SEARCH · PubMed Health

Results for “TRANSPORTATION”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources

PubMed1988

Effects of insulin on glucose transport and glucose transporters in rat heart.

The effect of insulin on glucose transport and glucose transporters was studied in perfused rat heart. Glucose transport was measured by the efflux of labelled 3-O-methylglucose from hearts preloaded with this hexose. Insulin stimulated 3-O-methylglucose transport by: (a) doubling the maximal velocity (Vmax); (b) decreasing the Kd from 6.9 to 2.7 mM; (c) increasing the Hill coefficient toward 3-O-methylglucose from 1.9 to 3.1; (d) increasing the efficiency of the transport process (k constant). Glucose transporters in enriched plasma and microsomal membranes from heart were quantified by the [3H]cytochalasin-B-binding assay. When added to normal hearts, insulin produced the following changes in the glucose transporters: (a) it increased the translocation of transporters from an intracellular pool to the plasma membranes; (b) it increased (from 1.6 to 2.7) the Hill coefficient of the transporters translocated into the plasma membranes toward cytochalasin B, suggesting the existence of a positive co-operativity among the transporters appearing in these membranes; (c) it increased the affinity of the transporters (and hence, possibly, of glucose) for cytochalasin B. The data provide evidence that the stimulatory effect of insulin on glucose transport may be due not to the sole translocation of intracellular glucose transporters to the plasma membrane, but to changes in the functional properties thereof.

3-O-Methylglucose↗

PubMed1979

Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system.

We have isolated mutants of Escherichia coli that have an altered beta-galactoside transport system. This altered transport system is able to transport a sugar, maltose, that the wild-type beta-galactoside transport system is unable to transport. The mutation that alters the specificity of the transport system is in the lacY gene, and we refer to the allele as lacYmal. The lacYmal allele was detected originally in strains in which the lac genes were fused to the malF gene. Thus, as a result of gene fusion and isolation of the lacYmal mutation, a new transport system was evolved with regulatory properties and specificity similar to those of the original maltose transport system. Maltose transport via the lacYmal gene product is independent of all of the normal maltose transport system components. The altered transport system shows a higher affinity than the wild-type transport system for two normal substrates of the beta-galactoside transport system, thiomethyl-beta-D-galactoside and o-nitrophenyl-beta-D-galactoside.

Biological Transport, Active↗

PubMed1986

Transport of butyric acid in vascularly perfused anuran small intestine: importance of pH and anion transport.

Butyric acid transport was studied in the isolated, vascularly perfused frog small intestine. At luminal butyric acid concentrations of 5-50 mM, absorption was a nonlinear function of the luminal concentration, whereas the relationship of absorption to concentration remained linear at 0-1,000 microM. The most important factor regulating the rate and direction of butyric acid transport was the pH. We used unidirectional flux analysis to determine net transport across the epithelium while the pH of the luminal or vascular compartments was changed. We found a four- to fivefold decrease in butyric acid transport into the portal circulation as the lumen pH was increased from 6.0 to 8.0. The pH of the vascular perfusate influenced the vascular-to-lumen transport of butyric acid in the same proportions. The second important regulatory factor of butyric acid transport was the 4,4'-diisothiocyananostilbene-2,2'-disulfonic acid (DIDS)-sensitive anion transport protein. DIDS added to the lumen at 10(-6) M decreased butyric acid transport by approximately 40% at pH 7.4. DIDS also inhibited butyric acid transport when added to the vascular perfusate or when transport was measured in a vascular-to-lumen direction. We suggest that, at the relatively low pH of the proximal small intestine, butyric acid becomes protonated and lipophilic and is mainly transported directly through the cell membrane. At the more alkaline pH of the distal small intestine butyric acid is in the ionized form and transport by the DIDS-sensitive anion transport protein may predominate.

3-O-Methylglucose↗

PubMed1992

Transport of fatty acids into human and rat peroxisomes. Differential transport of palmitic and lignoceric acids and its implication to X-adrenoleukodystrophy.

The different topology of palmitoyl-CoA ligase (on the cytoplasmic surface) and of lignoceroyl-CoA ligase (on the luminal surface) in peroxisomal membranes suggests that these fatty acids may be transported in different form through the peroxisomal membrane (Lazo, O., Contreras, M., and Singh, I. (1990) Biochemistry 29, 3981-3986), and this differential transport may account for deficient oxidation of lignoceric acid in X-adrenoleukodystrophy (X-ALD) (Singh, I., Moser, A. B., Goldfisher, S., and Moser, H. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4203-4207). To define the transport mechanism for these fatty acids through the peroxisomal membrane and its possible implication to lignoceric acid metabolism in X-ALD, we examined cofactors and energy requirements for the transport of palmitic and lignoceric acids in isolated peroxisomes from rat liver and peroxisomes isolated from X-ALD and control fibroblasts. The similar rates of transport of palmitoyl-CoA (87.6 +/- 6.3 nmol/h/mg protein) and palmitic acid in the fatty acid activating conditions (83.4 +/- 5.1 nmol/h/mg protein) and lack of transport of palmitic acid (4% of palmitoyl-CoA transport) when ATP and/or CoASH were removed or substituted by alpha,beta-methyleneadenosine-5'-triphosphate (AMPCPOP) and/or desulfoCoA-agarose from assay medium clearly demonstrate that transport of palmitic acid requires prior synthesis of palmitoyl-CoA by palmitoyl-CoA ligase on the cytoplasmic surface of peroxisomes. The 10-fold higher rate of transport of lignoceric acid (5.3 +/- 0.6 nmol/h/mg protein) as compared with lignoceroyl-CoA (0.41 +/- 0.11 nmol/h/mg protein) and lack of inhibition of transport of lignoceric acid when ATP and/or CoASH were removed or substituted with AMPCPOP or desulfoCoA-agarose suggest that lignoceric acid is transported through the peroxisomal membrane as such. Moreover, the lack of effect of removal of ATP or substitution with AMPOPCP (a nonhydrolyzable substrate) demonstrates that the translocation of palmitoyl-CoA and lignoceric acid across peroxisomal membrane does not require energy. The transport, activation, and oxidation of palmitic acid are normal in peroxisomes from X-ALD. The deficient lignoceroyl-CoA ligase (13% of control) and oxidation of lignoceric acid (10% of control) as compared with normal transport of lignoceric acid into peroxisomes from X-ALD clearly demonstrates that pathogenomonic accumulation of very long chain fatty acids (greater than C22) in X-ALD is due to the deficiency of peroxisomal lignoceroyl-CoA ligase activity.

Acyl Coenzyme A↗

PubMed2005

A species difference in the transport activities of H2 receptor antagonists by rat and human renal organic anion and cation transporters.

A clinical drug-drug interaction between famotidine (a H2 receptor antagonist) and probenecid has not been reproduced in rats. The present study hypothesized that the species-dependent probenecid sensitivity is due to a species difference in the contribution of renal organic anion and cation transporters. The transport activities of the H2 receptor antagonists (cimetidine, famotidine, and ranitidine) by rat and human basolateral organic anion and cation transporters [human organic anion transporter (hOAT) 1, hOAT2, r/hOAT3, rat organic cation transporter (rOct) 1, and r/hOCT2] were compared using their cDNA transfectants. The transport activities (Vmax/Km) of famotidine (Km, 345 microM) by rOat3 were 8- and 15-fold lower than those of cimetidine (Km, 91 microM) and ranitidine (Km, 155 microM), respectively, whereas the activity by hOAT3 (Km, 124 microM) was 3-fold lower than that of cimetidine (Km, 149 microM) but similar to that of ranitidine (Km, 234 microM). Comparison of the relative transport activity with regard to that of cimetidine suggests that famotidine was more efficiently transported by hOAT3 than rOat3, and vice versa, for ranitidine. Only ranitidine was efficiently transported by hOAT2 (Km, 396 microM). rOct1 accepts all of the H2 receptor antagonists with a similar activity, whereas the transport activities of ranitidine and famotidine (Km, 61/56 microM) by r/hOCT2 were markedly lower than that of cimetidine (Km, 69/73 microM). Probenecid was a potent inhibitor of r/OAT3 (Ki, 2.6-5.8 microM), whereas it did not interact with OCTs. These results suggest that, in addition to the absence of OCT1 in human kidney, a species difference in the transport activity by hOAT3 and rOat3 accounts, at least in part, for the species difference in the drug-drug interaction between famotidine and probenecid.

Animals↗

PubMed1996

Structure-function analysis of liver-type (GLUT2) and brain-type (GLUT3) glucose transporters: expression of chimeric transporters in Xenopus oocytes suggests an important role for putative transmembrane helix 7 in determining substrate selectivity.

The liver-type (GLUT2) and brain-type (GLUT3) human facilitative glucose transporters exhibit distinct kinetics (K(m) values for deoxyglucose transport of 11.2 +/- 1.1 and 1.4 +/- 0.06 mM, respectively) and patterns of substrate transport (GLUT2 is capable of D-fructose transport, GLUT3 is not) [Gould, G. W., Thomas, H. M., Jess, T. J., & Bell, G. I. (1991) Biochemistry 30, 5139-5145]. We have generated a range of chimeric glucose transporters composed of regions of GLUT2 and GLUT3 with a view to identifying the regions of the transporter which are involved in substrate recognition and binding. The functional characteristics of these chimeras were determined by expression in Xenopus oocytes after microinjection of cRNA. Replacement of the region from the start of putative transmembrane helix 7 to the C-terminus of GLUT3 with the corresponding region from GLUT2 results in a chimera with the ability to transport fructose and exhibits a K(m) for 2-deoxyglucose transport of close to that observed for wild-type GLUT2 (8.3 +/- 0.3 mM compared to 11.2 +/- 1.1 mM). Replacement of the region in GLUT3 from the end of helix 7 to the C-terminus with the corresponding region from GLUT2 resulted in a species which was unable to transport fructose and whose K(m) for 2-deoxyglucose was indistinguishable from wild-type GLUT3. We have determined the affinity for 2-deoxyglucose, D-fructose, and D-galactose of these and other chimeras. In addition, the Ki for maltose, a competitive inhibitor of 2-deoxyglucose transport, which binds to the exofacial sugar binding site was determined for these chimeras. The results obtained support a model in which the seventh putative transmembrane-spanning helix is intimately involved in the selection of transported substrate and in which this region plays an important role in determining the K(m) for 2-deoxyglucose. Additional data is presented which suggests that a region between the end of putative transmembrane helix 7 and the end of helix 10, together with sequences in the N-terminal half of the protein may also participate in substrate recognition and transport catalysis.

Amino Acid Sequence↗

PubMed1997

Phorbol esters increase dopamine transporter phosphorylation and decrease transport Vmax.

Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport Vmax in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK1, cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport Vmax by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter Vmax suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.

Alanine↗

PubMed1999

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes.

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.

3-O-Methylglucose↗

PubMed1999

Drug transport and targeting. Intestinal transport.

A wide variety of transporters are found in the intestine, and are involved in the membrane transport of daily nutrients as well as drugs. These intestinal transporters are located in the brush border membrane as well as basolateral membrane. Each transporter exhibits its own substrate specificity, and some have broader specificities than others. In addition, the distribution and characteristics of the intestinal transporters exhibit regional differences along the intestine, implying diverse physiologic functions and in some cases pathologic responses. Indeed several genetic disorders have been shown to result from deficient intestinal transporters. The development of prodrugs that target to intestinal transporters has been successful in improving oral absorption. For example, the intestinal peptide transporter is utilized in order to increase the bioavailability of several classes of peptidomimetic drugs, especially ACE inhibitors and beta-lactam antibiotics. The bioavailability of poorly absorbed drugs can be improved by utilization of the transporters responsible for the intestinal absorption of various solutes and/or by inhibiting the transporter involved in the efflux system. Recent advances in gene cloning and molecular biology techniques make it possible to study the characteristics and distribution of transporters at the molecular level. Based on molecular characterizations of membrane transporters and accumulated biochemical data on their specificities and kinetics, structural modification and targeting of a specific transporter is a promising strategy for the design of drugs that improve bioavailability and tissue distribution.

Biological Availability↗

PubMed1990

DeltapH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet Leaves: I. Evidence for Carrier-Mediated, Electrogenic Flux through Multiple Transport Systems.

Amino acid transport into plasma membrane vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves was investigated. The transport of alanine, leucine, glutamine, glutamate, isoleucine, and arginine was driven by a trans-membrane proton concentration difference. DeltapH-Dependent alanine, leucine, glutamine, and glutamate transport exhibited simple Michaelis-Menten kinetics, and double-reciprocal plots of the data were linear with apparent K(m) values of 272, 346, 258, and 1981 micromolar, respectively. These results are consistent with carrier mediated transport. DeltapH-Dependent isoleucine and arginine transport exhibited biphasic kinetics, suggesting these amino acids may be transported by at least two transport systems. Symport mediated alanine transport was electrogenic as demonstrated by the effect of membrane potential (DeltaPsi) on DeltapH-dependent flux. In the absence of significant charge compensation, a low rate of alanine transport was observed. When DeltaPsi was held at 0 millivolt with symmetric potassium concentrations and valinomycin, the rate of flux was stimulated fourfold. In the presence of a negative DeltaPsi, alanine transport increased sixfold. These results are consistent with an electrogenic transport process which results in a net flux of positive charge into the vesicles. The effect of changing DeltaPsi on the kinetics of alanine transport altered V(max) with no apparent change in K(m). Amino acid transport was inhibited by the protein modifier diethyl pyrocarbonate, but was insensitive to N-ethylmaleimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, p-chloromercuribenzenesulfonic acid, phenylglyoxal, and N,N'-dicyclohexylcarbodiimide. Four amino acid symport systems, two neutral, one acidic, and one basic, were resolved based on inter-amino acid competition experiments. One neutral system appears to be active for all neutral amino acids while the second exhibited a low affinity for isoleucine, threonine, valine, and proline. Although each symport was relatively specific for a given group of amino acids, each system exhibited some crossover specificity for amino acids in other groups.

Journal Article↗

PubMed1994

An Arabidopsis peptide transporter is a member of a new class of membrane transport proteins.

An Arabidopsis peptide transport gene was cloned from an Arabidopsis cDNA library by functionally complementing a yeast peptide transport mutant. The Arabidopsis plant peptide transporter (AtPTR2) allowed growth of yeast cells on dipeptides and tripeptides but not peptides four residues and higher. The plant peptide transporter also conferred sensitivity to a number of ethionine-containing, toxic peptides of chain length three or less and restored the ability to take up radiolabeled dileucine at levels similar to that of the wild type. Dileucine uptake was reduced by the addition of a variety of growth-promoting peptides. The sequence of a cDNA insert of 2.8 kb indicated an open reading frame encoding a 610-amino acid polypeptide (67.5 kD). Hydropathy analysis predicted a highly hydrophobic protein with a number of potential transmembrane segments. At the amino acid level, the Arabidopsis plant peptide transporter shows 24.6, 28.5, and 45.2% identity to the Arabidopsis nitrate-inducible nitrate transporter (CHL1), the rabbit small intestine oligopeptide transporter (PepT1), and the yeast peptide transporter (Ptr2p), respectively, but little identity to other proteins known to be involved in peptide transport. Root growth of Arabidopsis seedlings exposed to ethionine-containing toxic peptides was inhibited, and growth was restored by the addition of certain peptides shown to compete with dileucine uptake in yeast expressing the Arabidopsis transport gene. Consistent with the observed inhibition of root growth by toxic peptides, the peptide transporter is expressed in the roots of Arabidopsis seedlings. This study represents the characterization of a plant peptide transporter that is a member of a new class of related membrane transport proteins.

Amino Acid Sequence↗

PubMed1976

Density regulation of amino acid transport in cultured, androgen-responsive tumour cells.

In androgen-responsive cells, activity of a sodium-dependent transport system for neutral amino acids is shown to be density-dependent, whereas activity of a sodium-independent system is not. Transport of the non-metabolizable amino acid, alpha-aminoisobutyric acid (alpha-aib) was largely sodium-dependent, whereas transport of cycloleucine (cyleu) was by both sodium-dependent and sodium-independent systems. The Km for cyleu transport changed with density because only the sodium-dependent portion of this transport was density-dependent. By contrast, the Vmax for alpha-aib transport changed with density, indicating that either the amount of functional membrane carrier or the activity of the sodium pump was affected.

Amino Acids↗

PubMed1996

Regulation of expression of taurine transport in two continuous renal epithelial cell lines and inhibition of taurine transporter by a site-directed antibody.

UNLABELLED: The renal tubular epithelium adapts to changes in the sulfur amino acid composition of the diet, particularly in terms of reabsorption of taurine. The adaptive response is expressed by enhanced or decreased NaCl-dependent taurine transport by rat renal brush border membrane vesicles (BBMV). Taurine transport activity in two cultured renal epithelial cell lines (MDCK and LLC-PK1) is up- or down-regulated by extracellular taurine concentration as the result of reciprocal changes in the Vmax of the transporter. In MDCK cells, abundance of taurine transporter mRNA (pNCT mRNA) was up- or down-regulated after incubation in media containing 0, 50, or 500 microM taurine. Decreased mRNA was observed in both cell lines after 12 h, and it was appreciably reduced after 72 h exposure to 500 microM taurine. Northern blot analysis of mRNA from LLC-PK1 cells using pNCT cDNA as a riboprobe showed that two transcripts, 9.6 kb and 7.2 kb, were expressed; the abundance of mRNA was increased or decreased after incubation in taurine-free or high taurine medium, respectively. Down-regulation was observed primarily in the 7.2 kb transcript after 24 h incubation. Rapid up-regulation occurred in the 9.6 kb transcript within 12 h of transfer from high to low taurine. Nuclear run-off assays showed that the gene for pNCT is induced at the transcriptional level by taurine. Regulation of expression of the taurine transporter was also studied by injection of pNCT cRNA into Xenopus laevis oocytes. Expression of transport activity was significantly reduced (64%) when oocytes were incubated in 50 microM taurine as compared to 0 microM taurine. Transport activity was totally blocked when pNCT cRNA-injected oocytes were exposed to an active phorbol ester, PMA (10(-6) M). Inhibition of uptake was reversed by staurosporine, an inhibitor of protein kinase C activity. An inactive phorbol ester, 4 alpha-phorbol, had no effect on taurine transport. A polyclonal antibody directed a highly conserved intracellular segment between homologous transmembrane domains VI and VII inhibited taurine transport activity in both pNCT cRNA-injected oocytes and BBMV. Incubation of oocytes with 10 micrograms/ml antibody (Ab) reduced taurine uptake to 46% of control, and 20-80 micrograms/ml Ab reduced uptake to 20% of control. In BBMV, active taurine uptake (10 microM) was inhibited approximately 30% by 10 pg Ab/mg protein, whereas none specific IgG had no significant effect. Proline uptake (20 microM) by BBMV was not inhibited by the Ab, nor was GABA uptake (50 microM). Two pNCT proteins, approximately 70 kD and approximately 30 kD, were detected by Western blot, and the abundance of both was regulated by medium taurine. IN CONCLUSION: (i) regulation of taurine transport activity in LLC-PK1 cells by medium taurine occurs at a level of mRNA transcription; (ii) regulation of pNCT occurs at both transcriptional and translational levels; (iii) pNCT expression is regulated by protein kinase C-dependent phosphorylation; and (iv) the intracellular segment between domains VI and VII may be required for activation of the taurine transporter; this segment may function as a gate in taurine transport.

Amino Acid Sequence↗

PubMed1988

Heart glucose transport and transporters in rat heart: regulation by insulin, workload and glucose.

Aspects of the regulation of the glucose transport by perfused hearts of normal rats have been studied by measuring glucose transport (via the efflux of labelled 3-O-methyl-D-glucose) and glucose transporters (via the labelled cytochalasin B binding assay). Similarly to what is observed with insulin, increasing workload (by raising perfusion pressure from 50 to 100 mm Hg) stimulated glucose transport 7 to 8-fold. Glucose (via its analog 3-O-methylglucose, used at 15 mmol/l) stimulated its own transport 4-fold. The three stimuli favored the translocation of glucose transporters from an intracellular pool (microsomes) to the plasma membrane. Insulin increased the apparent affinity (decreased dissociation constant values) of plasma membrane transporters for cytochalasin, as well as the Hill coefficient, indicating the occurrence of a positive cooperativity amongst plasma membrane transporters. Workload increased only the Hill coefficient, glucose only the apparent affinity for cytochalasin of plasma membrane transporters. This study shows that insulin, workload and glucose itself stimulate glucose transport by favouring the translocation process of glucose transporter as well as by changing, albeit by a different mechanism, the functional properties of the transporters once translocated to the plasma membrane.

3-O-Methylglucose↗

PubMed1998

The pharmacological profile of L-glutamate transport in human NT2 neurones is consistent with excitatory amino acid transporter 2.

The human teratocarcinoma cell line NTera2/D1 can be differentiated to produce post-mitotic neurones (NT2-N cells) by prolonged (> 3 week) exposure to retinoic acid. In this study, we describe the characterisation of high-affinity Na+-dependent L-glutamate transport activity in post-mitotic differentiated NT2-N cells. NT2-N cells, but not the undifferentiated precursor cells, transported L-glutamate in a Na+-dependent manner, as determined by equimolar replacement of Na+ with choline. L-glutamate uptake was saturable and Eadie-Hofstee transformation of the saturation data revealed a Km of 10.6+/-0.8 microM, and a maximum transport capacity (Vmax) of 100.3+/-12.3 pmol min(-1) mg(-1) protein. Pharmacological characterisation of the transport activity in NT2-N cells produced a rank order of inhibitory activity which was identical to that determined for the human excitatory amino acid transporter 2 which we have analysed in a stable mammalian cell line (Madin Darby Canine Kidney (MDCK) cells). Of particular note, L-glutamate transport by NT2-N cells was sensitive to both dihydrokainate and kainate. The expression of human excitatory amino acid transporter mRNAs was studied using reverse transcriptase polymerase chain reaction. NT2-N cells expressed transcripts for excitatory amino acid transporters 2 and 3, but not for the subtypes 1, 4 and 5. We conclude that although the mRNA expression studies suggest the presence of transcripts for both excitatory amino acid transporter 2 and 3 in NT2-N cells, the sensitivity to dihydrokainate and kainate determined in the pharmacological analysis indicates that, of the known transporter subtypes, excitatory amino acid transporter 2 contributes to the bulk of the L-glutamate transport activity present in these cells.

Animals↗

PubMed2003

Efflux ratio cannot assess P-glycoprotein-mediated attenuation of absorptive transport: asymmetric effect of P-glycoprotein on absorptive and secretory transport across Caco-2 cell monolayers.

PURPOSE: The purpose of this work was to determine whether P-glycoprotein (P-gp) modulates absorptive and secretory transport equally across polarized epithelium (i.e., Caco-2 cell monolayers) for structurally diverse P-gp substrates, a requirement for the use of the efflux ratio to quantify P-gp-mediated attenuation of absorption across intestinal epithelium. METHODS: Studies were performed in Caco-2 cell monolayers. Apparent permeability (P(app)) in absorptive (P(app,AB)) and secretory (P(app,BA)) directions as well as efflux ratios (P(app,BA)/P(app,AB)) were determined for substrates as a function of concentration. Transport of these compounds (10 microM) was measured under normal conditions and in the presence of the P-gp inhibitor, GW918 (1 microM), to dissect the effect of P-gp on absorptive and secretory transport. Apparent biochemical constants of P-gp-mediated efflux activity were calculated for both transport directions. RESULTS: Efflux ratios for rhodamine 123 and digoxin were comparable (approx. 10). However, transport studies in the presence of GW918 revealed that P-gp attenuated absorptive transport of digoxin by approx. 8-fold but had no effect on absorptive transport of rhodamine 123 (presumably because absorptive transport of rhodamine 123 occurs via paracellular route). The apparent Km for P-gp-mediated efflux of digoxin was > 6-fold larger in absorptive vs. secretory direction. For structurally diverse P-gp substrates (acebutolol, colchicine, digoxin, etoposide, methylprednisolone, prednisolone, quinidine, and talinolol) apparent Km was approximately 3 to 8-fold greater in absorptive vs. secretory transport direction, whereas apparent J(max) was somewhat similar in both transport directions. CONCLUSIONS: P-gp-mediated efflux activity observed during absorptive and secretory transport was asymmetric for all substrates tested. For substrates that crossed polarized epithelium via transcellular pathway in both directions, this difference appears to be caused by greater apparent Km of P-gp-mediated efflux activity in absorptive vs. secretory direction. These results clearly suggest that use of efflux ratios could be misleading in predicting the extent to which P-gp attenuates the absorptive transport of substrates.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Glucose regulates its transport in L8 myocytes by modulating cellular trafficking of the transporter GLUT-1.

The effect of culture conditions simulating hypo- and hyper-glycaemia on glucose transport and on the subcellular localization of the glucose transporter GLUT-1 was studied in L8 myocytes. Incubation of the cells with 20 mM-glucose for 25 h decreased the rate of 2-deoxy-D-[3H]glucose (dGlc) uptake to 0.106 +/- 0.016 nmol/min per 10(6) cells compared with 0.212 +/- 0.025 in cells maintained at 2 mM-glucose (final glucose concentrations at the end of the incubation period were 16-17 mM and 0.7-1.0 mM respectively). An additional 5 h incubation of these cells with medium containing the opposite glucose concentration (i.e. change from 17 mM to 1 mM and from 1 mM to 17 mM) increased the transport rate to 0.172 +/- 0.033 nmol/min per 10(6) cells in cultures initially conditioned at high glucose, and decreased the transport to 0.125 +/- 0.029 in those conditioned at low glucose. Plasma-membrane- and microsomal-membrane-enriched fractions were prepared from these cells for [3H]cytochalasin B (CB) binding and Western-blot analysis with antibodies against GLUT-1 and GLUT-4. A decrease in glucose concentration increased the number of D-glucose-displaceable CB-binding sites and GLUT-1 protein in the plasma-membrane fraction to the same extent as the increase in dGlc transport. Under downregulatory conditions, the lower dGlc-transport capacity could be accounted for by a decreased number of transporters in the plasma membrane of the cells. No apparent modification of the intrinsic activity of the glucose transporters was observed in up- or down-regulated cells. Under downregulatory conditions, the CB-binding data indicated a large increase in the number of transporters in the intracellular membranes of the myocytes. Western blots of the same membranes also indicated an increase in GLUT-1 content. However, the interaction of the intracellular GLUT-1 protein with the polyclonal antibodies was much weaker than that of the plasma-membrane-associated GLUT-1. The GLUT-4 concentration was too low to permit quantification in membrane fractions. Our findings suggest that autoregulation of glucose transport in L8 myocytes is accompanied by parallel changes in the number of GLUT-1 transporters in the plasma membrane, and that the rate of transporter degradation may be augmented in the upregulated myocytes. These glucose-induced changes are fully reversible.

Animals↗

PubMed2001

Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity.

The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.

Amino Acid Sequence↗