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PubMed1995

A role for gamma-glutamyl transpeptidase and the amino acid transport system xc- in cystine transport by a human pancreatic duct cell line.

1. The roles of the gamma-glutamyl cycle and the anionic amino acid transport system xc- in mediating L-cystine uptake were investigated in cultured human pancreatic duct PaTu 8902 cells. This cell line exhibits morphological features of normal pancreatic duct cells and expresses gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2), an enzyme involved in the metabolism and regulation of intracellular glutathione (GSH). 2. Uptake of L-cystine (10 microM) was linear for up to 10 min, temperature dependent, Na+ independent, saturable (Michaelis-Menten constant (Km), 86 +/- 25 microM; maximal velocity (Vmax), 109 +/- 33 nmol (mg protein)-1 h-1) and reduced by 80-90% by a 50-fold excess concentration of L-glutamate and L-homocysteic acid, but not L-aspartate. These transport properties resemble those described for system xc-, which exchanges cystine for intracellular glutamate. 3. Acivicin, a known inhibitor of gamma-GT, decreased gamma-GT activity from 2.58 +/- 0.96 to 0.97 +/- 0.11 mU (mg protein)-1 and decreased the initial rates of L-cystine and L-glutamine uptake by 41-55%. Anthglutin (1-gamma-L-glutamyl-2-(2-carboxyphenylhyl)hydrazine), a structurally different inhibitor of gamma-GT, also caused a concentration-dependent (0.01-1 mM) decrease in gamma-GT activity and L-cystine uptake. 4. Neither acivicin nor anthglutin inhibited the uptake of L-glutamate, a poor substrate for gamma-GT. 5. In the presence of a 500-fold excess concentration of glutamate, which should abolish entry of cystine via system xc-, the remaining fraction of cystine transport was inhibited by 50% by acivicin, suggesting that transport is, in part, dependent on the activity of gamma-GT. 6. Cystine transport was also 60-80% inhibited by a series of gamma-glutamyl amino acids (5 mM) including gamma-glutamyl-glutamate, gamma-glutamyl-glutamine and gamma-glutamyl-glycine. alpha-Dipeptides inhibited cystine transport by only 6-22%. 7. These findings demonstrate that in human pancreatic duct PaTu 8902 cells, cystine uptake is mediated by system xc- (50-60%) and the gamma-glutamyl cycle. Our results provide the first evidence linking gamma-GT with cystine transport in human epithelial cells and are of relevance in view of the importance of cystine as a sulphur amino acid source for GSH synthesis in cells exposed to oxidative stress.

Adenosine Triphosphate↗
PubMed1991

Plasma membrane H(+)-HCO3- transport in rat hepatocytes: a principal role for Na(+)-coupled HCO3- transport.

Na(+)-coupled HCO3- transport has been demonstrated in the basolateral membrane of hepatocytes, but there is uncertainty regarding its stoichiometry or capacity compared with other mechanisms of H(+)-HCO3- transport. After preincubation in medium free of Na+, either in the presence or absence of HCO3(-)-CO2, rat hepatocytes in primary culture were reexposed to Na+ or HCO3(-)-CO2 alone or in combination. Transporter electrogenicity was assessed by measuring membrane potential difference (PD), and the relative capacities of Na(+)-coupled HCO3- transport, Cl(-)-HCO3- exchange, and Na(+)-H+ exchange were assessed by measuring the magnitude and rate of change of intracellular pH (pHi) using BCECF. In the absence of Na+, exposure to HCO3- alone had no consistent effect on PD or pHi. In the absence of HCO3-, reexposure to Na+ depolarized cells by 3 +/- 1 mV and caused an amiloride-inhibitable increase in pHi of 0.031 +/- 0.02 units/min. In the presence of HCO3-, reexposure to Na+ hyperpolarized cells by -14 +/- 5 mV and increased pHi at a rate of 0.133 +/- 0.11 units/min; both the hyperpolarization and alkalinization were inhibited by SITS but unaffected by amiloride. These changes in PD indicate that Na(+)-coupled HCO3- transport is electrogenic, consistent with coupling of more than one HCO3- to each Na+. Furthermore, SITS-inhibitable Na(+)-dependent alkalinization exceeds amiloride-inhibitable Na(+)-dependent alkalinization by an order of magnitude, suggesting that the transport capacity of Na(+)-coupled HCO3- transport exceeds that of Na(+)-H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Uneven distribution of nucleoside transporters and intracellular enzymatic degradation prevent transport of intact [14C] adenosine across the sheep choroid plexus epithelium as a monolayer in primary culture.

BACKGROUND: Efflux transport of adenosine across the choroid plexus (CP) epithelium might contribute to the homeostasis of this neuromodulator in the extracellular fluids of the brain. The aim of this study was to explore adenosine transport across sheep CP epithelial cell monolayers in primary culture. METHODS: To explore transport of adenosine across the CP epithelium, we have developed a method for primary culture of the sheep choroid plexus epithelial cells (CPEC) on plastic permeable supports and analysed [14C] adenosine transport across this cellular layer, [14C] adenosine metabolism inside the cells, and cellular uptake of [14C] adenosine from either of the chambers. The primary cell culture consisted of an enriched epithelial cell fraction from the sheep fourth ventricle CP and was grown on laminin-precoated filter inserts. RESULTS AND CONCLUSION: CPEC grew as monolayers forming typical polygonal islands, reaching optical confluence on the third day after the seeding. Transepithelial electrical resistance increased over the time after seeding up to 85 +/- 9 Omega cm2 at day 8, while permeability towards [14C] sucrose, a marker of paracellular diffusion, simultaneously decreased. These cells expressed some features typical of the CPEC in situ, including three nucleoside transporters at the transcript level that normally mediate adenosine transport across cellular membranes. The estimated permeability of these monolayers towards [14C] adenosine was low and the same order of magnitude as for the markers of paracellular diffusion.However, inhibition of the intracellular enzymes, adenosine kinase and adenosine deaminase, led to a significant increase in transcellular permeability, indicating that intracellular phosphorylation into nucleotides might be a reason for the low transcellular permeability. HPLC analysis with simultaneous detection of radioactivity revealed that [14C] radioactivity which appeared in the acceptor chamber after the incubation of CPEC monolayers with [14C] adenosine in the donor chamber was mostly present as [14C] hypoxanthine, a product of adenosine metabolic degradation. Therefore, it appears that CPEC in primary cultures act as an enzymatic barrier towards adenosine. Cellular uptake studies revealed that concentrative uptake of [14C] adenosine was confined only to the side of these cells facing the upper or apical chamber, indicating uneven distribution of nucleoside transporters.

Journal Article↗
PubMed1994

Transport mechanism of an H1-antagonist at the blood-brain barrier: transport mechanism of mepyramine using the carotid injection technique.

The blood-brain barrier (BBB) permeability of mepyramine was measured by the carotid injection technique to elucidate the transport mechanism of an H1-antagonist in the central nervous system. Mepyramine was found to enter the brain by saturable and carrier-mediated transport. The in vivo kinetic parameters were estimated as follows: the maximum uptake rate (Jmax) was 7.12 +/- 1.37 mumol/min/g of brain, the Michaelis constant (Kt) was 4.40 +/- 2.00 mM, and the nonsaturable first order rate (Kd) was 0.28 +/- 0.02 ml/min/g of brain. The mepyramine transport was not inhibited either by nutrients or by choline, hemicholinium-3, though it was inhibited by the classical H1-antagonists such as diphenhydramine, diphenylpyraline, and also by propranolol. The above inhibitory effects suggest that a transport system different from the amine transport system exists for the BBB transport of mepyramine, and that this transporter is common not only for H1-antagonists but also for basic drugs.

Animals↗
PubMed1990

[Transport of drugs through human erythrocyte membranes. Change of transport by introduction of amino group or amino acids in benzoic acid].

The transporting properties of benzoic acid (BA) and its derivatives such as hippuric acid (HPA), p-aminohippuric acid (AHPA), N-benzoyl-beta-alanine (NBA), p-amino-N-benzoyl-beta-alanine (ANBA), N-benzoyl-6-aminocaproic acid (NBC), p-amino-N-benzoyl-6-amino-caproic acid (ANBC), o-, m- or p-hydroxybenzoic acid (o-, m- or p-HBA) and alpha- or gamma-resorcylic acid (alpha- or gamma-RA) through erythrocyte membranes were examined in two aspects of the inward direction from a drug-containing medium into the erythrocyte and the outward direction from the drug-containing erythrocyte to the drug-free medium. The significant difference in the rate of transport was observed between both directions. The introduction of a few methylene groups into the amino acid moieties of BA derivatives was slower in the rate of transport than that of more methylene groups. The rate of transport was slowed down by the introduction of amino group at p-position: NBC greater than NBA greater than HPA much greater than ANBC greater than ANBA greater than AHPA. The rate of transport in these drugs was correlated with the changes in partition coefficients. The same correlation was also observed in the drugs to which hydroxyl groups were introduced except alpha- or gamma-RA. This transport of alpha- or gamma-RA suggested the participation of the band 3 anion transporter protein.

Aminohippuric Acids↗
PubMed1989

Mechanism of IGF-I-stimulated glucose transport in human adipocytes. Demonstration of specific IGF-I receptors not involved in stimulation of glucose transport.

We demonstrate the presence of specific insulinlike growth factor I (IGF-I) receptors in human adipocytes. Competition studies with 125I-labeled IGF-I and unlabeled IGF-I, IGF-II, and insulin showed the specificity of 125I-IGF-I binding to the IGF-I receptors in adipocytes, membranes, and partially purified detergent-solubilized extracts. The monoclonal antibody to the IGF-I receptor (alpha-IR3) inhibits 125I-IGF-I binding and immunoprecipitates the IGF-I receptor. In addition, the alpha-subunit of IGF-I receptor is approximately 10,000 Mr larger than the alpha-subunit of insulin receptor, and IGF-I stimulates phosphorylation of the beta-subunit of the IGF-I receptor. IGF-I stimulates basal glucose transport in human adipocytes, but the concentrations of IGF-I required for half-maximal and maximal stimulation of glucose transport are 800- and 1000-fold greater than that of insulin. The possibility of IGF-I stimulating glucose transport by interacting predominantly with insulin receptors is suggested by data showing that 1) IGF-I competes with insulin-binding sites, 2) there is a lack of an additive effect with IGF-I and insulin in stimulating glucose transport, 3) alpha-IR3, which specifically inhibits IGF-I binding, does not inhibit IGF-I or insulin-stimulated glucose transport, 4) insulin-receptor antibody MA-10 inhibits IGF-I and insulin-stimulated glucose transport, and 5) IGF-I stimulates insulin-receptor autophosphorylation, although its effect is markedly decreased compared with insulin. In summary, human adipocytes possess specific IGF-I receptors. However, IGF-I stimulates glucose transport predominantly by interacting with the insulin receptor.

Adipose Tissue↗
PubMed1998

A Novel Ventilated Transport Container for Transporting Laboratory Animals.

A novel ventilated transport container was developed to allow safe transport of laboratory animals among biocontainment areas, conventional animal holding areas, or laboratories. This specialized device allowed simultaneous transport and concurrent isolation of 2 unanesthetized nonhuman primates carried in standard transport boxes as the primary enclosure. Its use substantially reduced the risk to the primates by eliminating the need for anesthesia during transport between containment areas and, additionally, improved the margin of safety during transport. The device was easily decontaminated and was also appropriate to transport other small laboratory animals between biocontainment areas.

Journal Article↗
PubMed1998

Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, Spgp, in endotoxemic rat liver.

Endotoxin-induced cholestasis is mainly caused by an impaired canalicular secretion. Mrp2, the canalicular multispecific organic anion transporter, is strongly down-regulated in this situation, and canalicular bile salt secretion is also reduced. We hypothesized that other adenosine triphosphate-binding cassette (ABC) transporters may compensate for the decreased transport activity to protect the cell from cytokine-induced oxidative damage. Therefore, we examined the expression of ABC-transport proteins in membrane fractions of whole liver and of isolated hepatocytes of endotoxin-treated rats and performed reverse-transcriptase polymerase chain reaction (RT-PCR) on mRNA isolated from these livers. In addition, the localization of these transporters was examined using confocal scanning laser microscopy. By 6 hours after endotoxin administration, we found a clear increase of mrp1 mRNA and protein, whereas mrp2 mRNA and protein were decreased. This was confirmed in isolated hepatocytes. In addition, mdr1b mRNA was strongly increased, whereas mdr1a and mdr2 mRNA did not change significantly. Both the mRNA and protein levels of the sister of P-glycoprotein (spgp), the recently cloned bile salt transporter, decreased. After endotoxin treatment, the normally sharply delineated canalicular staining of mrp2 and spgp had changed to a fuzzy pattern, suggesting localization in a subapical compartment. We conclude that endotoxin-induced cholestasis is caused by decreased mrp2 and spgp levels, as well as an abnormal localization of these proteins. The simultaneous up-regulation of mrp1 and mdr1b may confer resistance to hepatocytes against cytokine-induced metabolic stress.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

Regulation of the A system of amino acid transport in Chinese hamster ovary cells, CHO-K1: the difference in specificity between the apo-repressor inactivator (apo-ri) and the transporter and the characterization of the proposed apo-ri.

When amino acids that are generally transported through the A system are added to derepressed cultures of CHO-K1 cells or to cultures that are undergoing starvation-derepression, as in the co-repressor (co-r), co-inactivator (co-i), (co-ri) assay, the A system undergoes trans-inhibition, inactivation, and repression. The effect of inactivation and repression is not related to the ability of amino acids to bind to the A system transporter but supports a model in which these amino acids act as co-r's/co-i's, and by binding to a aporepressor/inactivator (apo-ri), the product of gene R1, convert it into a repressor/inactivator (ri). For example, beta-alanine acts as a strong co-r but does not inhibit proline transport through the A system. Hydroxyproline and histidine, although poor inhibitors of proline transport, are very effective as co-ri's. Diaminobutyrate, phenylalanine, alpha-keto-glutarate, pyro-glutamate, isoleucine, and valine, compounds that inhibit A system transport, listed in decreasing order of effectiveness, are all equally poor as co-ri's. Also the Km for the transport of 2-(methylamino)isobutyric acid (MeAIB) through the A system is two times the concentration of MeAIB required to produce one-half inactivation. Amino acid effectors and mutation can modify the conversion of the apo-ri to repressor (r) and inactivator (i). The apo-ri is converted by alanine, serine, proline, and MeAIB to ri, by beta-alanine and tryptophane to r, and by hydroxyproline to r and reduced i. The full constitutive and partial constitutive mutants alar4 and alar2, respectively, are in the same complementation group. Alar4 has no active apo-ri while the rate of derepression of alar2 is twice and the inactivation rate is equal to that of the parent culture.

Amino Acid Transport Systems, Neutral↗
PubMed1999

ATP-Dependent colchicine transport by human erythrocyte glutathione conjugate transporter.

We have recently demonstrated mutually inhibitory ATP-dependent transport of dinitrophenyl-S-glutathione (DNP-SG) and doxorubicin by DNP-SG ATPase purified from human erythrocyte membranes (S. Awasthi et al., 1998a,b). Our previous studies indicate a broad substrate specificity for this transport mechanism, including some P-glycoprotein substrates. Present studies were carried out to determine whether colchicine (COL), a classical P-glycoprotein substrate, could be transported by purified human erythrocyte DNP-SG ATPase reconstituted in artificial liposomes. We also investigated whether leukotriene C4 (LTC4), an endogenous proinflammatory glutathione-conjugate derived from arachidonic acid, would inhibit colchicine transport. Uptake of COL was compared in proteoliposomes reconstituted with the purified DNP-SG ATPase as well as control liposomes in the presence or absence of ATP. Increased colchicine uptake was observed upon addition of ATP to proteoliposomes, but not control liposomes. Uptake was linear with respect to the amount of vesicle protein used. Sensitivity to osmolarity was consistent with intravesicular COL accumulation. The ATP-dependent colchicine uptake was sensitive to temperature in a manner consistent with a protein-mediated transport process with activation energy of 7.3 kcal/mol. Time-dependent COL uptake by proteoliposomes in the presence of ATP was consistent with a single compartment model with an apparent rate constant of 0.21 +/- 0.02 min-1. Kinetic studies indicated a saturable behavior with respect to ATP (Km 2.3 +/- 0.7 mM) and colchicine (Km 4.3 +/- 0.2 microM). LTC4 was found to be a competitive inhibitor of COL transport (Kis 16.4 microM). Since DNP-SG ATPase is present in many tissues, it may play an important role in determining colchicine accumulation in cells. Increased LTC4 would tend to increase cellular COL accumulation.

Adenosine Triphosphatases↗
PubMed2005

A general channel model accounts for channel, carrier, counter-transport and co-transport kinetics.

In this work we propose a unifying model of mediated membrane transport, based upon the idea that the integral membrane proteins involved in these processes operate via complex channel mechanisms. In the first part, we briefly review literature about the structural aspects of membrane transporters. We conclude that there is a substantial amount of evidence suggesting that most membrane proteins performing transport are embodied with channel-like structures that may constitute the translocation paths. This includes cases where the phenomenological transport kinetics do not correspond to the classical channel behavior. In the second part of this article we introduce the general channel model of mediated transport and employ it to derive specific examples, like simple one- or two-ligand channels, water-ligand channels, simple carriers, co- and counter-transport systems and more complex water-ligand carriers. We show that, for the most part, these particular cases can be obtained by the application of the techniques of diagram reduction to the full model. The necessary conditions for diagram reduction reflect physical properties of the protein and its surroundings.

Biological Transport, Active↗
PubMed2005

The remarkable transport mechanism of P-glycoprotein: a multidrug transporter.

Human P-glycoprotein (ABCB1) is a primary multidrug transporter located in plasma membranes, that utilizes the energy of ATP hydrolysis to pump toxic xenobiotics out of cells. P-glycoprotein employs a most unusual molecular mechanism to perform this drug transport function. Here we review our work to elucidate the molecular mechanism of drug transport by P-glycoprotein. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Development of novel spin-labeled transport substrates has allowed for quantitative and rigorous measurements of drug transport in real time by EPR spectroscopy. We have developed a new drug transport model of P-glycoprotein from the results of mutagenic, quantitative thermodynamic and kinetic studies. This model satisfactorily accounts for most of the unusual kinetic, coupling, and physiological features of P-glycoprotein. Additionally, an atomic detail structural model of P-glycoprotein has been devised to place our results within a proper structural context.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Glycosylation of the human erythrocyte glucose transporter: a minimum structure is required for glucose transport activity.

The involvement of the carbohydrate moiety of the human erythrocyte glucose transporter in glucose transport activity was previously demonstrated (Feugeas et al. (1990) Biochim. Biophys. Acta 1030, 60-64): N-glycanase treatment of the transport glycoprotein reconstituted in proteoliposomes resulted in a dramatic decrease of the Vmax. In this study, kinetic measurements of glucose equilibrium influx confirm our previous results. In order to investigate that a minimum glycosidic structure is required to maintain glucose transport activity, proteoliposomes were respectively treated with either sialidase, or sialidase and endo-beta-galactosidase, or a pool of exo-glycosidases which allows the release of all the sugar residues, except the proximal N-acetylglucosamine. Kinetic measurements of zero-trans influx made on sialidase- and (sialidase + endo-beta-galactosidase)-treated proteoliposomes did not reveal any significant changes in the glucose transport activity. On the contrary, treatment of the same proteoliposomes by a pool of exoglycosidases led to a complete abolition of activity, suggesting that a minimum glycosidic structure is required for glucose transport activity.

Biological Transport↗
PubMed2004

Novel inter- and intrasubunit contacts between transport-relevant residues of the homodimeric mitochondrial phosphate transport protein.

Ser158 is located near the middle of the matrix loop connecting transmembrane helices C and D of the mitochondrial phosphate transport protein (PTP). The mutant Ser158Thr PTP is transport-inactive. His32 is located near the middle of transmembrane helix A and Thr79 is located 5 residues away from transmembrane helix B and its N-terminal (matrix end). Single site mutant PTPs that have either residue replaced with Ala are transport-inactive. Based on the high resolution structure of a subunit of the bovine ADP/ATP translocase, on sequence similarities between members of the mitochondrial transport protein family, and on the PTP subunit/subunit contact site between transmembrane A helices, it is now suggested that the Ser158 site is at the PTP subunit/subunit contact site. This contact site is essential for keeping the transport cycles catalyzed by the two PTP subunits 180 degrees out of phase. The data also suggest that His32 and Thr79 of the same subunit interact and couple the phosphate and the proton transport paths.

Amino Acid Sequence↗
PubMed2006

Organic anion transporting polypeptide 2B1 is a high-affinity transporter for atorvastatin and is expressed in the human heart.

BACKGROUND: The cardiac effects of statins are subject to controversial discussion, and the mechanism of their uptake into the human heart is unknown. A candidate protein is the organic anion transporting polypeptide (OATP) 2B1 (SLCO2B1), because related transporters are involved in the uptake of statins into the human liver. In this study we examine OATP2B1 expression in the human heart and describe statins as inhibitors and substrates of OATP2B1. METHODS: The expression of OATP2B1 was analyzed in 46 human atrial and 15 ventricular samples, including samples from hearts with dilated cardiomyopathy and hearts with ischemic cardiomyopathy. RESULTS: Significant messenger ribonucleic acid expression was found in all samples, with no difference in the diseased hearts. However, patients who had taken atorvastatin exhibit decreased OATP2B1 messenger ribonucleic acid expression compared with patients with no statin treatment. OATP2B1 protein was detected at approximately 85 kd in atrial samples, as well as ventricular samples, and could be localized to the vascular endothelium. Furthermore, estrone-3-sulfate transport into OATP2B1-overexpressing Madin-Darby canine kidney II cells was inhibited by various drugs, including atorvastatin, simvastatin, cerivastatin, glyburide (INN, glibenclamide), and gemfibrozil, with the most pronounced effect being found for atorvastatin (inhibition constant, 0.7 +/- 0.4 micromol/L). Whereas simvastatin (lactone) itself was not transported by OATP2B1, atorvastatin was identified as a high-affinity substrate for OATP2B1 (Michaelis-Menten constant, 0.2 micromol/L) by direct transport measurement via liquid chromatography-tandem mass spectrometry. CONCLUSION: OATP2B1 is a high-affinity uptake transporter for atorvastatin and is expressed in the vascular endothelium of the human heart, suggesting its involvement in cardiac uptake of atorvastatin.

Aged↗
PubMed2005

SLC5A8 (SMCT1)-mediated transport of butyrate forms the basis for the tumor suppressive function of the transporter.

The identification of SLC5A8 as a tumor suppressor gene in colorectal cancer marks, for the first time, the association of a plasma membrane transporter with tumor suppressive properties. The subsequent establishment of the functional identity of SLC5A8 as a Na+-coupled transporter for short-chain monocarboxylates provides a mechanism for the tumor suppressive function of the transporter. Butyrate, a substrate for the transporter, is a histone deacetylase inhibitor and protective against colorectal cancer. This fatty acid is produced in the colonic lumen by bacterial fermentation of dietary fiber. SLC5A8 mediates the concentrative entry of butyrate from the lumen into colonocytes. Consequently, the transport function of SLC5A8 has the ability to influence the acetylation status of histones and hence gene expression in colonocytes. The ability of SLC5A8 to deliver butyrate into colonic epithelial cells most likely underlies the tumor suppressive role of this transporter.

Animals↗
PubMed2006

Role of dopamine transporter (DAT) in dopamine transport across the nasal mucosa.

Dopamine is a catecholamine neurotransmitter necessary for motor functions. Its deficiency has been observed in several neurological disorders, but replacement of endogenous dopamine via oral or parenteral delivery is limited by poor absorption, rapid metabolism and the inability of dopamine to cross the blood-brain barrier. The intranasal administration of dopamine, however, has resulted in improved central nervous system (CNS) bioavailability compared to that obtained following intravenous delivery. Portions of the nasal mucosa are innervated by olfactory neurons expressing dopamine transporter (DAT) which is responsible for the uptake of dopamine within the central nervous system. The objective of these studies was to study the role of DAT in dopamine transport across the bovine olfactory and nasal respiratory mucosa. Western blotting studies demonstrated the expression of DAT and immunohistochemistry revealed its epithelial and submucosal localization within the nasal mucosa. Bidirectional transport studies over a 0.1-1 mM dopamine concentration range were carried out in the mucosal-submucosal and submucosal-mucosal directions to quantify DAT activity, and additional transport studies investigating the ability of GBR 12909, a DAT inhibitor, to decrease dopamine transport were conducted. Dopamine transport in the mucosal-submucosal direction was saturable and was decreased in the presence of GBR 12909. These studies demonstrate the activity of DAT in the nasal mucosa and provide evidence that DAT-mediated dopamine uptake plays a role in the absorption and distribution of dopamine following intranasal administration.

Animals↗
PubMed2006

α2 isoform-specific activation of 5'adenosine monophosphate-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside at a physiological level activates glucose transport and increases glucose transporter 4 in mouse skeletal muscle.

5'Adenosine monophosphate-activated protein kinase (AMPK) has been implicated in exercise-induced stimulation of glucose metabolism in skeletal muscle. Although skeletal muscle expresses both the alpha1 and alpha2 isoforms of AMPK, the alpha2 isoform is activated predominantly in response to moderate-intensity endurance exercise in human and animal muscles. The purpose of this study was to determine whether activation of alpha2 AMPK plays a role in increasing the rate of glucose transport, promoting glucose transporter 4 (GLUT4) expression, and enhancing insulin sensitivity in skeletal muscle. To selectively activate the alpha2 isoform, we used 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), which is metabolized in muscle cells and preferentially stimulates the alpha2 isoform. Subcutaneous administration of 250 mg/kg AICAR activated the alpha2 isoform for 90 minutes, but not the alpha1 isoform in hind limb muscles of the C57/B6J mouse. The maximal activation of the alpha2 isoform was observed 30 to 60 minutes after administration of AICAR and was similar to the activation induced by a 30-minute swim in a current pool. The increase in alpha2 activity paralleled the phosphorylation of Thr(172), the essential residue for full kinase activation, and the activity of acetyl-coenzyme A carboxylase beta, a known substrate of AMPK in skeletal muscle. Subcutaneous injection of AICAR rapidly increased, by 30%, the rate of 2-deoxyglucose (2DG) transport into soleus muscle; 2DG transport increased within 30 minutes and remained elevated for 4 hours after administration of AICAR. Repeated intraperitoneal injection of AICAR, 3 times a day for 4 to 7 days, increased soleus GLUT4 protein by 30% concomitant with a significant 20% increase in insulin-stimulated 2DG transport. These data suggest that moderate endurance exercise promotes glucose transport, GLUT4 expression, and insulin sensitivity in skeletal muscle at least partially via activation of the alpha2 isoform of AMPK.

AMP-Activated Protein Kinases↗