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V Ganapathy

Publications and source records attributed to V Ganapathy.

At least 181 records · Page 10Linked to original sources
PubMed1992

The gamma-aminobutyric acid transporter and its interaction with taurine in the apical membrane of the bovine retinal pigment epithelium.

The characteristics of gamma-aminobutyric acid (GABA) uptake were investigated in apical membrane vesicles prepared from the bovine retinal pigment epithelium. An inwardly directed NaCl gradient stimulated GABA uptake markedly, and the time course of uptake exhibited an overshoot phenomenon indicating the presence of an active transport mechanism for GABA in these membranes. Other monovalent cations were not capable of substituting for Na+. In addition to this obligatory requirement for Na+, the GABA uptake also exhibited a Cl(-)-dependence, evident from the observations that the uptake was negligible in the presence of NaF or sodium gluconate in place of NaCl. NO3- and SCN- could substitute for Cl- to some extent. The uptake process was electrogenic, with a Na+/Cl-/GABA stoichiometry of 2:1:1 or 3:1:1. Substrate-specificity studies showed that the beta-amino acids such as taurine, hypotaurine and beta-alanine interacted with the GABA uptake process. Uptake of GABA could be completely inhibited by an excess of taurine and, similarly, uptake of taurine could be completely inhibited by an excess of GABA, suggesting that common transport processes operate in the uptake of these two compounds. However, a number of compounds which are specific inhibitors of GABA uptake inhibited taurine uptake only to a maximum of 50%. Kinetic analysis of GABA uptake in the concentration range 0.1-10 microM revealed that the uptake occurred via a single system and that taurine was a competitive inhibitor of this system. The Michaelis-Menten constant (Kt) for GABA was 0.94 microM and the apparent inhibition constant (Ki) for taurine was 230 microM. On the contrary, even though the kinetic analysis of taurine uptake in the concentration range 25-150 microM revealed participation of a single system in the uptake process, the inhibition of taurine uptake by GABA was not competitive. The presence of GABA decreased the maximal velocity of the taurine uptake process and also decreased the Kt for taurine. Based on these data, it is proposed that: (i) there are two distinct transport systems, namely the GABA transporter and the taurine transporter, in these membranes which accept both GABA and taurine as substrates, (ii) the affinities of these systems for taurine are very similar and cannot be kinetically distinguished under the experimental conditions employed, and (iii) the difference between the affinities of these system for GABA is much greater than for taurine.

Amino Acids↗
PubMed1992

Evidence for an ATP-driven H(+)-pump in the plasma membrane of the bovine corneal epithelium.

In a highly enriched plasma membrane fraction isolated from the bovine corneal epithelium, MgATP dependent intravesicular acidification was identified by measuring Acridine Orange quenching. The rate of acidification was increased 2.7-fold by pre-exposure of the membranes to 1% cholate which was subsequently removed by Sephadex G-50 column chromatography. However, in a lysosomal fraction whose enrichment with respect to the homogenate was 82-fold in N-acetyl-beta-D-glucosaminidase, cholate pre-exposure had no significant effect on the rate of intralysosomal acidification. This difference is assumed to reflect reorientation by cholate of the H(+)-pump's normally inaccessible ATP-binding site in right-side-out vesicles of the plasma membrane-enriched fraction to a configuration in which this site becomes accessible to externally added ATP. In contrast, the ATP-binding site of the H(+)-pump in the lysosomal fraction is completely exposed to the exterior even in the absence of cholate treatment. The characteristics of the H(+)-pump in the plasma membrane fraction was subsequently determined using cholate-pretreated membrane vesicles. The rank order of nucleotide support of the H(+)-pump activity was: ATP >> GTP > ITP. However, UTP and CTP were totally inactive. The pump is electrogenic because the activity of the pump was enhanced in voltage-clamped membrane vesicles. Substitution of Mg2+ with Mn2+ did not change the acidification rate but Co2+ only partly activated whereas Ca2+ and Zn2+ were ineffective as activators. The H(+)-pump was relatively unaffected by oligomycin, azide or vanadate but completely inhibited by 10 microM NEM or NBD-Cl and 92% inhibited by 20 microM DCCD.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

Active transport of dopamine in human placental brush-border membrane vesicles.

Brush-border membrane vesicles isolated from normal human term placentas were found to accumulate dopamine against a concentration gradient when an inwardly directed NaCl gradient was imposed across the membrane. The activity of the transport system was obligatorily dependent on Na+ as well as Cl-. Intravesicular H+ and intravesicular K+ stimulated the transport activity. The system possessed high affinity for dopamine and norepinephrine, with considerably lower affinity for serotonin. The stoichiometry of Na(+)-Cl(-)- dopamine was 1:1:1. The system was electrogenic because the NaCl-dependent dopamine uptake was stimulated by an inside-negative membrane potential, and this characteristic was observed in the presence and in the absence of intravesicular K+. Kinetic analysis revealed that the uptake was due to a carrier-mediated component plus a diffusion/binding component. The apparent Michaelis-Menten constant for dopamine for the carrier-mediated component was 19 +/- 7 nM. The transport system was clearly distinct from the serotonin transporter. Analysis of the inhibition of dopamine uptake by various monoamine uptake inhibitors showed that the uptake occurred via a transport system that is similar to the neuronal norepinephrine transporter.

Anions↗
PubMed1992

Constitutive expression of the taurine transporter in a human colon carcinoma cell line.

The human colon carcinoma cell line HT-29, when grown to confluence, was found to take up taurine and accumulate it against a concentration gradient from a NaCl-containing uptake medium. Replacement of NaCl with choline chloride almost totally abolished the uptake. Taurine uptake was dependent not only on Na+ but also on Cl-, because other anions failed to support the uptake in the presence of Na+. The uptake process was specific for beta-amino acids such as taurine, hypotaurine, and beta-alanine. Apparently, a single transport system with a Michaelis-Menten constant of 10.6 +/- 0.3 microM was responsible for the uptake. Stoichiometric analyses revealed that the Na+:taurine coupling ratio was 2:1, whereas the Cl-:taurine coupling ratio was 1:1. Culture of the cells in the presence of taurine caused downregulation of the uptake system. These cells were also capable of accumulating beta-alanine against a concentration gradient in the presence of NaCl. Beta-Alanine uptake occurred via a single transport system with an apparent Michaelis-Menten constant of 36 +/- 2 microM. Taurine and beta-alanine exhibited mutual interaction during uptake. Kinetic experiments strongly suggested that a common transporter was responsible for the uptake of these two beta-amino acids. It is concluded that the HT-29 cells constitutively express the taurine transporter and that this cell line may be a suitable model for investigations of intestinal taurine transporter.

Alanine↗
PubMed1992

Selective impairment of taurine transport by cyclosporin A in a human placental cell line.

We investigated, using a human placental choriocarcinoma cell line as a model, the effects of the immunosuppressive drug cyclosporin A on several placental transport systems mediating the transfer of glucose and amino acids from mother to fetus. The results of the investigation show that the transport system responsible for the transfer of taurine is selectively impaired by the drug, whereas the other transport systems are either stimulated or not affected. The inhibitory effect of the drug on taurine transport appears to be due to interference with calmodulin-dependent processes because calmodulin antagonists such as W-7, calmidazolium, and CGS 9343B mimic the effects of cyclosporin A. FK506, another immunosuppressive drug that is currently undergoing clinical trials, does not have this inhibitory effect.

Biological Transport, Active↗
PubMed1991

Relative sensitivity to inhibition by cimetidine and clonidine differentiates between the two types of Na(+)-H+ exchangers in cultured cells.

Available evidence indicates that there are two types of Na(+)-H+ exchangers, type A (housekeeping type) and type B (epithelial or apical type), in mammalian cells. We have recently reported, using isolated membrane vesicles, that these two types can be differentiated by their relative sensitivities to inhibition by clonidine and cimetidine [Kulanthaivel, Leibach, Mahesh, Cragoe & Ganapathy (1990) J. Biol. Chem. 264, 1249-1252]. The present study was undertaken to determine whether this approach is also effective in cultured cells. The JAR human placental choriocarcinoma cell line and the opossum kidney (OK) cell line, when grown as confluent monolayer cultures on an impermeable plastic support, express Na(+)-H+ exchanger activity which is measurable by determining Na+ uptake into the cells from the culture medium. The JAR cell Na(+)-H+ exchanger was found to be about 100 times more sensitive to inhibition by dimethylamiloride than the OK cell Na(+)-H+ exchanger. Inhibition studies with clonidine and cimetidine were able to differentiate between these two exchangers very clearly. Cimetidine was 18 times more potent than clonidine in inhibiting the JAR cell Na(+)-H+ exchanger. In contrast, clonidine was at least 8 times more potent than cimetidine in inhibiting the Na(+)-H+ exchanger of the OK cell. The results show that the JAR cell expresses the type A Na(+)-H+ exchanger, whereas the OK cell expresses the type B Na(+)-H+ exchanger. This approach also proved to be very effective in correctly identifying the type of Na(+)-H+ exchanger in a third cell line (HeLa). It is concluded that the relative susceptibility to inhibition by clonidine and cimetidine offers an easy and efficient means of differentiating between the two types of Na(+)-H+ exchangers in cultured cells.

Animals↗
PubMed1991

Transport of peptides in renal brush border membrane vesicles. Suitability of 125I-labelled tyrosyl peptides as substrates.

Three tyrosine-containing peptides (Tyr-Pro, Tyr-Pro-Phe and Tyr-Pro-Phe-Pro) were [125I]iodo-labelled and their uptake characteristics in renal brush border membrane vesicles was investigated to assess the suitability of these peptides as substrates in peptide transport studies. Hydrolysis of these peptides during uptake measurements was avoided by using brush border membrane vesicles prepared from the kidneys of Japan Fisher 344 rats which genetically lack dipeptidylpeptidase IV activity. The uptake of 125I-Tyr-Pro and 125I-Tyr-Pro-Phe in these membrane vesicles was found to be stimulated by an inwardly directed H+ gradient. In both cases, the time course of uptake exhibited an overshoot, an indication for active transport. The uptake processes were electrogenic since they were stimulated by an inside-negative membrane potential. Free amino acids had no effect on the uptake of Tyr-Pro and Tyr-Pro-Phe whereas many di- and tripeptides effectively blocked the uptake. These results demonstrated that the [125I]iodo-labelled Tyr-Pro and Tyr-Pro-Phe are suitable substrates for peptide transport studies because their uptake characteristics clearly suggest participation of the peptide transporter in their uptake. In contrast, the uptake of 125I-Tyr-Pro-Phe-Pro was not active, and was not stimulated by the H+ gradient or by the membrane potential. In addition, competition experiments with unlabelled amino acids and peptides indicated that the uptake did not occur via the peptide transporter. However, when allowed to be hydrolyzed to 125I-Tyr-Pro and Phe-Pro by using dipeptidylpeptidase IV-positive renal brush border membrane vesicles prepared from USA Fisher 344 rats, the radiolabel from the [125I]iodo-labelled tetrapeptide was found to be taken up into the vesicles via the peptide transporter. These data provide direct evidence to show that intact tetrapeptides are not substrates for the renal peptide transporter.

Amino Acid Sequence↗
PubMed1991

Expression and cyclic AMP-dependent regulation of a high affinity serotonin transporter in the human placental choriocarcinoma cell line (JAR).

The JAR human placental choriocarcinoma cell line transports serotonin, accumulating the monoamine inside the cell against a concentration gradient. The transport is energized by an NaCl gradient. Tricyclic (imipramine and desipramine) and non-tricyclic (paroxetine and fluoxetine) antidepressants inhibit the transporter markedly, but reserpine and 5-hydroxytryptophan do not. Ouabain, gramicidin, and nigericin, which reduce or abolish the transmembrane Na+ gradient, and phloridzin, which interferes with glucose transport into the cells, inhibit the transport. Preincubation of the cells with glucose-free medium also causes similar inhibition. The activity of the serotonin transporter in this cell line is stimulated in response to overnight (16-h) incubation with increasing concentrations of cholera toxin (0.1-1,000 ng/ml). Under these conditions the stimulation is maximal at 10 ng/ml cholera toxin (3.1 +/- 0.2-fold). Cholera toxin increases the cAMP content of these cells by several hundredfold within 2 h. Isobutylmethylxanthine (100 microM), dibutyryl cAMP (100 microM), and forskolin (100 microM) mimic the action of cholera toxin, eliciting a 1.6-2.5-fold stimulation of the serotonin transporter activity. The stimulatory effect of cholera toxin is antagonized significantly by simultaneous incubation of the cells with 50 microM N-(2-aminoethyl)-5-isoquinolinesulfonamide, a protein kinase inhibitor. The effect of cholera toxin on serotonin transport is specific because, under similar conditions, cholera toxin inhibits 3-O-methyl-D-glucose transport and does not influence taurine transport in this cell line. There is also no significant change in the protein content of the cells after cholera toxin treatment. Kinetic analysis reveals that cholera toxin causes an increase in the maximal velocity (7.89 +/- 0.67 to 17.55 +/- 1.06 pmol/mg of protein/5 min) and a decrease in the Michaelis-Menten constant (0.52 +/- 0.09 to 0.29 +/- 0.04 microM). These data show that the JAR human placental choriocarcinoma cell line expresses a high affinity serotonin transporter that is sensitive to inhibition by antidepressants and that the activity of the transporter is under cAMP-dependent regulation.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Transport of taurine and its regulation by protein kinase C in the JAR human placental choriocarcinoma cell line.

The JAR human placental choriocarcinoma cell line transports taurine, concentrating it over 1000-fold inside the cell. The transport system is energized by a Na+ gradient and exhibits an absolute requirement for Cl-. Neutral beta-amino acids such as beta-alanine and hypotaurine effectively compete with the system, whereas neutral alpha-amino acids such as alanine, leucine and alpha-aminoisobutyric acid do not. The transport system interacts with gamma-aminobutyric acid to an appreciable extent. Kinetic analysis reveals that the taurine transport system in this cell line is of a high-affinity and low-capacity type (apparent dissociation constant 2.3 +/- 0.3 microM; maximal velocity 88.5 +/- 5.0 pmol/3 min per mg of protein). Pretreatment of the JAR choriocarcinoma cells with phorbol 12-myristate 13-acetate results in the inhibition of the taurine transport system in a dose-dependent manner. The inhibition is blocked by co-treatment of the cells with staurosporine, an inhibitor of protein kinase C. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, has no effect on the transport system. These data show that the choriocarcinoma cells express a taurine transporter with characteristics similar to those of the taurine transporter described in the normal human placenta, and that the activity of the transporter in these cells is under the regulatory control of protein kinase C.

Alkaloids↗
PubMed1991

Functional expression of the intestinal peptide-proton co-transporter in Xenopus laevis oocytes.

The expression of the intestinal peptide-proton cotransporter was examined in Xenopus laevis oocytes by microinjection of poly(A)+ mRNA prepared from rabbit intestinal mucosal cells. The concomitant expression of the glucose-sodium co-transporter was used as the control for the effectiveness of the expression technique. There was significant endogenous activity of Gly-Sar uptake in water-injected oocytes, but the uptake activity increased nearly 3-fold in poly(A)+ mRNA-injected oocytes. The expression of the peptide transporter was time-dependent. There was no detectable expression on day 1 after injection. The expression became noticeable on day 2 and increased with time, reaching a maximum on day 4. There was no further change on days 5 and 6. The endogenous uptake rate measured in water-injected oocytes, on the contrary, showed a slight decrease during this time. The expressed peptide transporter retained its substrate specificity, having affinity for the dipeptides, Gly-Sar and Gly-Pro, and no or little affinity for the free amino acids, Gly and Sar. The expressed peptide transporter also showed a dependence on a transmembrane H+ gradient for maximal activity. These data demonstrate that the mammalian intestinal peptide-proton co-transporter can be successfully expressed in Xenopus laevis oocytes. This expression system can provide an effective assay procedure to clone the gene encoding the transporter.

Animals↗
PubMed1991

Proton-coupled solute transport in the animal cell plasma membrane.

This review focuses primarily on the progress made in the last couple of years in the understanding of the intestinal peptide transporter, a prototype for H(+)-coupled solute transport systems in the animal cell plasma membrane. The impressive number of transport systems currently known to be energized by the components of the proton-motive force indicates that the role of H+ as the coupling ion for active transport has not been lost during evolution.

Adenosine Triphosphate↗
PubMed1991

Taurine uptake in apical membrane vesicles from the bovine retinal pigment epithelium.

Characteristics of taurine uptake were investigated in apical membrane vesicles isolated from bovine retinal pigment epithelium. Uptake of taurine into these vesicles was stimulated markedly by the presence of an inwardly directed NaCl gradient across the membrane. The time course of the NaCl gradient-dependent uptake showed an overshoot, indicating a transient accumulation of taurine in the vesicles against a concentration gradient. Monovalent cations other than Na+ did not support taurine uptake. There was also an anion requirement for the uptake system, and Cl- was the most potent among several monovalent anions tested. The NaCl-dependent taurine uptake was stimulated by inside-negative, K+, and H+ diffusion potentials, demonstrating the electrogenic nature of the system. Stoichiometric analysis revealed that two Na+ and one Cl- ions were involved in the translocation of one taurine molecule. The system had an apparent dissociation constant (Kt) of 96 +/- 17 microM and a maximal velocity of 267 +/- 24 pmol/mg of protein over 15 sec. Uptake of radiolabeled taurine was inhibited by excess amounts of unlabeled taurine, hypotaurine, beta-alanine, and gamma-aminobutyric acid. The relative potencies of the amino acids to inhibit taurine uptake were compared in three tissues, the bovine retinal pigment epithelium, the rat kidney, and the human placenta. In the apical membrane vesicles of the pigment epithelium, gamma-aminobutyric acid was many times more potent than taurine itself in inhibiting radiolabeled taurine uptake. However, in the brush border membrane vesicles of the kidney and the placenta, taurine was many times more potent than gamma-aminobutyric acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Calcium-induced inhibition of taurine transport in brush-border membrane vesicles from rabbit small intestine.

The influence of Ca2+ on the activity of the taurine transport system was investigated in rabbit small intestinal brush-border membrane vesicles. Preincubation of the brush-border membrane vesicles with Ca2+ prepared by the Mg2(+)-aggregation method markedly decreased the NaCl gradient-dependent uptake of taurine in these vesicles. Uptake of glucose and alanine, both dependent on a Na+ gradient, were also decreased by Ca2(+)-treatment, but their reduction was very small compared with that of taurine uptake. The inhibitory effect of Ca2+ was dose- and time-dependent. The inhibition was reduced by the presence of ethylene glycol-bis(beta-amino ethyl ether)-N,N,N'-N'-tetraacetic acid during treatment of the membrane vesicles with Ca2+. Neomycin partially protected the taurine transporter activity from the Ca2(+)-induced inhibition, but indomethacin did not. 5-Nitro-2-(3-phenylpropylamino)benzoate, a Cl(-)-channel blocker, did not increase taurine uptake in the Ca2(+)-treated membrane vesicles. It is concluded that the Ca2(+)-induced inhibition of taurine uptake in rabbit intestinal brush-border membrane vesicles is not due to accelerated dissipation of the ion gradient driving forces across the membrane but rather to a direct effect on the transporter, most likely mediated by the activation of the membrane-bound phospholipase C.

Alanine↗
PubMed1990

The ATP-binding site of the human placental H+ pump contains essential tyrosyl residues.

Transient exposure of human placental brush-border membrane vesicles to cholate reorients the ATP-driven H+ pump, enabling the pump to transport H+ into the vesicles upon addition of ATP to the external medium. H+ uptake can be measured in these vesicles by following the decrease in the absorbance of acridine orange, a delta pH indicator. We investigated the role of tyrosyl residues in the catalytic function of the H+ pump by studying the effects of tyrosyl group specific reagents on ATP-driven H+ uptake in cholate-pretreated membrane vesicles. The reagents tested were 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), N-acetylimidazole, tetranitromethane, and p-nitrobenzenesulfonyl fluoride. Treatment of the membrane vesicles with these reagents resulted in the inhibition of the ATP-driven H+ uptake, and the inhibitory potency was in the following order: NBD-Cl greater than tetranitromethane greater than p-nitrobenzenesulfonyl fluoride greater than N-acetylimidazole. The inhibition of the H+ pump by NBD-Cl was reversible by 2-mercaptoethanol, and the inhibition by N-acetylimidazole was reversible by hydroxylamine. Since these reagents are not absolutely specific for tyrosyl groups and can also react with thiol groups, we studied the interaction of N-acetylimidazole with the H+ pump whose triol groups were masked by reaction with p-(chloromercuri)benzenesulfonate. The SH-masked pump was totally inactive, but the activity could be restored by dithiothreitol. On the contrary, the activity of the SH-masked H+ pump which was subsequently treated with N-acetylimidazole could not be restored by dithiothreitol, suggesting that thiol groups were not involved in the inhibition of the H+ pump by N-acetylimidazole.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Interaction of fluoxetine with the human placental serotonin transporter.

The interaction of fluoxetine, a non-tricyclic antidepressant, with the human placental serotonin transporter was investigated by studying its influence on [3H]paroxetine binding to the transporter and on [3H]serotonin uptake via the transporter. These studies were done using brush-border membrane vesicles purified from normal term human placentas. Fluoxetine inhibited binding of paroxetine to the membrane vesicles in a concentration-dependent manner, with a Ki value of 3 nM. Kinetic analysis revealed that the inhibition was competitive because the presence of 10 nM fluoxetine increased the Kd for paroxetine from 72 to 461 pM, but had no effect on the Bmax. Fluoxetine also caused a time-dependent dissociation of paroxetine already bound to the transporter. The dissociation followed first-order kinetics. Uptake of serotonin in these membrane vesicles was also inhibited by fluoxetine. The inhibition was concentration dependent with a Ki value of 66 nM at pH 7.5 and 80 nM at pH 6.5. The effect of fluoxetine on the uptake kinetics was to increase the apparent dissociation constant (Kt) for serotonin without influencing the maximal transport capacity (Vmax). The results demonstrate that fluoxetine is a high-affinity ligand and a potent inhibitor of the serotonin transporter found in the human placental brush-border membrane.

Carrier Proteins↗
PubMed1990

Kinetic evidence for a common transporter for glycylsarcosine and phenylalanylprolylalanine in renal brush-border membrane vesicles.

We investigated the interaction between glycylsarcosine (Gly-Sar), a neutral dipeptide, and phenylalanylprolylalanine (Phe-Pro-Ala), a neutral tripeptide, for transport into renal brush-border membrane vesicles isolated from Japanese F344 rats. This rat strain is genetically deficient in dipeptidylpeptidase IV. Owing to the absence of this enzyme, Phe-Pro-Ala was found to be completely resistant to hydrolysis by the brush-border membranes, and this enabled us to study the uptake of the intact tripeptide without interference by hydrolysis. Gly-Sar was also resistant to hydrolysis by these membrane preparations. Transport of Gly-Sar as well as that of Phe-Pro-Ala in these vesicles was driven by an inwardly directed H+ gradient. Gly-Sar transport was blocked completely by increasing concentrations of Phe-Pro-Ala and vice versa. Gly-Sar inhibited Phe-Pro-Ala transport competitively; and similarly, Phe-Pro-Ala inhibited Gly-Sar transport competitively. The dissociation constant (Kt) for Gly-Sar transport (94 +/- 5 microM) was very similar to the inhibition constant (Ki) for Gly-Sar to inhibit Phe-Pro-Ala transport (107 +/- 13 microM). The Kt for Phe-Pro-Ala transport (36 +/- 3 microM) was equal to the Ki for Phe-Pro-Ala to inhibit Gly-Sar transport (36 +/- 6 microM). Furthermore, linear correlation was exhibited by various neutral di- and tripeptides, which were also resistant to hydrolysis, in their abilities to inhibit the transport of Gly-Sar and that of Phe-Pro-Ala. These results strongly suggest that a common carrier system participates in the transport of neutral di- and tripeptides in renal brush-border membrane vesicles.

Amino Acid Sequence↗