Study on the effect of hypergravity stress on L-glutamate uptake by rat brain nerve endings

Using rat brain synaptosomes, we have investigated the effect of hypergravity on the kinetic parameters of Na(+)-dependent, high-affinity L-glutamate transport activity. The time-course of L-[14C]-glutamate uptake and dependence of L-[14C]-glutamate uptake velocity on glutamate concentrations were analyzed. K(m) and Vmax of this process have been determined. The hypergravity stress was created by centrifugation of rats for 1 hour at 10 g. We observed no differences in K(m) values between the control rats (10.7 +/- 2.5 microM) and animals exposed to hypergravity (6.7 +/- 1.5 microM). The similarity of this parameter for the two studied groups of animals showed that affinity of glutamate transporter to substrate was not sensitive to hypergravity stress. In contrast, the maximal velocity of glutamate uptake changed in hypergravity conditions. Vmax reduced from 12.5 +/- +/- 3.2 nmol/min per 1 mg of protein (control group) to 5.6 +/- 0.9 nmol/min per 1 mg of protein (animals, exposed to hypergravity stress). The possible mechanisms of attenuation of the glutamate transporter activity without modifying K(m) of glutamate uptake were discussed.     

Energy requirement for L-glutamate uptake and utilization by Hansenula subpelliculosa cells

Shieh, K. Z. (Illinois Institute of Technology, Chicago), and L. R. Hedrick. Energy requirement for l-glutamate uptake and utilization by Hansenula subpelliculosa cells. J. Bacteriol. 92:1638-1644. 1966.-Cells of the yeast Hansenula subpelliculosa require an energy source for the uptake of glutamate. A lag period of 20 to 40 min was required after the addition of glucose to the cells before glutamate uptake was initiated. When cells were preincubated in glucose, and washed with distilled water prior to the addition of glutamate, there was no lag period. Preincubation in glucose and glutamate lowered both the rate and the total uptake of glutamate as compared with cells preincubated in glucose alone. This is attributed to the partial utilization of the glucose-metabolite by glutamate or to the partial saturation of binding sites by glutamate during the preincubation period. Transport of glutamate by these yeast cells appears to be via a carrier, where energy is required for the binding of the amino acid to nonspecific binding sites. In addition to total uptake, some aspects of the C(14)-glutamate utilization were measured. Of the total uptake, 58% was metabolized and converted to CO(2), 25.2% remained in the soluble pool, and 16.8% was incorporated into trichloroacetic acid-insoluble products. When the available energy source was depleted, the processes of uptake, metabolism, and incorporation ceased, even though there was an ample supply of glutamate present within the cells. Removal of cells from glutamate and addition of glucose reinitiated the incorporation of glutamate into proteins and other trichloroacetic acid-insoluble compounds. Therefore, an additional energy source is required with this species of yeast for glutamate uptake, for the priming of mechanisms required for its metabolism, and for its incorporation.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

Glucose Metabolism in Sediments of a Eutrophic Lake: Tracer Analysis of Uptake and Product Formation

The uptake of glucose and the formation of end products from glucose catabolism have been measured for sediments of eutrophic Wintergreen Lake with a combination of tritiated and ¹⁴C-labeled tracers. Time course analyses of the loss of [³H]glucose from sediments were used to establish rate constants for glucose uptake at natural substrate concentrations. Turnover times from these analyses were about 1 min for littoral and profundal sediments. No seasonal or site differences were noted in turnover times. Time course analyses of [U-¹⁴C]glucose uptake and ¹⁴C-labeled end product formation indicated that glucose mass flow could not be calculated from end product formation since the specific activity of added [¹⁴C]glucose was significantly diluted by pools of intracellular glucose and glucose metabolites. Mass flow could only be accurately estimated by use of rates of uptake from tracer studies. Intermediate fermentation end products included acetate (71%), propionate (15%), lactate (9%), and only minor amounts of butyrates or valerates. Addition of H₂ to sediments resulted in greater production of lactate (28%) and decreased formation of acetate (50%), but did not affect glucose turnover. Depth profiles of glucose uptake indicated that rates of uptake decreased with depth over the 0- to 18-cm interval and that glucose uptake accounted for 30 to 40% of methanogenesis in profundal sediments.     

Metabolism of [2-14C]acetate and its use in assessing hepatic Krebs cycle activity and gluconeogenesis

To examine the fate of the carbons of acetate and to evaluate the usefulness of labeled acetate in assessing intrahepatic metabolic processes during gluconeogenesis, [2-14C]acetate, [2-14C]ethanol, and [1-14C]ethanol were infused into normal subjects fasted 60 h and given phenyl acetate. Distributions of 14C in the carbons of blood glucose and glutamate from urinary phenylacetylglutamine were determined. With [2-14C]acetate and [2-14C]ethanol, carbon 1 of glucose had about twice as much 14C as carbon 3. Carbon 2 of glutamate had about twice as much 14C as carbon 1 and one-half to one-third as much as carbon 4. There was only a small amount in carbon 5. These distributions are incompatible with the metabolism of [2-14C]acetate being primarily in liver. Therefore, [2-14C]acetate cannot be used to study Krebs cycle metabolism in liver and in relationship to gluconeogenesis, as has been done. The distributions can be explained by: (a) fixation of 14CO2 from [2-14C]acetate in the formation of the 14C-labeled glucose and glutamate in liver and (b) the formation of 14C-labeled glutamate in a second site, proposed to be muscle. [1,3-14C]Acetone formation from the [2-14C]acetate does not contribute to the distributions, as evidenced by the absence of 14C in carbons 2-4 of glutamate after [1-14C]ethanol administration.     

Transport of 1,5-anhydro-D-glucitol into human polymorphonuclear leukocytes.

1,5-Anhydro-D-glucitol (AG) is one of the main polyols and its structure resembles glucose. It has been proposed that decreased serum AG concentrations in diabetic patients are a novel indicator of diabetic metabolic derangement. However, the pathway of AG metabolism still remains to be clarified. In this study we investigated the transport of AG into human polymorphonuclear leukocytes (PMNLs) isolated from healthy volunteers and found that 0.1 mM 3-O-methy-D-glucose (3OMG) was equilibrated with a half saturation time of 10 s, while the uptake rate of AG was much slower. The concentration dependence of AG uptake revealed that the AG transport velocity reached a plateau, with a Km of about 50 mM and Vmax of about 25 nmol/min/10(7) cells. Transport of 14C-labeled 3OMG was inhibited by unlabeled D-glucose or AG in a dose-dependent manner. The mean inhibition constant (Ki) for D-glucose and for AG were 1.06 and 4.93 mM, respectively. Cytochalasin B (20 microM) inhibited 3OMG transport by 90% but AG transport by only 50%. S/V for 14C-labeled AG transport plotted against the concentration of unlabeled 3OMG showed a non-linear and biphasic pattern. These results suggest that AG influx into PMNLs is mediated not only by the cytochalasin B-sensitive glucose transport system but also via another facilitated transport system.     

Glutamate transport,glutamine synthetase and phosphate-activated glutaminase in rat CNS white matter. A quantitative study

Glutamatergic signal transduction occurs in CNS white matter, but quantitative data on glutamate uptake and metabolism are lacking. We report that the level of the astrocytic glutamate transporter GLT in rat fimbria and corpus callosum was approximately 35% of that in parietal cortex; uptake of [3H]glutamate was 24 and 43%, respectively, of the cortical value. In fimbria and corpus callosum levels of synaptic proteins, synapsin I and synaptophysin were 15-20% of those in cortex; the activities of glutamine synthetase and phosphate-activated glutaminase, enzymes involved in metabolism of transmitter glutamate, were 11-25% of cortical values, and activities of aspartate and alanine aminotransferases were 50-70% of cortical values. The glutamate level in fimbria and corpus callosum was 5-6 nmol/mg tissue, half the cortical value. These data suggest a certain capacity for glutamatergic neurotransmission. In optic and trigeminal nerves, [3H]glutamate uptake was < 10% of the cortical uptake. Formation of [14C]glutamate from [U-14C]glucose in fimbria and corpus callosum of awake rats was 30% of cortical values, in optic nerve it was 13%, illustrating extensive glutamate metabolism in white matter in vivo. Glutamate transporters in brain white matter may be important both physiologically and during energy failure when reversal of glutamate uptake may contribute to excitotoxicity.     

Glutamate transporters of blood platelets as potential peripheral markers to analyze changes of glutamate transport activity in brain under altered gravity conditions

Activity of high-affinity glutamate transporters was altered in brain nerve terminals under artificial gravity conditions. Blood platelets contain glutamate transporters and are able to uptake glutamate. The goal of the research was to analyze comparatively L-[14C]glutamate transport in neuronal and non-neuronal tissues. The kinetic characteristics of transporters, [Na+]-dependence and influence of competitive transporter inhibitor DL-threo-beta-hydroxyaspartate (DL-THA) on the glutamate uptake by synaptosomes and platelets were determined. Km value of the L-[14C]glutamate uptake was very similar for preparations. Controversy, Vmax was three orders lower for platelets as compared with synaptosomes. It seems to correlate with reduced number of glutamate transporters in the plasma membrane of platelets in comparison with nerve terminals. It was concluded that the glutamate uptake process was primarily similar for both objects studied. Thus, it is reasonable to use platelets as potential peripheral diagnostic markers to analyze changes of glutamate transport activity in brain under altered gravity conditions.     

Sodium-Dependent Proline Uptake in the Rat Hippocampal Formation: Association with Ipsilateral-Commissural Projections of CA3 Pyramidal

Na+-dependent uptake of L-[3H]proline was measured in a crude synaptosomal preparation from the entire rat hippocampal formation or from isolated hippocampal regions. Among hippocampal regions, Na+-dependent proline uptake was significantly greater in areas CA1 and CA2-CA3-CA4 than in the fascia dentata, whereas there was no marked regional difference in the distribution of Na+-dependent gamma-[14C]aminobutyric acid ([14C]GABA) uptake. A bilateral kainic acid lesion, which destroyed most of the CA3 hippocampal pyramidal cells, reduced Na+-dependent proline uptake by an average of 41% in area CA1 and 52% in area CA2-CA3-CA4, without affecting the Na+-dependent uptake of GABA. In the fascia dentata, neither proline nor GABA uptake was significantly altered. Kinetic studies suggested that hippocampal synaptosomes take up proline by both a high-affinity (KT = 6.7 microM) and a low-affinity (KT = 290 microM) Na+-dependent process, whereas L-[14C]glutamate is taken up predominantly by a high-affinity (KT = 6.1 microM) process. A bilateral kainic acid lesion reduced the Vmax of high-affinity proline uptake by an average of 72%, the Vmax of low-affinity proline uptake by 44%, and the Vmax of high affinity glutamate uptake by 43%, without significantly changing the affinity of the transport carriers for substrate. Ipsilateral-commissural projections of CA3 hippocampal pyramidal cells appear to possess nearly as great a capacity for taking up proline as for taking up glutamate, a probable transmitter of these pathways. Therefore proline may play an important role in transmission at synapses made by the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers.     

Glycolytic enzymes and a GLUT-1 glucose transporter in the outer segments of rod and cone photoreceptor cells

The presence of glycolytic enzymes and a GLUT-1-type glucose transporter in rod and cone outer segments was determined by enzyme activity assays, glucose uptake measurements, Western blotting, and immunofluorescence microscopy. Enzyme activities of six glycolytic enzymes including hexokinase, phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase, were found to be present in purified rod outer segment (ROS) preparations. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase have confirmed that these enzymes are present in rod and cone outer segments and not simply contaminants from the inner segments or other cells. Rod outer segments were also found to contain glucose transport activity as detected by 3-O-[14C]methylglucose uptake and exchange. The glucose transporter had a Km of 6.3 mM and a Vmax of 0.15 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for net uptake and a Km of 29 mM and a Vmax of 1.06 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for equilibrium exchange. These Km values for net uptake and equilibrium exchange are similar to values obtained for human red blood cells and are characteristic of GLUT-1-type glucose transporter. The transport was inhibited by both cytochalasin B and phloretin. Western blot analysis and immunofluorescence microscopy using type-specific glucose transporter antibodies indicated that both rod and cone outer segment plasma membranes have a GLUT-1 glucose transporter of Mr 45K as found in red blood cells and brain microsomal membranes. Solid-phase radioimmune competitive inhibition studies indicated that rod outer segment plasma membranes contained 15% the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per micron2 of plasma membrane. These studies support the view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes.     

Alterations of Amino Acid Transmitter Systems in Spinal Cords of Chronic Paraplegic Dogs

The high-affinity uptake of [3H]serotonin, [3H]glutamate, and [3H]gamma-aminobutyric acid [( 3H]GABA) and the Na+-independent binding of [3H]glutamate and [3H]GABA were studied using spinal cord preparations obtained from normal mongrel dogs and from dogs made paraplegic by midthoracic spinal cord crush. Lumbosacral regions of the spinal cord were removed either before (1 week) or after (3 to 8 weeks) onset of spasticity. A myelin-free synaptosomal fraction was obtained by centrifugation and used for studying high-affinity uptake and for preparing synaptic plasma membranes for Na+-independent binding experiments. For the paraplegic groups, the uptake of 30 nM [3H]serotonin was 66 and 18% of control values after 1 and 3 weeks, respectively. Eadie-Hofstee analysis of [3H]serotonin uptake showed a 90% reduction in Vmax for the paraplegic group relative to control values, thereby indicating the expected loss of descending serotonergic pathways. The high-affinity uptakes of 1 microM [3H]glutamate and [3H]GABA were the same in both the control and nonspastic paraplegic groups after 1 week. However, after 3 weeks, the uptakes of [3H]glutamate and [3H]GABA were 60-70% higher for the spastic group than for the control animals. For both amino acids, Eadie-Hofstee plots revealed no difference in Km and higher Vmax for the spastic group relative to control values. After 1 and 3 weeks, the Na+-independent binding of 5 nM [3H]glutamate was 40-85% higher and the binding of 10 nM [3H]GABA was 40-60% lower for the paraplegic groups relative to the values for the control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of glutamate in Corynebacterium glutamicum. 1. Kinetic properties and regulation by internal pH and potassium

The active uptake system for glutamate in Corynebacterium glutamicum is inducible by growth on glutamate as sole energy and carbon source and is also susceptible to catabolite repression by glucose. The basic level of uptake activity is low in glucose-grown cells (1.5 nmol.mg dry mass-1.min-1), it is intermediate when acetate is the carbon source (3.8 nmol.mg dry mass-1.min-1) and becomes fully induced by glutamate (15 nmol.mg dry mass-1.min-1). In all cases the uptake has, except for different Vmax values, identical kinetic and energetic properties, and is characterized by a low apparent Km value of 0.5-1.3 microM and by high substrate specificity. The transported substrate species is the deprotonated form which can also be concluded from the extremely high pH optimum of transport above pH 9. Glutamate uptake in cells grown in media with low K+ concentration is not influenced by external Na+ but is drastically stimulated by addition of K+. Stimulation by K+ could be separated into two different mechanisms. (a) Addition of K+ increases the internal pH, thereby stimulating glutamate uptake which is regulated by the internal pH in C. glutamicum. The apparent pK of the internal 'pH switch' is 6.6; below this value, uptake of glutamate is inhibited. (b) Internal K+ also directly promotes glutamate uptake. Effective uptake of glutamate can be observed only when the cytosolic K+ concentration exceeds a threshold value of about 200 mM. Stimulation of glutamate uptake by external K+ is not due to functional coupling of K+ and glutamate transport but reveals the necessity to replenish the internal K+ pool.     

Cellobiose transport by mixed ruminal bacteria from a Cow.

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 microM for the Km and 10 nmol/min/mg of protein for the Vmax. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher Vmax values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.     

Studies on a wide-spectrum intestinal dipeptide uptake system in the monkey and in the human.

1. The intestinal transport of glycine and leucine residues of glycyl-L-leucine was studied in the monkey and in the human in vitro. Uptake of both [14C]glycyl-L-leuine and glycyl-L-[14C]leucine show similar Kt values, but there is a marked difference in the Vmax. values. Preliminary studies suggest that this anomalous difference in the Vmax. values may be due to the greater efflux rate of glycine from the tissue. 2. Arrhenius plots of both [14C]glycyl-L-leucine uptake and glycyl-L-[14C]leucine uptake in the monkey intestine show a discontinuity at about 20 degrees C. The activation energies above and below the discontinuity are similar for both [14C]glycyl-L-leucine uptake and glycyl-L-[14C]leucine uptake. These similarities in uptake characteristics suggest that the dipeptide glycyl-L-leucine is transported as one unit. 3. In the monkey intestine, glycyl-L-leucine uptake is inhibited by a wide variety of dipeptides, including those containing acidic and basic amino acids. The inhibition was shown to be competitive by using four representative dipeptides namely: L-alanyl-L-alanine, L-alanyl-L-leucine, L-glutamyl-L-glutamic acid and L-lysyl-L-lysine. The results strongly suggest that in the monkey intestine there may be a dipeptide-uptake system with an extremely broad specificity. These results were also confirmed in the human in a limited way.     

The accumulation and metabolism of [C]hypoxanthine by slices of rabbit renal medulla

The uptake of hypoxanthine by rabbit renal medulla has been studied with in vitro conditions. Unlike the uptake by renal cortex slices reported earlier, no evidence was found for involvement of an organic cation transport system. Medullary accumulation of the ¹⁴C-labeled material occurred in the absence of O₂ if glucose was present as substrate. Uptake of the ¹⁴C label was not supported by other sugars or metabolic intermediates, however. Metabolic inhibitors reduced both aerobic and anaerobic uptake. Tissue extracts were subjected to high-voltage electrophoresis and gel filtration for identification of possible metabolites. These studies indicated that most of the uptake of ¹⁴C-labeled material was accounted for by its conversion to inosine-like and/or inosinic acid-like compounds. That is, when experimental conditions were designed to retard slice metabolism of hypoxanthine, tissue to medium ratios for ¹⁴C approximated 1.0. A third metabolite was found occasionally, but remains unidentified.     

The uptake and metabolism of L-glutamate by tissue slices of the cestode Hymenolepis diminuta

The in vitro uptake of L-[3H]glutamate by tissue slices of the cestode Hymenolepis diminuta, denuded of tegument, was investigated. Two sodium concentration-dependent mechanisms, one of high affinity (Kt 1.8 X 10(-5) M; Vmax 4.76 pMoles/min/mg wet weight) and another of low affinity (Kt 2.2 X 10(-4) M; Vmax 50.7 pMoles/min/mg wet weight), were identified, in addition to a sodium insensitive component. Exchange of preloaded [3H]glutamate did not occur in tissue slices incubated in dilute unlabelled glutamate. Acidic amino acids, imipramine and fluoxetine were effective inhibitors of high and low affinity uptake, while glutamate receptor ligands, neurotransmitters and some antihelminthics generally were not. The concentrations present in, and the metabolism of glutamate by, tissue slices was examined by HPLC. The significance of the three modes of glutamate uptake and their possible role in the physiology of H. diminuta are discussed.     

Heterotrophic Potential for Amino Acid Uptake in a Naturally Eutrophic Lake

The uptake of sixteen (14)C-labeled amino acids by the indigenous heterotrophic microflora of Upper Klamath Lake, Oregon, was measured using the kinetic approach. The year-long study showed a seasonal variation in the maximum uptake velocity, V(max), of all the amino acids which was proportional to temperature. The maximum total flux of amino acids by the heterotrophic microflora ranged from 1.2 to 11.9 mumol of C per liter per day (spring to summer). Glutamate, asparagine, aspartate, and serine had the highest V(max) values and were respired to the greatest extent. The percentages of the gross (net + respired) uptake of the amino acids which were respired to CO(2) ranged from 2% for leucine to 63% for glutamate. Serine, lysine, and glycine were the most abundant amino acids found in Upper Klamath Lake surface water; at intermediate concentrations were alanine, aspartate, and threonine; and the remaining amino acids were always below 7.5 x 10(-8) M (10 mug/liter). The amino acid concentrations determined chemically appear to be the sum of free and adsorbed amino acids, since the values obtained were usually greater than the (K(t) + S(n)) values obtained by the heterotrophic uptake experiments.     

Mechanism of glutamate uptake in Zymomonas mobilis.

The energetics of the anaerobic gram-negative bacterium Zymomonas mobilis, a well-known ethanol-producing organism, is based solely on synthesis of 1 mol of ATP per mol of glucose by the Entner-Doudoroff pathway. When grown in the presence of glucose as a carbon and energy source, Z. mobilis had a cytosolic ATP content of 3.5 to 4 mM. Because of effective pH homeostasis, the components of the proton motive force strongly depended on the external pH. At pH 5.5, i.e., around the optimal pH for growth, the proton motive force was about -135 mV and was composed of a pH gradient of 0.6 pH units (internal pH 6.1) and a membrane potential of about -100 mV. Measurement of these parameters was complicated since ionophores and lipophilic probes were ineffective in this organism. So far, only glucose transport by facilitated diffusion is well characterized for Z. mobilis. We investigated a constitutive secondary glutamate uptake system. Glutamate can be used as a nitrogen source for Z. mobilis. Transport of glutamate at pH 5.5 shows a relatively high Vmax of 40 mumol.min-1.g (dry mass) of cells-1 and a low affinity (Km = 1.05 mM). Glutamate is taken up by a symport with two H+ ions, leading to substantial accumulation in the cytosol at low pH values.     

Effects of Dorsal Bilateral Rhizotomy Treatment on Transmitter Systems in the Spinal Cord of Normal and Spastic Dogs

The high-affinity uptakes of [3H]serotonin, [3H]-glutamate, and gamma-[3H]aminobutyric acid were studied using a myelin-free crude synaptosomal fraction prepared from the spinal cords of normal dogs and spastic dogs following sham treatment or dorsal bilateral rhizotomy surgery. Compared to sham-operated controls, rhizotomy surgery of normal dogs produced, after 1 week, a 30% reduction in the Vmax value of [3H]glutamate, but did not alter the uptake of gamma-[3H]aminobutyric acid. This treatment also produced a 60% decrease in the Vmax value of [3H]serotonin. Comparison of the effect of rhizotomy surgery on normal and spastic dogs revealed that the spastic group had 60% higher Vmax values for uptakes of [3H]glutamate and gamma-[3H]aminobutyric acid. Comparison of sham-operated spastic dogs and rhizotomy-treated spastic animals showed that there was a 25% decrease in the uptake of both amino acids in the rhizotomy-treated spastic group. Overall, the data (a) support the hypothesis that glutamate is the neurotransmitter from some of the primary afferents, and (b) suggest that sprouting of interneuronal amino acid transmitter systems may occur in the spinal cords of spastic dogs.     

Hexose transport in normal and SV40-transformed human endothelial cells in culture

The mechanism of glucose entry into human vascular endothelial cells was studied in monolayer cultures of normal (primary) and virally (SV40) transformed umbilical vein endothelium. Radioisotopic uptake studies with the glucose analogues 2-deoxy-D-glucose, and 3-O-methyl-D-glucose, and the nonmetabolizable stereoisomer L-glucose, indicated the presence of a saturable, stereospecific hexose carrier mechanism in both cell types. In other experiments with D-glucose and 3-O-methyl-D-glucose, the phenomenon of countertransport was demonstrable. Hexose transport was not affected by KCN, dinitrophenol, or ouabain, but was inhibited by phloretin and phlorizin in a pattern consistent with facilitated diffusion. Kinetic constants were obtained for both 2-deoxy-D-glucose and 3-O-methyl-D-glucose uptake. Similar Km values (range, 3.3-4.7 mM) were noted with normal and transformed cells, whereas the apparent Vmax was 0.56 nmol/microliter cytosol/minute for primary cells and 1.7-2.5 nmol/mu cytosol/minute for transformed cells. Under standard culture conditions, as well as following 18 hours of serum deprivation, insulin at concentrations up to 10(-5) M did not appear to influence hexose uptake in either cell type. Metabolism of 14C(U)-D-glucose to 14CO2 also was not stimulated by insulin. The presence of an insulin-insensitive, facilitated transport system for glucose in vascular endothelium has relevance for glucose metabolism in this tissue, and potentially for the association of certain vascular diseases (e.g., diabetic microangiopathy, atherosclerosis) with altered glucose homeostasis.