Antibody raised against the human erythrocyte glucose transporter identified a recombinant Xgtll bacteriophage in a cDNA library prepared from immunoselected polysomal RNA from adult rat brain. The cDNA predicts a 492-amino acid protein that demonstrates 97.6% identity to the human hepatoma hexose carrier. The tissue distribution of the transporter mRNA is identical to that of immunologically identifiable protein and transport activity, except in liver in which high levels of transport are associated with little or no transporter mRNA or protein. As assayed by blot-hybridization analysis, mRNA from insulin-responsive and nonresponsive tissues are indistinguishable. These data suggest that a genetically unrelated protein is responsible for hexose transport in normal liver.

Elevation of glucose transport is an alteration common to most virally induced tumors. Rat fibroblasts transformed with wild-type or a temperature-sensitive Fujinami sarcoma virus (FSV) were studied in order to determine the mechanisms underlying the increased transport. Five- to tenfold increases in total cellular glucose transporter protein in response to transformation were accompanied by similar increases in transporter messenger RNA levels. This, in turn, was preceded by an absolute increase in the rate of glucose transporter gene transcription within 30 minutes after shift of the temperature-sensitive FSV-transformed cells to the permissive temperature. The transporter messenger RNA levels in transformed fibroblasts were higher than those found in proliferating cells maintained at the nonpermissive temperature. The activation of transporter gene transcription by transformation represents one of the earliest known effects of oncogenesis on the expression of a gene encoding a protein of well-defined function.

No abstract

The accumulation of the lipophilic cation, triphenylmethylphosphonium, has been employed to determine the resting membrane potential in human erythrocytes, turkey erythrocytes, and rat white adipocytes. The triphenylmethylphosphonium cation equilibrates rapidly in human erythrocytes in the presence of low concentrations of the hydrophobic anion, tetraphenylborate. Tetraphenylborate does not accelerate the uptake of triphenylmethylphosphonium ion by adipocytes. The cell associated vs. extracellular distribution of the triphenylmethylphosphonium ion is proportional to changes in membrane potential. The distribution of this ion reflects the membrane potential determining concentration of the ion with dominant permeability in a "Nernst" fashion. The resting membrane potentials for the human erythrocyte, turkey erythrocyte, and rat white adipocyte were found to be -8.4 +/- 1.3, -16.8 +/- 1.1, and -58.3 +/- 5.0 mV, respectively, values which compare favorably with values obtained by other methods. In addition, changes in membrane potential can be assessed by following triphenylmethylphosphonium uptake without determining the intracellular water space. The method has been successfully applied to a study of hormonally induced changes in membrane potential of rat white adipocytes.

1,3-dipyrenylpropane (PC3P) and bis(4-biphenylmethyl)ether, two molecules that form intramolecular excimers, were embedded in phospholipid vesicles and biological membranes to monitor dynamic properties of membrane lipids. Excimer formation was evaluated from determinations of excimer to monomer emission intensity ratios (ID/IM). ID/IM values of PC3P and bis(4-biphenylmethyl)ether were reduced when cholesterol was added to egg lecithin vesicles. PC3P was sensitive to the temperature-induced crystalline to liquid-crystalline phase transition in dimyristoyl phosphatidylcholine vesicles. For studies of cellular membranes, membranes, PC3P was used exclusively, because of the fluorescence of tryptophan residues of membrane proteins interferes with the responses bis(4-biphenylmethyl)ether. Microviscosities of membrane interiors were calculated from standard curves of IM/ID plotted against solvent viscosity. Microviscosity values of egg lecithin vesicles and biological membranes, especially those obtained with PC3P, were more than an order of magnitude lower than values obtained by other techniques. We concluded that the intramolecular process leading to the formation of the excimer is influenced differently in isotropic solvents than in anisotropic environments, such as lipid bilayers. Although distinguishable ID/IM ratios can be obtained for different biological membranes (mitochondrial, microsomal, and plasma membranes were studied), this parameter may be phenomenological and not simply related to membrane microviscosity. As such, fluorescent probes that form intramolecular excimers are of value in making qualitative comparisons of different membranes and in studying the relative effects of physical changes and chemical agents on membrane structure. These probes may also be valuable for studying structural anisotropy of biological membranes.

Antibody to the carboxyl-terminal of hexose transporter protein GLUT-1 was used to localize this carrier in normal rat kidney (NRK) cells during D-glucose (Glc) deprivation. Glc-deprivation of NRK cells induces increased hexose transport, inhibits the glycosylation of GLUT-1, and increases the content of both native, 55,000 apparent mol wt (Mr) and aglyco, 38,000 Mr GLUT-1 polypeptides. The distribution of GLUT-1 protein in subcellular fractions isolated from Glc-fed NRK cells shows that the 55,000 Mr polypeptide is most abundant in intracellular membrane fractions. Glc-fed cells that have been tunicamycin treated contain principally the 38,000 Mr GLUT-1 polypeptide, which is found predominantly in intracellular membrane fractions. In Glc-deprived cells the 55,000 Mr GLUT-1 polypeptide localizes predominantly in the Golgi and plasma membrane fractions, whereas the more abundant 38,000 Mr GLUT-1 polypeptide is distributed throughout all membrane fractions. In Glc-deprived but fructose-fed cells only the 55,000 Mr GLUT-1 polypeptide is detected, and it is found predominantly in the plasma membrane and Golgi fractions. The localization of GLUT-1 protein was directly and specifically visualized in NRK cells by immunofluorescence microscopy. Glc-fed cells show little labeling of cell borders and a small punctate juxtanuclear pattern suggestive of localization to the Golgi and, perhaps, endoplasmic reticulum. Glc-fed cells that have been tunicamycin treated show large punctate intracellular accumulations suggestive of localization to distended Golgi and perhaps endoplasmic reticulum. Glc-deprived cells exhibited intense labeling of cell borders as well as intracellular accumulations. Glc-deprived but fructose-fed cells show fewer intracellular accumulations, and the labeling is, in general, limited to the cell borders. Our results suggest that Glc deprivation induces the selective accumulation of GLUT-1 in the plasma membrane of NRK cells.

No abstract

Barbiturates reduce cerebral blood flow, metabolism, and Glc transfer across the blood-brain barrier. The effect of barbiturates on hexose transport in cultured mammalian cell lines and human erythrocytes was studied. Pentobarbital inhibits [3H]-2-dGlc uptake in 3T3-C2 murine fibroblasts by approximately 95% and approximately 50% at 10 and 0.5 mM, respectively. Uptake of [3H]-2-dGlc is linear with time in the presence or absence of pentobarbital, and the percent inhibition is constant. This suggests that hexose transport, not phosphorylation, is inhibited by barbiturates. Inhibition by pentobarbital of hexose transport in 3T3-C2 cells is rapid (< 1 min), is not readily reversible, is not altered by the presence of albumin [1% (w/v)], and is independent of temperature (4-37 degrees C) and the level of cell surface GLUT-1. The IC50's for inhibition of hexose transport in 3T3-C2 cells by pentobarbital, thiobutabarbital, and barbital are 0.8, 1.0, and 4 mM, respectively. This is consistent with both the Meyer-Overton rule and the pharmacology of barbiturates. Neither halothane (< or = 10 mM) nor ethanol [< or = 0.4% (v/v)] significantly inhibits hexose transport. Inhibition by pentobarbital (0.5 mM) of [3H]-2-dGlc uptake by 3T3-C2 cells decreases the apparent Vmax (approximately 50%) but does not alter the apparent Km (approximately 0.5 mM). Inhibition of hexose transport by barbiturates, but not ethanol [< or = 0.4% (v/v)], is also observed in human erythrocytes and four other cultured mammalian cell lines. Pentobarbital quenches (Qmax approximately 75%) the intrinsic fluorescence of purified and reconstituted GLUT-1 (Kd approximately 3 mM). Quenching is independent of Glc occupancy, is unchanged by mild proteolytic inactivation, and does not appear to directly involve perturbations of the lipid bilayer. We propose that barbiturates can interact directly with GLUT-1 and inhibit the intrinsic activity of the carrier. Glc crosses the blood-brain barrier primarily via the GLUT-1 of the endothelial cells of cerebral capillaries. Partial inhibition of this process by barbiturates may be of significance to cerebral protection.