Biomedical subjects

V Ganapathy

Publications and source records attributed to V Ganapathy.

At least 199 records · Page 11Linked to original sources

PubMed1990

An essential role for vicinal dithiol groups in the catalytic activity of the human placental Na(+)-H+ exchanger.

We examined the effects of phenylarsine oxide, a reagent specific for vicinal dithiol groups, on the catalytic activities, Na+ influx and H+ efflux, of the human placental Na(+)-H+ exchanger. Treatment of the placental brush-border membrane vesicles with the reagent markedly inhibited both the activities. The inhibition was partially reversible by dithiols. The effect of phenylarsine oxide was to reduce the maximal velocity of the exchanger without influencing its affinity for Na+. The exchanger was partially protected from this inhibition by amiloride but not by cimetidine even though both these compounds interacted with the Na(+)-binding site. The data demonstrate that vicinal dithiol groups are essential for the catalytic function of the placental Na(+)-H+ exchanger and that the critical dithiol groups are located at a site distinct from the Na(+)-binding site.

Amiloride↗

PubMed1990

Evidence for tripeptide/H+ co-transport in rabbit renal brush-border membrane vesicles.

L-Phe-L-Pro-L-Ala is a tripeptide which is hydrolysable almost exclusively by dipeptidyl peptidase IV in rabbit renal brush-border membrane vesicles. In order to delineate the mechanism of the transport of an intact tripeptide across the brush-border membrane, we studied the characteristics of the uptake of [3H]Phe-Pro-Ala in membrane vesicles in which the activity of dipeptidylpeptidase IV was completely inhibited by treatment with di-isopropyl fluorophosphate. In these vesicles, uptake of radiolabel from the tripeptide was found to be Na(+)-independent, but was greatly stimulated by an inwardly directed H+ gradient. The H(+)-gradient-dependent radiolabel uptake appeared to be an active process, because the time course of uptake exhibited an overshoot phenomenon. The process was also electrogenic, being stimulated by an inside-negative membrane potential. Under the uptake-measurement conditions there was no detectable hydrolysis of [3H]Phe-Pro-Ala in the incubation medium when di-isopropyl fluorophosphate-treated membrane vesicles were used. Analysis of intravesicular contents revealed that the radiolabel inside the vesicles was predominantly (greater than 90%) in the form of intact tripeptide. These data indicate that the uptake of radiolabel from [3H]Phe-Pro-Ala in the presence of an inwardly directed H+ gradient represents almost exclusively uptake of intact tripeptide. Uphill transport of the tripeptide was also demonstrable in the presence of an inwardly directed Na+ or K+ gradient, but only if nigericin was added to the medium. Under these conditions, nigericin, an ionophore for Na+, K+ and H+, was expected to generate a transmembrane H+ gradient. Uptake of Phe-Pro-Ala in the presence of a H+ gradient was inhibited by di- and tri-peptides, but not by free amino acids. It is concluded that tripeptide/H+ co-transport is the mechanism of Phe-Pro-Ala uptake in rabbit renal brush-border membrane vesicles.

Amino Acid Sequence↗

PubMed1990

Modulation of serotonin uptake kinetics by ions and ion gradients in human placental brush-border membrane vesicles.

The modulation of serotonin uptake kinetics by Na+, Cl-, H+, and K+ was investigated in brush-border membrane vesicles prepared from normal human term placentas. The presence of Na+ and Cl- in the external medium was mandatory for the function of the serotonin transporter. In both cases, the initial uptake rate of serotonin was a hyperbolic function of the ion concentration, indicating involvement of one Na+ and one Cl- per transport of one serotonin molecule. The apparent dissociation constant for Na+ and Cl- was 145 and 79 mM, respectively. The external Na+ increased the Vmax of the transporter and also increased the affinity of the transporter for serotonin. The external Cl- also showed similar effects on the Vmax and the Kt, but its effect on the Kt was small compared to that of Na+. The presence of an inside-acidic pH, with or without a transmembrane pH gradient, stimulated the NaCl-dependent serotonin uptake. The effect of internal [H+] on the transport function was to increase the Vmax and decrease the affinity of the transporter for serotonin. The presence of K+ inside the vesicles also greatly stimulated the initial rates of serotonin uptake, and the stimulation was greater at pH 7.5 than at pH 6.5. This stimulation was a hyperbolic function of the internal K+ concentration at both pH values, indicating involvement of one K+ per transport of one serotonin molecule. The apparent dissociation constant for K+ was 5.6 mM at pH 6.5 and 4.0 mM at pH 7.5. The effects of internal [K+] on the uptake kinetics were similar to those of internal [H+].(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorides↗

PubMed1990

Evidence for tripeptide-proton symport in renal brush border membrane vesicles. Studies in a novel rat strain with a genetic absence of dipeptidyl peptidase IV.

We have investigated the transport characteristics of L-phenylalanyl-L-prolyl-L-alanine in renal brush-border membrane vesicles isolated from Japan Fisher 344 rats. This particular rat strain genetically lacks dipeptidyl peptidase IV. Owing to the absence of this enzyme, the tripeptide was found to be completely resistant to hydrolysis by the renal brush-border membrane vesicles. Uptake of the tripeptide into these membrane vesicles in the presence of an inwardly directed Na+ gradient was slightly greater than in the presence of a K+ gradient, but there was no evidence for active transport. On the contrary, uptake was very rapid in the presence of an inside-alkaline transmembrane pH gradient, and accumulation of the tripeptide inside the vesicles against a concentration gradient could be demonstrated under these conditions. The uptake was drastically reduced by dissipation of the pH gradient. The uptake was stimulated by an inside-negative membrane potential and inhibited by an inside-positive membrane potential. Moreover, the uptake was greater in voltage-clamped membrane vesicles than in control vesicles. Many di- and tripeptides inhibited this pH gradient-stimulated uptake of Phe-Pro-Ala. The apparent dissociation constant for the tripeptide was 48 microM. High performance liquid chromatography analysis of the intravesicular content at the peak of the overshoot revealed that the tripeptide was transported across the membrane almost entirely in the intact form. These data provide the first direct evidence for the presence of an electrogenic tripeptide-proton symport in renal brush-border membranes.

Amino Acid Sequence↗

PubMed1990

Hydrolysis and transport of proline-containing peptides in renal brush-border membrane vesicles from dipeptidyl peptidase IV-positive and dipeptidyl peptidase IV-negative rat strains.

In this investigation, we have demonstrated that the renal brush-border membrane of Fischer 344 rats from the Japanese Charles River Inc. specifically lacks dipeptidyl peptidase IV (DPP IV) activity, whereas the renal brush-border membrane of Fischer 344 rats from three different sources within the United States possesses normal levels of DPP IV activity. Comparison of the brush-border proteins between Charles River (U.S.A.) Fischer 344 rats (DPP IV positive) and Japanese Charles River Fischer 344 rats (DPP IV negative) revealed that a protein band (Mr = 100,000), apparently identical with DPP IV, was absent in the membranes from Japanese Charles River Fischer 344 rats. We examined the handling of radiolabeled beta-casomorphin fragment 1-5 (Tyr-Pro-[3H]Phe-Pro-Gly), a specific substrate for DPP IV, in renal brush-border membrane vesicles isolated from DPP IV-positive and DPP IV-negative rats. Although the membrane vesicles from DPP IV-positive rats were able to hydrolyze the pentapeptide to di- and tripeptides with the subsequent active transport of these products via the H+ gradient-dependent peptide transport system, the membrane vesicles from DPP IV-negative rats failed to hydrolyze the pentapeptide and hence lacked the ability to transport the radiolabel actively from the parent peptide. The H+ gradient-dependent glycyl-sarcosine uptake and the Na+ gradient-dependent proline uptake, however, were normal in DPP IV-negative rats. Urine analysis revealed that the DPP IV-negative rats excreted proline- and hydroxyproline-containing peptides in significantly increased amounts in their urine compared with control rats. Furthermore, following intravenous administration of Tyr-Pro-Phe-Pro-NH2, a peptide that is exclusively hydrolyzed by DPP IV, urinary excretion of the peptide in the intact form was many-fold greater in DPP IV-negative rats than in control rats. These data provide conclusive evidence for the obligatory role of DPP IV in the renal handling of proline (and hydroxyproline)-containing peptides.

Animals↗

PubMed1990

The Na(+)-H+ exchanger of the placental brush-border membrane is pharmacologically distinct from that of the renal brush-border membrane.

We have compared the pharmacological properties of the human placental brush-border membrane Na(+)-H+ exchanger with those of the rabbit renal brush-border membrane Na(+)-H+ exchanger. The exchanger activity in both preparations was inhibited by cimetidine, clonidine, and harmaline. Cimetidine was found to be 4-5 times more potent than clonidine in inhibiting the placental Na+-H+ exchanger. However, the order of potency was reversed for the renal exchanger, in which case clonidine was 3-4 times more potent than cimetidine as an inhibitor. There was, however, no difference between the potencies of harmaline to inhibit the two exchangers. When amiloride and four of its analogs were tested as inhibitors, the Na(+)-H+ exchanger of the placental brush-border membrane exhibited greater sensitivity to inhibition by all of these compounds than the Na(+)-H+ exchanger of the renal brush-border membrane. The difference between the two exchangers was more prominent with the 5-amino-substituted amiloride derivatives than with amiloride. The greatest difference between the Ki values was for dimethylamiloride (the kidney/placenta ratio was 185), followed by ethylisopropyl amiloride, hexamethylene amiloride, and t-butyl amiloride. These results indicate that the two Na+-H+ exchangers are pharmacologically distinct.

Amiloride↗

PubMed1990

High-affinity paroxetine binding to the human placental serotonin transporter.

We investigated the interaction of paroxetine, a nontricyclic antidepressant, with the serotonin transporter of the human placental brush-border membrane. Paroxetine bound to the purified placental brush-border membranes with a high affinity [dissociation constant (Kd) = 72 pM]. The maximal binding capacity (Bmax) was 3.9 pmol/mg protein. Imipramine, desipramine, and serotonin inhibited the binding in a dose-dependent manner with inhibition constant (Ki) values of 4.4 nM, 48.7 nM, and 1.77 microM, respectively, whereas reserpine, ketanserin, and 5-hydroxytryptophan did not have any effect. Imipramine and serotonin inhibited paroxetine binding by increasing the Kd with essentially no effect on Bmax. Binding of paroxetine to the membranes increased hyperbolically with increasing concentrations of Na+ in the assay medium. Cl- had little effect on the binding. The effect of Na+ was primarily to increase the affinity of the transporter for paroxetine with no effect on Bmax. The association constant (Ka) increased hyperbolically as the concentration of Na+ increased, indicating a 1Na+:1paroxetine stoichiometry. The maximal value for Ka was 12.1 +/- 2.5 x 10(12) M-1, and Kd for Na+ was 10.0 +/- 3.5 mM. Treatment of the membranes with tyrosyl group-specific reagents reduced the Na(+)-dependent binding, suggesting the involvement of tyrosyl residues in the binding process. This inhibition was, however, significantly reduced when treatment with the reagent was performed in the presence of Na+, suggesting that the reactive tyrosyl residues were located at or near the Na(+)-binding site. Paroxetine inhibited NaCl gradient-dependent serotonin uptake in placental brush-border membrane vesicles both at pH 6.5 and 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloro-7-nitrobenzofurazan↗

PubMed1990

Uphill transport of beta-alanine in intestinal brush-border membrane vesicles.

The characteristics of beta-alaline uptake were studied in brush-border membrane vesicles isolated from the proximal small intestine of rabbits and were compared with those of L-alpha-alanine uptake. The uptake of beta-alanine as well as L-alpha-alanine was significantly stimulated by imposing an inwardly directed Na+ gradient. Studies on transstimulation and substrate specificity provide evidence that the transport system serving beta-alanine is distinct from the system serving alpha-alanine. The beta-system also accepts taurine as a substrate. The Na(+)-dependent uptakes of beta-alanine and L-alpha-alanine were differentially influenced by anions. The order in which anions supported uptake was Cl- = SCN- greater than F- greater than NO3- = SO2(-4) for beta-alanine, whereas it was SCN- greater than F- = Cl- = NO3- greater than SO2(-4) for L-alpha-alanine. Cl- appeared to be the preferred anion to support the uptake of beta-alanine. beta-Alanine uptake was greater in the presence of an inwardly directed Cl- gradient than in the presence of Cl- at equal concentrations on both sides of the membrane. The uptake was maximal when a Na+ gradient and a Cl- gradient were present simultaneously. The NaCl gradient-driven beta-alanine uptake was stimulated by an inside-negative K(+)-diffusion potential induced by valinomycin, showing that the uptake process is electrogenic. Stoichiometric analyses suggest that multiple Na+ and one Cl- are associated with the uptake of one beta-alanine molecule. The kinetic study shows that the transporter for beta-alanine is a high-affinity, low-capacity system (Kt = 46 +/- 1 microM; Vmax = 30 +/- 1 pmol.mg protein-1.15 s-1).

Alanine↗

PubMed1990

Peptidases and peptide transport in renal brush-border membrane vesicles from rats with acute renal failure.

We investigated the influence of acute renal failure induced by injection of uranyl nitrate on renal handling of peptides in rats. Urinary excretion of brush-border peptidases increased significantly as a result of uranyl nitrate treatment. H+-coupled Gly-Sar uptake was reduced in renal brush-border vesicles from uranyl nitrate-treated rats compared to control rats. The impairment of dipeptide uptake was evident whether the uptake was measured in the presence or absence of a H+ gradient. Kinetic analysis indicated that the impairment was due mainly to a decrease in the maximal velocity of the transporter. beta-Casomorphin, a pentapeptide, was hydrolyzed to di- and tripeptides by dipeptidylpeptidase IV and the hydrolytic products were taken up actively into control brush-border vesicles via the peptide transporter. But in uranyl nitrate-treated rats, the capacity to hydrolyze and transport beta-casomorphin was greatly reduced. These results show that the ability of the kidneys to process peptides is significantly impaired as a result of uranyl nitrate-induced acute renal failure.

Acute Kidney Injury↗

PubMed1989

Tyrosine residues are essential for the activity of the human placental taurine transporter.

Treatment of human placental brush-border membrane vesicles with four tyrosine group-specific reagents, N-acetylimidazole, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tetranitromethane and p-nitrobenzesulfonyl fluoride, inhibited NaCl gradient-driven taurine uptake in these vesicles without affecting the vesicle integrity. The relative potency of these reagents to inhibit the transporter was in the following order: tetranitromethane greater than NBD-Cl greater than p-nitrobenzenesulfonyl fluoride greater than N-acetylimidazole. The inhibition by N-acetylimidazole was reversible with hydroxylamine and the inhibition by NBD-Cl was reversible with 2-mercaptoethanol. Kinetic analysis of taurine uptake in control and in N-acetylimidazole-treated membrane vesicles revealed that the inhibition was primarily due to a reduction in the maximal velocity. There was no change in the affinity of the transporter for taurine in control and treated vesicles. The transporter could be protected from the N-acetylimidazole-induced inhibition by Na+. The dependence of taurine uptake rate on extravesicular Na+ concentration was sigmoidal and analysis of the data revealed that two Na+ ions were involved per transport of one taurine molecule. It is concluded that tyrosine residues are essential for optimal transport function of the human placental taurine transporter and that these critical tyrosine residues are located at or near the Na+-binding site of the transporter.

4-Chloro-7-nitrobenzofurazan↗

PubMed1989

Evidence for an imipramine-sensitive serotonin transporter in human placental brush-border membranes.

Serotonin is actively transported into brush-border membrane vesicles isolated from normal human term placentas and an inward-directed NaCl gradient provides the driving force for this process. Uptake is negligible if Na+ is replaced by Li+, K+, Rb+, Cs+ or choline. The presence of Cl- seems necessary for the maximal activity of this Na+-dependent uptake system. Intravesicular K+ (20-40 mM) stimulates serotonin uptake, the stimulation being considerably greater at pH 7.5 than at pH 6.5. But, in the absence of K+, uptake at pH 6.5 was twice the uptake at pH 7.5. Unlabeled serotonin and dopamine inhibit the uptake of radiolabeled serotonin and the IC50 values are 70 nM and 20 microM, respectively. Histamine and 5-hydroxytryptophan do not significantly interact with the system (IC50 greater than 1 mM). Kinetic analysis reveals that serotonin uptake in these vesicles occurs via a single, saturable, high affinity system (Kt = 51 +/- 2 nM; Vmax = 6.4 +/- 0.1 pmol/mg of protein/15 s). The transporter is highly sensitive to inhibition by imipramine (IC50 = 32 nM) and desipramine (IC50 = 160 nM) but relatively insensitive to reserpine and hydralazine.

Carrier Proteins↗

PubMed1989

Involvement of thiol groups in the function of the dipeptide/proton cotransport system in rabbit renal brush-border membrane vesicles.

The role of thiol groups in H+-gradient-dependent dipeptide transport in rabbit renal brush-border membrane vesicles was investigated using glycylsarcosine as the substrate. Treatment of the membrane vesicles with a thiol-group-reducing agent, dimercaptopropanol, stimulated Gly-Sar transport. On the other hand, treatment with thiol group oxidants such as 5,5'-dithiobis(2-nitrobenzoic acid), plumbagin and phenazine methosulfate inhibited Gly-Sar transport. These effects were irreversible, because washing the membranes after treatment failed to reverse the effects. Incubation of the membrane vesicles with phenylarsine oxide, a reagent which interacts specifically with vicinal dithiols, significantly inhibited Gly-Sar transport. In all cases, the stimulation or the inhibition of the dipeptide transport was primarily due to changes in the maximal velocity of the transport system, the apparent affinity constant remaining unaltered. These results demonstrate the involvement of one or more vicinal dithiol groups in the function of the renal dipeptide transport system and that these thiol groups must exist in reduced form to maintain maximal transport activity. In addition, these data indirectly suggest that a dithiol-disulfide interchange may play a role in the function of the renal dipeptide transport system.

Animals↗

PubMed1989

Receptors for IGF-I, but not for IGF-II, on proximal colon epithelial cell apical membranes.

Rabbit proximal colon epithelial cell apical membranes, which are known to contain receptors for insulin, were isolated by a Ca2+-precipitation technique. Binding assays with 125I-insulin-like growth factor I (IGF-I) revealed the presence of specific high-affinity binding sites, with 50% inhibition of binding observed at a concentration of 13.7 ng/ml IGF-I. In contrast, 50% inhibition of 125I-IGF-I binding was observed at an insulin concentration of 1.37 micrograms/ml, suggesting that 125I-IGF-I was not binding to insulin receptors present in this tissue. Cross-linking studies revealed an 125I-IGF-I binding subunit of relative molecular weight (Mr) of 130,000 under reducing conditions on docecyl sulfate-polyacrylamide gel electrophoresis that was similar to the IGF-I binding subunit in human placental membranes (Mr 140,000). Binding and cross-linking studies with 125I-insulin-like growth factor II (IGF-II), however, failed to reveal a specific receptor for this peptide in colon epithelial cell membranes. These results establish the coexistence of receptors for IGF-I and insulin, but not IGF-II, on rabbit proximal colon epithelial cell apical membranes and demonstrate that colon epithelial cells are capable of selective synthesis of various peptide hormone receptors.

Animals↗

PubMed1989

Active transport of taurine in rabbit jejunal brush-border membrane vesicles.

The characteristics of taurine uptake in rabbit jejunal brush-border membrane vesicles were investigated. Taurine was transiently accumulated inside the vesicles against a concentration gradient when an inwardly directed NaCl gradient was imposed across the membrane. Uptake of taurine showed an absolute requirement for both Na+ and Cl-. The NaCl gradient-dependent taurine uptake was stimulated by a valinomycin-induced, inside-negative, K+-diffusion potential, suggesting that the uptake process was electrogenic. The uptake system exclusively interacted with beta-amino acids of small size, but had no affinity for alpha-amino acids. Kinetic analysis revealed that the system exhibited high affinity for taurine (Kt = 14.4 +/- 0.5 microM). Taurine uptake was greatly influenced by extravesicular concentrations of Na+ and Cl-. The Cl- stoichiometry was found to be one. In the presence of Cl-, taurine uptake was sigmoidally related to Na+ concentration, and the Na+ stoichiometry was calculated to be three. Thus three Na+ and one Cl- were involved per transport of one taurine molecule. The uptake process was not affected by other transport inhibitors such as amiloride, harmaline, furosemide, and 4,4'-diisothiocyanostilbene-2,2'-disulfonate.

Animals↗

PubMed1989

Multiple transport systems for organic cations in renal brush-border membrane vesicles.

The characteristics of guanidine uptake in brush-border membrane vesicles isolated from rabbit renal cortex were investigated. Guanidine uptake was markedly stimulated by an outwardly directed H+ gradient, resulting in a transient uphill transport. This stimulation was not due to an inside-negative, H+-diffusion potential because an ionophore-induced H+-diffusion potential and a K+-diffusion potential (both inside-negative) failed to enhance guanidine uptake. The H+ gradient itself appeared to be the driving force for the uptake. These data suggest that guanidine-H+ antiport (or guanidine-OH- symport) is the mechanism of guanidine uptake in these membrane vesicles. Guanidine uptake was only minimally inhibited by organic cations such as tetraethylammonium, N1-methylnicotinamide, and choline, but many other organic cations such as amiloride, clonidine, imipramine, and harmaline caused considerable inhibition. Uptake of radiolabeled guanidine was inhibited more effectively by guanidine than by tetraethylammonium, whereas uptake of radiolabeled tetraethylammonium was inhibited more effectively by tetraethylammonium than by guanidine. beta-Lactam antibiotics did not inhibit guanidine uptake but did inhibit tetraethylammonium uptake. Kinetic analysis showed that there were at least two kinetically distinct carrier systems for guanidine uptake, whereas tetraethylammonium uptake occurred via a single carrier system. These data provide evidence that renal brush-border membranes possess multiple carrier systems for organic cations.

Animals↗

PubMed1989

Inactivation of the human placental serotonin transporter by tyrosyl group-specific reagents.

Treatment of human placental brush-border membrane vesicles with tyrosyl group-specific reagents, N-acetylimidazole, 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole and tetranitromethane, inhibited NaCl gradient-driven serotonin uptake in these vesicles without affecting vesicle integrity. The concentrations of these reagents causing 50% inhibition of serotonin uptake were 3.75 mM, 10 microM and 5 microM, respectively. The inhibition of N-acetylimidazole was reversible with hydroxylamine and the inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was reversible with 2-mercaptoethanol. Kinetic analysis of serotonin uptake in control and in N-acetylimidazole-treated membrane vesicles revealed that the treatment decreased the maximal velocity of the transport system with virtually no effect on the affinity of the transporter for serotonin. Similarly, the treatment did not change the affinity of the transporter for Na+. Even though serotonin uptake was reduced in treated vesicles compared with control vesicles at all concentrations of Na+, in both cases the dependence of the uptake rate on Na+ concentration was hyperbolic, indicating the involvement of one Na+ per transport of one serotonin molecule. The serotonin transporter could be protected from the N-acetylimidazole-induced inhibition by Na+. It is concluded that tyrosyl residues are essential for optimal transport function of the human placental serotonin transporter and that these critical tyrosyl residues are located at or near the Na+-binding site.

4-Chloro-7-nitrobenzofurazan↗

PubMed1988

A proton gradient, not a sodium gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles.

An inward-directed H+ gradient markedly stimulated lactate uptake in rabbit intestinal brush-border membrane vesicles, and uphill transport against a concentration gradient could be demonstrated under these conditions. Uptake of lactate was many-fold greater in the presence of a H+ gradient than in the presence of a Na+ gradient. Moreover, there was no evidence for uphill transport of lactate in the presence of a Na+ gradient. The H+-gradient-dependent stimulation of lactate uptake was not due to the effect of a H+-diffusion potential. The uptake process in the presence of a H+ gradient was saturable [Kt (concn. giving half-maximal transport) for lactate 12.7 +/- 4.5 mM] and was inhibited by many monocarboxylates. It is concluded that a H+ gradient, not a Na+ gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1988

A proton gradient is the driving force for uphill transport of lactate in human placental brush-border membrane vesicles.

The characteristics of lactate transport in brush-border membrane vesicles isolated from normal human full-term placentas were investigated. Lactate transport in these vesicles was Na+-independent, but was greatly stimulated when the extravesicular pH was made acidic. In the presence of an inwardly directed H+ gradient ([H+]o greater than [H+]i), transient uphill transport of lactate could be demonstrated. This H+ gradient-dependent stimulation was not a result of a H+ diffusion potential. Transport of lactate in the presence of the H+ gradient was not inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or by furosemide, ruling out the participation of an anion exchanger in placental lactate transport. Many monocarboxylates strongly interacted with the lactate transport system, whereas, with the single exception of succinate, dicarboxylates did not. The monocarboxylates pyruvate and lactate, but not the dicarboxylate succinate, when present inside the vesicles, were able to exert a trans-stimulatory effect on the uptake of radiolabeled lactate. Kinetic analyses provided evidence for a single transport system with a Kt of 4.1 +/- 0.4 mM for lactate and a Vmax of 54.2 +/- 9.9 nmol/mg of protein/30 s. Pyruvate inhibited lactate transport competitively, by reducing the affinity of the system for lactate without altering the maximal velocity. It is concluded that human placental brush-border membranes possess a transport system specific for lactate and other monocarboxylates and that this transport system is Na+-independent and is energized by an inwardly directed H+ gradient. Lactate-H+ symport rather than lactate-OH- antiport appears to be the mechanism of the H+ gradient-dependent lactate transport in these membranes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗