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.     

Regulation of Human Trophoblast GLUT1 Glucose Transporter by Insulin-Like Growth Factor I (IGF-I)

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.     

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

Summary 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.     

Amino acid uptake by Saccharomyces cerevisiae plasma membrane vesicles

A procedure is described which allows for the efficient separation of Saccharomyces cerevisiae plasma membranes from other cellular membranes by discontinuous sucrose density gradient centrifugation. After vesiculization in an osmotic stabilization buffer the plasma membrane vesicles retain the ability to transport amino acids. Amino acid uptake was affected by the proton gradient dissipator $\text{m-}\text{chlorocarbonylcyanide}$ phenylhydrazone and was dependent, in some cases, on the presence of sodium ion.     

The apical and basal plasma membranes of the human placental syncytiotrophoblast contain different erythrocyte membrane protein isoforms. Evidence for placental forms of band 3 and spectrin

Using immunochemical techniques, we identified forms of erythrocyte membrane proteins in apical and basal plasma membranes of human placental trophoblast. A wheat germ agglutinin-binding intrinsic protein was present in the microvillous (maternal facing) but not the basal (fetal facing) membrane of the syncytiotrophoblast epithelium. Conversely, erythrocyte-related proteins of the basal membrane included two intrinsic membrane proteins, a 95,000 Mr band 3 isoform and a form of spectrin. These four proteins were all absent from the microvillous membrane. The basal membrane spectrin isoform was also present in basal membrane skeletons. A 70,000 Mr polypeptide which reacted with antibodies to band 3 was present in both microvillous and basal plasma membranes. Therefore, certain isoforms of red cell membrane proteins are polarized between the two surfaces of the human placental syncytiotrophoblast. We propose that the localization of spectrin to the basal membrane is related to the less bundled organization of microfilaments at this membrane compared with that of the microvillous membrane. The band 3 isoforms are candidates for participation in maternofetal anion transport.     

Alterations in labeling of cell-surface glycoproteins from normal and diabetic rat intestinal microvillous membranes

The effect of chronic streptozotocin-induced diabetes was studied on intestinal microvillous membrane surface carbohydrate groups. After 7 weeks of diabetes, purified microvillous membranes were prepared from rat small intestine and surface galactoproteins identified by labeling with galactose oxidase/sodium boro[³H]hydride. Membrane surface sialic acid residues were labeled using the sodium metaperiodate/sodium boro[³H]hydride technique. Membranes were solubilized in SDS and protein labeling analyzed by acrylamide electrophoresis. Membranes from diabetic rats showed an 81% increase in galactoprotein labeling ($\text{P< 0.02}$) while labeling of sialic acid residues was unchanged. The greatest increase in galactoprotein labeling occurred in protein monomers of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 116 000–200 000, where there was a 155% increase in labeling ($\text{P< 0.005}$). These results indicate that intestinal microvillous membrane protein glycosylation is altered in chronic diabetes. This increase in surface membrane carbohydrates could explain the decreased rates of proteolytic degradation previously described for at least one microvillous protein. An increase in membrane galactose groups has also been noted in hepatocyte and kidney glomerular basement membranes, which suggests the presence of a systematic change in membrane protein glycosylation occurring as a result of the diabetic state.     

Ca2+ transport mediated by a synthetic neutral Ca2+-ionophore in biological membranes

The effect of a synthetic neutral ligand on the Ca²⁺ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca²⁺-ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca²⁺ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate or carbonylcyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$ (FCCP) are present, presumably to facilitate the diffusion of the charged Ca²⁺-ionophore complex across the hydrophobic core of the membrane.In mitochondria, the neutral ionophore promotes the active transport of Ca²⁺ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.     

Glucose transport from mother to fetus—A theoretical study

The factors that affect and govern the glucose transfer from maternal blood to the fetus are not completely deciphered. We present a steady state, one dimensional mathematical simulation which integrates the main mechanisms that have been shown to exist: metabolic consumption of the placenta, simple and facilitated diffusion via the two membranes of the microvillous and simple diffusion within the placenta. The model uses all available physiologic data we could collect. Numerical results indicate that the most crucial factor in determining the fetal glucose concentration is the facilitated diffusion process at the basal membrane or, more specifically: the permeability of the basal membrane and the density of the transporter GLUT1 on its faces. The gradient between the maternal and the fetal glucose concentration is important as is the metabolic consumption of the placenta. The diffusion within the placenta and the conditions that prevail at the apical microvillous plasma membrane are much less significant. Intrasyncytial concentration of glucose is close to that of maternal blood. The adjustment of the fetal glucose concentration to abrupt changes of its surrounding is estimated to be quite rapid hence for all practical purposes this steady state model can serve as a reasonable approximation. Parameters that await experimental determination are identified.     

Glycodeoxycholate transport in brush border membrane vesicles isolated from rat jejunum and ileum

The transport of the bile salt, glycodeoxycholate, was studied in vesicles derived from rat jejunal and ileal brush border membranes using a rapid filtration technique. The uptake was osmotically sensitive, linearly related to membrane protein and resembled d-glucose transport. In ileal, but not jejunal, vesicles glycodeoxycholate uptake showed a transient vesicle/medium ratio greater than 1 in the presence of an initial sodium gradient. The differences between glycodeoxycholate uptake in the presence and absence of a Na⁺ gradient yielded a saturable transport component. Kinetic analysis revealed a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value similar to that described previously in everted whole intestinal segments and epithelial cells isolated from the ileum. These findings support the existence of a transport system in the brush border membrane that: (1) reflects kinetics and characteristics of bile salt transport in intact intestinal preparations, and (2) catalyzes the co-transport of Na⁺ and bile salt across the ileal membrane in a manner analogous to d-glucose transport.     

Inhibition by dexamethasone of the in vitro transport of 3-O-methylglucose into rat thymocytes

Reduced glucose transport across the plasma membrane and reduced phosphorylation may both be responsible for the early inhibitory effect of physiological concentrations of glucocorticoids on glucose uptake by rat thymocytes.The early inhibitory effects of glucocorticoids (5 · 10^?7 M dexamethasone) on glucose consumption and ¹⁴CO₂ formation from d-[U-¹⁴C]glucose were reproduced.The total uptake curve of 4.8 μM $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyl-}\text{d}\text{-glucose}$ was biexponential with $\text{t}\text{1}\text{2}$ of 1.1 min and 36 min, respectively, the rapid part comprising about 50% of the equilibrated intracellular water space. The latency of the effect of 5 · 10^?7 M dexamethasone on $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyl-}\text{d}\text{-glucose}$ uptake ranged from 15 to 100 min and the inhibition varied from 15 to 55% independently of the lag period. The effect of 3-O-methylglucose concentration on the initial uptake by steroid-responsive cell preparations was tested after 45 min of preincubation with or without 5 · 10^?7 M dexamethasone. In 12 experiments dexamethasone reduced V from 1.36 ± 0.16 mmol · min^?1 · l^?1 cell water to 0.81 ± 0.10 mmol · min^?1 · l^?1 cell water with insignificant change of K_m (6.0 mM versus 5.9 mM). Dexamethasone had similar effect after 90 or 120 min.The variabilities of control cell transport capacity, the lag period and the magnitude of the dexamethasone effect could not be accounted for by changes in pH, effects of cell density, concentrations of albumin, ethanol, nucleosides, pyruvate or correlated to age and sex of the rats. In conclusion the inhibition of glucocorticoids on glucose consumption by thymocytes appears to be an inhibited plasma membrane transport capacity.     

Evidence against proton gradient formation being the cause of chlorophyll fluorescence quenching by N-methylphenazonium methosulfate

In strong illumination, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-poisoned chloroplasts exhibit a high yield of chlorophyll fluorescence while $\text{P-700}$ turnover, proton uptake, and phosphorylation are inhibited and a pH gradient is undetectable. When 10 μM $\text{N-}\text{methylphenazonium}$ methosulfate (PMS) is included, the fluorescence yield in light is substantially reduced, and when 100 μM ascorbate is also included, the yield is diminished approximately to the level in darkness. Only very slight increases in $\text{P-700}$ turnover and proton uptake (but no detectable pH gradient) accompany the fluorescence yield decline.When 10 μM PMS and 15 mM ascorbate are added to poisoned chloroplasts (the oxygen concentration being greatly reduced), $\text{P-700}$ turnover, proton uptake, the pH gradient and phosphorylation all reach high levels. In this case, the yield of chlorophyll fluorescence is low and is the same in both light and dark. Further addition of an uncoupler eliminates proton uptake, the pH gradient and phosphorylation but does not significantly elevate the fluorescence yield. From these observations we suggest that, in DCMU-poisoned chloroplasts, the fluorescence quenching with PMS occurs by a mechanism unrelated to the generation of a phosphorylation potential.With chloroplasts unpoisoned by DCMU, PMS quenches fluorescence and considerably stimulates proton uptake, the pH gradient and phosphorylation. However, in this case, PMS serves to restore net electron transport.     

Selective fetal malnutrition: The effect of ethanol and acetaldehyde upon in vitro uptake of alpha amino isobutyric acid by human placenta

Uptake of alpha amino isobutyric acid was measured in human placental villus tissue exposed $\text{in vitro}$ to ethyl alcohol (ethanol) (0.3 g/dl–2 g/d1) or acetaldehyde (50 μM-20 mM). Ethanol and acetaldehyde significantly inhibited uptake of amino acid at higher, pharmacologic concentrations (2 g/dl and 2–20 mM respectively). Inhibition by 10 mM acetaldehyde was partially reversible. The results suggest that the human placenta is resistant to acute ethanol-associated effects upon amino acid transport $\text{in vitro}$. However, both ethanol and its major circulating metabolite, acetaldehyde, may still alter placental function during $\text{in vivo}$ chronic exposure.     

Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases of Neurospora crassa

A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. $\text{N,N′-}\text{Dicyclohexylcarbodiimide}$, octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, $\text{N-1-}\text{napthylphthalamate}$, triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H⁺ transport across their respective membranes.     

Partial characterization of the glucose transport activity in the golgi-rich fraction of fat cells

The glucose transport activity solubilized from the basal and plus insulin forms of the Golgi-rich fraction of adipocytes was partially characterized, and the results were compared with those of the activity obtained from the plus insulin form of the plasma membrane-rich fraction. The transport activity was determined in a cell-free, reconstituted, system. Prior to reconstitution, the activities in the three preparations were all (a) stable at 0°C for at least 4 h, but not at 37°C or above; (b) most stable at pH 7–9, and (c) less stable in Tes than in Tris buffer. After reconstitution, the three activities were all (d) stable at 0°C, (e) most active at pH 5.5, (f) mildly stimulated by divalent cations, (g) unaffected by insulin or 1 mM of several SH-blocking agents, (h) inhibited by heavy metal ions, 10–100 mM of monovalent salts, organic solvents, several sugar isomers, and specific sugar-transport inhibitors. The rates of d-glucose uptake by the three liposome preparations were all inhibited more strongly by 2-deoxy-d-glucose or $\text{3}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ than by d-glucose. These data indicate that the general properties of the glucose transport activity in the Golgi-rich fraction are similar to those of the activity in the plasma membrane-rich fraction.     

Transfer of sodium ions across the erythrocyte membrane in manic — Depressive illness: Treatment with lithium carbonate

The efflux of ²²Na from erythrocytes was measured $\text{in vitro}$ under experimental conditions such that rate constants due to efflux either by active transport, passive diffusion, or exchange diffusion could be calculated. No significant differences were found in the rate constants for ²²Na efflux between seven male bipolar depressed patients and eight male control subjects, who had no personal or family history of psychiatric illness. Treatment of patients with lithium carbonate either for less than one week or for 4–5 weeks produced no changes in the rate constants describing the efflux of ²²Na from red cells. Also, addition of 1mM LiCl $\text{in vitro}$ did not alter the active transport of ²²Na from erythrocytes. These data provide no evidence for either an abnormality in ²²Na transfer across the red cell membrane of bipolar depressives or an effect of the lithium ion upon such transfer.     

Role of the plasma membrane in the mechanism of cholesterol efflux from cells

In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [³H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu₅AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg / ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for at least one-third of the cell cholesterol of 3.2 ± 0.6 and 14.3 ± 1.5 h, respectively. Plasma membrane vesicles (0.5–5.0 μm diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values for plasma membrane vesicles of Fu₅AH and WIRL cells are 3.9 ± 0.5 and 11.2 ± 0.7 h, respectively. These $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rate indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu₅AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu₅AH and WIRL cells were the same, with values of 3.1 ± 0.1 and 2.9 ± 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values for cholesterol efflux from these cell lines.     

Phenylalanine uptake in isolated renal brush border vesicles

The uptake of l-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques.Brush border microvilli but not basolateral plasma membrane vesicles take up l-phenylalanine by an Na⁺-dependent, saturable transport system. The apparent affinity of the transport system for l-phenylalanine is 6.1 mM at 100 mM Na⁺ and for Na⁺ 13 mM at 1 mM l-phenylalanine. Reduction of the Na⁺ concentration reduces the apparent affinity of the transport system for l-phenylalanine but does not alter the maximum velocity.In the presence of an electrochemical potential difference for Na⁺ across the membrane (η_Na0 >η_Na1) the brush border microvilli accumulate transiently l-phenylalanine over the concentration in the incubation medium (overshoot phenomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K⁺ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient.These results indicate that the entry of l-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na⁺ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na⁺ and on the electrical potential difference. The exit of l-phenylalanine across the basolateral plasma membranes is Na⁺-independent and probably involves facilitated diffusion.