with a histidyl-group-specific reagent, diethyl pyrocarbonate and with a carboxy-group-specific reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. In both cases the inhibition was irreversible and non-competitive in nature. But, if the membrane vesicles were treated with these reagents in the presence of amiloride, cimetidine or clonidine, there was no inhibition. Since amiloride, cimetidine and clonidine all interact with the active site of the exchanger in a mutually exclusive manner, the findings provide evidence for the presence of essential histidyl and carboxy groups at or near the active site of the human placental Na+-H+ exchanger. This conclusion was further substantiated by the findings that Rose Bengal-catalysed photo-oxidation of histidine residues as well as covalent modification of carboxy residues with NN'-dicyclohexylcarbodi-imide irreversibly inhibited the Na+-H+ exchanger and that amiloride protected the exchanger from inhibition caused by NN'-dicyclohexylcarbodi-imide.

Amiloride↗
PubMed1987

Role of dipeptidyl peptidase IV in uptake of peptide nitrogen from beta-casomorphin in rabbit renal BBMV.

We examined the handling of radiolabeled beta-casomorphin, Tyr-Pro-[3H]Phe-Pro-Gly, by rabbit renal brush-border membrane vesicles (BBMV). The uptake of radiolabel into the vesicles was Na+-independent, but an inward-directed H+ gradient stimulated the uptake. The H+ gradient-dependent uptake was further accelerated by an interior-negative membrane potential, but inhibited in the presence of a protonophore. Treatment of the membrane vesicles with diisopropylfluorophosphate (DFP) greatly reduced the uptake of the radiolabel. Control as well as DFP-treated vesicles exhibited H+ gradient-dependent Gly-Sar uptake. Unlabeled beta-casomorphin inhibited Gly-Sar uptake in control vesicles, but the inhibition was significantly reduced in DFP-treated vesicles. DFP inhibited the activity of dipeptidyl peptidase IV in these vesicles and there was a direct correlation between the activity of the enzyme and the capacity of beta-casomorphin to inhibit Gly-Sar uptake. Many di- and tripeptides reduced the uptake of Gly-Sar and the uptake of radiolabel from beta-[3H]casomorphin to a similar extent. We conclude that beta-casomorphin is hydrolyzed by dipeptidyl peptidase IV and the products are transported into the vesicles by the H+ gradient-driven peptide transport system. This conclusion is supported by the results from the analysis of the incubation medium by high-performance liquid chromatography that showed rapid hydrolysis of the pentapeptide by brush-border membranes to di- and tripeptides.

Animals↗
PubMed1987

H+ gradient-driven dipeptide reabsorption in proximal tubule of rat kidney. Studies in vivo and in vitro.

Microinfusion of glycylsarcosine into superficial nephron sections showed that the dipeptide was reabsorbed mainly in late portions of the rat proximal tubule. In vivo microperfusion data demonstrated a saturable, high-capacity, low-affinity dipeptide reabsorption mechanism that was inhibited by other peptides but not by amino acids or peptidase inhibitors. The reabsorption was enhanced by lowering the luminal pH from 7.5 to 5.5. In vitro studies with rat cortical brush-border vesicles showed that glycylsarcosine uptake was independent of a Na+ gradient and greater uptake occurred when the extravesicular pH was acidic compared with the intravesicular pH. An inward-directed H+ gradient stimulated glycylsarcosine uptake and caused a transient accumulation of the dipeptide inside the vesicles above the equilibrium value. The presence of a proton ionophore abolished the H+ gradient-dependent uptake. An inside-negative membrane potential stimulated the initial uptake of the dipeptide. The uptake process was saturable and inhibited by other peptides but not by amino acids. The vesicle studies also showed that there are at least two peptide transport systems functioning in these vesicles, one a high-affinity, low-capacity type and the other a low-affinity, high-capacity type.

Amino Acids↗
PubMed1987

Development of dipeptide transport in rat renal brush border membranes: studies with glycylsarcosine.

Dipeptide-proton cotransport was studied in rat renal brush border membrane vesicles from animals of different ages, starting at 7 days after birth. In the presence of an inward-directed proton gradient, glycyl-sarcosine uptake exhibited the overshoot phenomenon in all age groups studied. The magnitude of the "overshoot" increased with age and maximum accumulation of glycyl-sarcosine was observed in 42-day-old rat renal brush border membrane vesicles. Kinetic studies with glycyl-sarcosine indicated changes only in maximal velocity without any significant change in the apparent affinity value during the postnatal development of the renal brush border membrane. Since the Na+-H+ exchanger located at the brush border membrane is primarily responsible for the generation of the proton gradient, the driving force for dipeptide transport, we also measured the Na+-H+ exchanger activity in rat renal brush border membrane vesicles from suckling and adult animals. The exchanger activity was significantly greater in the adult rats compared to the suckling rats. In addition, we have confirmed in the present study previous findings from other laboratories that the activities of the Na+ gradient-driven glucose and amino acid transport systems in renal brush border membrane vesicles were higher in the adult than in the suckling rats.

Age Factors↗
PubMed1987

Inhibition of brush-border membrane Na+-H+ exchanger by loperamide.

Loperamide, an opiate agonist, inhibited the Na+-H+ exchanger in brush-border membrane vesicles isolated from term human placenta, rabbit renal cortex and rabbit small intestine in a dose dependent manner. Because the placental Na+-H+ exchanger was the most sensitive to inhibition by loperamide (IC50 = 60 microM), characterization of the inhibition was done with the placental Na+-H+ exchanger. The inhibition of the placental Na+-H+ exchanger by loperamide was instantaneous and freely reversible. Kinetic analyses demonstrated that the inhibition was of a mixed type. Loperamide (70 microM) reduced the maximal velocity (Vmax) from 46.4 +/- 1.2 to 34.5 +/- 1.1 nmol/mg of protein/15 s and increased the apparent affinity constant (Kt) for Na+ from 12.3 +/- 1.0 to 16.5 +/- 1.5 mM. Loperamide interacted with the exchanger protein at more than one site. The effects of loperamide were not antagonized by naloxone, suggesting the noninvolvement of opiate receptors in this process. These results differentiate loperamide from other Na+-H+ exchanger inhibitors such as amiloride, cimetidine and clonidine, which interact with the exchanger at a single site in a strictly competitive manner.

Amiloride↗
PubMed1987

Driving force for peptide transport in mammalian intestine and kidney.

Transport of small intact peptides across the brush-border membrane of the epithelial cells of small intestine and renal proximal tubule is a well-established phenomenon. This peptide transport system recognizes dipeptides and tripeptides as substrates and it is distinct from the transport systems available for absorption of free amino acids. While there is no doubt that active transport of amino acids is energized by a Na+ gradient, the nature of the driving force that energizes peptide transport has been a subject of controversy for many years. Numerous studies with intact tissue preparations have shown that the dependence of peptide transport on a Na+ gradient is at best partial. Recent investigations with isolated brush-border membrane vesicles from small intestine and kidney have clearly established that a H+ gradient rather than a Na+ gradient drives the active transport of intact peptides in these tissues. Since the transport mechanism involves peptide-H+ cotransport, thus rendering the process electrogenic, a membrane potential also plays a role in the energization of this transport system. Nutrient transport driven by an electrochemical H+ gradient is of common occurrence in microorganisms, but peptide transport represents the first example of this kind in mammalian systems. There is strong evidence that a H+ gradient actually exists in vivo across the brush-border membrane of intestinal and renal epithelial cells. The activity of the Na+-H+ exchanger which is located predominantly in the brush-border membrane and which, under physiological conditions, couples the influx of Na+ into the cell with the efflux of H+ from the cell, is primarily responsible for the existence of this H+ gradient. The driving force for the exchanger, a transmembrane Na+ gradient, is in turn generated and maintained at the expense of metabolic energy by the (Na+-K+) ATPase, which is present in the basal-lateral membrane of these cells. Accordingly, peptide transport in small intestine and kidney can be classified as a tertiary active transport system.

Animals↗
PubMed1986

Identification of histidyl and thiol groups at the active site of rabbit renal dipeptide transporter.

Active transport of dipeptides in rabbit renal brush-border membrane vesicles is energized by an inward-directed H+ gradient rather than a Na+ gradient. We examined the effects of treatment of membrane vesicles with diethylpyrocarbonate (DEP), a reagent specific for histidyl groups, on this H+ gradient-dependent dipeptide uptake. DEP inhibited the uptake of all three dipeptides studied, Gly-sarcosine, Gly-Gly, and Gly-Pro (Ki = 0.6-0.9 mM), and the inhibition was noncompetitive. The dipeptide transporter could be protected from DEP inhibition by the presence of dipeptide substrates during the treatment of the vesicles with the inhibitor, whereas leucine plus Na+ failed to offer the protection. Na+-dependent leucine uptake was also inhibited by DEP (Ki = 2.5 mM) and the amino acid transporter could be protected from the inhibition by leucine plus Na+, but not by dipeptides. Treatment of membrane vesicles with the thiol group-specific reagents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole,3-bromopyruvate, p-chloromercuribenzenesulfonic acid, and N-ethylmaleimide, also inhibited the H+ gradient-dependent dipeptide uptake. The potency of their inhibition was in the order: 7-chloro-4-nitrobenz-2-oxa-1,3-diazol greater than p-chloromercuribenzenesulfonic acid greater than 3-bromopyruvate greater than N-ethylmaleimide. The inhibition could be reversed in some cases by treatment of the membrane vesicles with reducing agents such as 2,3-dimercaptopropanol following incubation with the inhibitors. Dipeptide substrates could protect the dipeptide transporter from the inhibition. We conclude that histidyl and thiol groups are present at or near the substrate-binding site of the rabbit renal dipeptide transporter.

4-Chloromercuribenzenesulfonate↗
PubMed1986

Interaction of clonidine with human placental Na+ -H+ exchanger.

The effect of clonidine, an alpha 2-adrenergic receptor agonist, on the Na+ -H+ exchanger in human placental brush-border membrane vesicles was examined. The exchanger was inhibited by clonidine. The inhibition was freely reversible, and the apparent inhibition constant for the process was 250 microM. The nature of inhibition was found to be competitive with respect to Na+. The Dixon plot (1/v versus clonidine concentration) was linear (r2 = 0.998), indicating the interaction of the drug with a single site on the exchanger protein. Similar kinetic analyses with amiloride, a potassium-sparing diuretic, and cimetidine, a histamine type II receptor antagonist, revealed that these drugs also inhibited the Na+ -H+ exchanger by interacting with a single site on the protein. The presence of clonidine increased the intercepts without affecting the slopes of the l/v versus amiloride concentration and the l/v versus cimetidine concentration plots. These results demonstrate that all three drugs, amiloride, cimetidine and clonidine, interact with the human placental Na+-H+ exchanger at a single site in a mutually exclusive manner, and the site of interaction is identical with the Na+-binding site on the external surface of the exchanger protein.

Amiloride↗
PubMed1986

Characterization of tryptophan transport in human placental brush-border membrane vesicles.

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.

Amino Acids↗