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PubMed1997

The hemopoietic growth factor, interleukin-3, promotes glucose transport by increasing the specific activity and maintaining the affinity for glucose of plasma membrane glucose transporters.

Most mammalian cells rely on an external supply of glucose for survival, proliferation, and function. Glucose enters cells through specific transporter molecules at the plasma membrane by a facilitative process that does not expend energy. Regulation of glucose transport into cells is thought to occur largely through transporter expression at the cell surface, but the extent to which the intrinsic properties of glucose transporters are regulated is at present controversial. Using a bone marrow-derived cell line that responds to the hemopoietic growth factor, interleukin-3 (IL-3), we investigated IL-3 regulation of glucose transport. IL-3 significantly increased 2-deoxyglucose (2-DOG) uptake within 1 h (26 +/- 8.0%, n = 11) with a maximum 73% increase after 6 h. Withdrawal of IL-3 resulted in decreased uptake within 1 h and this continued to decline to 43% of initial uptake by 16 h. To determine whether these changes in 2-DOG uptake were associated with corresponding changes in glucose transporter expression, subtype-specific antisera against Glut-1 and Glut-3 were used. Little change in membrane expression of these transporters was observed prior to 16 h. Fractionation of cell membranes on Nycodenz gradients showed that the majority of each transporter subtype was associated with the plasma membrane (63-93%) and that transporter distribution did not change markedly in response to addition or withdrawal of IL-3. These results demonstrate that IL-3 regulates glucose uptake by modulating the intrinsic transporting ability of glucose transporters. Decreased transporter affinity for 2-DOG and 3-O-methylglucose was observed following IL-3 withdrawal. Similar affinity changes were observed with 2-DOG following exposure of IL-3-stimulated cells to the protein kinase inhibitors, genistein and staurosporine. In contrast, the tyrosine phosphatase inhibitor, vanadate, acted like IL-3 to increase transporter affinity for glucose. Together these results demonstrate that IL-3 acts to maintain the intrinsic transport properties of glucose transporters without markedly affecting their expression or translocation.

3-O-Methylglucose↗

PubMed1997

Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid.

Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1-5 and SGLT1. The apparent Km of DHA transport via GLUT1 and GLUT3 was 1.1 +/- 0.2 and 1.7 +/- 0.3 mM, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2-4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, D-glucose, and 3-O-methylglucose among other hexoses while fructose and L-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-alpha-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2-8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA.

Animals↗

PubMed1989

Effects of three proposed inhibitors of adipocyte glucose transport on the reconstituted transporter.

Three compounds which inhibit glucose transport in rat adipocytes have been proposed to act directly on the glucose transporter protein. We tested these proposals by examining the effects of the compounds on the stereospecific glucose uptake catalyzed by adipocyte membrane proteins after reconstitution into liposomes. Effects on the transport activity reconstituted from human erythrocyte membranes were also examined. Glucose 6-phosphate, which was suggested to inhibit the transporter noncompetitively (Foley, J.E. and Huecksteadt, T.P. (1984) Biochim. Biophys. Acta 805, 313-316), had no effect on either type of reconstituted transporter, even when present at 5 mM on both sides of the liposomal membranes. Thus, it is unlikely to act directly on the transporter. The metalloendoproteinase substrate dipeptide Cbz-Gly-Phe-NH2, which inhibited insulin-stimulated but not basal glucose uptake in adipocytes (Aiello, L.P., Wessling-Resnick, M. and Pilch, P.F. (1986) Biochemistry 25, 3944-3950), inhibited the reconstituted erythrocyte transporter noncompetitively with a Ki of 1.5-2 mM. The inhibition of the erythrocyte transporter was identical in liposomes of soybean and egg lipid. Transport reconstituted using adipocyte membrane fractions was also inhibited by the dipeptide, with the activity from basal microsomes more sensitive than that from insulin-stimulated plasma membranes. These results indicate that the dipeptide interacts directly with the transporter, and may be a potentially useful probe for changes in transporter structure accompanying insulin action. Phenylarsine oxide, which was suggested to act directly on the adipocyte transporter (Douen, A.G., and Jones, M.N. (1988) Biochim. Biophys. Acta 968, 109-118), produced only slight (about 10%) inhibition of the reconstituted adipocyte and erythrocyte transporters, even when present at 100-200 microM and after 30 min of pretreatment. These results suggest that the major actions of phenylarsine oxide observed in adipocytes are not direct effects on the transporter, but rather effects on the pathways by which insulin regulates glucose transport activity (Frost, S.C. and Lane, M.D. (1985) J. Biol. Chem. 260, 2646-2652).

Adipose Tissue↗

PubMed1992

Trafficking of glucose transporters in 3T3-L1 cells. Inhibition of trafficking by phenylarsine oxide implicates a slow dissociation of transporters from trafficking proteins.

We have compared the rates of insulin stimulation of cell-surface availability of glucose-transporter isoforms (GLUT1 and GLUT4) and the stimulation of 2-deoxy-D-glucose transport in 3T3-L1 cells. The levels of cell-surface transporters have been assessed by using the bismannose compound 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy) propyl-2-amine (ATB-BMPA). At 27 degrees C the half-times for the appearance of GLUT1 and GLUT4 at the cell surface were 5.7 and 5.4 min respectively and were slightly shorter than that for the observed stimulation of transport activity (t 1/2 8.6 min). This lag may be due to a slow dissociation of surface transporters from trafficking proteins responsible for translocation. When fully-insulin-stimulated cells were subjected to a low-pH washing procedure to remove insulin at 37 degrees C, the cell-surface levels of GLUT1 and GLUT4 decreased, with half-times of 9.2 and 6.8 min respectively. These times correlated well with decrease in 2-deoxy-D-glucose transport activity that occurred during this washing procedure (t1/2 6.5 min). When fully-insulin-stimulated cells were treated with phenylarsine oxide (PAO), a similar decrease in transport activity occurred (t1/2 9.8 min). However, surface labelling showed that this corresponded with a decrease in GLUT4 only (t1/2 7.8 min). The cell-surface level of GLUT1 remained high throughout the PAO treatment. Light-microsome membranes were isolated from cells which had been cell-surface-labelled with ATB-BMPA. Internalization of both transporter isoforms to this pool occurred when cells were maintained in the presence of insulin for 60 min. In contrast with the surface-labelling results, we have shown that the transfer to the light-microsome pool of both transporters occurred in cells treated with insulin and PAO. These results suggest that both transporters are recycled by fluid-phase endocytosis and exocytosis. PAO may inhibit this recycling at a stage which involves the re-emergence of internalized transporters at the plasma membrane. The GLUT1 transporters that are recycled to the surface in insulin- and PAO-treated cells appear to have low transport activity. This may be because of a failure to dissociate fully from trafficking proteins at the cell surface. GLUT4 transporters appear to have a greater tendency to remain internalized if the normal mechanisms that commit transporters to the cell surface, such as dissociation from trafficking proteins, are uncoupled.

3T3 Cells↗

PubMed2001

Na+- and Cl--coupled active transport of carnitine by the amino acid transporter ATB(0,+) from mouse colon expressed in HRPE cells and Xenopus oocytes.

1. ATB(0,+) is an amino acid transporter energized by transmembrane gradients of Na+ and Cl(-) and membrane potential. We cloned this transporter from mouse colon and expressed the clone functionally in mammalian (human retinal pigment epithelial, HRPE) cells and Xenopus laevis oocytes to investigate the interaction of carnitine and its acyl esters with the transporter. 2. When expressed in mammalian cells, the cloned ATB(0,+) was able to transport carnitine, propionylcarnitine and acetylcarnitine. The transport process was Na(+) and Cl(-) dependent and inhibitable by the amino acid substrates of the transporter. The Michaelis constant for carnitine was 0.83 +/- 0.08 mM and the Hill coefficient for Na(+) activation was 1.6 +/- 0.1. 3. When expressed in Xenopus laevis oocytes, the cloned ATB(0,+) was able to induce inward currents in the presence of carnitine and propionylcarnitine under voltage-clamped conditions. There was no detectable current in the presence of acetylcarnitine. Carnitine-induced currents were obligatorily dependent on the presence of Na(+) and Cl(-). The currents were saturable with carnitine and the Michaelis constant was 1.8 +/- 0.4 mM. The analysis of Na(+)- and Cl(-)-activation kinetics revealed that 2 Na(+) and 1 Cl(-) were involved in the transport of carnitine via the transporter. 4. These studies describe the identification of a novel function for the amino acid transporter ATB(0,+). Since this transporter is expressed in the intestinal tract, lung and mammary gland, it is likely to play a significant role in the handling of carnitine in these tissues. 5. A Na(+)-dependent transport system for carnitine has already been described. This transporter, known as OCTN2 (novel organic cation transporter 2), is expressed in most tissues and transports carnitine with high affinity. It is energized, however, only by a Na(+) gradient and membrane potential. In contrast, ATB(0,+) is a low-affinity transporter for carnitine, but exhibits much higher concentrative capacity than OCTN2 because of its energization by transmembrane gradients of Na(+) and Cl(-) as well as by membrane potential.

Amino Acid Sequence↗

PubMed2004

The hFbpABC transporter from Haemophilus influenzae functions as a binding-protein-dependent ABC transporter with high specificity and affinity for ferric iron.

Pathogenic Haemophilus influenzae, Neisseria spp. (Neisseria gonorrhoeae and N. meningitidis), Serratia marcescens, and other gram-negative bacteria utilize a periplasm-to-cytosol FbpABC iron transporter. In this study, we investigated the H. influenzae FbpABC transporter in a siderophore-deficient Escherichia coli background to assess biochemical aspects of FbpABC transporter function. Using a radiolabeled Fe3+ transport assay, we established an apparent Km=0.9 microM and Vmax=1.8 pmol/10(7)cells/min for FbpABC-mediated transport. Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide m-chlorophenyl hydrazone had no effect. Metal competition experiments demonstrated that the transporter has high specificity for Fe3+ and selectivity for trivalent metals, including Ga3+ and Al3+, over divalent metals. Metal sensitivity experiments showed that several divalent metals, including copper, nickel, and zinc, exhibited general toxicity towards E. coli. Significantly, gallium-induced toxicity was specific only to E. coli expressing FbpABC. A single-amino-acid mutation in the gene encoding the periplasmic binding protein, FbpA(Y196I), resulted in a greatly diminished iron binding affinity Kd=5.2 x 10(-4) M(-1), approximately 14 orders of magnitude weaker than that of the wild-type protein. Surprisingly, the mutant transporter [FbpA(Y196I)BC] exhibited substantial transport activity, approximately 35% of wild-type transport, with Km=1.2 microM and Vmax=0.5 pmol/10(7)cells/min. We conclude that the FbpABC complexes possess basic characteristics representative of the family of bacterial binding protein-dependent ABC transporters. However, the specificity and high-affinity binding characteristics suggest that the FbpABC transporters function as specialized transporters satisfying the strict chemical requirements of ferric iron (Fe3+) binding and membrane transport.

ATP-Binding Cassette Transporters↗

PubMed2001

Differential kinetics of transport of 2',3'-dideoxyinosine and adenosine via concentrative Na+ nucleoside transporter CNT2 cloned from rat blood-brain barrier.

The concentrative Na+ nucleoside transporter type 2 (CNT2), cloned from a rat blood-brain barrier cDNA library, yields very high flux ratios for purine nucleosides after expression in frog oocytes. This high activity of the rat CNT2 produced from the blood-brain barrier-derived cDNA, designated clone A-11, enabled a kinetic analysis of 2',3'-dideoxyinosine transport via the rat CNT2. CNT2 transported both adenosine and 2',3'-dideoxyinosine. The 2',3'-dideoxyinosine transport parameters included a Km of 29.2 +/- 8.3 microM, a V(max) of 0.40 +/- 0.11 pmol/oocyte/min, and a constant of nonsaturable transport (KD) of 15.7 +/- 0.6 nl/oocyte/min. The 2',3'-dideoxyinosine Vmax was 27-fold lower than the adenosine Vmax and the 2',3'-dideoxyinosine KD was >15-fold greater than the KD of adenosine transport. Adenosine inhibited both the saturable component of 2',3'-dideoxyinosine transport with a K(I) of 14.8 +/- 1.6 microM, and inhibited the nonsaturable component of 2',3'-dideoxyinosine transport. Both the saturable and nonsaturable components of 2',3'-dideoxyinosine transport were sodium-dependent with a sodium K0.5 of 8.7 +/- 0.9 mM, and a Hill coefficient of 1.00 +/- 0.10. The transport of 2',3'-dideoxyinosine was strongly inhibited by thymidine, whereas thymidine was a weak inhibitor of adenosine transport via rat CNT2. Thymidine was transported by rat CNT2 with a Km = 130 +/- 44 microM and a Vmax = 1.7 +/- 0.5 pmol/oocyte/min. These studies provide evidence for asymmetric transport sites on rat CNT2, where 2',3'-dideoxyinosine and thymidine compete selectively at a low Vmax site on the transporter, whereas adenosine is transported at a high Vmax site.

Adenosine↗

PubMed1998

Serine-O-sulphate transport by the human glutamate transporter, EAAT2.

1. Expression of the recombinant human excitatory amino aid transporters, EAAT1 and EAAT2, in Xenopus laevis oocytes allows electrogenic transport to be studied under voltage clamp conditions. 2. We have investigated the transport of the pharmacological substrate, L-serine-O-sulphate transport by EAAT1 and EAAT2. The EC50 values for L-serine-O-sulphate transport by EAAT2 showed a steep voltage-dependence, increasing from 152+/-11 microM at - 100 mV to 1930+/-160 microM at 0 mV. In contrast to EAAT2, EC50 values for L-serine-O-sulphate transport by EAAT1 were relatively constant over the membrane potential range of - 100 mV to 0 mV. The EC50 values for L-glutamate and D-aspartate transport, by EAAT2, were also relatively constant over this membrane potential range. 3. Chloride ions modulated the voltage-dependent changes in EC50 values for transport by EAAT2. This effect was most apparent for L-serine-O-sulphate transport, and to a lesser extent for L-glutamate and not at all for D-aspartate transport by EAAT2. 4. Extracellular sodium and proton concentrations also modulated the voltage-dependence of L-serine-O-sulphate EC50 values for EAAT2. 5. We speculate that these different properties of L-serine-O-sulphate transport by EAAT2 compared to other substrates may be due to the much stronger acidity of the sulphate group of L-serine-O-sulphate compared to carboxyl groups of L-glutamate or D-aspartate. 6. These results highlight some of the differences in the way different glutamate transporter subtypes transport substrates. This may be used to understand further the transport process and develop subtype selective inhibitors of glutamate transport.

Animals↗

PubMed1997

A comparison of the association of helicopter and ground ambulance transport with the outcome of injury in trauma patients transported from the scene.

INTRODUCTION: Comprehensive emergency medical services and helicopter aeromedical transport systems have been developed based on the principle that early definitive care improves outcome. The purpose of this study was to compare outcomes between patients transported by helicopter and those transported by ground. METHODS: Data were obtained from the North Carolina Trauma Registry for the period between 1987 and 1993 on all patients transported by helicopter and ground admitted to one of the eight state designated trauma centers. Study patients included only those who were transported directly from the scene of injury to the trauma center (interhospital transfers were excluded). Mortality (outcome) was compared after patient stratification by injury severity and transport time, using Cochran-Mantel-Haenszel statistics and logistic regression-derived probabilities of survival. RESULTS: One thousand three hundred forty-six patients (7.3% of the total) were transported from scene to trauma center by helicopter and 17,144 were transported by ground. In patients transported by helicopter, the mean Trauma Score was lower (12 +/- 3.6) versus 14.3 +/- 3.6 (p < 0.001) and the mean Injury Severity Score was higher (17 +/- 11.1) versus 10.8 +/- 8.4 (p < 0.001). A trend toward increased survival was observed among patients transported by helicopter with a higher Injury Severity Score. Statistical significance was achieved only for patients with a Trauma Score between 5 and 12 and Injury Severity Score between 21 and 30. CONCLUSION: The large majority of trauma patients transported by both helicopter and ground ambulance have low injury severity measures. Outcomes were not uniformly better among patients transported by helicopter. Only a very small subset of patients transported by helicopter appear to have any chance of improved survival based on their helicopter transport. This study suggests that further effort should be expended to try to better identify patients who may benefit from this expensive and risky mode of transport.

Air Ambulances↗

PubMed2000

Vesicular transport as a new paradigm in short-term regulation of transepithelial transport.

The vectorial transepithelial transport of water and electrolytes in the renal epithelium is achieved by the polarized distribution of various transport proteins in the apical and basolateral membrane. The short-term regulation of transepithelial transport has been traditionally thought to be mediated by kinetic alterations of transporter without changing the number of transporters. However, a growing body of recent evidence supports the possibility that the stimulus-dependent recycling of transporter-carrying vesicles can alter the abundance of transporters in the plasma membrane in parallel changes in transepithelial transport functions. The abundance of transporters in the plasma membrane is determined by net balance between stimulus-dependent exocytic insertion of transporters into and endocytic retrieval of them from the plasma membrane. The vesicular recycling occurs along the tracts of the actin microfilaments and microtubules with associated motors. This review is to highlight the importance of vesicular transport in the short-term regulatory process of transepithelial transport in the renal epithelium. In the short-term regulation of many other renal transporters, vesicular transport is likely to be also involved. Thus, vesicular transport is now emerged as a wide-spread general regulatory mechanism involved in short-term regulation of renal functions.

Animals↗

PubMed1990

Dendritic transport: quantitative analysis of the time course of somatodendritic transport of recently synthesized RNA.

We have previously reported that recently synthesized RNA is selectively transported into the dendrites of hippocampal neurons grown in culture (Davis et al., 1987). The present study provides further details about this transport process, focusing especially on the velocity of transport, by comparing the velocity of dendritic transport of RNA in neurons of different ages and in the branched and unbranched dendrites of individual neurons. In our previous study, we recognized that calculations of transport velocity could be compromised because transport was being evaluated in a population of dendrites of varying lengths. The present study uses a mathematical modeling approach to determine how the morphology of the population of dendrites would affect the analysis of transport velocity. Focusing first on a simple model, we compared the distribution of transported material at various times when all dendrites were of the same length and when the population included dendrites of different lengths. We found that the distance of labeling increased linearly over time when all dendrites were of the same length, but increased with a negatively accelerating curve when dendrites were of different lengths. We then determined the actual distribution of dendritic lengths in cultured hippocampal neurons, based on immunostaining with an antibody directed against the selective dendritic marker, microtubule-associated protein 2 (MAP2). Using a computer model, we calculated the mean distance of transport as a function of time in this population of dendrites, assuming different velocities of transport. The velocity that best fit the measured distances of RNA transport in both 7- and 15-d-old neurons was 11 microns/hr (0.26 mm/d). However, for the dendrites exhibiting the longest distance of labeling, the best-fitting curve assumed a velocity of 21 microns/hr in both 7- and 15-d-old neurons (0.50 mm/d). Comparisons of transport in branched and unbranched dendrites revealed that the distance of labeling over branched dendrites was consistently longer than over unbranched dendrites of individual neurons. However, neurons with a larger proportion of branched dendrites did not exhibit a greater mean distance of transport. The density of silver grains was higher over branched than over unbranched dendrites, suggesting that a greater amount of recently synthesized RNA may be transported into branched dendrites. Taken together, these results suggest that RNA transport into dendrites is regulated differentially in the dendrites of individual neurons.

Animals↗

PubMed1985

Evidence that anion transport by band 3 proceeds via a ping-pong mechanism involving a single transport site. A 35 Cl NMR study.

Band 3 catalyzes the one-for-one exchange of monovalent anions across the red cell membrane. At least two anion binding sites have been postulated to exist on the transport unit: 1) a transport site that has been observed by saturation kinetics and by 35 Cl NMR studies of chloride binding, and 2) a 35Cl NMR-invisible inhibitory site that has been proposed to explain the inhibition of anion exchange at large anion concentrations. A number of independent studies have indicated that the transport site is alternately exposed to different sides of the membrane during the transport cycle. Yet the role, if any, of the postulated inhibitory site in the transport cycle is not known. Here it is shown that: 1) when the [Cl-], [Br-], or pH is varied, the band 3 transport sites on both sides of the membrane behave like a homogeneous population of simple anion binding sites in 35Cl NMR experiments, and 2) when the [Cl-] is varied, the outward-facing transport site behaves like a simple anion binding site. These results indicate that the postulated inhibitory site has no effect on chloride binding to the transport site. Instead, the results are quantitatively consistent with the ping-pong model (Gunn, R. B., and Frölich, O. (1979) J. Gen. Physiol. 74, 351-374), which states that the transport site is the only site involved in the transport cycle. Expressions are derived for the macroscopically observed characteristics of a ping-pong transporter: these characteristics are shown to be weighted averages of the microscopic properties of the inward- and outward-facing conformations of the transport site. In addition to supporting the simplicity of the transport mechanism, the high pH titration curve for chloride binding to the transport site provides insight into the structure of the site. The macroscopically observed pKA = 11.1 +/- 0.1 in the leaky ghost system indicates that an arginine must provide the essential positive charge in the inward- or outward-facing conformation of the transport site, or in both conformations.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1997

Interaction of dipyridamole, a nucleoside transport inhibitor, with the renal transport of organic cations by LLCPK1 cells.

Dipyridamole is a well-known inhibitor of nucleoside transport by various cell membranes and is frequently used in in vitro studies that characterize nucleoside transport properties. Because interactions between the renal transport of organic cations and nucleosides have previously been suggested, we studied the effect of dipyridamole on the renal transport of the typical organic cations cimetidine and N1-methylnicotinamide by LLCPK1 monolayer cells grown on a permeable support. [14C]Mannitol was used to correct for extracellular flux. Basolateral to apical transcellular flux (transepithelial flux-extracellular flux) of [3H]cimetidine was significantly reduced by the monolayer cells (90%) in the presence of 50 microM dipyridamole. In addition, the effect of dipyridamole on cimetidine renal transport was dose dependent (IC50 = 7.7 microM). The dipyridamole inhibitory effect was nearly comparable with the effect of 1 mM quinine (a typical organic cation transport inhibitor), which led to 95% inhibition of cimetidine renal transport over time. The dipyridamole effect on N1-methylnicotinamide renal transport was less potent. The effect of 1 mM of typical probes of the nucleoside transporters (i.e., thymidine, adenosine, uridine) and the effect of 100 nM of another nucleoside transport inhibitor, dilazep, were also studied on cimetidine transport by LLCPK1 monolayer cells. These compounds did not exert any significant effect. These results suggest that dipyridamole, a widely used nucleoside transport inhibitor, is also an inhibitor of organic cation renal transport and they alert us to possible interactions between the renal transport of nucleosides and organic cations. This finding also has relevance to the interpretation of in vitro studies using this agent as a nucleoside membrane transport inhibitor.

Animals↗

PubMed1996

Catecholamine transporters and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity: studies comparing the cloned human noradrenaline and human dopamine transporter.

The uptake and cytotoxicity of 1-methyl-4-phenylpyridinium (MPP+), the toxic metabolite of the parkinsonism inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), were studied in COS-7 cells transiently transfected with the cloned human noradrenaline and dopamine transporters and in permanently transfected SK-N-MC neuroblastoma cells. MPP+ had a 10- to 20-fold lower K(m) value for the noradrenaline than for the dopamine transporter. In dopamine transporter expressing cells, the maximal transport rate (Vmax) of MPP+, dopamine and noradrenaline was the same, but in noradrenaline transporter expressing cells the Vmax of MPP+ and dopamine was only one-half of the Vmax of noradrenaline. The turnover numbers (Vmax of uptake/maximal binding sites of binding) were 5 times higher for the dopamine transporter (as measured with [3H]dopamine and [3H]-2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane than for the noradrenaline transporter (as measured with [3H]noradrenaline and [3H]nisoxetine). In SK-N-MC cells with similar Vmax values for both catecholamines, noradrenaline transporter expressing cells were killed by lower concentrations of MPP+ in the medium than dopamine transporter expressing cells. Desipramine blocked the toxicity of MPP+ toward the noradrenaline transporter, but not the dopamine transporter expressing cells. We conclude that the toxic effect of MPTP at the striatal dopamine system in the MPTP primate model of Parkinson's disease is not correlated with the affinity profile of MPP+ for catecholamine transporters, but rather with the higher turnover number of MPP+ at the dopamine transporter. In contradistinction, the toxicity of MPTP at the noradrenaline neurons in the primate cerebral cortex (Pifl et al., 1991) may involve the higher affinity of MPP+ for the noradrenaline transporter.

1-Methyl-4-phenylpyridinium↗

PubMed1996

Functional expression of the plasma membrane serotonin transporter but not the vesicular monoamine transporter in human placental trophoblasts and choriocarcinoma cells.

We investigated the functional expression of the plasma membrane serotonin transporter and the vesicular monoamine transporter in choriocarcinoma cells and normal trophoblasts. The RBL 2H3 cells, a rat basophilic leukaemia cell line, which express both transporters were used for comparison. The choriocarcinoma cells JAr and BeWo were found to possess the plasma membrane serotonin transporter as assessed by the presence of serotonin transport activity in intact cells that was Na(+)-dependent and was sensitive to inhibition by tricyclic and non-tricyclic antidepressants. The activity of the vesicular monoamine transporter in these cells was determined by measuring serotonin transport in digitonin-permeabilized cells. The transport in permeabilized cells was very slow, was not stimulated by ATP and was insensitive to inhibition by reserpine. Under similar conditions, the vesicular monoamine transporter activity was demonstrable in RBL cells, which was stimulated by ATP and was inhibitable by reserpine, bafilomycin A1 (an inhibitor of the V-type H(+)-pump) and carbonyl cyanide p-trifluoromethoxy phenylhydrazone (a protonophore which dissipates transmembrane H+ gradients). In corroboration with these findings, mRNA transcripts hybridizable to the vesicular monoamine transporter cDNA probe were detectable in RBL cells but not in JAr choriocarcinoma cells. Similarly, there was no evidence for the expression of the vesicular monoamine transporter as assessed by Northern blot analysis in normal trophoblasts which were maintained in culture to differentiate to form multinucleated syncytial cells. It is concluded that the trophoblasts and choriocarcinoma cells express the plasma membrane serotonin transporter but not the vesicular monoamine transporter.

Adenosine Triphosphate↗

PubMed1990

Glycolipid and glycoprotein transport through the Golgi complex are similar biochemically and kinetically. Reconstitution of glycolipid transport in a cell free system.

Glycolipid transport between compartments of the Golgi apparatus has been reconstituted in a cell free system. Transport of lactosylceramide (galactose beta 1-4-glucose-ceramide) was followed from a donor to an acceptor Golgi population. The major glycolipid in CHO cells is GM3 (sialic acid alpha 2-3 galactose beta 1-4-glucose-ceramide). Donor membranes were derived from a Chinese hamster ovary (CHO) cell mutant (Lec2) deficient in the Golgi CMP-sialic acid transporter, and therefore contained lactosylceramide as the predominant glycolipid. Acceptor Golgi apparatus was prepared from another mutant, Lec8, which is defective in UDP-Gal transport. Thus, glucosylceramide is the major glycolipid in Lec8 cells. Transport was measured by the incorporation of labeled sialic acid into lactosylceramide (present originally in the donor) by transport to acceptor membranes, forming GM3. This incorporation was dependent on ATP, cytosolic components, intact membranes, and elevated temperature. Donor membranes were prepared from Lec2 cells infected with vesicular stomatitus virus (VSV). These membranes therefore contain the VSV membrane glycoprotein, G protein. Donor membranes derived from VSV-infected cells could then be used to monitor both glycolipid and glycoprotein transport. Transport of these two types of molecules between Golgi compartments was compared biochemically and kinetically. Glycolipid transport required the N-ethylmaleimide sensitive factor previously shown to act in glycoprotein transport (Glick, B. S., and J. E. Rothman. 1987. Nature [Lond.]. 326:309-312; Rothman, J. E. 1987. J. Biol. Chem. 262:12502-12510). GTP gamma S inhibited glycolipid and glycoprotein transport similarly. The kinetics of transport of glycolipid and glycoprotein were also compared. The kinetics of transport to the end of the pathway were similar, as were the kinetics of movement into a defined transport intermediate. It is concluded that glycolipid and glycoprotein transport through the Golgi occur by similar if not identical mechanisms.

Animals↗

PubMed1998

Effect of ethanol on intracellular vesicular transport from Golgi to the apical cell membrane: role of phosphatidylinositol 3-kinase and phospholipase A2 in Golgi transport vesicles association and fusion with the apical membrane.

The study of ethanol effects on intracellular transport and membrane biogenesis in rat hepatocytes revealed that, during synthesis of transport vesicles, the cytosolic phosphatidylinositol 3-kinase incorporated into the membrane of Golgi transport vesicles and a portion of the vesicular phosphatidylinositol was phosphorylated to phosphatidylinositol 3-phosphate. Association of the enzyme with Golgi transport vesicles and the transport to the apical portion of the cell membrane was not affected by 0 to 120 mM ethanol, but was dependent on the presence of the p85 subunit of the phosphatidylinositol 3-kinase. In the presence of ATP-enriched cytosol and calcium ions, association of Golgi transport vesicles with the apical membrane was followed by phospholipase A2-specific hydrolysis of phosphatidylinositol 3-phosphate and incorporation of the transport vesicle membrane into the apical membrane. Association of Golgi transport vesicles with apical membranes was not affected by preincubation of the cell membrane or Golgi transport vesicles with 0 to 120 mM ethanol, but was inhibited when the p85 phosphatidylinositol 3-kinase was incorporated into the membrane before incubation with Golgi transport vesicles. The fusion of Golgi transport vesicles with the apical membrane and generation of lysophosphatidylinositol 3-phosphate and arachidonate was inhibited with EGTA or after depletion of ATP from cytosol. Results of these studies provide evidence that phosphatidylinositol 3-kinase and phospholipase A2 activities are crucial for the final step of exocytotic transport. The process consists of two stages. First, the p85 subunit of phosphatidylinositol 3-kinase is involved in the specific association of the vesicle with membrane receptor, and that is followed by phospholipase A2-specific lysophospholipid generation, perturbation of the membranes, and fusion of the transport vesicle membrane with the apical membrane. Addition of ethanol to the in vitro transport system decreased production of Golgi transport vesicles, but had no effect on their association with apical membrane or fusion with the membrane.

Animals↗

PubMed1989

Regulation of glucose transport activity and expression of glucose transporter mRNA by serum, growth factors and phorbol ester in quiescent mouse fibroblasts.

We have investigated the effects of growth factors such as serum, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) on glucose transport activity in quiescent mouse Swiss 3T3 cells. DNA synthesis was synchronously induced by either calf serum, or platelet-poor plasma in combination with PDGF or FGF. Early stimulation of glucose transport in the quiescent cells was also caused by serum, or by either PDGF or FGF. The time courses for the stimulation of transport were identical for serum, PDGF and FGF, and the stimulated uptake in each case was associated with a 5-6-fold increase in Vmax. There were no detectable changes in apparent Km. Expression of glucose transporter mRNA was also enhanced by these growth factors. By contrast, EGF, insulin and platelet-poor plasma had little effect on glucose transport and transporter-gene expression, although uridine uptake was enhanced by all of these growth factors. These results suggest that cell cycle-dependent stimulation of glucose transport and expression of the transporter mRNA are regulated by a specific class of growth factors such as PDGF and FGF. The tumor promoter phorbol 12-myristate 13-acetate (PMA) also stimulated glucose transport and expression of transporter mRNA in quiescent 3T3 cells. These stimulations were absent in PMA-pretreated cells. However, serum, PDGF and FGF were able to stimulate glucose transport as well as expression of the transporter mRNA in PMA-pretreated cells, suggesting that there are at least two independent pathways for regulating glucose transport and glucose transporter mRNA level in quiescent fibroblasts.

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