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V Ganapathy

Publications and source records attributed to V Ganapathy.

At least 235 records · Page 13Linked to original sources
PubMed1986

Na+-H+ exchanger of human placental brush-border membrane: identification and characterization.

Syncytiotrophoblast brush-border membrane vesicles isolated from full-term human placentas were shown to transport Na+ against a concentration gradient in the presence of an outward proton gradient [( H+i] greater than [H+]o). This proton gradient-coupled Na+ uptake was markedly inhibited and the uphill transport abolished when the electrochemical proton gradient was dissipated by carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone. The presence of nigericin also eliminated the concentrative uptake of Na+ in these vesicles. Dimethylamiloride and harmaline inhibited the proton gradient-induced Na+ uptake. The apparent inhibition constant for this process was 0.32 microM for dimethylamiloride was freely reversible and the inhibitor reduced the Na+ uptake by directly interacting with the exchanger protein rather than by dissipating the H+ gradient. The dimethylamiloride-sensitive Na+ uptake was saturable with respect to Na+. The affinity constant for Na+ was 7.8 +/- 1.2 mM and the maximal velocity was 38.7 +/- 2.4 nmol X mg protein-1 X min-1. The dimethylamiloride-insensitive Na+ uptake was not saturable and probably represented simple diffusion. The diffusional component accounted for only 10% of the total uptake. Li+ strongly competed with Na+ for the uptake process and the apparent inhibition constant was 3.6 +/- 0.4 mM. Tetraethylammonium also caused significant inhibition of Na+ uptake, whereas K+, Rb+, Cs+, and choline had no effect. These data provide evidence for the existence of a Na+-H+ exchanger in human placental brush-border membrane, and the properties of this exchanger are similar to those of the Na+-H+ exchanger identified in the brush-border membrane of mammalian kidney and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1986

Carrier-mediated reabsorption of small peptides in renal proximal tubule.

Recent studies with a variety of tissue preparations in the kidney have demonstrated that proximal tubular cells possess specific transport systems for di- and tripeptides. In contrast to the well-known amino acid and glucose transport systems, active transport of peptides in these cells is energized by an H+ gradient rather than an Na+ gradient. Like amino acid-Na+ and glucose-Na+ cotransport systems, peptide-H+ cotransport is electrogenic and hence a membrane potential also contributes to the uphill transport of peptides in these cells. Di- and tripeptides that are filtered at the glomerulus, as well as those that are produced in the tubular lumen from larger polypeptides by the action of brush-border peptidases, serve as substrates for the renal peptide transport system under physiological conditions. The H+ gradient that is necessary to drive renal peptide transport is generated in vivo by concerted action of the basolateral Na+-K+-ATPase and the brush-border Na+-H+ exchanger. The peptidases and the peptide transport system in the renal brush-border membrane play a significant role in the reabsorption of peptide-bound amino acids as well as in the regulation of plasma levels of small peptides.

Absorption↗
PubMed1986

Inhibition of human placental Na+-H+ exchanger by cimetidine.

The Na+-H+ exchanger of human placental brush-border membrane was strongly inhibited by cimetidine, a histamine type II receptor antagonist. The inhibition was freely reversible and the apparent inhibition constant for the process was 38 microM. The nature of inhibition was found to be competitive with respect to Na+. Cimetidine inhibition was specific as other related compounds had no inhibitory effect. Treatment of the membrane vesicles with diethylpyrocarbonate inactivated the exchanger, but the presence of cimetidine during the treatment provided complete protection from this inhibition. These results show that histidine residues are located at the external Na+-binding site of the exchanger and cimetidine interacts with this site. The Na+-H+ exchanger of rabbit renal brush-border membrane was also susceptible to inhibition by the drug. However, transport of proline and glucose in placental brush-border membrane vesicles was not affected by cimetidine. The placental Na+-H+ exchanger was also inhibited by amiloride and the inhibition was freely reversible and competitive with respect to Na+. These results show that the characteristics of the inhibition of Na+-H+ exchanger by cimetidine and amiloride are very similar.

Amiloride↗
PubMed1985

Proton gradient-coupled uphill transport of glycylsarcosine in rabbit renal brush-border membrane vesicles.

An inward-directed proton gradient energizes the transport of intact glycylsarcosine against a concentration gradient in rabbit renal brush-border membrane vesicles. Dissipation of the proton gradient abolishes the uphill transport. Generation of an inside-negative membrane potential nearly doubles the intravesicular concentration of the dipeptide at the peak of the overshoot without altering the equilibrium value. These data provide direct evidence for peptide-proton cotransport in the renal brush-border membrane.

Animals↗
PubMed1985

Dipeptide transport in brush-border membrane vesicles isolated from normal term human placenta.

We studied the transport of glycylsarcosine by the brush-border membrane vesicles isolated from normal term human placentas. This dipeptide resisted hydrolysis by placental membrane vesicles and was transported intact into an osmotically responsive intravesicular space. The transport process was Na+-independent and probably occurred down a concentration gradient. Many peptides inhibited the transport of glycylsarcosine whereas amino acids had no effect. These results demonstrate, for the first time, the presence of a dipeptide transport system in the human placenta.

Amino Acids↗
PubMed1985

Peptide transport in rabbit intestinal brush-border membrane vesicles studied with a potential-sensitive dye.

Peptide transport in purified rabbit intestinal brush-border membrane vesicles has been studied using a potential-sensitive fluorescent dye, di-S-C3(5). Transport of dipeptides is accompanied by an increase in the fluorescence of the dye in the presence and absence of Na+, indicating electrogenic, Na+-independent peptide transport. Dipeptides containing D-amino acids also increase the fluorescence, showing that these peptides too possess significant affinity for the peptide transport system. beta-Alanylglycylglycine and prolylglycylglycine, very much like the dipeptides, increase the fluorescence even in the absence of Na+ which demonstrates the Na+-independent, electrogenic transport of tripeptides. However, concentrations needed for half-maximal fluorescence changes are higher for tripeptides than for dipeptides suggesting different affinities for the carriers. The studies, in addition, provide evidence for the existence of more than one carrier system for translocation of small peptides in rabbit intestinal brush-border membrane.

Animals↗
PubMed1985

Identification of insulin receptors on the mucosal surface of colon epithelial cells.

Brush-border membranes were isolated from the mucosal surface of rabbit proximal colon epithelial cells by a procedure involving Ca2+ precipitation. Ouabain-insensitive K+-phosphatase, a marker enzyme for the colon brush-border membrane, was enriched 17-fold by this technique, while no enrichment was observed in the activity of ouabain-sensitive K+-phosphatase, a marker for the basal-lateral membrane. Insulin binding studies revealed a dose-dependent inhibition of 125I-insulin binding with porcine insulin and approximately 4 X 10(-9) M insulin was required to produce 50% inhibition of 125I-insulin binding, while desoctapeptide insulin, insulin-like growth factor I, and A chain of insulin had less effect on 125I-insulin binding. This is the first demonstration of the existence of high-affinity insulin binding sites on the brush-border membrane of mammalian colon epithelial cells. Subsequent studies with the cross-linking agent disuccinimidyl suberate confirmed the presence of insulin binding sites in these membranes and autoradiography of polyacrylamide gels revealed that the binding subunit of the colon epithelial cell brush-border insulin receptor is similar in size to that observed in hepatic tissue. Interestingly, the insulin binding capacity/mg of protein of this preparation is high, suggesting that large numbers of insulin receptors are present in vivo on the mucosal surface of colon epithelial cells. The potential physiological role of these previously unrecognized insulin receptors is discussed.

Animals↗
PubMed1985

Inhibition of rat and human prolidases by captopril.

We examined the effects of captopril on prolidase activity of crude homogenates of various tissues in the rat and in the human. It was found that captopril caused significant inhibition of prolidases from liver, kidney, and intestine in both species, whereas it showed minimal inhibitory effect on erythrocyte prolidase. In the rat, Ki for the inhibition of kidney and liver prolidases was in the range of 25-35 microM while, in the human, the value was considerably higher. The nature of inhibition was found to be competitive both in the rat and in the human. Oral administration of captopril (daily dose, 40 mg/kg) for 7 days in the rat resulted in increased urinary excretion of peptide-bound 4-hydroxy-L-proline compared to controls, indicating in vivo inhibition of tissue prolidases by captopril.

Animals↗
PubMed1984

Characteristics of glycylsarcosine transport in rabbit intestinal brush-border membrane vesicles.

Glycylsarcosine was found to be very resistant to hydrolysis by brush-border membrane vesicles from rabbit intestine. The dipeptide was transported intact into an osmotically responsive intravesicular space. The initial uptake rate of glycylsarcosine into these vesicles was greater in mannitol medium compared to that in the presence of an inward gradient of either Na+ or other monovalent cations. When vesicles preloaded with glycylsarcosine were incubated in a peptide-free medium, there was a rapid efflux of the dipeptide and the t1/2 for the process was less than 2 min. An inside-negative K+ diffusion potential generated by valinomycin stimulated glycylsarcosine uptake even in the absence of Na+. Experiments with the potential-sensitive dye DiS-C3 (5) showed that glycylsarcosine depolarized the brush-border membrane in the presence and absence of Na+. Imposition of an inward proton gradient stimulated the initial uptake rates of glycylsarcosine while the equilibrium uptake was not affected. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone decreased this proton gradient-induced stimulation. An inward proton gradient increased the Vmax of the transport system (10.8 +/- 0.8 nmol/min/mg of protein when [pH]o = [pH]i = 5.5; and 20.8 +/- 2.2 nmol/min/mg of protein when [pH]o = 5.5 and [pH]i = 7.8), without significantly affecting the apparent Kt (17.3 +/- 1.4 mM versus 19.5 +/- 2.0 mM). Glycyl-L-proline uptake was inhibited by glycylsarcosine and KI for the process was 20.8 +/- 3.0 mM. A relatively lower KI (2.8 +/- 1.2 mM) was obtained for the inhibition of glycylsarcosine uptake by glycyl-L-proline. The uptake of glycyl-L-proline and glycylsarcosine was strongly inhibited by L-carnosine, glycyl-L-leucine, and L-prolylglycine. With each inhibitory peptide, the KI values for the inhibition of glycyl-L-proline uptake and of glycylsarcosine uptake were comparable. Preloading the vesicles with unlabeled glycylsarcosine stimulated the uptake of labeled glycyl-L-proline. These data suggest that in rabbit intestinal brush-border membrane vesicles (i) glycylsarcosine and proton(s) are co-transported, (ii) this process results in a net transport of positive charge across the membrane and, (iii) a single transport system is involved in the translocation of glycyl-L-proline and glycylsarcosine.

Animals↗
PubMed1983

Role of pH gradient and membrane potential in dipeptide transport in intestinal and renal brush-border membrane vesicles from the rabbit. Studies with L-carnosine and glycyl-L-proline.

We examined the role of pH gradient and membrane potential in dipeptide transport in purified intestinal and renal brush-border membrane vesicles which were predominantly oriented right-side out. With an intravesicular pH of 7.5, changes in extravesicular pH significantly affected the transport of glycyl-L-proline and L-carnosine, and optimal dipeptide transport occurred at an extravesicular pH of 5.5-6.0 in both intestine and kidney. When the extravesicular pH was 5.5, glycyl-L-proline transport was accelerated 2-fold by the presence of an inward proton gradient. A valinomycin-induced K+ diffusion potential (interior-negative) stimulated glycyl-L-proline transport, and the stimulation was observed in the presence and absence of Na+. A carbonyl cyanide p-trifluoromethoxyphenylhydrazone-induced H+ diffusion potential (interior-positive) reduced dipeptide transport. It is suggested that glycyl-L-proline and proton(s) are cotransported in intestinal and renal brush-border membrane vesicles, and that the process results in a net transfer of positive charge.

Animals↗
PubMed1983

Dipeptide metabolism in the isolated perfused rat kidney.

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.

Animals↗
PubMed1983

Electrogenic transport of 5-oxoproline in rabbit renal brush-border membrane vesicles. Effect of intravesicular potassium.

The Na+-dependent transport of 5-oxoproline into rabbit renal brush-border vesicles was stimulated by a K+ diffusion potential (interior-negative) induced by valinomycin. Na+ salts of two anions of different epithelial permeabilities also affected 5-oxoproline transport. These results show that the Na+-dependent 5-oxoproline transport in renal brush-border vesicles is an electrogenic process which results in a net transfer of positive charge. Maximum transport of 5-oxoproline occurred at an extravesicular pH of 6.0 to 8.0 and over that pH range, 5-oxoproline exists completely as an anion with a negative charge. The simplest stoichiometry consistent with this process is, therefore, the cotransport of one 5-oxoproline anion with two sodium ions. The presence of K+ inside the vesicles stimulated the Na+-dependent transport of 5-oxoproline. This stimulatory effect was specific for K+ and required the presence of Na+. The presence of Na+ gradient was not mandatory for the K+ action. The stimulation by the intravesicular K+ was seen in the presence as well as in the absence of a K+ gradient. Therefore, the increased influx of 5-oxoproline was not coupled to the simultaneous efflux of K+. The presence of K+ in the extravesicular medium alone did not affect the Na+-dependent transport of 5-oxoproline, showing that the site of K+ action was intravesicular. Glutamate did not interact with the Na+-dependent 5-oxoproline transport even in the presence of an outward K+ gradient.

Animals↗
PubMed1982

Transport and utilization of methionine sulfoxide in the rabbit.

Methionine sulfoxide is transported into purified intestinal and renal brush border membrane vesicles from rabbit by an Na+-dependent mechanism and is accumulated inside the vesicles against the concentration gradient. Both in intestine and kidney, the rate of transport is enhanced with increasing concentrations of Na+ in the external medium. Increasing the Na+ gradient reduces the apparent Kt for methionine sulfoxide without causing any change in Vmax. With an outward K+ gradient (vesicle greater than medium), valinomycin stimulates the Na+-gradient-dependent transport of methionine sulfoxide in the kidney, showing the electrogenicity of the transport process. A number of amino acids inhibit methionine sulfoxide transport in both the intestine and kidney. An enzymatic activity capable of reducing methionine sulfoxide to methionine is present in the intestinal mucosa, renal cortex and liver. The activity is highest in renal cortex and lowest in intestine. The methionine sulfoxide-reducing activity is stimulated by NADH, NADPH, glutathione and dithiothreitol and the potency of the stimulation is in the order: dithiothreitol greater than NADPH greater than glutathione greater than NADH.

Amino Acids↗
PubMed1982

Prolidase contamination in commercial bovine serum albumin.

Bovine serum albumin from a number of commercial sources were screened for the presence or absence of peptidase contamination. Peptidase activity was monitored using various peptides as substrates. Two commercial preparations were found to have peptidase activity, and the enzyme was identified, on the basis of its substrate specificity, as prolidase (EC 3.4.14.9). The contaminating activity was in the order of 3-4 units/g albumin.

Animals↗
PubMed1982

Peptide transport in rabbit kidney. Studies with L-carnosine.

L-Carnosine was shown to be transported into rabbit renal brush-border membrane vesicles by an Na+ -independent mechanism. The transport was competitively inhibited by glycyl-L-proline. Various di- and tripeptides inhibited L-carnosine transport, whereas free amino acids did not. Inhibition studies showed that blocking the free amino and carboxyl groups of the peptide reduced its affinity for the transport carrier. Under the conditions in which there was no detectable hydrolysis of L-carnosine in the medium, intravesicular contents showed a 30% hydrolysis of the peptide within the vesicles. Disruption of membrane vesicles with deoxycholate resulted in a 3-fold increase in L-carnosine hydrolyzing activity over untreated intact vesicles. Based on these observations, a model for peptide transport is proposed in which transport of the intact peptide across the membrane is followed by its partial or complete hydrolysis by a membrane peptidase whose active site is on the cytoplasmic side of the membrane.

Animals↗