The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity

We used an improved cryosectioning technique in combination with quantitative immunoelectron microscopy to study GLUT4 compartments in isolated rat white adipose cells. We provide clear evidence that in unstimulated cells most of the GLUT4 localizes intracellularly to tubulovesicular structures clustered near small stacks of Golgi and endosomes, or scattered throughout the cytoplasm. This localization is entirely consistent with that originally described in brown adipose tissue, strongly suggesting that the GLUT4 compartments in white and brown adipose cells are morphologically similar. Furthermore, insulin induces parallel increases (with similar magnitudes) in glucose transport activity, approximately 16-fold, and cell-surface GLUT4, approximately 12-fold. Concomitantly, insulin decreases GLUT4 equally from all intracellular locations, in agreement with the concept that the entire cellular GLUT4 pool contributes to insulin-stimulated exocytosis. In the insulin-stimulated state, GLUT4 molecules are not randomly distributed on the plasma membrane, but neither are they enriched in caveolae. Importantly, the total number of GLUT4 C-terminal epitopes detected by the immuno-gold method is not significantly different between basal and insulin-stimulated cells, thus arguing directly against a reported insulin-induced unmasking effect. These results provide strong morphological evidence (1) that GLUT4 compartments are similar in all insulin-sensitive cells and (2) for the concept that GLUT4 translocation almost fully accounts for the increase in glucose transport in response to insulin.

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Effect of Magnesium and Calcium Ions on the Photoelectron Transport Activity of Low-Salt Suspended Hydrilla verticillata Thylakoids: Possible Sites of Cation Interaction

Zeitschrift für Naturforschung C ◽

10.1515/znc-1998-9-1011 ◽

1998 ◽

Vol 53 (9-10) ◽

pp. 849-856

Author(s):

Sujata R. Mishra ◽

Surendra Chandra Sabat

Keyword(s):

Electron Transport ◽

Photosystem Ii ◽

Electron Donor ◽

Water Oxidation ◽

Electron Flow ◽

Hydrilla Verticillata ◽

Transport Activity ◽

Low Salt ◽

Electron Transport Activity ◽

Stimulation Of

Stimulatory effect of divalent cations like calcium (Ca2+) and magnesium (Mg2+) was investigated on electron transport activity of divalent cation deficient low-salt suspended (LS) thylakoid preparation from a submerged aquatic angiosperm, Hydrilla verticillata. Both the cations stimulated electron transport activity of LS-suspended thylakoids having an intact water oxidation complex. But in hydroxylamine (NH2OH) - or alkaline Tris - washed thylakoid preparations (with the water oxidation enzyme impaired), only Ca2+ dependent stimulation of electron transport activity was found. The apparent Km of Ca2+ dependent stimulation of electron flow from H2O (endogenous) or from artificial electron donor (exogenous) to dichlorophenol indophenol (acceptor) was found to be identical. Calcium supported stimulation of electron transport activity in NH2OH - or Tris - washed thylakoids was electron donor selective, i.e., Ca2+ ion was only effective in electron flow with diphenylcarbazide but not with NH2OH as electron donor to photosystem II. A magnesium effect was observed in thylakoids having an intact water oxidation complex and the ion became unacceptable in NH2OH - or Tris - washed thylakoids. Indirect experimental evidences have been presented to suggest that Mg2+ interacts with the water oxidation complex, while the Ca2+ interaction is localized betw een Yz and reaction center of photosystem II.

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A rapid method for the functional reconstitution of amino acid transport systems from rat liver plasma membranes. Partial purification of System A

Biochemical Journal ◽

10.1042/bj2550963 ◽

1988 ◽

Vol 255 (3) ◽

pp. 963-969 ◽

Cited By ~ 8

Author(s):

A R Quesada ◽

J D McGivan

Keyword(s):

Amino Acid ◽

Rapid Method ◽

Amino Acid Transport ◽

Plasma Membranes ◽

Transport Systems ◽

Partial Purification ◽

Acid Transport ◽

Transport Activity ◽

System A ◽

System L

A rapid method for the functional reconstruction of amino acid transport from liver plasma-membrane vesicles using the neutral detergent decanoyl-N-glucamide (‘MEGA-10’) is described. The method is a modification of that previously employed in this laboratory for reconstitution of amino acid transport systems from kidney brush-border membranes [Lynch & McGivan (1987) Biochem. J. 244, 503-508]. The transport activities termed ‘System A’, ‘System N’, and ‘System L’ are all reconstituted. The reconstitution procedure is rapid and efficient and is suitable as an assay for transport activity in studies involving membrane fractionation. By using this reconstitution procedure, System A transport activity was partially purified by lectin-affinity chromatography.

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Electrogenic Na+-dependentl-alanine transport in the lizard duodenum. Involvement of systems A and ASC

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.2001.280.3.r612 ◽

2001 ◽

Vol 280 (3) ◽

pp. R612-R622

Author(s):

Virtudes Medina ◽

Antonio Lorenzo ◽

Mario Dı́az

Keyword(s):

Amino Acid ◽

Amino Acid Transport ◽

Short Circuit ◽

Duodenal Mucosa ◽

Neutral Amino Acid ◽

Acid Transport ◽

Transport Activity ◽

System A ◽

Alanine Transport ◽

Gallotia Galloti

l-Alanine transport across the isolated duodenal mucosa of the lizard Gallotia galloti has been studied in Ussing chambers under short-circuit conditions. Net l-alanine fluxes, transepithelial potential difference (PD), and short-circuit current ( Isc) showed concentration-dependent relationships. Na+-dependent l-alanine transport was substantially inhibited by the analog α-methyl aminoisobutyric acid (MeAIB). Likewise, MeAIB fluxes were completely inhibited byl-alanine, indicating the presence of system A for neutral amino acid transport. System A transport activity was electrogenic and exhibited hyperbolic relationships for net MeAIB fluxes, PD, and Isc, which displayed similar apparent K m values. Na+-dependentl-alanine transport, but not MeAIB transport, was partially inhibited by l-serine and l-cysteine, indicating the participation of system ASC. This transport activity represents the major pathway for l-alanine absorption and seemed to operate in an electroneutral mode with a negligible contribution to the l-alanine-induced electrogenicity. It is concluded from the present study that the active Na+-dependent l-alanine transport across the isolated duodenal mucosa of Gallotia galloti results from the independent activity of systems A and ASC for neutral amino acid transport.

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Selective Stimulation of Cat Sciatic Nerve Using an Array of Varying-Length Microelectrodes

Journal of Neurophysiology ◽

10.1152/jn.2001.85.4.1585 ◽

2001 ◽

Vol 85 (4) ◽

pp. 1585-1594 ◽

Cited By ~ 228

Author(s):

Almut Branner ◽

Richard B. Stein ◽

Richard A. Normann

Keyword(s):

Sciatic Nerve ◽

Spinal Cord Injuries ◽

Electrode Array ◽

Selective Stimulation ◽

System A ◽

Antagonist Muscles ◽

Cuff Electrodes ◽

Varying Length ◽

Muscle Groups ◽

Stimulation Of

Restoration of motor function to individuals who have had spinal cord injuries or stroke has been hampered by the lack of an interface to the peripheral nervous system. A suitable interface should provide selective stimulation of a large number of individual muscle groups with graded recruitment of force. We have developed a new neural interface, the Utah Slanted Electrode Array (USEA), that was designed to be implanted into peripheral nerves. Its goal is to provide such an interface that could be useful in rehabilitation as well as neuroscience applications. In this study, the stimulation capabilities of the USEA were evaluated in acute experiments in cat sciatic nerve. The recruitment properties and the selectivity of stimulation were examined by determining the target muscles excited by stimulation via each of the 100 electrodes in the array and using force transducers to record the force produced in these muscles. It is shown in the results that groups of up to 15 electrodes were inserted into individual fascicles. Stimulation slightly above threshold was selective to one muscle group for most individual electrodes. At higher currents, co-activation of agonist but not antagonist muscles was observed in some instances. Recruitment curves for the electrode array were broader with twitch thresholds starting at much lower currents than for cuff electrodes. In these experiments, it is also shown that certain combinations of electrode pairs, inserted into an individual fascicle, excite fiber populations with substantial overlap, whereas other pairs appear to address independent populations. We conclude that the USEA permits more selective stimulation at much lower current intensities with more graded recruitment of individual muscles than is achieved by conventional cuff electrodes.

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

Biochemical Journal ◽

10.1042/bj2810809 ◽

1992 ◽

Vol 281 (3) ◽

pp. 809-817 ◽

Cited By ~ 32

Author(s):

J Yang ◽

A E Clark ◽

R Harrison ◽

I J Kozka ◽

G D Holman

Keyword(s):

Cell Surface ◽

Glucose Transport ◽

Glucose Transporter ◽

Fluid Phase ◽

Transport Activity ◽

Phenylarsine Oxide ◽

Fluid Phase Endocytosis ◽

Surface Labelling ◽

Slow Dissociation ◽

Stimulation Of

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.

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Glucose Elevates NITRATE TRANSPORTER2.1 Protein Levels and Nitrate Transport Activity Independently of Its HEXOKINASE1-Mediated Stimulation of NITRATE TRANSPORTER2.1 Expression

PLANT PHYSIOLOGY ◽

10.1104/pp.113.230599 ◽

2013 ◽

Vol 164 (1) ◽

pp. 308-320 ◽

Cited By ~ 35

Author(s):

F. de Jong ◽

K. Thodey ◽

L. V. Lejay ◽

M. W. Bevan

Keyword(s):

Nitrate Transport ◽

Transport Activity ◽

Protein Levels ◽

Stimulation Of

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Effects of phenylarsine oxide on stimulation of glucose transport in rat skeletal muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1990.258.4.c648 ◽

1990 ◽

Vol 258 (4) ◽

pp. C648-C653 ◽

Cited By ~ 32

Author(s):

E. J. Henriksen ◽

J. O. Holloszy

Keyword(s):

Skeletal Muscle ◽

Glucose Transport ◽

Cytochalasin B ◽

Contractile Activity ◽

Transport Activity ◽

Rat Skeletal Muscle ◽

Phenylarsine Oxide ◽

Muscle Glucose Transport ◽

Glucose Analogue ◽

Stimulation Of

The trivalent arsenical phenylarsine oxide (PAO) inhibits insulin-stimulated glucose transport in adipocytes and skeletal muscle through direct interactions with vicinal sulfhydryls. In muscle, glucose transport is also activated by contractile activity and hypoxia. It was therefore the purpose of the present study to investigate whether vicinal sulfhydryls are involved in the stimulation of glucose transport activity in the isolated rat epitrochlearis muscle by hypoxia or contractions. PAO (greater than 5 microM) caused a twofold increase in rate of transport of the nonmetabolizable glucose analogue 3-O-methylglucose (3-MG) that was completely prevented by cytochalasin B, the vicinal dithiol dimercaptopropanol, dantrolene, or 9-aminoacridine, both inhibitors of sarcoplasmic reticulum Ca2+ release, or omission of extracellular Ca2+. Although PAO treatment (greater than or equal to 20 microM) prevented approximately 80% of the increase in 3-MG transport caused by insulin, it resulted in only a approximately 50% inhibition of the stimulation of 3-MG transport by either hypoxia or contractile activity. PAO treatment (40 microM) of muscles already maximally stimulated by insulin, contractile activity, or hypoxia did not reverse the enhanced rate of 3-MG transport. These data suggest that vicinal sulfhydryls play a greater role in the activation of glucose transport by insulin than by muscle contractions or hypoxia. The finding that PAO inhibits the stimulation of glucose transport, but does not affect glucose transport after it has been stimulated, provides evidence that vicinal sulfhydryls are involved in the pathways for glucose transport activation in muscle, but not in the glucose transport mechanism itself.

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Transport and regulatory properties of the apical Na-K-2Cl cotransporter of macula densa cells

AJP Renal Physiology ◽

10.1152/ajprenal.1998.275.5.f703 ◽

1998 ◽

Vol 275 (5) ◽

pp. F703-F709 ◽

Cited By ~ 5

Author(s):

M. Anuar Laamarti ◽

P. Darwin Bell ◽

Jean-Yves Lapointe

Keyword(s):

Macula Densa ◽

Rabbit Kidney ◽

Detectable Effect ◽

Transport Activity ◽

Dibutyryl Camp ◽

Intracellular Acidification ◽

Nacl Concentration ◽

Regulatory Properties ◽

Hill Number ◽

Stimulation Of

[Formula: see text]/NH3fluxes were used to probe apical Na-K-2Cl transport activity of macula densa (MD) cells from rabbit kidney. In the presence of 25 mM NaCl and 5 mM Ba2+, addition of 20 mM[Formula: see text] to the lumen produced a profound intracellular acidification, and ∼80% of the initial acidification rate was bumetanide sensitive. The[Formula: see text]-induced acidification rate was dependent on luminal Cl− and Na+ with apparent affinities of 17 ± 4 mM (Hill number 1.45) and 1.0 ± 0.3 mM, respectively. In the presence of saturating luminal NaCl concentration ([NaCl]L), blockade of basolateral Cl− efflux with 10 μM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) reduced the [Formula: see text]-induced acidification rate by 51 ± 6% ( P> 0.01, n = 5). Under similar conditions, dibutyryl-cAMP (DBcAMP) + forskolin increased the[Formula: see text]-induced acidification rate by 27%, whereas it produced no detectable effect at low luminal NaCl concentration. Most of the observed DBcAMP + forskolin effect was probably due to the stimulation of the basolateral Cl− conductance, since, in the presence of basolateral NPPB, this activation was changed to a 17.1% and 16.6% inhibition of the[Formula: see text]-induced acidification rate observed at high or low [NaCl]L, respectively. We conclude that the cotransporter found in MD cells displays, with respect to other Na-K-2Cl cotransporters, a relatively high affinity for luminal Na+ and luminal Cl− and can be specifically inhibited by increases in intracellular Cl− and cAMP concentrations.

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