Reversible independent alterations in glucose transport and metabolism in cultured human cells deprived of glucose

During cell division in the Xenopus egg (diameter 1.25 mm) new cell membrane is formed in the furrow region (rate of growth approx 4-10(4) mum2/min). Freeze-fracture electron microscopy has produced the following data. Preexisting plasma membrane faces show a reversed polarity with respect to particle distribution, i.e. more particles are attached to the E-face (density 1600-2200 particles/mum2) than to the P-face (300 particles/mum2). A frequency histogram of 2331 measured intramembranous particles does not show a continuous range of sizes. The following sizes were very obvious: 95 A (12%), 125 A (30%) and 180 A (6%). At the tips of surface protrusions both the E- and the P- face are particle-free. Nascent cell membrane fracture faces are more difficult to obtain. The particle density is low (E-face 300-500 particles/mum2). Lowering the ambient temperature to 5 degrees C for approx. 5 mins does not change the normal particle pattern, but it improves the output in nascent membrane fracture faces. The fact that in the Xenopus egg preexisting and nascent membrane regions are continuous but nevertheless maintain their highly different particle densities is noteworthy. The freeze-fracture data are discussed in relation to, among other things, the known values of the specific resistances of these membrane regions.     

Proteins and glycoproteins of membranes from developing chick red cells

Membrane vesicles produced when chick erythroid cells are disrupted by nitrogen cavitation were isolated by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Sodium dodecyl sulfate-polyacrylamide gel analysis of isolated membranes shows eight to ten proteins and four to five glycoproteins. Membranes must be prepared with protease inhibitors, otherwise an endogenous activity degrades high molecular weight polypeptide components. Red cells from several stages of development (5- and 17-day embryos and adult chickens) all appear to have the same major embrane proteins. However, primitive erythroid cells from 5-day embryos lack a Mr = 40,000 glycoprotein that is present in definitive erythrocytes from 17-day embryos and from adult chickens; erythrocytes from young chicks show a decrease in the amount of a glycoprotein of Mr = 50,000. Lactoperoxidase-catalyzed iodination of intact 5-day embryonic red cells detects three surface components which comigrate with the membrane glycoproteins on sodium dodecyl sulfate polyacrylamide gels.     

In vitro modulation of L-arginine transport in trophoblast cells by glucose

BACKGROUND: The uptake of the semi-essential amino acid, L-arginine, into trophoblast cells was measured with the aim of determining the effect of different glucose concentrations on L-arginine influx kinetics. METHODS: This study used a novel superfused microcarrier culture system of BeWo cells (an established choriocarcinoma cell line with many characteristics of normal human trophoblast) and a rapid, paired-tracer dilution technique to measure unidirectional influx into the cells. RESULTS: At 10 mmol L-1 D-glucose, L-arginine unidirectional influx across the microvillous border of the cells was saturable with a Km of 1.14 +/- 0.14 mmol L-1 and a Vmax of 121. 36 +/- 5.89 nmol mg-1 protein min-1. When cells were preincubated for 24 h in the presence of 30 mmol L-1 D-glucose, there was a significant increase in the Vmax for L-arginine of nearly 30%. Similarly, preincubation in the presence of 1 mmol L-1 D-glucose and 12.5 mIU mL-1 human insulin reduced the Km for L-arginine influx by over 55%. CONCLUSION: These data suggest that the modulation of placental transport of L-arginine by glucose and insulin could contribute to the fetal macrosomia observed in diabetic mothers.     

Contraction of developing avian heart muscle

26 patients with at least one clinical symptom that could indicate a tear of the scapho-lunate interosseous ligament but normal static and dynamic radiographs were examined by arthroscopy. We found that a diagnosis of scapho-lunate instability could be established by dynamic manoeuvres during radio-carpal and mid-carpal arthroscopy. Five patients were found to have true scapholunate instability. Six tears of the interosseous ligament without instability were also detected but it was doubtful if the tear was the origin of the painful symptomatology. This experience suggests that dynamic manoeuvres during arthroscopy are superior to other methods in diagnosing scapholunate instabilities at the pre-radiographic stage.     

Intracellular glucose concentration in derepressed yeast cells consuming glucose is high enough to reduce the glucose transport rate by 50%

1. This study describes the in vitro characterization of two potent and selective 5-HT6 receptor antagonists at the rat and human recombinant 5-HT6 receptor. 2. In binding assays with [3H]-LSD, 4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) and 4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulphonamide (Ro 63-0563) had mean pKi values +/-s.e.mean at the rat 5-HT6 receptor of 7.35+/-0.04 and 7.83+/-0.01, respectively and pKi values at the human 5-HT6 receptor of 7.26+/-0.06 and 7.91+/-0.02, respectively. 3 .Both compounds were found to be over 100 fold selective for the 5-HT6 receptor compared to 23 (Ro 04-6790) and 69 (Ro 63-0563) other receptor binding sites. 4. In functional studies, neither compound had any significant effect on basal levels of cyclicAMP accumulation in Hela cells stably expressing the human 5-HT6 receptor, suggesting that the compounds are neither agonists nor inverse agonists at the 5-HT6 receptor. However, both Ro 04-6790 and Ro 63-0563 behaved as competitive antagonists with mean +/-s.e.mean pA2 values of 6.75+/-0.07 and 7.10+/-0.09, respectively. 5. In rats habituated to observation cages, Ro 04-6790 produced a behavioural syndrome similar to that seen following treatment with antisense oligonucleotides designed to reduce the expression of 5-HT6 receptors. This behavioural syndrome consisted of stretching, yawning and chewing. 6. Ro 04-6790 and Ro 63-0563 represent valuable pharmacological tools for the identification of 5-HT6 receptors in natural tissues and the study of their physiological function.     

Membrane dynamics of the water transport protein aquaporin-1 in intact human red cells

Aquaporin-1 (AQP1) is the prototype integral membrane protein water channel. Although the three-dimensional structure and water transport function of the molecule have been described, the physical interactions between AQP1 and other membrane components have not been characterized. Using fluorescein isothiocyanate-anti-Co3 (FITC-anti-Co3), a reagent specific for an extracellular epitope on AQP1, the fluorescence photobleaching recovery (FPR) and fluorescence imaged microdeformation (FIMD) techniques were performed on intact human red cells. By FPR, the fractional mobility of fluorescently labeled AQP1 (F-alphaAQP1) in the undeformed red cell membrane is 66 +/- 10% and the average lateral diffusion coefficient is (3.1 +/- 0.5) x 10(-11) cm2/s. F-alphaAQP1 fractional mobility is not significantly affected by antibody-induced immobilization of the major integral proteins band 3 or glycophorin A, indicating that AQP1 does not exist as a complex with these proteins. FIMD uses pipette aspiration of individual red cells to create a constant but reversible skeletal density gradient. F-alphaAQP1 distribution, like that of lipid-anchored proteins, is not at equilibrium after microdeformation. Over time, approximately 50% of the aspirated F-alphaAQP1 molecules migrate toward the membrane portion that had been maximally dilated, the aspirated cap. Based on the kinetics of migration, the F-alphaAQP1 lateral diffusion coefficient in the membrane projection is estimated to be 6 x 10(-10) cm2/s. These results suggest that AQP1 lateral mobility is regulated in the unperturbed membrane by passive steric hindrance imposed by the spectrin-based membrane skeleton and/or by skeleton-linked membrane components, and that release of these constraints by dilatation of the skeleton allows AQP1 to diffuse much more rapidly in the plane of the membrane.     

Short-term exercise enhances insulin-stimulated GLUT-4 translocation and glucose transport in adipose cells

This investigation examined the effects of short-term exercise training on insulin-stimulated GLUT-4 glucose transporter translocation and glucose transport activity in rat adipose cells. Male Wistar rats were randomly assigned to a sedentary (Sed) or swim training group (Sw, 4 days; final 3 days: 2 x 3 h/day). Adipose cell size decreased significantly but minimally (approximately 20%), whereas total GLUT-4 increased by 30% in Sw vs. Sed rats. Basal 3-O-methyl-D-[14C]glucose transport was reduced by 62%, whereas maximally insulin-stimulated (MIS) glucose transport was increased by 36% in Sw vs. Sed rats. MIS cell surface GLUT-4 photolabeling was 44% higher in the Sw vs. Sed animals, similar to the increases observed in MIS glucose transport activity and total GLUT-4. These results suggest that increases in total GLUT-4 and GLUT-4 translocation to the cell surface contribute to the increase in MIS glucose transport with short-term exercise training. In addition, the results suggest that the exercise training-induced adaptations in glucose transport occur more rapidly than previously thought and with minimal changes in adipose cell size.     

Purinergic inhibition of glucose transport in cardiomyocytes

ATP is known to act as an extracellular signal in many organs. In the heart, extracellular ATP modulates ionic processes and contractile function. This study describes a novel, metabolic effect of exogenous ATP in isolated rat cardiomyocytes. In these quiescent (i.e. noncontracting) cells, micromolar concentrations of ATP depressed the rate of basal, catecholamine-stimulated, or insulin-stimulated glucose transport by up to 60% (IC50 for inhibition of insulin-dependent glucose transport, 4 microM). ATP decreased the amount of glucose transporters (GLUT1 and GLUT4) in the plasma membrane, with a concomitant increase in intracellular microsomal membranes. A similar glucose transport inhibition was produced by P2 purinergic agonists with the following rank of potencies: ATP approximately ATPgammaS approximately 2-methylthio-ATP (P2Y-selective) > ADP > alpha,betameATP (P2X-selective), whereas the P1 purinoceptor agonist adenosine was ineffective. The effect of ATP was suppressed by the poorly subtype-selective P2 antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid but, surprisingly, not by the nonselective antagonist suramin nor by the P2Y-specific Reactive Blue 2. Glucose transport inhibition by ATP was not affected by a drastic reduction of the extracellular concentrations of calcium (down to 10(-9) M) or sodium (down to 0 mM), and it was not mimicked by a potassium-induced depolarization, indicating that purinoceptors of the P2X family (which are nonselective cation channels whose activation leads to a depolarizing sodium and calcium influx) are not involved. Inhibition was specific for the transmembrane transport of glucose because ATP did not inhibit (i) the rate of glycolysis under conditions where the transport step is no longer rate-limiting nor (ii) the rate of [1-14C]pyruvate decarboxylation. In conclusion, extracellular ATP markedly inhibits glucose transport in rat cardiomyocytes by promoting a redistribution of glucose transporters from the cell surface to an intracellular compartment. This effect of ATP is mediated by P2 purinoceptors, possibly by a yet unknown subtype of the P2Y purinoceptor family.     

The effect of ATP-depletion on the inhibition of glucose exits from human red cells

Proliferation and differentiation of epithelial cells are thought to be regulated by soluble factors in extracellular fluid and insoluble components of the extracellular matrix. We have examined the combined effects of soluble factors and an extracellular matrix (EHS matrix) on DNA synthesis, cell proliferation, and surfactant protein gene expression in primary cultures of alveolar type II epithelial cells. Cells on EHS matrix cultured in DMEM containing insulin, cholera toxin, EGF, aFGF, 5% rat serum, and 15-fold concentrated bronchoalveolar lavage fluid (D-GM) formed larger aggregates than cells cultured on the same substratum in DMEM containing 5% rat serum (D-5). Cells cultured in D-GM on EHS matrix incorporated more [3H]-thymidine than cells on the same substratum in D-5, with an eight-fold increase seen on day 4 of culture. This increase in [3H]-thymidine incorporation was accompanied by a labeling index of greater than 65% of the cells. Cell counts showed that exposure of type II cells on EHS matrix to D-GM resulted in increased cell number on day 4 of culture. [3H]-thymidine autoradiography combined with immunostaining with anti-cytokeratin, anti-SP-A, and anti-vimentin antibodies demonstrated that the proliferating cells were epithelial cells that contained SP-A. Type II cells cultured on plastic in D-GM also showed increased [3H]-thymidine incorporation compared to cells cultured in D-5. The level of [3H]-thymidine incorporation by cells on plastic, however, was significantly less than that seen in cells cultured in the same medium on EHS matrix. Type II cells cultured on EHS matrix in D-GM had a decreased abundance of mRNAs for SP-A and SP-C than cells cultured on EHS matrix in D-5 as determined by Northern analysis. This inhibition was reversed by switching from D-GM to D-5 on day 4 and culturing the cells for an additional 4 days. In contrast, SP-B mRNA was increased in response to D-GM. This increase was not reversed by switching from D-GM to D-5 on day 4. These results suggest that the interaction of soluble factors and extracellular matrix components has a strong influence on type II cell proliferation, which were partially associated with the reversible inhibition of lung tissue-specific protein mRNAs. Their dynamic interplay among the type II cell, the extracellular matrix, and growth factors may determine multicellular functions and play an important role in normal lung development and in the repair of the lung epithelium following injury.     

Hypergravity effects on normal and avulsed developing avian radii

Rhode Island Red female chicks were subjected to complete closed fracture of the right radius at 2 weeks post-hatching. The animals were allowed to heal for 1 week at either earth gravity or 2 G hypergravity state with control and estrogen-injected groups. Intact and fractured radial length, weight, average epiphysial-diaphysial diameters, and length, width, and weight of healing fracture callus were measured. Daily 2000 IU estrogen administration for 7 d increased intact radial length. Estrogen augments the effects of the 2-G state by inhibiting growth and depleting the mass of both intact and fractured radii and by decreasing the average distal epiphysial diameter of fractured bones. Animals exposed to the hypergravity state without hormonal treatment showed decreased fractured radial length, weight, and smaller proximal epiphysial diameters. The measurable parameters of the fracture callus (width, length, and weight) were depressed by the hypergravity state regardless of whether the animal was untreated or supplemented with estrogen.     

Hereditary variation in uric acid transport by avian kidney slices

Uric acid transport in renal cortical slices from a selected line of hyperuricemic chickens was investigated. Slices from the hyperuricemic (HUA) line accumulated less than half as much uric acid as slices from a control (LUA) line when uric acid in the medium varied from 0.01 to 5 mM. Uric acid uptake by both lines increased as the uric acid concentration in the medium was raised from 0.1 to 0.5 mM, but was markedly inhibited in the HUA line at 3-5 mM. Omission of sodium or potassium from the incubation medium inhibited uric acid uptake by slices from both lines. Ouabain inhibited uric acid uptake in the LUA line. The sodium and potassium requirements for initiation of uric acid uptake were higher, and the potassium requirement for maximal uptake was lower, for slices of the HUA line. No genetic differences in potassium or sodium contents of slices were observed when the potassium content of the incubation medium was altered or when the medium contained ouabain. These studies indicate that hereditary hyperuricemia in chickens may be due to a qualitative change in renal uric acid transport which involves the interaction of cations in the transport process.     

Differential distribution of agrin isoforms in the developing and adult avian retina

At the developing and regenerating neuromuscular junction, agrin is responsible for the formation of aggregates containing the acetylcholine receptor (AChR) and other molecules. Multiple isoforms of agrin are generated by alternative splicing, and the presence of an 8, 11, or 19 (8 + 11) amino acid insert at splice site B is required for agrin's AChR aggregation activity. An antiserum was generated against the 19 amino acid peptide which reacted specifically with the B11 and B19 agrin isoforms. The antiserum blocked the formation of agrin-induced AChR aggregates on myotubes, but the peptide itself had no aggregation activity, suggesting that agrin's active site is close to the splice site, but not the peptide itself. In the embryonic and adult retina anti-peptide immunoreactivity was concentrated in the synapse-containing layers. In contrast, the inner limiting membrane and radial cells, which express strong immunoreactivity with a pan-specific anti-agrin antiserum, were not stained by the anti-peptide antiserum, showing that agrin isoforms are differentially distributed in the retina. In addition, agrin B11 and B19 isoforms were associated with ganglion cell axons, particular at early developmental stages before synapse formation, indicating additional functions for these isoforms in the developing CNS.     

Long-term culture of avian embryonic cells in vitro

The pH of the embryonic blood, one of the most important environmental factors for embryonic cells, was found to range from 8.1 to 8.5 in chick embryos until 108 h after incubation. Based on these results, the culture medium adjusted to pH 8.0 was used to culture embryonic chick and quail cells. They were easily subcultured for a long period of time at pH 8.0. This pH culture condition may have wide application for manipulating embryonic cells or tissues and establishing cell lines from avian embryos.     

D-Glucose transport in cultured cells of neural origin: the membrane as possible control point of glucose utilization

The function of plasma membrane as control point of glucose metabolism has been studied in confluent monolayer of C1300 neuroblastoma (N2A) and glioma (C6) cells. In neuroblastoma, steady state intracellular glucose concentration reached the extracellular levels, while intracellular contents in C6 glioma cells remained very low. In C6 glial cells the amount of glycogen as source of energy was much higher than that found in C1300 neuroblastoma cells. Influx rates of D-glucose in C6 glioma cells were only half those found in neuroblastoma cells. During the influx period (0-40 s) the transport of glucose in these cells did not exceed the phosphorylation rate, whereas a steady, time-dependent increase in glucose content was observed in neuroblastoma cells. While glucose uptake in neuroblastoma cells seems to be regulated at the level of phosphorylating enzymes, the control point in C6 glioma is believed to be membrane transport.     

Cerebral glucose transport and metabolism in preterm human infants

Few data regarding early developmental changes in cerebral (blood-to-brain) glucose transport (CTXglc) and CMRglc are available for humans. We measured CBF, CTXglc, and CMRglc with positron emission tomography at 4 to 7 days of life in six preterm human infants whose estimated gestational age was 25 to 34 weeks. The Michaelis-Menten constants Kt and Tmax were estimated from CTXglc and the calculated cerebral capillary plasma glucose concentration. Mean CMRglc was 8.8 mumol 100 g-1 min-1. The CMRglc did not correlate with plasma glucose concentration (r = .315, P = .543), whereas CTXglc showed a significant correlation with plasma glucose concentration (r = .836, P = .038). Estimation of the Michaelis-Menten constants from the best fit to the measured data produced values of Kt = 6.0 mumol mL-1 and Tmax = 32.6 mumol 100 g-1 min-1. These values for Kt in the developing human brain are similar to those that have been reported for the mature brain of adolescent and adult humans and adult nonhuman primates, indicating the affinity of the glucose transport protein for D-glucose is similar. However, Tmax is approximately one third to one half of the comparable values for mature brain, indicating a reduced number of available luminal transporters.     

Specificity of the glucose transport system in Leishmania tropica promastigotes

The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2-deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has a Q10 of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (alpha-methyl-D-glucoside, 1,5-anhydroglucitol) and carbon 3 (3-0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature--they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident; therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.     

Hydroxyurea affects cell morphology, cation transport, and red blood cell adhesion in cultured vascular endothelial cells

Hydroxyurea (HU) significantly increases fetal hemoglobin (Hb) production and concomitantly affects passive erythrocyte K transport and cell volume in patients homozygous for Hb S, thus decreasing disease severity. Red blood cells (RBCs) with Hb S display a greater adherence to vascular endothelial cells (VECs) than do Hb A cells, thus increasing the probability of vaso-occlusive crisis. The effect of HU on the structure and function of VECs is still unknown. In the present study, HU significantly changed, in a dose-dependent manner, the morphology and monovalent cation composition of cultured VECs after incubation in normal culture medium for up to 10 days in the absence and presence of 0.3 (therapeutic dose) and 3.0 (toxic dose) mmol/L HU. Treated cells showed significant morphologic changes such as an increase in apparent cell size and the formation of multinucleated giant cells. The protein content per dish decreased by 50% and 80% at 0.3 and 3.0 mmol/L HU, respectively, accompanied by an increase in cell Na (maximum, approximately 200%) and cell K (maximum, approximately 50%) contents at about days 4 to 6 and 8 to 10, respectively. In addition, HU decreased RBC adherence to VECs in experiments with 51Cr-loaded Hb A or Hb S RBCs. The HU-induced changes in VEC morphology, cation composition, and RBC adherence may be caused or accompanied by alterations in cell membrane permeability, transformation of endothelial cells, or decreased number/density of VEC adhesion molecules. Precise mechanisms of the HU effects warrant further investigation in light of the reported beneficial effects of HU in the treatment of sickle cell anemia.