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Biomedical subjects

A Klip

Publications and source records attributed to A Klip.

At least 181 records · Page 10Linked to original sources
PubMed1984

Stimulation of hexose uptake in rat thymic lymphocytes by phorbol ester. A role for Ca2+ and Na+/H+ exchange?

The tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) stimulates hexose uptake into rat thymocytes. This study explores two possible messengers of this stimulation: changes in cytosolic [Ca2+], and activation of the Na+/H+ antiport. The cytosolic level of Ca2+, determined by the fluorescence of quin-2, was elevated by TPA, and this rise required extracellular Ca2+. In contrast, stimulation of hexose uptake was still observed in Ca2+ -free media even when cytoplasmic [Ca2+] was buffered with quin-2. TPA also raised the cytoplasmic pH, presumably through activation of the Na+/H+ exchange. However, replacement of extracellular Na+ by N-methylglucamine+ or choline+ which prevents the cytoplasmic alkanization did not prevent stimulation of hexose uptake by TPA. Moreover, amiloride, at concentrations that inhibit Na+/H+ exchange in these cells, did not interfere with stimulation of hexose uptake by TPA. In conclusion, stimulation of hexose uptake by phorbol ester in rat thymocytes does not appear to be mediated by changes in cytosolic free Ca2+ or in the activity of the Na+/H+ antiport.

Animals↗
PubMed1984

Chemical identity of the glucose transporter with the [3H]cytochalasin B-photolabelled component of human erythrocyte membranes. Equal sensitivity to trypsin and endoglycosidase F.

The protein photolabelled by [3H]cytochalasin B and band 4.5, which contains the human erythrocyte hexose transporter, were compared by electrophoretically monitoring the effect of digestion with endoglycosidase F and trypsin. Band 4.5 was found to consist of two minor components, Mr 58,000 and 52,000, and one main component, Mr 60,000-50,000. Deglycosylation by endoglycosidase F converted both the [3H]-labelled species and the main polypeptide of band 4.5 from a mixture of polypeptides of Mr 50,000-60,000 to a sharp component of Mr 46,000. Tryptic cleavage of the photolabelled protein produced a [3H]-labelled peptide of 19,000 daltons, which corresponded to an analogous tryptic fragment of the main component of band 4.5. Endoglycosidase F treatment of trypsin-treated samples had no effect on the 19,000 dalton fragment or the labelled 19,000 component, indicating that both species lack the carbohydrate moiety of the parent protein. This parallel chemical behaviour indicates that the photolabelled polypeptide is representative of the main constituent of band 4.5. Photolabelling may be used with confidence to quantitate glucose transporters in other cells.

Blood Proteins↗
PubMed1984

Labelling of the human erythrocyte glucose transporter with 3H-labelled cytochalasin B occurs via protein photoactivation.

Irradiation of human erythrocyte membranes with 3H-labelled cytochalasin B results in specific photolabelling of the glucose transporter. The action spectrum of photolabelling has a maximum at approx. 280 nm, whereas the absorption spectrum of cytochalasin B is maximal at 210 nm. By irradiating with narrow-band-width light centered at 280 nm for 2 h, 8% of the transporters become covalently labelled and 47% of the remaining cytochalasin B-binding sites are obliterated. We conclude that photolabelling driven by narrow-bandwidth irradiation proceeds via photoactivation of an aromatic amino acid residue on the transporter molecule, and when compared to wide-bandwidth irradiation, permits more efficient incorporation of the label without causing additional photodamage to the remaining transporters.

Carrier Proteins↗
PubMed1984

Identification and characterization of the glucose transporter of the blood-brain barrier by cytochalasin B binding and immunological reactivity.

[3H]Cytochalasin B was used as a ligand to identify and characterize the glucose transporter in cerebral microvessels of the rat and the pig. Specific cytochalasin B binding, defined as that fraction of the total binding that is stereospecifically displaced by excess (500 mM) D-glucose, is saturable. Kinetic studies of this specific binding to cerebral microvessel preparations showed a dissociation constant (Kd) of 0.65-0.88 microM and a maximal binding (Bmax) of 60-80 pmol/mg of protein. In comparison, the Bmax of particulate fractions of the cerebral cortex was about one-tenth that of cerebral microvessels. The ability of various hexoses to displace specific cytochalasin B binding to cerebral microvessels in vitro correlated well with the capability of these hexoses to cross the blood-brain barrier in vivo. Irreversible photoaffinity labeling of the glucose transporter of cerebral microvessels with cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis labeled a polypeptide(s) with a molecular weight of about 53,000. Antibodies prepared against the glucose transporter of human erythrocytes also reacted with a polypeptide(s) with a molecular weight of about 53,000 on electrophoresed preparations of cerebral microvessels. These results indicate that cerebral microvessels are richly endowed with a glucose transporter moiety of similar molecular weight and antigenic characteristics as the glucose transporter of human erythrocytes and other mammalian tissues.

Animals↗
PubMed1984

Is intracellular Ca2+ involved in insulin stimulation of sugar transport? Fact and prejudice.

Insulin stimulates the rate of glucose transport in muscle and fat tissue by incorporation of transporters from internal membranes into the plasma membrane. It is conceivable that cell Ca2+ ions could play a role in transporter translocation. Indeed Ca2+ has been thought to mediate insulin action, but the evidence remains highly controversial. Experiments to this effect include (i) determinations of a requirement for extracellular Ca2+ in the hormonal response, (ii) stimulation of glucose transport by agents thought to elevate cytosolic Ca2+, [Ca2+]i, and (iii) determinations of Ca2+ efflux. Actual measurements of the effect of insulin on [Ca2+]i were missing until recently. Current methods to measure [Ca2+]i include Ca2+-selective intracellular electrodes, metallochromic dyes, and photoproteins. Main drawbacks of these procedures have been the requirement of microinjection for their incorporation, which restricts their use to large cells, and their interaction with cytoplasmic Mg2+ and H+. Recently, a fluorescent Ca2+ chelator, quin-2, has been devised, which circumvents these difficulties. A permeable, nonchelating precursor of quin-2 penetrates cells and once in the cytosol becomes an impermeant, fluorescent Ca2+ chelator. With this technique it was shown that insulin does not change [Ca2+]i while stimulating glucose transport in L6 muscle cells.

Adipose Tissue↗
PubMed1984

Induction of sugar uptake response to insulin by serum depletion in fusing L6 myoblasts.

The L6 muscle cell line is proposed as an excellent cell culture system for studying glucose transport and its regulation by serum and insulin throughout myogenesis. The rate of hexose uptake decreased after cell fusion. Fetal calf serum (5 h) stimulated hexose uptake, and serum deprivation depressed it. Both effects were prevented by cycloheximide. Insulin stimulated the uptake of either 2-deoxy-D-glucose or 3-O-methyl-D-glucose provided that the basal rate was previously depressed by serum deprivation. The response to the hormone was significant only after the onset of cell fusion. Stimulation of transport was observed within the first 10 min after insulin administration and was insensitive to cycloheximide. Half-maximal stimulation of uptake was obtained with 5 X 10(-8) M insulin in 1% albumin solutions. Basal and insulin-stimulated uptake showed equal sensitivity to cytochalasin B. However, they differed in their sensitivity to extracellular Ca2+: basal transport was depressed, whereas insulin-stimulated transport was further enhanced in Ca2+-free media. Advantages of the cell culture system are discussed.

3-O-Methylglucose↗
PubMed1984

Role of calcium ions in insulin action on hexose transport in L6 muscle cells.

It has been proposed that Ca2+ ions mediate the stimulation by insulin of glucose uptake in muscle (Clausen, T., Cell Calcium 1:311-325, 1980). However, absolute measurements of the concentration of cytosolic free Ca2+, [Ca2+]i, during the course of insulin action have not been made. The stimulation of hexose uptake by insulin was studied in an in vitro model system of muscle cells, the L6 cell line. The following evidence suggests that Ca2+ ions are not likely to fulfill the purported role. 1) Insulin in Ca2+-free media induced stimulation of 2-deoxy-D-glucose uptake. 2) Elevation of [Ca2+]i with the ionophore A23187 did not enhance hexose uptake. 3) Insulin action was not diminished when the hormone was added to Ca2+-depleted cells in Ca2+-free media with A23187. 4) Hexose uptake was not affected by a number of agents thought to modify [Ca2+]i including epinephrine, caffeine, 2,4-dinitrophenol, hyperosmolar mannitol, salicylate, vanadate, veratrine, and trypsin. 5) Direct determinations of [Ca2+]i by fluorescence of the novel indicator Quin-2 did not show differences between basal and insulin-stimulated cells; under identical conditions hexose uptake was stimulated by the hormone. 6) Chelation of [Ca2+]i with Quin-2 in Ca2+-free media did not affect the response to insulin. 7) Low concentrations of trypsin (7.5 micrograms/ml) elevated [Ca2+]i but did not increase the rate of hexose uptake.

Aminoquinolines↗
PubMed1983

Role of calcium in serum-stimulation of hexose transport in muscle cells.

Serum stimulates glucose uptake into several cells in culture. In intact muscle, an increase in cytosolic free Ca2+ has been proposed to mediate the activation of glucose uptake by hormones and other stimuli [Cell Calcium (1980) 1, 311-325]. We report that hexose (2-deoxy-D-glucose) uptake into L6 muscle cells in culture is enhanced several-fold by fetal calf serum. The increase in uptake is due to stimulation of transmembrane transport, since serum also stimulated uptake of the non-metabolizable hexose 3-O-methyl-D-glucose. The role of Ca2+ in this stimulation was assessed: (i) stimulation of transport by serum was independent of the presence of extracellular Ca2+ during the incubation with serum; (ii) the intracellular levels of free Ca2+, measured by the fluorescence of the novel Ca-indicator quin-2, were identical in serum-stimulated and control cells. It is concluded that hexose transport can increase in muscle cells without concomitant changes in cytoplasmic free Ca2+.

Animals↗
PubMed1983

Identification of the glucose transporter in rat skeletal muscle.

The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [3H]cytochalasin B in the presence of L- and D-glucose. [3H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by D-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000-50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Animals↗
PubMed1983

Na+ -dependent proline transport in isolated membrane vesicles from the L6 muscle cell line. Stimulation of uptake by intravesicular proline.

Membrane vesicles of L6 myoblasts were prepared in order to study the amino acid transport system A. The role of the membrane in the adaptive response of transport to amino acid-supplementation was assessed. The membranes, prepared by N2 cavitation, displayed Na+ (but not K+)-dependent L-proline uptake. An overshoot of L-[3H]proline uptake was observed after exposure of the vesicles to an inward Na+ gradient. Isolated membrane vesicles loaded with 50 microM proline displayed countertransport (stimulation of proline uptake). It is concluded that the adaptive decrease of proline uptake observed in amino acid-supplemented cells cannot be accounted for by trans-inhibition of transport.

Animals↗
PubMed1983

The glucose transport system of muscle plasma membranes: characterization by means of [3H]cytochalasin B binding.

A membrane-rich preparation was isolated from adult rat skeletal muscle in low salt media and further fractionated in sucrose gradients. Fraction F2, with a relative density of 1.092-1.119, consisted of sealed membrane vesicles which were enriched in plasma membrane markers. These vesicles were capable of stereospecific D-glucose uptake which was sensitive to cytochalasin B (CB). The membranes were also enriched in high affinity [3H]CB binding activity (Kd of 0.28 microM). [3H]CB binding to the glucose carrier of these plasma membranes, estimated as the fraction of binding protectable by D-glucose, ranged between 2.5 and 7.4 pmol/mg protein in several membrane preparations. The amount of [3H]CB binding to muscle membranes from newborn and adult rats was not markedly different. Trypsin, at low concentrations, altered the molecular weight of several membrane components, without affecting [3H]CB binding. Higher concentrations of trypsin abolished [3H]CB binding. Both 2,4-dinitrofluorobenzene (0.1 mM) and N-ethylmaleimide (15 mM) inhibited [3H]CB binding; inhibition by these reagents was prevented by inclusion of micromolar concentrations of CB in the reaction mixture. Several procedures that extracted specific proteins enriched the D-glucose-sensitive [3H]CB binding to the protein-depleted membranes. Antibody raised against the glucose carrier of human red cell membranes cross-reacted with a polypeptide of Mr about 45K of muscle membranes which might represent the glucose carrier.

Animals↗
PubMed1983

Insulin binding to differentiating muscle cells in culture.

Saturable binding of 125I-labelled insulin was detected on L6 myoblasts grown in monolayers. Binding was proportional to the number of cells and was pH sensitive, decreasing above pH 7.0. Binding also decreased with increasing temperature in the range 22-37 degrees C. Binding of insulin over a range of hormone concentrations (10(-10)-10(-6) M) was analyzed by Scatchard plots. The data are compatible with the coexistence of two components with Kd of 3 and 190 nM. The number of both types of binding sites increased with cell differentiation (upon alignment and cell fusion), when expressed either per unit protein, DNA, or surface membrane cholesterol. On the other hand, their association constants did not vary. Binding of insulin to a nonfusing mutant of L6 cells resembled binding to the parent myoblasts before alignment.

Animals↗
PubMed1982

Hexose transport in L6 muscle cells. Kinetic properties and the number of [3H]cytochalasin B binding sites.

(1) Myoblasts in culture (L6 cell line) were used as an in vitro model system, to study the kinetic and pharmacological properties of hexose transport in skeletal muscle tissue. (2) Uptake of 2-deoxy-D-[3H]glucose into L6 cells grown in monolayer culture was judged rate limiting since: (2) The time course of sugar uptake extrapolated to zero, (b) a parallel inhibition of hexose uptake and phosphorylation was caused by cytochalasin B, and (c) very little backflow of the hexose was detected. (3) Uptake of 2-deoxy-D-[3H]glucose by cells in monolayers was linear for at least 20 min and it was stimulated by countertransport. The Kt value was 0.83 mM. Cytochalasin B inhibited uptake non-competitively, and half maximal inhibition was achieved at 0.3 microM. Cytochalasin E (up to 5 microM) did not affect 2-deoxy-D-[3H]glucose uptake. (4) L6 myoblasts, detached by trypsinization, retained the hexose transport activity. Kt in detached cells was 0.96 mM. V was 3.2 nmol/min per mg protein, and half maximal inhibition was observed with 0.25 microM cytochalasin B. (5) [3H]Cytochalasin B binding to detached cells showed saturable and non-saturable components. The former could be further separated into cytochalasin E-sensitive binding (probably associated to cytoskeletal proteins) and cytochalasin E-insensitive binding, a fraction of which was inhibited by D-glucose. The D-glucose sensitive sites amount to 16.3 pmol/mg protein, and showed a Kd of 0.49 microM, which is in close agreement with the Ki of cytochalasin B inhibition of hexose uptake. These sites probably are equivalent to the hexose carrier molecules, and are present at a density of 6.8 . 10(6) sites/cell.

3-O-Methylglucose↗
PubMed1982

Regulation of amino acid transport in L6 myoblasts. II. Different chemical properties of transport after amino acid deprivation.

The mechanism of stimulation of amino acid transport system A caused by amino acid deprivation in L6 cells was investigated. In cells loaded with alpha-aminoisobutyric acid (AIB), amino acid deprivation increased the rate of proline uptake only after the intracellular [AIB] dropped below 7 mM. Efflux of proline was not sensitive to the presence of proline in the outer medium (with or without external Na+), suggesting that efflux through system A (and possibly uptake) is not susceptible to transinhibition. Transport (stimulated uptake) into amino acid-deprived cells and that into amino acid-supplemented cells differed in several chemical properties: 1) In the former group, transport was higher at lower pH values than in the latter, and the optimum pH values were 7.5 and 7.8, respectively. 2) Unlike proline uptake in supplemented cells, uptake in deprived cells was inhibited by 50% with N-ethylmaleimide (1 mM) or by 50 microM p-chloromercuribenzoate (PCMBS). Inhibition by PCMBS was not due to collapse of the Na+ gradient. The mercurial inhibited only the deprivation-induced stimulation of transport, bringing the rate of proline uptake to the "basal" uptake level observed in amino acid-supplemented cells. Proline uptake was not stimulated by a second deprivation following treatment with PCMBS and a supplementation-deprivation cycle. However, in untreated cells, or by reversing mercaptide formation with dithiotreitol, the second deprivation stimulated transport. Deprivation at 4 degrees C did not elicit stimulation of proline uptake. Cycloheximide prevented the stimulation and decreased the rate of proline uptake in deprived cells more efficiently than in supplemented cells. Actinomycin D prevented stimulation when added at the onset of deprivation. The above data indicate that stimulation of transport by deprivation is protein synthesis-dependent and that the stimulated transport had chemical properties distinct from the "basal" transport in supplemented cells. The evidence presented is consistent with a model of activation of a finite pool of transporters upon deprivation, the chemical characteristics of which differ from those of the "basal" transport system.

4-Chloromercuribenzenesulfonate↗
PubMed1980

Interaction of the sugar carrier of intestinal brush-border membranes with HgCl2.

HgCl2 was used as an inhibitor and potential label for the glucose carrier of intestinal brush-border membranes. Half-maximal inhibition of Na+-dependent D-glucose uptake was reached with micromolar concentrations of HgCl2 when the protein concentration was 1.2 mg/ml. Similar concentrations were found to inhibit the binding of [3H]phlorizin, a reversible competitive inhibitor of sugar transport. Inhibition was reversed by dithioerythritol but only marginally by EDTA. The data support the involvement of a sulfhydryl group in the inhibitory process. Deoxycholate-extracted membranes, which are enriched in specific phlorizin binding activity, were used for labeling studies using 203HgCl2. The polypeptides were separated by gel electrophoresis and analyzed by protein staining and autoradiography. Non-specific 203HgCl2 labeling was minimized by pre-treatment with sulfhydryl reagents which do not inhibit phlorizin binding. Several bands, which are lost from the autoradiographic pattern during a negative purification of the phlorizin binding sites, could be ruled out as essential components of the sugar carrier. The polypeptide profile was also analyzed following proteolysis, which abolished phlorizin binding. Those radioactive bands of which apparent Mr values were alterd by the treatment were considered as possible candidates. Finally, samples in which inhibition was reversed by thiols were also studied. The possible identity of the polypeptide(s) involved in glucose translocation is disussed in the light of these observations.

Animals↗