Effects of anesthetic alcohols on membrane transport processes in human erythrocytes

1. 1. Anesthetic alcohols (pentanol, hexanol and heptanol) were found to increase the fluidity of red cell membrane lipids as monitored by the fluorescence depolarization of diphenylhexatriene. The relative potency of the alcohols was found to be parallel to their relative membrane/water partition coefficients.

2. 2. Hexanol had biphasic effect on erythritol uptake by simple diffusion by red cells. At concentrations less than 9 mM, hexanol had no significant effect. At concentrations greater than 9 mM, there was an approximately linear increase in erythritol permeability with increasing alcohol concentration.

3. 3. The facilitated transport of uridine was markedly inhibited by hexanol. Hexanol at 6 mM produced a 65% inhibition of uridine (4 mM) uptake. Hexanol decreased both the apparent K_m and V values for the equilibrium exchange of uridine.

4. 4. The facilitated transport of galactose was only slightly inhibited by hexanol.

5. 5. Hexanol was without effect on the passive and active fluxes of Na⁺ and K⁺ in red cells with altered cation contents. Cells that were slightly depleted of K⁺ and cells that were highly K⁺-depleted were both insensitive to hexanol.

Characterization and regulation of galactose transport in Neurospora crassa

Two galactose uptake systems were found in the mycelia of Neurospora crassa. In glucose-grown mycelia, galactose was transported by a low-affinity (Km = 400 mM) constitutive system which was distinct from the previously described glucose transport system I (R. P. Schneider and W. R. Wiley, J. Bacteriol. 106:479--486, 1971). In carbon-starved mycelia or mycelia incubated with galactose, a second galactose transport activity appeared which required energy, had a high affinity for galactose (Km = 0.7 mM), and was shown to be the same as glucose transport system II. System II also transported mannose, 2-deoxyglucose, xylose, and talose and is therefore a general monosaccharide transport system. System II was derepressed by carbon starvation, completely repressed by glucose, mannose, and 2-deoxyglucose, and partially repressed by fructose and xylose. Incubation with galactose yielded twice as much activity as starvation. This extra induction by galactose required protein synthesis, and represented an increase in activity of system II rather than the induction of another transport system. Glucose, mannose, and 2-deoxyglucose caused rapid degradation of preexisting system II; fructose and xylose caused a slower degradation of activity.     

Carbohydrate Transport in Trypanosoma gambiense*

SYNOPSIS. The uptake of ¹⁴C-labeled carbohydrates by Trypanosoma gambiense was studied. Glucose, mannose, glycerol, 2-deoxyglucose and fructose were rapidly absorbed by the parasite, and all had saturation kinetics. The glucose analog 3-O-methylglucose was not taken up by T. gambiense. Competitive inhibition experiments indicate that there are 2 transport loci for the tested substrates. It is suggested that there is a “glucose site” thru which glucose, mannose and glycerol, but not fructose, are transported and a “fructose site” thru which only fructose is transported. The specificity of the glucose-transporting mechanism appears to differ from those of other animals.     

Further evidence for the multiplicity of carriers for free glucalogues in hamster small intestine

The ratios between the Na⁺-dependent unidirectional fluxes of glucose and 6-deoxyglucose change along the small intestine of hamsters. This obsevation provides further evidence for the existence of more than a single common carrier for the Na⁺-dependent transport of free monosaccharides into the small intestinal mucosa.     

2-Deoxyglucose transport by intestinal epithelial cells isolated from the chick

Summary Characteristics of 2-deoxyglucose uptake (2DG) by intestinal epithelial cells isolated from chickens were evaluated as a means of discriminating between the concentrative transport system for monosaccharides, associated with the mucosal brush border, and other possible routes of monosaccharide entry. 2DG was chosen as it is not a substrate for the mucosal transport system. The deoxysugar enters via a saturable pathway which is not Na⁺-dependent, is not inhibited by K⁺, does not accumulate solute against a concentration gradient; exhibits a high sensitivity to inhibition by phloretin; is relatively insensitive to phlorizin inhibition; and has low affinity [but high capacity relative to Na⁺-dependent mucosal transport of 3-O-methylglucose (3-OMG) and other monosaccharides]. These characteristics confirm those established in an earlier report for Na⁺-independent uptake of 3-OMG. Complications encountered in the use of 2DG as a test sugar include significant rates of metabolic conversion to an anionic form which presumably is a phosphorylated species. Methods for distinguishing between transport and subsequent metabolism are described. Inhibition of 2DG entry by several other sugars is described and inhibitory constants (K's) given for each.     

Carbohydrate transport in Moniliformis dubius (Acanthocephala). I. The kinetics and specificity of hexose absorption.

The uptakes of 14C-glucose, -2-deoxyglucose, -mannose, -N-acetylglucosamine, -3-0-methylglucose, -fructose, and -galactose by female Moniliformis dubius were nonlinear, saturable functions of hexose concentration. Kinetic and inhibition studies indicated that glucose and 2-deoxyglucose were absorbed via a single common transport locus. Mannose, N-acetylglucosamine, 3-0-methylglucose, fructose, and galactose (in decreasing order of effectiveness) inhibited the uptake of glucose in a completely competitive manner; their absorptions appeared to be mediated by the glucose transport locus and, to some degree, by one or more additional transport systems. Kinetic studies suggested that the apparent inhibitions of 14C-glucose uptake by maltose and glucose-6-phosphate were due to free glucose liberated through the action of surface hydrolases. The uptake of 14C-glucose was also inhibited by salicin, alpha-methylglucoside, and beta-methylglucoside, but not by pentoses, L-hexoses, sugar alcohols, disaccharides (except maltose), gluconic acid, glucuronic acid, phlorizin, or ouabain. Glucose uptake was not Na+-dependent.     

Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius

Streptomyces peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. peucetius var. caesius.     

[3H]2-deoxyglucose transport by slices of cerebral cortex: Apparent dependence upon mitochondrial energy

The transport of [³H]2-deoxyglucose by brain slices was studied. Cerebral cortex slices were incubated in vitro in the presence of [³H]2-deoxyglucose, orl-[³H] glucose as a marker for diffusion. Transport was defined as the difference between [³H]2DG uptake andl-[³H]glucose uptake. Half-maximal velocity was seen at 2.0 mM 2DG and [³H]2DG transport was not inhibited by 20-fold higher concentrations ofl-glucose. Net [³H]2DG transport was unchanged in media deficient in Na⁺, K⁺, Mg²⁺, Ca²⁺ or Cl^–. Uptake was significantly inhibited by 1.0 mM 2,4-DNP and a suggestion of inhibition by azide was seen. These data are consistent with a hypothesis that hexose transport in the brain depends to some extent upon mitochondrial energy.     

Effects of Barbiturates on Facilitative Glucose Transporters are Pharmacologically Specific and Isoform Selective

Barbiturates inhibit GLUT-1-mediated glucose transport across the blood-brain barrier, in cultured mammalian cells, and in human erythrocytes. Barbiturates also interact directly with GLUT-1. The hypotheses that this inhibition of glucose transport is (i) selective, preferring barbiturates over halogenated hydrocarbon inhalation anesthetics, and (ii) specific, favoring some GLUT-# isoforms over others were tested. Several oxy- and thio-barbiturates inhibited [³H]-2-deoxyglucose uptake by GLUT-1 expressing murine fibroblasts with IC₅₀s of 0.2–2.9 mm. Inhibition of GLUT-1 by barbiturates correlates with their overall lipid solubility and pharmacology, and requires hydrophobic side chains on the core barbiturate structure. In contrast, several halogenated hydrocarbons and ethanol (all ≤10 mm) do not significantly inhibit glucose transport. The interaction of these three classes of anesthetics with purified GLUT-1 was evaluated by quenching of intrinsic protein fluorescence and displayed similar specificities and characteristics. The ability of barbiturates to inhibit other facilitative glucose transporters was determined in cell types expressing predominantly one isoform. Pentobarbital inhibits [³H]-2-deoxyglucose and [¹⁴C]-3-O-methyl-glucose uptake in cells expressing GLUT-1, GLUT-2, and GLUT-3 with IC₅₀s of ∼1 mm. In contrast, GLUT-4 expressed in insulin-stimulated rat adipocytes was much less sensitive than the other isoforms to inhibition by pentobarbital (IC₅₀ of >10 mm). Thus, barbiturates selectively inhibit glucose transport by some, but not all, facilitative glucose transporter isoforms. Received: 10 November 1998/Revised: 3 February 1999     

SPECIFICITY AND KINETIC PROPERTIES OF MONOSACCHARIDE UPTAKE INTO GUINEA PIG CEREBRAL CORTEX IN VITRO

Abstract— The uptake into the non-raffinose space of cerebral cortex slices of a number of ¹⁴C-labelled glucose analogues has been studied. Evidence on competition with glucose for the transport process has been used to derive information on the substrate specificity of sugar uptake to the brain. The kinetic properties of the uptake of 2-deoxygIucose indicate that the transport is a facilitated process rather than diffusion. Classical competition between glucose and 2-deoxyglucose for transport is shown and arguments are advanced for regarding glucose as a competitive inhibitor of 2-deoxyglucose transport. The apparent K_m for deoxyglucose is 10 mM and for glucose is suggested to be of the order of 5 mm , The value of such a kinetic approach to sugar transport in various conditions is discussed.     

Cyclic AMP regulation of glucose transport in germinating Pilobolus longipes spores

Pilobolus longipes spores were activated by either glucose or 6-deoxyglucose. Glucose-induced spore activation was previously shown to follow an increase in intracellular cyclic AMP. Concurrent with glucose-induced spore activation, were shifts in 6-deoxyglucose transport kinetics towards higher V _max and K _m values. Cyclic AMP derivatives also caused spore activation and similar changes in the kinetic parameters of 6-deoxyglucose transport. The time course of activation was paralleled by changes in transport activity. Inhibition of phosphodiesterase alone did not cause activation or induce changes in transport activity, but in combination with sub-optimal levels of either 6-deoxyglucose or cAMP derivatives, it amplified the germination signals to produce large increases in both spore activation and 6-deoxyglucose transport activity. These results support the conclusion that glucose transport in germinating spores is regulated by cAMP.Abbreviations IBMX 3-isobutyl-1-methylxanthine; monobutyryl cyclic AMP - N⁶ monobutyryladenosine 3:5-cyclic monophosphate - 8-bromo cyclic AMP 8-bromoadenosine 3:5-cyclic monophosphate     

How hexoses and inhibitors influence the malate transport system in Zygosaccharomyces bailii

When grown in fructose or glucose the cells of Zygosaccharomyces bailii were physiologically different. Only the glucose grown cells (glucose cells) possessed an additional transport system for glucose and malate. Experiments with transport mutants had lead to the assumption that malate and glucose were transported by one carrier, but further experiments proved the existence of two separate carrier systems. Glucose was taken up by carriers with high and low affinity. Malate was only transported by an uptake system and it was not liberated by starved malate-loaded cells, probably due to the low affinity of the intracellular anion to the carrier. The uptake of malate was inhibited by fructose, glucose, mannose, and 2-DOG but not by non metabolisable analogues of glucose. The interference of malate transport by glucose, mannose or 2-DOG was prevented by 2,4-dinitrophenol, probably by inhibiting the sugar phosphorylation by hexokinase. Preincubation of glucose-cells with metabolisable hexoses promoted the subsequent malate transport in a sugar free environment. Preincubation of glucose-cells with 2-DOG, but not with 2-DOG/2,4-DNP, decreased the subsequent malate transport. The existence of two separate transport systems for glucose and malate was demonstrated with specific inhibitors: malate transport was inhibited by sodium fluoride and glucose transport by uranylnitrate. A model has been discussed that might explain the interference of hexoses with malate uptake in Z. bailii.Abbreviations 2,4-DNP 2,4-dinitrophenol - 2-DOG 2-deoxyglucose - 6-DOG 6-deoxyglucose - pCMB para-hydroxymercuribenzoate     

Effect of lithium ion on melibiose transport inEscherichia coli

Summary Both Li⁺ and Na⁺ stimulated the uptake of thiomethylgalactoside by the melibiose transport system ofEscherichia coli. On the other hand, Li⁺ inhibited the growth of cells on melibiose as a sole source of carbon. This inhibition was specific for melibiose, and Li⁺ had no effect on growth of cells on glucose, galactose, lactose, or glycerol. The effect of the cation on melibiose transport was investigated in a mutant which cannot utilize glucose. After entry into this cell, melibiose is cleaved into glucose and galactose by -galactosidase, and the resulting glucose is excreted. Since the entry step was found to be rate-limiting, glucose production could be taken as a measure of melibiose transport. Li⁺ inhibited the transport of melibiose, but not the induction of the melibiose operon nor the activity of -galactosidase. Li⁺ was found to inhibit the entry ofp-nitrophenyl--d-galactoside, but notp-nitrophenyl--d-galactoside entry. Thus, the cation specificity for the melibiose membrane carrier varies with different transport substrates.     

Enzymes related to galactose utilization inPseudomonas cepacia

Pseudomonas cepacia produced a characteristic green sheen on EMB-galactose plates owing to production of galactonic acid by the constitutive membrane-associated glucose dehydrogenase of this bacterium. Mutants isolated as glucose dehydrogenase deficient (Gcd^–) also were deficient in membrane-associated galactose dehydrogenase. A strain that formed glucose dehydrogenase at 30°C but not at 40°C was also temperature sensitive with respect to formation of galactose dehydrogenase. The Gcd^– strains still utilized galactose. A second, NAD-specific, galactose dehydrogenase (not membrane associated) along with a transport system for galactose were induced during growth on galactose and constituted an alternative pathway of conversion of galactose to galactonate. Enzymes of the De Ley-Doudoroff pathway of conversion of galactonate to pyruvate and glyceraldehyde-3-phosphate were induced during growth on galactose. Unexpectedly, growth on galactose also elicited formation of enzymes of the Entner-Doudoroff (ED) route. Furthermore, mutants blocked in the ED pathway grew poorly on galactose. One interpretation of these findings is that glyceraldehyde-3-phosphate formed from galactose via the De Ley-Doudoroff route (by cleavage of 2-keto-3-deoxy-6-phosphogalaconate) is reconverted to hexose phosphate and metabolized via the ED pathway.     

Kinetic constants for intestinal transport of four monosaccharides determined under conditions of variable effective resistance of the unstirred water layer

Summary Theoretical considerations have suggested that variations in the resistance of the unstirred water layer (UWL) have a profound effect on the kinetic constants of intestinal transport. In this study, a previously validatedin vitro technique was employed to determine the unidirectional flux rate of glucose, galactose, 3-O-methyl glucose and fructose into the rabbit jejunum under carefully-defined conditions of stirring of the bulk phase known to yield different values for the effective resistance of the UWL. For each monosaccharide, uptake is much greater when the resistance of the UWL is low than when high. The maximal transport rate,J _d ^m , of glucose was half as large as theJ _d ^m of galactose and 3-O-methyl glucose (3-O-MG), and was twice as great as theJ _d ^m of fructose. The apparent affinity constant,K _m ^* ,of glucose is less than that of fructose, which was lower than theK _m ^* of galactose and 3-O-MG. The use of the Lineweaver-Burk double reciprocal plot is associated with an overestimation of bothJ _d ^m andK _m ^* .This discrepancy between the true and apparent values of the kinetic constants is much greater for lower than for higher values ofJ _d ^m andK _m ^* ;variations in the resistance of the unstirred layer influences the magnitude and direction of the discrepancy. The apparent passive permeability coefficient is similar for each sugar, but because of the different values ofJ _d ^m , passive permeation contributes relatively more to the uptake of glucose and fructose than of galactose or 3-O-MG. Under conditions of high unstirred layer resistance, differences in uptake rates of the sugars are due to differences in theirJ _d ^m rather than theirK _m ^* .Kinetic analysis is compatible with the suggestion that the glucose carriers are predominantly near the tip of the villus, whereas those for galactose and 3-O-MG are located along the entire villus and theK _m ^* of their carriers at the tip is lower than theirK _m ^* towards the base of the villus. It is proposed that there are multiple or heterogeneous intestinal carriers for glucose, galactose and 3-O-methyl glucose in the jejunum of the rabbit.Abbreviations Used in this Paper C ₁ Concentration of the probe molecule in the bulk phase - C ₂ Concentration of the probe molecule at the aqueous-membrane interface - d Effective thickness of the intestinal unstirred water layer - D Free diffusion coefficient of the probe molecule     

Endogenousd-glucose transport in oocytes ofXenopus laevis

Summary Endogenous glucose uptake by the oocytes ofXenopus laevis consists of two distinct components: one that is independent of extracellular Na⁺, and the other one that represents Na⁺-glucose cotransport. The latter shows similar characteristics as 2 Na⁺-1 glucose cotransport of epithelial cells: The similarities include the dependencies on external concentrations of Na⁺, glucose, and phlorizin, and on pH. As in epithelial cells, the glucose uptake in oocytes can also be stimulated by lanthanides. Both the electrogenic cotransport and the inhibition by phlorizin are voltage-dependent; the data are compatible with the assumption that the membrane potential acts as a driving force for the reaction cycle of the transport process. In particular, hyperpolarization seems to stimulat transport by recruitment of substrate binding sites to the outer membrane surface. The results described pertain to oocytes arrested in the prophase of the first meiotic division; maturation of the oocytes leads to a downregulation of both the Na⁺-independent and the Na⁺-dependent transport systems. The effect on the Na⁺-dependent cotransport is the consequence of a change of driving force due to membrane depolarization associated with the maturation process.     

The role of the pentose phosphate shunt in glucose-induced insulin release: In vitro studies with 6-aminonicotinamide, methylene blue, NAD, NADH, NADP, NADPH and nicotinamide on isolated pancreatic rat islets

The possible role of the pentose phosphate shunt in insulin release was investigated in vitro with collagenase isolated pancreatic islets of rats. Parameters measured were insulin released into the medium and measured by an immunoassay and formation of ¹⁴CO₂ from glucose labeled either in the C-1 or C-6 position. The in vitro effect of the following substances was studied:

1. 1. 6-Aminonicotinamide, an antimetabolite in the synthesis of pyridine nucleotides. In islets of animals pretreated with 6-amino nicotinamide 6 h previously and in the presence of 3 mg/ml glucose in the incubation medium, 6-aminonicotinamide markedly reduced oxidation of [1-¹⁴C]glucose but did not affect that of glucose labeled in C-6. Concomitantly there was a marked decrease in insulin release. This action of 6-aminonicotinamide did not take place when it was added only to the incubation medium. Pretreatment with 6-aminonicotinamide did not change the insulin concentration of the islets, making it unlikely that it interfered with insulin synthesis. The effect of 6-aminonicotinamide is consistent with partial inhibition of the pentose shunt.

2. 2. Methylene blue: this agent was selected because it is known from studies with red blood cells that it will oxidize NADPH and thus stimulate activity of the pentose shunt. In concentrations of 0.5 and 2 μg/ml, methylene blue markedly stimulated oxidation of [1-¹⁴C]glucose but not that of C-6. Simultaneously there was a dose related decrease of insulin released.

3. 3. Pyridine nucleotides: in the absence of glucose only NADPH exhibited a significant effect of insulin release. If glucose (3 mg/ml) was present 1 or 10 mM of NAD⁺ or NADH exhibited a significant effect, NADP⁺ or NADPH were less effective. If the pentose shunt was blocked by pretreatment with 6-aminonicotinamide, all 4 pyridine nucleotides stimulated insulin release. Similarly there was an increase in oxidation of [1-¹⁴C]glucose, consitent with restimulation of the pentose shunt.

4. 4. Nicotinamide by itself exhibited a small effect; however, it was much less than the one produced by equimolar concentrations of the pyridine nucleotides.

Conclusion: Restricted availability of NADPH either less production or by fast removal leads to a decrease in glucose-induced insulin release. Pyridine nucleotides will restimulate 6-aminonicotinamide blockade insulin release and glucose oxidation by the pentose shunt. Recently it has been proposed by others that the polyol pathway may play a key role in insulin release, our data are consistent with such a hypothesis. Furthermore they do support a major role of the pentose shunt in insulin release.