Facilitated Diffusion Transport Research Articles

Cloning, characterization, and functional expression of cDNAs encoding glucose transporter proteins from the human parasite Schistosoma mansoni.

The blood-dwelling adult form of the parasitic worm, Schistosoma mansoni, consumes prodigious quantities of host glucose following its transport across the tegument. Immunologic or pharmacologic targeting of the relevant transporter proteins might form the basis of a schistosome control strategy. Here we report the isolation and characterization of three different full-length cDNAs whose predicted protein sequences show a high degree of sequence and structural similarity to the facilitated diffusion transporter proteins of other animals, plants, and bacteria. Functional expression of two cDNAs has been achieved by injection of Xenopus oocytes with in vitro derived sense strand RNA. Injected oocytes have a significantly increased ability to take up radiolabeled glucose analogues over controls. S. mansoni glucose transporters expressed in oocytes exhibit stereospecificity for D-glucose, have relaxed specificity for different hexoses, exhibit sodium independence, and are markedly inhibited by phloretin and cytochalasin B. These two transporters, expressed in oocytes, have a Km for 3-O-methylglucose of 1.3 and 2 mM. A third glucose transporter homologue cDNA appears to derive from a recent pseudogene. Both of the functional S. mansoni glucose transporter genes are expressed in larval and adult male and female schistosomes.

Functional reconstitution of the Trypanosoma brucei plasma-membrane D-glucose transporter.

The D-glucose transporter of Trypanosoma brucei was solubilized from the plasma membrane and reconstituted into proteoliposomes. Using the reconstitution of D-glucose transport as the assay and non-specific L-glucose uptake as control, we have purified a membrane protein fraction from T. brucei bloodstream-form ghosts by EDTA/alkali treatment and solubilization with the detergents octylglucoside or octylthioglucoside. Upon removal of the detergent by dialysis, the solubilized protein fraction was reconstituted in sonicated liposomes by a freeze/thaw-sonication step. The reconstituted transporter catalyzed specific D-glucose uptake and was compared in several characteristics with the native facilitated-diffusion transporter as present in live trypanosomes [Seyfang, A. & Duszenko, M. (1991) Eur. J. Biochem. 202, 191-196]. As in vivo, the uptake is time dependent and Na+ independent. Transporter substrate affinity and inhibitor specificity are completely retained and it is inhibited by mercuric ions, phloretin and cytochalasin B, but only partially inhibited by phlorizin. The reconstituted transporter also demonstrates trans-stimulation properties indicative of the carrier-mediated transport of D-glucose. In contrast to the human erythrocyte-type glucose transporter, in T. brucei D-fructose uptake was also catalyzed by the same reconstituted protein fraction and specific D-glucose or D-fructose transport were mutually competitive. Both the inhibitor studies and the fructose transport capacity in the reconstituted system are in good agreement with the native transport in live trypanosomes. The specific activity of D-glucose transport was 1.9 +/- 0.3 nmol.min-1.mg protein-1 at 0.2 mM D-glucose and the yield was about 0.8% of total ghost protein after removal of the variant-surface-glycoprotein coat. The successful functional reconstitution of a protozoan glucose transporter represents an important step towards its purification and detailed characterization. This is especially interesting since bloodstream-form trypanosomes depend entirely upon glycolysis for their ATP production.

Differential regulation of glucose transport and transporters by glucose in vascular endothelial and smooth muscle cells.

Hyperglycemia has been implicated in the pathogenesis of both micro- and macrovascular complications in diabetes. Little is known, however, about glucose transporters and their regulation in the vascular system. In this study, the regulation of glucose transporters by glucose was examined in cultured BAECs and BSMCs, and in human arterial smooth muscle cells. Both BAECs and BSMCs transported glucose via the facilitated diffusion transport system. Glucose-transport activity in vascular smooth muscle cells was inversely and reversibly regulated by glucose. Exposure of BSMCs and HSMCs to high glucose decreased Vmax for 2DG and 3-O-MG uptake, whereas Km remained unchanged. The hexose-transport system of BAECs exhibited lower 2DG and 3-O-MG uptake compared with BSMCs and showed little or no adaptation to changes in ambient glucose. Northern blot analysis demonstrated that GLUT1 mRNA levels in BAECs and BSMCs were unaffected by the concentration of glucose in the medium. GLUT2-5 mRNA could not be detected by Northern blot analysis. GLUT1 protein, quantified by Western blot analysis, was more abundant in BSMCs than in BAECs and was decreased by approximately 50% when medium glucose was elevated from 1.2 to 22 mM for 24 h. The alterations in the level of GLUT1 protein correlated with the changes observed in transport activity. These observations suggest differential regulation of glucose transporter in response to glucose between smooth muscle and endothelial cells. The sites of autoregulation may involve translational control and/or the stability of the protein in the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The glomerular mesangium in diabetes mellitus

Like the renal glomerular mesangium in patients with diabetic nephropathy, glomerular mesangial cell cultures grown in 30 mM glucose accumulate increased amounts of the extracellular matrix (ECM) proteins fibronectin, laminin, and type IV collagen. This is due to increased ECM protein synthesis and mRNA levels. Similar to other cells types that are affected by the diabetic state (such as, vascular cells and peripheral nerve), mesangial cells transport glucose by an insulin-independent, facilitated diffusion transport system. Kinetic studies reveal that intracellular glucose levels may reach the ambient glucose concentrations achieved in diabetes. Growth studies reveal that glucose does not exert its effect on mesangial cell ECM accumulation by affecting cell growth, but rather it causes an increase in diacylglycerol (DAG) mass and activates protein kinase C. Agents such as phorbol myristate acetate (PMA) and the cell permeable DAG analogue, oleoyl acetyl glycerol (OAG) which activate protein kinase C also increase ECM mRNAs. These results implicate protein kinase C activation in the increased ECM accumulation observed in mesangial cell cultures grown in high glucose.

Probing the structure and function of the human erythrocyte glucose transporter.

Introduction Recent I )NA cloning studies have revealed the existence of a large family o f homologous solute transporters (the ‘sugar transporter’ family) in organisms as diverse as mammals. plants, fungi and bacteria [ 11. These proteins catalyse the transport of ;I mide range o f substrates. including not only monosaccharides and disaccharides, but also iionsugar molecules such as tricx-boxylic acids and a variety of antibiotics and antiseptic agents [2] . Interestingly, the fhii ly contains examples of both passive transporters, typified by the human erythrocyte monosaccharide transport protein [ 3 1, and active trmsporters such ;IS the galactose/I I + symporter of Escherichiu coli [ 4 I , and the Mg’+ -tetracycline/l I + antiporters o f (;ram-negative bacteria [ 21. The most intensively studied member of the family is the facilitated diffusion transporter for glucose and other monosaccharides that is found in the human rrythrocyte membrane (GI,[ J‘I’I in the terminology of Fukunioto et al. 151) 101. This 492-residue integral membrane protein is widely distributed in mammalian tissues. but is most abundant in those w i t h a barrier function. such as the placental syncytiotrophoblast 171 and the blood-brain barrier [ X 1. l1nlike other members of the family. it can readily be purified in functionally active form on a large scale [ 0 I , and so represents an ideal model system for investigating the molecular mechanism of transport in the family as a w hole. I Iere. I describe recent advances in our understanding of the transport mechanism, derived both from examination of the aligned amino acid sequences of the family niemhers. and from biochemical experiments on the purified human protein. Although such approaches

Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi.

The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.

Facilitated diffusion and sodium-dependent transport of purine and pyrimidine nucleosides in rat liver

In mammalian cells, nucleoside transport usually is mediated by facilitated diffusion. In addition, a Na +-dependent, concentrative nucleoside transport system has been detected in several tissues but not the liver. To further clarify hepatic nucleoside transport mechanisms, we measured the uptake of [2- 14C]uridine (2 to 100 μmol/L) and of [8- 14C]adenosine (10 to 75 μmol/L) by the isolated perfused rat liver in the presence or absence of extracellular sodium or specific inhibitors of facilitated nucleoside diffusion. Uridine transport and metabolism were monitored by the release of labeled catabolites including 14CO 2, which indicated complete degradation of the pyrimidine. Adenosine, uridine and uridine catabolites were measured in the effluent perfusate by reversed-phase high-performance liquid chromatography and a radioactivity flow monitor. The existence of a Na +-dependent nucleoside transport system could be inferred from the following observations: (a) Sodium depletion caused a strong inhibition of nucleoside transport reflected by an up to threefold and 15-fold increase in extracellular uridine and adenosine, respectively. The sodium-dependent transport of uridine was saturated when the influent uridine concentration was raised beyond 20 μmol/L. No such saturation was observed for much higher concentrations of adenosine used (10 to 75 μmol/L). (b) Na +-free perfusion resulted in a strong suppression of the release of uridine catabolites by the liver. Complete uridine breakdown was depressed to 7% of the amount of 14CO 2 released in the presence of sodium and at influent uridine concentrations below 20 μmol/L. (c) Inhibition of uridine (10 μmol/L) transport and degradation was observed after coperfusion with adenosine, deoxyadenosine, guanosine and deoxyguanosine. No effect was obtained with cytidine, deoxycytidine, thymidine or hypoxanthine (100 μmol/L each), indicating a specificity identical to that reported for one of the sodium-dependent transport systems from other tissues. (d) Na +-free perfusion further enhanced the effects on nucleoside uptake of specific inhibitors of the nucleoside facilitated-diffusion transporter (dipyridamol, S-(4-nitrobenzyl)-6-thioinosine; 50μmol/L). The results of our experiments demonstrate the presence of a sodium-dependent and a sodium-independent nucleoside transport system in the isolated perfused rat liver.

Glycerol utilization in Fusarium oxysporum var. lini: regulation of transport and metabolism

Glycerol was transported in the fungus Fusarium oxysporum var. lini by a facilitated diffusion transport system with a half-saturation constant, Ks, of 0.5 mM and a maximum velocity, Vmax, of 0.9 mmol (g dry wt)-1 h-1 at pH 5 and 25 degrees C. 1,2-Propanediol was a competitive inhibitor of glycerol transport, but the cells did not actively accumulate 1,2-propanediol. The transport system was partially constitutive. In cells grown in the presence of glucose, glycerol was not transported, indicating that the synthesis of the system was under glucose repression. Glycerol kinase and NADP(+)-dependent glycerol dehydrogenase activities were present under all physiological conditions tested. A flavin-dependent glycerol phosphate dehydrogenase was induced only when glycerol was the sole energy source in the medium. This enzyme, together with the transport system, constitute the regulated steps in the glycerol metabolic pathway.

Differential regulation of the HepG2 and adipocyte/muscle glucose transporters in 3T3L1 adipocytes. Effect of chronic glucose deprivation

Glucose transport in 3T3L1 adipocytes is mediated by two facilitated diffusion transport systems. We examined the effect of chronic glucose deprivation on transport activity and on the expression of the HepG2 (GLUT 1) and adipocyte/muscle (GLUT 4) glucose transporter gene products in this insulin-sensitive cell line. Glucose deprivation resulted in a maximal increase in 2-deoxyglucose uptake of 3.6-fold by 24 h. Transport activity declined thereafter but was still 2.4-fold greater than the control by 72 h. GLUT 1 mRNA and protein increased progressively during starvation to values respectively 2.4- and 7.0-fold greater than the control by 72 h. Much of the increase in total immunoreactive GLUT 1 protein observed later in starvation was the result of the accumulation of a non-functional or mistargeted 38 kDa polypeptide. Immunofluorescence microscopy indicated that increases in GLUT 1 protein occurred in presumptive plasma membrane (PM) and Golgi-like compartments during prolonged starvation. The steady-state level of GLUT 4 protein did not change during 72 h of glucose deprivation despite a greater than 10-fold decrease in the mRNA. Subcellular fractionation experiments indicated that the increased transport activity observed after 24 h of starvation was principally the result of an increase in the 45-50 kDa GLUT 1 transporter protein in the PM. The level of the GLUT 1 transporter in the PM and low-density microsomes (LDM) was increased by 3.9- and 1.4-fold respectively, and the GLUT 4 transporter content of the PM and LDM was 1.7- and 0.6-fold respectively greater than that of the control after 24 h of glucose deprivation. These data indicate that newly synthesized GLUT 1 transporters are selectively shuttled to the PM and that GLUT 4 transporters undergo translocation from an intracellular compartment to the PM during 24 h of glucose starvation. Thus glucose starvation results in an increase in glucose transport in 3T3L1 adipocytes via a complex series of events involving increased biosynthesis, decreased turnover and subcellular redistribution of transporter proteins.

The high Km glucose transporter of islets of Langerhans is functionally similar to the low affinity transporter of liver and has an identical primary sequence.

The liver has been shown to contain a facilitated diffusion glucose transporter with high Km for glucose that is structurally distinct from the low Km glucose transporters found in most other tissues. We find that 3-O-methyl glucose is greater than 90% equilibrated across dispersed islet cells within 60 s, consistent with a facilitated diffusion transport mechanism. L-Glucose uptake was minimal throughout the time course, indicating stereospecificity. Measurement of glucose transport over a range of 3-O-methyl glucose concentrations from 0.05 to 60 mM revealed the presence of a component of glucose transport with an apparent Km of 17 mM, a value essentially identical to that previously reported for liver. Interestingly, a second component of glucose transport was also observed with an apparent Km of 1.4 mM, as has been reported for other tissues such as erythrocytes that are known to contain the "HepG2" or "erythroid/brain" type glucose transporter. Further evidence for the existence of two transport components is provided by the observation that a low concentration of cytochalasin B (0.4 microM) completely inhibits the low Km transport activity but has no effect on the high Km transporter. The kinetic similarity of high Km glucose transport in liver and islets is readily understood in light of our structural analysis. Sequence analysis of cDNA clones indicates that the liver and islet glucose transporters have identical sequences and, thus, are the products of the same gene.

Oxidative stress in the brain: A new model

1.(1) Sheep erythrocytes possess a facilitated diffusion transport system selective for small neutral amino acids of intermediate size (C-system). The effects of seven thiol-reactive agents on this system were investigated.2.(2) L-Alanine influx by this route was inhibited by HgCl2, p-chloromercuriphenylsulphonate (PCMBS), azodicarboxylic acid bisdimethylamide (diamide), N-ethylmaleimide and t-butylhydroperoxide. Iodoacetamide and 5,5′-dithiobis(2-nitrobenzoate) had no effect.3.(3) Detailed analysis of these inhibitor effects suggested the presence of three distinct classes of cellular thiol groups essential for normal transport function.4.(4) Class 1 thiols react with PCMBS and are located on the outer surface of the cell membrane in the region of the transport site. Class 2 thiols react with N-ethylmaleimide and diamide but are not affected by t-butylhydroperoxide. Class 3 thiols are oxidized during t-butylhydroperoxide treatment and are presumably also attacked by diamide and N-ethylmaleimide.5.(5) Class 3 thiols are either intracellular GSH or reactive thiols which readily form mixed disulphides with GSSG. Any direct involvement of GSH in amino acid transport is not mediated by the γ-glutamyl cycle.

Sugar transport in rat liver lysosomes. Direct demonstration by using labelled sugars.

Purified rat liver lysosomes ('tritosomes') were prepared from rats injected with Triton WR-1339. 2. The water space of tritosomes, measured by using [3H]water and [14C]sucrose, was 2.15 +/- 0.72 microliter/mg of protein (mean +/- S.E.M., n = 12). 3. Tritosomes, when compared with a crude preparation of normal lysosomes by an indirect method of study, showed sugar specificity but decreased stereospecificity of sugar uptake. 4. At 125 mM the relative rates of net uptake of D-[14C]ribose, D-[14C]- or D-[3H]glucose and 2-deoxy-D-[3H]glucose were the same as that inferred from the indirect study. 5. The entry of D-[3H]glucose into tritosomes showed concentration-dependence suggestive of saturation, with a Km of 48 +/- 18 mM (4). 6. D- and L-glucose, D-ribose, 2-deoxy-D-glucose and D-mannose competed with D-[14C]glucose or D-[14C]ribose for uptake. 7. Cytochalasin B inhibited D-[3H]glucose uptake. 8. Uptake of 1 mM-L-[14C]glucose was slower than for 1 mM-D-[14C]glucose. 9. It is concluded that a facilitated-diffusion transport system is present in purified rat liver lysosomes.

D-Glucose transport by membrane vesicles from quiescent, serum-stimulated, and SV40-transformed mouse 3T3 cells

Mixed membrane vesicle preparations from mouse embryo fibroblasts (Swiss 3T3) exhibited a facilitated diffusion transport system for d-glucose that showed many of the characteristics of the d-glucose transport system of whole cells: stereospecificity, counterflow, Michaelis-Menten kinetics with an apparent K m similar to that of whole cells, and sensitivity to inhibition by cytochalasin B. Comparison of the stereospecific d-glucose transport activities of membrane vesicles from quiescent, serum-stimulated, and SV40 virus-transformed 3T3 cells showed no significant differences in rates of d-glucose uptake or efflux. This is in contrast to whole cells; quiescent 3T3 cells transported 6-deoxy- d-glucose at a significantly lower rate than serum-stimulated or SV40-transformed cells. These results indicate that d-glucose transport in quiescent vs. actively growing cells is regulated by cellular factors that are not retained in membrane vesicle preparations.

Effect of Insulin and Dinoprostone on D-Glucose and 2-Deoxy-D-Glucose Uptake by Normal and Diabetic Rat Hepatocytes

□ Suspensions of deaggregated hepatocytes were prepared by a collagenase perfusion technique from livers of both normal rats and rats rendered diabetic by streptozocin treatment. Uptake of D-glucose and 2-deoxy-D-glucose was estimated by adding 14C- or 3H-labeled hexose to a stirring suspension of cells in Krebs-Henseleit buffer at 37°, separating the cells by rapid centrifugation, and measuring radioactivity in the packed cell pellet. Uptake, calculated after correction for entrapped extracellular fluid, includes any hexose bound to, transported into, or otherwise immobilized in the cells. High concentrations of glucose (5-20mM) establish an intracellular-extracellular distribution ratio near 1.0 within 1min, indicating a facilitated diffusion transport system. In contrast, a low level of glucose (71 nM) almost immediately {<15sec} establishes ratios of between 3 and 4, which suggests a significant amount of glucose binding to cell membrane. Such binding would not be detected at the high glucose levels because of its small magnitude. Hepatocytes from diabetic rats exhibit a decrease in this apparent binding to ~60% of normal; preincubation with 0.1IU/ml insulin increases this toward normal values, although it does not affect the binding by normal hepatocytes themselves. Preincubation with 0.1μM dinoprostone depresses glucose binding in cells from both normal and diabetic rats. A low concentration (1.2 nM) of 2-deoxy-D-glucose establishes even higher intracellular-extracellular distribution ratios of between 4 and 6, but the apparent binding of this sugar is identical in normal and diabetic rat hepatocytes and is not affected by preincubation with either insulin or dinoprostone alone. However, combined treatment with both agents causes a significant increase in 2-deoxyglucose binding. The results suggest that insulin promotes formation of hexose-binding sites and conversion of these to specific glucose-binding sites while dinoprostone may act by blocking the latter conversion.

Identification of the glucose transport protein of the microvillous membrane of human placenta by photoaffinity labelling

The initial step in transfer of glucose from mother to fetus is facilitated diffusion transport across the microvillous membrane of the placental syncytium (1). We have used 3H-cytochalasin B as a photoaffinity label to identify the transport protein involved. Two binding proteins were present, one of which is apparently the glucose transport protein and one of which is actin. The two were identified by competition labeling with D-glucose, and cytochalasin E. They were separated by selective extraction with dimethyl maleic anhydride. The glucose transport protein is apparently a single molecular species of 52,000 molecular weight.

A hypothesis for the role of dithiol-disulfide interchange in solute transport and energy-transducing processes.

We have recently shown that the physical mechanism for delta approximately mu H+-driven changes in the Km for three different transport systems is an oxidation-reduction reaction involving a dithiol-disulfide interconversion [Robillard, G.T. and Konings, W.N. (1981) Biochemistry, 20, 5025-5032; Konings, W.N. and Robillard, G.T. (1982) Proc. Natl Acad. Sci. USA, in the press]. Based on the similarities between the data from these three systems and published data from other systems, we now propose that dithiol-disulfide interchange may play a general role in membrane-related processes such as transport, energy transduction and hormone-receptor interactions. We propose that the affinities of the substrate-binding sites are regulated by a dithiol and a disulfide situated at different depths in the membrane. In addition we propose that the oxidation states of these two redox centers are coupled by dithiol-disulfide interchange such that, when one is oxidized, the other is reduced. Since a transmembrane electrical potential, delta psi, or a pH gradient, delta pH, can alter the redox state, it can change the affinity of the substrate-binding sites. The delta approximately mu H+-induced changes in affinity are sufficient to drive active transport (symport or antiport) and energy-transducing processes. A similar mechanism can be applied to transport systems driven by phosphorylated enzyme intermediates instead of delta approximately mu H+. Changes of the redox potential in a given compartment during metabolism could also control the affinity of ligand binding even in the absence of a delta approximately mu H+. The ligand-binding affinities of facilitated diffusion transport systems and receptor proteins may be regulated in this manner.

Uptake and release of choline in cultures of human glioma cells.

Human glioma cells (138MG) have a low-affinity uptake system for choline (Km = 20 microM; Vmax = 56 pmol/min/10(6) cells). The uptake is reduced by acetylcholine, hemicholinium-3, HgCl2, and phosphodiesterase inhibitors. Release of [3H]choline from preloaded cultures showed two pools with half-lives of 1.3 and 160 min. Choline release was stimulated by 8-bromo-cAMP or isobutylmethylxanthine. The results suggest that release of choline occurs by a facilitated diffusion transport system and is increased by elevations of intracellular cAMP.

Effects of thiol-reactive agents on amino acid transport by sheep erythrocytes

1. (1) Sheep erythrocytes possess a facilitated diffusion transport system selective for small neutral amino acids of intermediate size (C-system). The effects of seven thiol-reactive agents on this system were investigated. 2. (2) L-Alanine influx by this route was inhibited by HgCl 2, p- chloromercuriphenylsulphonate (PCMBS), azodicarboxylic acid bisdimethylamide (diamide), N- ethylmaleimide and t- butylhydroperoxide . Iodoacetamide and 5,5′-dithiobis(2-nitrobenzoate) had no effect. 3. (3) Detailed analysis of these inhibitor effects suggested the presence of three distinct classes of cellular thiol groups essential for normal transport function. 4. (4) Class 1 thiols react with PCMBS and are located on the outer surface of the cell membrane in the region of the transport site. Class 2 thiols react with N- ethylmaleimide and diamide but are not affected by t- butylhydroperoxide . Class 3 thiols are oxidized during t- butylhydroperoxide treatment and are presumably also attacked by diamide and N- ethylmaleimide . 5. (5) Class 3 thiols are either intracellular GSH or reactive thiols which readily form mixed disulphides with GSSG. Any direct involvement of GSH in amino acid transport is not mediated by the γ-glutamyl cycle.

Energetics of sugar transport by isolated intestinal epithelial cells: effects of cytochalasin B.

The capability of isolated intestinal epithelial cells to establish concentration gradients of 3-O-methylglucose (3-OMG) by a Na+-dependent transport system is limited by concomitant function of a Na+-independent, facilitated diffusion transport system. Monosaccharides accumulated by the active system are continuously lost via the passive system, which acts to lower steady-state sugar gradients maintained by the cell. Cytochalasin B is a potent inhibitor of the passive system and allows the cells to establish a sugar gradient that is much higher than normal. When extracellular [3-OMG] is 1 mM, cytochalasin induces sugar accumulation ratios of 30-fold (+/- phlorizin) in contrast to control ratios of approximately 10-fold. When [3-OMG] is 0.1 mM, cytochalasin (0.1 mM) induces 40-fold accumulation ratios. When changes in extracellular sugar concentration are considered, steady-state concentration gradients observed are 70-fold. For a Na:sugar coupling stoichiometry of 1:1, gradients of this magnitude represent the approximate theoretical maximum for a transport system driven exclusively by the transmembrane electrochemical potential for Na+.

Effects of phloretin and theophylline on 3-O-methylglucose transport by intestinal epithelial cells.

Phloretin and theophylline each exert an immediate inhibitory effect on the Na+-independent, facilitated-diffusion transport system for sugar associated with intestinal epithelial cells. Phloretin inhibits approximately 50% more of the total Na+-independent sugar flux than theophylline. Neither agent has an immediate effect on the Na+-dependent, concentrative sugar transport system, although preincubation of the cells with phloretin causes a significant inhibition. The slowly developing effect is correlated with a decrease in cellular adenosine triphosphate (ATP) and an elevation of intracellular Na+. Other agents which elevate cell Na+ also inhibit Na+-dependent sugar influx, even if ATP levels are not depleted. On the other hand, if ATP is depleted by phloretin under conditions in which the cells do not gain Na+, the inhibitory effect on Na+-dependent sugar flux tends to disappear. The slow-onset phloretin effects are due to transinhibition of the Na+-dependent sugar carrier by cellular Na+. When the passive sugar carrier is inhibited by phloretin or theophylline, the concentrative system can establish an enhanced sugar gradient. Because of the secondary metabolic effects of phloretin, theophylline induces a greater gradient enhancement despite its more limited effect on the passive sugar-transport system. Sugar gradients as large as 20-fold are induced by theophylline, in contrast to 12-fold gradients observed in the presence of phloretin and approximately 7- to 8-fold for untreated cells. These results are discussed in terms of conceptual questions regarding the energetics of Na+-dependent transport systems.