Chick kidney microsomal cytochrome P-450 involvement in the metabolism of 25-hydroxyvitamin D3

Suspensions of 2 to 5% rat thymocytes were incubated at 35 °C in buffered balanced salt solution (pH 7.3) with lactate and β-hydroxybutyrate as fuels. The dependence of 3-O-[Me-³H]methylglucose influx on external and internal 3-O-methylglucose concentrations was studied. Entry was almost rectilinear during the first minute. From the dependence of methylglucose entry (into sugar-free cells) on external methylglucose concentration, we judged the entry K_m to be about 7.7 mm and the entry V to be about 0.64 μmol · min^?1 · (ml of packed cell volume)^?1. Methylglucose inside the cell enhanced influx, hence equilibrium exchange was faster than entry. The dependence of equilibrium exchange on methylglucose concentration (inside and outside being equal) indicated a K_m of about 25 mm and a V of about 2.1 μmol · (min)^?1 · (ml of cell volume)^?1. This effect of internal sugar indicated that entry into sugar-free cells is limited mainly by the return of empty carrier to the outside surface and that loading the carrier on the inside enhances its outward mobility. The K_m and V for influx into cells containing 21 mm methylglucose were 5.9 mm and 1.17 μmol · min^?1 · (ml of packed cells)^?1. The effect of 21 mm internal sugar on lowering the influx K_m from about 7.7 mm to about 6 mm was reproducible and contributed to the evaluation of the constants of the transport rate law. It indicated that loading of the carrier at the external surface reduces its mobility, in contrast to the effect of loading on the inside. Mechanical explanations for this behavior are discussed.     

The inhibition of hexose transport and metabolism by small amounts of adenosine 5′-triphosphate in isolated rat adipocytes

The effects of ATP on glucose transport and metabolism were studied in rat adipocytes. Over a concentration range of 10–250 μm, ATP was found to inhibit several aspects of adipocyte glucose metabolism, particularly when stimulated by insulin. Much of the effect of ATP on glucose metabolism appeared related to impairment of glucose transport, reflected by inhibition of both basal and insulin-stimulated rates of 3-O-methylglucose transport. ATP inhibited the V of insulin-stimulated 3-O-methylglucose transport, but had no effect on the K_m. The inhibitory effects of ATP were much less apparent when cells were preincubated with insulin, suggesting that ATP inhibited only the components of hexose transport not yet activated by the hormone. At very high medium glucose concentrations, where transport was no longer rate limiting for metabolism, there was no inhibition of glucose oxidation by 250 μm ATP. However, when hexose transport was blocked with cytochalasin B (50 μm), a small inhibitory effect of ATP persisted on basal and insulin-stimulated glucose and fructose oxidation, suggesting that intracellular metabolism was impaired. The mechanism of the intracellular effect did not appear to be caused by uptake of exogenous ATP. These studies provide further evidence that energy metabolism may play an important role in the regulation of facilitated glucose transport.     

Effects of mebendazole on the absorption of low molecular weight nutrients by Ascaris suum

Van Den Bossche H. and De Nollin S. 1973. Effects of mebendazole on the absorption of low molecular weight nutrients by Ascaris suum. International Journal for Parasitology3: 401–407. The effect of the anthelmintic drug, mebendazole, on the uptake and/or transport of glucose, fructose, 3-O-methylglucose, glycine, proline, methionine and palmitic acid was studied on in vitro incubated Ascaris suum. The experiments presented indicate that mebendazole inhibits the uptake and/or transport of glucose by A. suum. This inhibition is followed by a marked decrease in the glycogen content of the ascaris muscle. The addition of glucose to the incubation medium significantly enhanced the rate of uptake and/or transport of 3-O-methylglueose, glycine, methionine, proline and palmitic acid indicating that the absorption mechanisms depend on energy.Therefore, the inhibitory effect of mebendazole on the glucose uptake also results in a decreased uptake of 3-O-methylglucose and of the amino acids and fatty acid studied. The fructose uptake was not affected by the addition of glucose.Although mebendazole decreased the uptake of the hexoses and of the amino acids whether or not glucose was added, the uptake of palmitic acid was not affected when glucose was omitted from the medium. Mebendazole failed to exhibit an effect on the uptake, transport and/or utilization of glucose in rat.     

Heat-induced stimulation of 3-O-methylglucose transport in rat thymocytes.

Cells incubated at 41–46 °C show a gradual increase in the initial rate of 3-O-methylglucose uptake when subsequently assayed at 37 °C. Cellular ATP levels remain constant throughout this temperature range, but at temperatures higher than 46 °C, ATP levels decline as does the extent of transport stimulation. Cells incubated at 45 °C for 5 min continue to show a gradual increase in transport activity throughout a subsequent 25-min incubation period at 37 °C. The increase in transport activity is characterized by an increase in the proportion of the rapid phase of 3-O-methylglucose uptake, with little or no change in the half-time of either the rapid phase or the slow phase. Transport stimulation at high temperatures is blocked by inhibitors of oxidative phosphorylation. Cells depleted of intracellular exchangeable Ca²⁺ by treatment with the ionophore A23187 in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid show nearly the same degree of stimulation at high temperatures as untreated cells, suggesting that exchangeable Ca²⁺ ions do not play an obligatory role in the mechanism of transport stimulation. It is suggested that structural changes occur at 41–46 °C in the membrane proteins controlling glucose transport activity.     

Insulin action on cardiac glucose transport Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na⁺/K⁺ pump. ⁸⁶Rb⁺-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40–60 min, and was used to monitor the activity of the Na⁺/K⁺ pump. Ouabain (10^?3 mol/I) inhibited the steady-state uptake of ⁸⁶Rb⁺ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive ⁸⁶Rb⁺-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. ⁸⁶Rb⁺-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na⁺/K⁺ pump activity by ouabain and a concomitant shift in the intracellular Na⁺:K⁺ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na⁺/K⁺ pump and the glucose transport system.     

Endotoxin-induced alterations in glucose transport in isolated adipocytes

2-Deoxyglucose and $\text{3-O-}\text{methyglucose}$ were used to assess endotoxin-induced changes in glucose transport in rat adipocytes. 6 h after Escherichia coli endotoxin injection insulin-stimulated 2-deoxyglucose uptake was significantly depressed ($\text{V}\text{decreased}\text{, K}{}_{\mathrm{<mtext>m</mtext>}}\text{unaltered}$), phosphorylation of 2-deoxyglucose was seemingly unimpaired; basal 3-methylglucose entry was significantly increased, insulin-stimulated uptake was unaltered. Insulin significantly reduced $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ in control and endotoxin-treated cells. Cytochalasin B-insensitive uptake of both 2-deoxyglucose and 3-methylglucose, a small fraction of total transport, increased significantly in endotoxic cells. Endotoxin reduced spermine- and insulin-stimulated 2-deoxyglucose uptake to a similar extent. Results are consistent with the hypotheses that (1) a site of endotoxin-induced insulin resistance is at the cell membrane level and may reflect a decrease in number or activity of effective carrier units, rather than alterations in affinity, (2) endotoxin does not compromise the hexokinase system, (3) the cell membrane-localized effect of endotoxin on hexose transport is not necessarily mediated by the insulin receptor and (4) the entry of 2-deoxyglucose and 3-methylglucose may involve two separate transport systems.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. III. Inhibition by cytochalasins, colchicine, and vinblastine.

The effect of cytochalasin A and B, colchicine and vinblastine on tumor cell killing by macrophages activated in vitro with lymphocyte mediators was examined. Both cytochalasins reversibly inhibited the killing of tumor cells by activated macrophages. Kinetic studies with cytochalasin B suggested that this drug exerts its effect on an early step of the cytotoxic process. Additional studies revealed that the drug inhibited the binding of tumor cells by activated macrophages.Colchicine inhibited both the binding and the killing of tumor cells by activated macrophages, whereas its structural analogue, lumicolchicine, had no effect on either macrophage function.Vinblastine also inhibited the binding and killing of tumor cells. However, this drug no longer inhibited tumor cell binding at low concentrations (<10^?6M) that still inhibited tumor cell killing. Further, vinblastine inhibited tumor cell killing when added late to an ongoing cytolytic reaction.These results suggest that the cytochalasins, colchicine and vinblastine inhibit macrophage mediated cytotoxicity by preventing intimate contact between the effector macrophages and their targets. In addition, vinblastine also appears to inhibit a later step of the cytolytic process, possibly the secretion of a cytotoxic macrophage product.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. IV. Role of energy metabolism

The role of energy metabolism on tumor cell killing by in vitro activated macrophages was studied. Depletion of extracellular glucose had little effect on the cytotoxic capacity of mediator-activated macrophages. Respiratory antagonists did not inhibit cytotoxicity regardless of whether or not the assays were carried out in low-glucose-containing medium. Sodium fluoride, a known inhibitor of glycolysis, inhibited the killing of tumor cells by activated macrophages. 2-Deoxyglucose, an analog of glucose, was found to be an effective inhibitor of cytotoxicity. Three other analogs, 5-thio-d-glucose, 3-O-methylglucose, and 2-deoxy-d-galactose, were without effect. The concentrations of 2-DG that inhibited cytotoxicity did not lower cellular ATP levels to an appreciable extent. The combined addition of inhibitors of glycolysis and respiration resulted in a marked reduction in ATP levels. Under these experimental conditions, macrophage-mediated cytotoxicity was also significantly inhibited.     

A Role for 3-O-Sulfated Heparan Sulfate in Promoting Human Cytomegalovirus Infection in Human Iris Cells

Human cytomegalovirus (HCMV) has emerged as a clinically opportunistic pathogen that targets multiple types of ocular cells and tissues, including the iris region of the uveal tract during anterior uveitis. In this report, we used primary cultures of human iris stroma (HIS) cells derived from human eye donors to investigate HCMV entry. The following lines of evidence suggested the role of 3-O-sulfated heparan sulfate (3-OS HS) during HCMV-mediated entry and cell-to-cell fusion in HIS cells. First, 3-O-sulfotransferase-3 (3-OST-3) expression in HIS cells promoted HCMV internalization, while pretreatment of HIS cells with heparinase enzyme or with anti-3-OS HS (G2) peptide significantly reduced the HCMV-mediated formation of plaques/foci. Second, coculture of the HCMV-infected HIS cells with CHO-K1 cells expressing 3-OS HS significantly enhanced cell fusion. Finally, a similar trend of enhanced fusion was observed with cells expressing HCMV glycoproteins (gB, gO, and gH-gL) cocultured with 3-OS HS cells. Taken together, these results highlight the role of 3-OS HS during HCMV plaque formation and cell-to-cell fusion and identify a novel target for future therapeutic interventions.     

Effect of physical training on glucose transporters in fat cell fractions

Physical training increases maximally insulin-stimulated glucose assimilation and 3-O-methylglucose transport in epididymal fat cells. In the present report, glucose-inhibitable cytochalasin B binding in subcellular fractions of epididymal adipocytes was measured to assess changes in number of glucose transporters induced by training. Groups of rats trained by swimming were compared to control groups of the same age, matched with respect to body weight by restricted feeding. It was found that in trained rats the number of glucose transporters in the low density microsome fractions from non-insulin-stimulated fat cells was larger than in untrained rats. In both groups of rats, insulin stimulation of adipocytes decreased the number of glucose transporters in low-density microsomes by about 60% and increased the number of glucose transporters in the plasma membrane fractions. The number of glucose transporters in the plasma membrane fractions from maximally insulin-stimulated fat cells was larger in trained rats than in control rats. [U-¹⁴C]Glucose incorporation into lipids varied in proportion to plasma membrane cytochalasin B binding per cell under all conditions tested. The results explain the enhancing effect of training on insulin responsiveness transport of hexose in fat cells.     

Sarcolemmal glucose transport in Ca2+-tolerant myocytes from adult rat heart. Calcium dependence of insulin action

Cardiac myocytes were isolated from adult rat ventricles by a method which preserves their functional integrity, including long survival in physiological concentrations of Ca²⁺. Sarcolemmal glucose transport was assessed by measuring linear initial uptake rates of the nonmetabolized glucose analog3-O-methyl-d-glucose. Transport was saturable and showed competition byd-glucose and other features of chemical and stereo-selectivity. Transport was stimulated by insulin in a dose-dependent manner, resulting in an almost 5-fold increase inV_max, with little change inK_m. Stimulation of 3-methylglucose transport by insulin was largely Ca²⁺ -dependent. Omission of Ca²⁺ from the incubation medium caused a minor rise in basal 3-methylglucose uptake but the insulin-stimulated rise inV_max was only 30%. The Ca²⁺ antagonist D600 also antagonized stimulation of hexose transport by insulin. In all the above respects, 3-methylglucose transport in myocytes is identical to that in intact heart muscle. In addition, the decrease in insulin response by Ca²⁺ emission was partially reversed by subsequent return to a Ca²⁺ -containing medium. ATP levels remained stable in the absence of Ca²⁺, showing that the Ca²⁺ dependence did not reflect nonspecific cell damage.     

The absorption of sugars and organic acids by the daughter sporocysts of Microphallus similis (Jäg.)

The kinetics of uptake, accumulation against a concentration gradient, competitive inhibition and metabolic inhibition suggest that d-glucose enters the daughter sporocysts of Microphallus similis by active transport.Studies of competitive inhibition suggest that galactose, fructose, d-fucose, α-methyl-d-glucoside, d-mannose and 3-O-methylglucose are actively absorbed at the same locus as d-glucose. Glucosamine, d-ribose and l-fucose, however, may be actively transported at a different locus or may be taken up by simple diffusion.Acetate, pyruvate, citrate and succinate are absorbed by simple diffusion.     

Investigations on isolated islets of langerhans in vitro: 16. Modification of the glucose-dependent inhibition of glucagon secretion

Investigation of glucagon secretion in isolated Wistar rat islets was carried out to elucidate further the regulatory function of glucose and arginine on pancreatic A-cells. The suppressive effect of D-glucose could also be demonstrated with L-glucose, D-mannose, D-fructose, D-galactose, D-glyceraldehyde and DL-dihyroxyacetone, but not in the presence of 3-O-methylglucose or mannitol. Sugars other than D-glucose inhibited glucagon secretion only at much higher concentrations than those at which D-glucose was effective. Furthermore, although 7.5 mM D-glucose caused up to 80% inhibition, the effects of other sugars appeared to level off at only 50–60% inhibition. The inhibitory action of D-glucose or D-glyceraldehyde on glucagon secretion could not be overcome by L-arginine, but 3-O-methylglucose, mannoheptulose, 2-deoxy-D-glucose, iodoacetamide, theophylline, epinephrine and acetylcholine were effective. The insulin secretion in response to glucose was inhibited by the metabolic inhibitors used, whereas the B-cell response in the presence of glyceraldehyde was diminished by iodoacetamide only. Like D-glucose, a variety of other sugars markedly reduced the stimulatory effect of L-arginine in glucagon release.The results show that the suppression of glucagon secretion is not specific for D-glucose and not strongly connected on a stimulated insulin secretion.     

Proton Fluxes Associated with Sugar Uptake in Vicia faba Leaf Tissues

Vicia faba leaf fragments bring the pH of their incubation medium to about 4.7, whatever the initial pH value. At this pH, addition of 20 millimolar sucrose causes a transient (20 to 40 minutes) alkalinization (0.05 to 0.10 pH unit) of the medium. The alkalinization is not observed in the presence of p-chloromercuribenzenesulfonic acid which blocks the sucrose carrier involved in phloem loading without affecting the ATPase (Delrot, Despeghel, Bonnemain 1980 Planta 149: 144-148). Addition of 20 millimolar glucose, fructose, or 3-O-methylglucose induces weaker alkalinization than sucrose. Sequential additions of sugars show that: (a) sucrose- and hexose-induced proton fluxes are nearly saturated at 20 millimolar sugar (b) there is no competition between sucrose and hexoses for inducing proton influxes whereas (c) glucose and 3-O-methylglucose are competing for a common system.     

P-glycoprotein possesses a 1,4-dihydropyridine-selective drug acceptor site which is alloserically coupled to a vinca-alkaloid-selective binding site.

[3H]Vinblastine bound with high affinity to surface membranes prepared from H69/LX4 cells which express P-glycoprotein (P-gp) and as a consequence are multidrug resistant (MDR). The KD was 9.8 +/- 1.5 nM and density of sites 31.2 +/- 8.6 pmol/mg of protein. [3H]Vinblastine binding was inhibited by cytotoxics and agents known to reverse MDR. 1,4-Dihydropyridine MDR reversing agents including nicardipine and nifedipine accelerated the dissociation of [3H]vinblastine from P-gp indicating a negative heterotropic allosteric effect. Cyclosporin A, vincristine and actinomycin D did not alter [3H]vinblastine dissociation kinetics. It is concluded that P-gp possesses at least two allosterically coupled drug acceptor sites, receptor site-1 that is selective for vinca alkaloids and cyclosporin A, and receptor site-2 that is selective for 1,4-dihydropyridines.     

Effect of sugars and amino acids on membrane potential in two clones of sugarcane

Sugarcane (Saccharum officinarum L.) leaf parenchyma cells bathed in 1X solution maintained an average membrane potential of −135 millivolts in the dark. No difference in membrane potential was found between clones 51 NG 97 and H50 7209. An electrogenic pump appears to contribute to membrane potential in these cells. Sugars (25 millimolar) added externally caused the following membrane potential depolarizations (in millivolts) in clone 51 NG 97: glucose, 18 ± 4; galactose, 24 ± 7; 3-O-methylglucose, 10 ± 4; sucrose, 22 ± 3; fructose, 21 ± 7; raffinose, 9 ± 3; mannitol, 0; lactose, 0; melibiose, 0; and 1-O-methyl-α-galactose, 0. Glycine (25 millimolar) and serine (10 millimolar) caused depolarizations of 47 ± 7 and 23 ± 2 millivolts, respectively. Depolarization shows saturation kinetics with respect to glucose concentration, with a K_m of 3 to 6 millimolar. The metabolic inhibitors KCN and salicyl hydroxamic acid together caused depolarization of the membrane potential and greatly inhibited depolarization by 25 millimolar glucose and 25 millimolar raffinose. In a series of substitution experiments, glucose (25 millimolar) caused almost total inhibition of depolarization by raffinose, sucrose, and 3-O-methylglucose (all 25 millimolar), but only partial inhibition of depolarization to 25 millimolar glycine. Glycine (25 millimolar), also, only partially inhibited depolarization by 25 millimolar glucose. Total depolarization to 25 millimolar glycine and 25 millimolar glucose was comparable to the amount of depolarization of membrane potential caused by 1 millimolar KCN plus 1 millimolar salicyl hydroxamic acid. The results are consistent with a co-transport mechanism of membrane transport, with sugars and amino acids being transported by separate carrier systems.     

Evidence for a plasmalemma redox system in sugarcane

A plasmalemma-bound NADH-dependent redox system has been identified in protoplasts isolated from cell suspensions of sugarcane. This system oxidized NADH as well as NADPH, increased O₂ consumption 3-fold, and increased the pH of the external medium while the cytoplasmic pH was decreased. In the presence of NADH, ferricyanide was rapidly reduced and the external medium was acidified. The uptake rates of K⁺, 3-O-methylglucose, leucine, and arginine were all decreased in the presence of NADH.     

Effects in adipocytes of diamide on GSH levels,glucose uptake and cell integrity

Concentrations of insulin and chemical agents (H₂O₂, vitamin K-5) which stimulate hexose transport in fat cells do not alter the cellular levels of glutathione (reduced form; GSH). Diamide, another agent used in studies of insulin action, markedly reduces GSH levels and increases the movement of sugar into the cell. However, unlike insulin, H₂O₂ or vitamin K-5, diamide causes a change in the permeability of fat cells that allows entry of compounds (inulin, sucrose, l-glucose) which are normally excluded by the plasma membrane. Moreover, the accelerated rate of methylglucose uptake produced by diamide treatment is not inhibited by cytochalasin B, an agent that blocks basal and insulin-stimulated methylglucose transport. These results indicate that diamide does not cause a stimulation of the glucose transport system and should not be used (or used with caution) in transport studies. Furthermore, oxidation of GSH does not appear to be necessary for the stimulation of hexose transport in adipocytes by insulin, H₂O₂ or vitamin K-5.     

A "Cysteineless" GLUT1 Glucose Transporter Has Normal Function When Expressed in Xenopus Oocytes

To test the role of cysteines in the function of GLUT1 glucose transporter, site-directed mutagenesis was used to replace all six GLUT1 cysteines with serine residues. When the individual and combined Cys →Ser mutants were expressed in Xenopus laevis oocytes, zero-trans uptake of 3-O-methylglucose was comparable to that seen in native GLUT1. The "cysteineless" construct also retained the kinetic features of GLUT1, including an asymmetric transport mechanism and similar substrate and inhibitor affinities. Whereas GLUT1 transport was inhibited by sulfhydryl reagents, that of the "cysteineless" construct was not. These results show that cysteines are not required for GLUT1 function or oligomer formation. The "cysteineless" construct may therefore serve as a template for reintroducing cysteines back into GLUT1 at sites useful for testing transporter structure and function.