Effect of 2-deoxy-d-Glucose on Aminoacids Metabolism in Rats’ Cerebral Cortex Slices

We studied the effect of different concentrations of 2-deoxy-d-glucose on the l-[U-¹⁴C]leucine, l-[1-¹⁴C]leucine and [1-¹⁴C]glycine metabolism in slices of cerebral cortex of 10-day-old rats. 2-deoxy-d-glucose since 0.5 mM concentration has inhibited significantly the protein synthesis from l-[U-¹⁴C]leucine and from [1-¹⁴C]glycine in relation to the medium containing only Krebs Ringer bicarbonate. Potassium 8.0 mM in incubation medium did not stimulate the protein synthesis compared to the medium containing 2.7 mM, and at 50 mM diminishes more than 2.5 times the protein synthesis compared to the other concentration. Only at the concentration of 5.0 mM, 2-deoxy-d-glucose inhibited the CO₂ production and lipid synthesis from l-[U-¹⁴C] leucine. This compound did not inhibit either CO₂ production, or lipid synthesis from [1-¹⁴C]glycine. Lactate at 10 mM and glucose 5.0 mM did not revert the inhibitory effect of 2-deoxy-d-glucose on the protein synthesis from l-[U-¹⁴C]leucine. 2-deoxy-d-glucose at 2.0 mM did not show any effect either on CO₂ production, or on lipid synthesis from l-[U-¹⁴C]lactate 10 mM and glucose 5.0 mM.     

Hydroxyurea,a natural metabolite and an intermediate in d-cycloserine biosynthesis in streptomyces garyphalus

It was found that hydroxyurea, l-arginine and l-citrulline respectively significantly stimulated the formation of d-cycloserine in Streptomyces garyphalus. The formation of [¹⁴C]-hydroxyurea by washed cells was demonstrated after incubation with l-[guanido-¹⁴C]-arginine and l-[ureido-¹⁴C]-citrulline. The ¹⁵N of H₂NCO¹⁵NHOH was incorporated to 40% in d-cycloserine. The mass spectrum as well as the ¹⁵N NMR spectrum of labelled N,2-dicarbobenzyloxy-d-cycloserine derived from [¹⁵N]-hydroxyurea showed that hydroxyurea was the source of the heterocyclic nitrogen in the biosynthesis of d-cycloserine.     

Inhibition of transporter mediated γ-aminobutyric acid (GABA) release by SKF 89976-A,a GABA uptake inhibitor,studied in a primary neuronal culture from chicken

The effect of SKF 89976-A, a lipophilic non-substrate inhibitor of the -aminobutyric acid (GABA) transporter, on the release of radioactive GABA andd-aspartate has been studied. Neuronal cultures from 8 day old chick embryos, grown for six days, served as a model. The cultures were incubated with [³H]d-aspartate and [¹⁴C] GABA with the subsequent addition of high or low concentrations of SKF 89976-A. Finally the cultures were exposed to differently composed media for either 30 or 300 seconds. The release was quantified, using liquid scintillation counting. The efflux of [³H]d-aspartate and [¹⁴C] GABA was increased by [K⁺] and time, and a minimum value was obtained at [Ca²⁺] 1.05 mM. The release of both [³H]d-aspartate and [¹⁴C] GABA was inhibited by SKF 89976-A. The obtained results indicate that transporter mediated processes are the major mechanisms of transmitter release in the investigated model.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

Differences in the release ofl-glutamate andd-aspartate from primary neuronal chick cultures

Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release ofd-[³H]aspartate andl-[³H]glutamate. Thed-[³H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependentl-[³H]glutamate release was calcium dependent, and furthermorel-[³H]glutamate release was optimal at potassium concentrations<30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1-aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux ofd-[³H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulatedl-glutamate release. We believe thatl-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture.d-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.     

Metabolic Effects of Blocking Lactate Transport in Brain Cortical Tissue Slices Using an Inhibitor Specific to MCT1 and MCT2

A novel inhibitor of lactate transport, AR-C122982, was used to study the effect of inhibiting the monocarboxylate transporters MCT1 and MCT2 on cortical brain slice metabolism. We studied metabolism of l-[3-¹³C]lactate, and d-[1-¹³C]glucose under a range of conditions. Experiments using l-[3-¹³C]lactate showed that the inhibitor AR-C122982 altered exchange of lactate. Under depolarizing conditions, net flux of label from d-[1-¹³C]glucose was barely altered by 10 or 100 nM AR-C122982. In the presence of AMPA or glutamate there were increases in net flux of label and metabolic pool sizes. These data suggest lactate may supply compartments in the brain not usually directly accessed by glucose. In general, it would appear that movement of lactate between cell types is not essential for metabolic activity, with the heavy metabolic workloads imposed being unaffected by inhibition of MCT1 and MCT2. Further experiments investigating the mechanism of operation of AR-C122982 are necessary to corroborate this finding.     

Partial purification of the Na+-dependentd-glucose transport system from renal brush border membranes

Summary A membrane extract enriched with the Na⁺-dependentd-glucose transport system was obtained by differential cholate solubilization of rat renal brush border membranes in the presence of 120mm Na⁺ ions. Sodium ions were essential in stabilizing the transport system during cholate treatment. This membrane extract was further purified with respect to its Na⁺-coupledd-glucose transport activity and protein content by the use of asolectin-equilibrated hydroxylapatite. The reconstituted proteoliposomes prepared from this purified fraction showed a transient accumulation ofd-glucose in response to a Na⁺ gradient. The observed rate of Na⁺-coupledd-glucose uptake by the proteoliposomes represented about a sevenfold increase as compared to that of the reconstituted system derived from an initial 1.2% cholate extract of the membranes. Other Na⁺-coupled transport systems such asl-alanine, -ketoglutarate and phosphate were not detected in these reconstituted proteoliposomes.     

[3H]d-Aspartic acid release in brain slices of adult and aged fischer 344 rats

Alterations in glutamate content and uptake have been reported to occur in aged animals. The present studies used [³H]d-Aspartic acid ([³H]-D-ASP) release as a marker for glutamate neurotransmission. Frequency dependent [³H]-D-ASP release was measured in adult (8 month) and aged (28–30 month) Fischer 344 rats. Relatively high stimulation frequencies (>10 Hz) were required to induce [³H]-D-ASP release in both adult and aged F344 rats in temporal cortex and hippocampus. In both brain areas aged animals showed significantly more [³H]-D-ASP release than adult animals Kainic acid 1 mM failed to induce the release of [³H]-D-ASP in either temporal cortex or hippocampus. Omega conotoxin GVIA (5×10^–9M) a N and L type voltage sensitive calcium channel antagonist failed to inhibit [³H]-D-ASP stimulated release. These results demonstrate an increase in [³H]-D-ASP release in aged compared to adult F344 rats. The data also suggest a novel calcium channel may be involved in [³H]-D-ASP release.     

The characteristics of arginine transport by rat cerebellar and cortical synaptosomes

The uptake ofl-[³H]arginine into synaptosomes prepared from rat cerebellum and cortex occurred by a high-affinity carrier-mediated process. The uptake of arginine appeared to be potentiated by removal of extracellular Na⁺, inhibited by high levels of extracellular K⁺, but not by depolarization with veratridine or 4-amino pyridine. The effect of Na⁺ removal or K⁺ elevation did not seem to be due to changes in intracellular Ca²⁺ or pH. In both brain regions, uptake was significantly inhibited byl-arginine,l-lysine,l-ornithine, andl-homoarginine, but not byd-arginine norl-citrulline. Uptake was also inhibited by N^G-monomethyl-l-arginine acetate, but not by N^G-nitro-l-arginine methyl ester nor N^G-nitro-l-arginine except in the cortex at a concentration of 1 mM. The results indicate that the carrier system operating in synaptosomes showed many of the characteristics of the ubiquitous y⁺ system seen in many other tissues, although its apparent sensitivity to variations in extracellular Na⁺ was unusual.     

Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

Effect of sodium on [3H]ethylketocyclazocine binding to opioid receptors in frog brain membranes

The specific binding of (³H)ethylketocyclazocine to frog brain membrane preparation was enhanced in the presence of sodium ions administered as NaCl, both at 0 °C and at room temperature. The optimal NaCl concentration was 25 mM at 0 °C and 50 mM at 24 °C. MgCl₂ inhibited the [³H]ethylketocyclazocine binding. Two binding sites (high and low affinity) were established with [³H]ethylketocyclazocine as ligand by equilibrium binding studies. Addition of NaCl increased the B_max of the low-affinity site more than that of the high-affinity site at both temperatures. Affinities were higher at 0 °C than at 24 °C. TheK _D values were not significantly influenced by sodium ions. The dissimilarities between the rat and frog brain opioid receptors in [³H]ethylketocyclazocine binding are attributed to the different lipid composition of the two membranes.Abbreviations used DAGO D-Ala²-(Me)Phe⁴-Gly-ol⁵-enkephalin - DALE d-Ala²-l-Leu⁵-enkephalin - DADLE d-Ala²-d-Leu⁵-enkephalin - EKC Ethylketocyclazocine - DHM Dihydromorphine - BIT 2-(p-ethoxybenzyl)1-diethylaminoethyl-5-isothiocyanobenzimidazole isothiocyanate - FIT Fentanyl isothiocyanate     

Transport and membrane binding of the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) in Xenopus laevis oocytes

Summary We have examined transport and membrane binding of 6-diazo-5-oxo-l-norleucine (DON, a photoactive diazo-analogue of glutamine) and their relationships to glutamine transport in Xenopus laevis oocytes. DON uptake was stereospecific and saturable (V _max of 0.44 pmol/oocyte · min and a K _m of 0.065 mm). DON uptake was largely Na^u+ dependent (80% at 50 m DON) and inhibited (>75%) by glutamine and arginine (substrates of the System B^0,+ transporter) at 1 mm. Glutamine and DON show mutual competitive inhibition of Na⁺-dependent transport. Preincubation of oocytes in medium containing 0.1 mm DON for 24 or 48 hr depressed the V _max for System B^0,+ transport (as measured by Na⁺-dependent glutamine uptake), this effect was highly specific (neither d-DON nor the System B^0,+ substrates glutamine and d-alanine showed any independent effect) and required Na⁺ ions. Glutamine (1 mm in preincubation medium) protected transport from inhibition by DON. The possibility that specific inactivation of System B^0,+ by DON reflects attachment of DON to the transporter was tested by examining the binding of [¹⁴C]DON to Xenopus oocyte membranes. Oocytes incubated in 100 mm NaCl in the presence of [¹⁴C]DON for up to 48 hr showed 2.4-fold higher ¹⁴C-binding to membranes than oocytes incubated in choline chloride. Na⁺-dependent DON binding (31 ± 11 fmol/g membrane protein) was suppressed by external glutamine, arginine or alanine and was largely confined to a membrane protein fraction of 48–65 kDa (as assessed by SDS-polyacrylamide gel electrophoresis). The present studies indicate that DON and glutamine uptake in oocytes are both mediated by System B^0,+ and demonstrate that DON binding to a particular membrane protein fraction is associated with inactivation of the transporter, offering the prospect of using [¹⁴C]DON as a covalent label for the transport protein in order to facilitate its isolation and subsequent biochemical characterization.This work was supported by The Wellcome Trust, Action Research for the Crippled Child, Ajinomoto GmbH, Pfrimmer GmbH, the Rank Prize Funds, the Medical Research Council and the University of Dundee. We are grateful to Dr. C.I. Pogson (Wellcome Research Laboratories) and Drs. J.C. Ellory and B. Elford (University of Oxford) for gifts of [¹⁴C]DON.     

Analysis of kinetic data in transport studies: New insights from kinetic studies of Na+-d-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling,rapid filtration apparatus

Summary Using the fast sampling, rapid filtration apparatus (FSRFA) recently developed in our laboratory (Berteloot et al., 1991.J. Membrane Biol. 122:111–125), we have studied the kinetic characteristics of Na⁺-d-glucose cotransport in brush-border membrane vesicles isolated from normal adult human jejunum. True initial rates of transport have been determined at both 20 and 35°C using a dynamic approach which involves linearregression analysis over nine time points equally spaced over 4.5 or 2.7 sec, respectively. When the tracer rate of transport was studied as a function of unlabeled substrate concentrations added to the incubation medium, a displacement curve was generated which can be analyzed by nonlinear regression using equations which take into account the competitive inhibition of tracer flux by unlabeled substrate. This approach was made imperative since at 20°C, in the presence of high substrate concentrations or 1mm phlorizin, no measurable diffusion was found and the resultant zero slope values cannot be expressed into a classicalv versus S plot. All together, our results support the existence of a single Na⁺-d-glucose cotransport system in these membranes for which Na⁺ is mandatory for uptake. This conclusion is at variance with that of a recent report using the same preparation (Harig et al., 1989.Am J. Physiol. 256:8618–8623). Since the discrepancy seems difficult to resolve on the consideration of experimental conditions alone, we have determined the kinetic parameters ofd-glucose transport using one time point measurements and linear transformations of the Michaelis-Menten equation, in order to investigate the potential problems of such a widely used procedure. Comparing these approaches, we conclude that: (i) the dynamic uptake measurements give a better understanding of the different uptake components involved; (ii) it does not matter whether a dynamic or a one time point approach is chosen to generate the uptake data provided that a nonlinear-regression analysis with proper weighting of the data points is performed; (iii) analytical procedures which rely on linearization of Michaelian process(es) are endowed with a number of difficulties which make them unsuitable to resolve multicomponent systems in transport studies. A more general procedure which uses a nonlinear-regression analysis and a displacement curve is proposed since we demonstrate that it is far superior in terms of rapidity, data interpretation, and visual information.     

D-penicillamine affects lipid peroxidation and iron content in the rat brain cortex

d-Penicillamine, a trifunctional aminoacid known for its ability to form metal complexes and for being a radical scavenger, has been investigated in vitro and in vivo in the rat brain cortex. At 50 M the drug facilitate lipid hydroperoxides and TBARS formation in brain cortex homogenates, while at higher concentrations a clear inhibition of the lipid peroxidative process was observed. The activity of thed-penicillamine (25 and 50 mg/Kg i.p) was evaluated in vivo after a 7-day treatment in rats in whose brain cortex a slow process of lipid peroxidation was induced by iron-saccharate injection. Lipid hydroperoxides, lipid soluble fluorescent compounds and the iron content of both iron-injected and contralateral hemicortices showed a significant decrease in comparison to rats untreated withd-penicillamine. The higher dose also induced in normal rats a significant decrease in basal TBARS and iron content of the brain cortex. In the iron-injected cortex the observed Fe²⁺/Fe³⁺ ratio was significantly different from that of normal rats. On the contrary ratios obtained formd-penicillamine treated animals were higher in comparison to both normal and iron-injected animals. These results suggest thatd-penicillamine, acting as a reducing agent, inhibits the iron redox system and, as a chelating agents, can remove metal from action sites where lipid peroxidation may occur.     

Effect ofd-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

Summary Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [³H]thymidine uptake, is very low. Inl-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured ind-valine medium was significantly lower than that of cells cultured inl-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured ind-valine medium was significantly higher than that of cells cultured inl-valine medium. Cytosine arabinoside decreased the number of labeled cells in bothl-valine andd-valine cultures. From these results, it appears thatd-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures. This research was sponsored by grants from the National Institute of Child Health and Human Development, Bethesda, MD (HD-03820, HD-11816, HD-05797), and the Mellon Foundation.     

Baclofen-induced,calcium-dependent stimulation of in vivo release ofd-[3H]aspartate from rat hippocampus monitored by intracerebral microdialysis

The release ofd-[³H]aspartate (used as a tracer for endogenous glutamate and aspartate) was studied at high K⁺ (100 mM) and under ischemia in rats implanted with 0.3 mm diameter dialysis tubing through the hippocampus. The effect on thed-[³H]aspartate release of the two -aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) and (±)--(p-chlorophenyl)GABA (baclofen), which specifically activate GABA_A and GABA_B receptors, respectively, was studied. Initial experiments employing HPLC analysis showed a coincident increase in the amounts of glutamate, aspartate and the amount of radioactivity following introduction of K⁺ (100 mM) or a period of ischemia suggesting that thed-[³H]aspartate labels the transmitter pools of the two amino acids under the present experimental conditions. The presence of 10 mM baclofen or 10 mM THIP in the perfusion medium did not inhibit ischemia inducedd-[³H]aspartate release. On the contrary, 10 mM baclofen alone (but not 0.1 or 1 mM) in the perfusion medium induced release ofd-[³H]aspartate in a calcium dependent manner, whereas 10 mM THIP had no significant releasing effect.Special issue dedicated to Dr. Elling Kvamme     

l-tyrosine-binding proteins on melanoma cells

Summary Crosslinking of [¹⁴C]l-tyrosine to at least five hamster melanoma cell surface proteins is reported. This effect was abolished by addition of nonradioactivel-tyrosine,l-phenylalanine, orl-dopa, but not byd-tyrosine, tyramine, dopamine, norepinephrine, or epinephrine. The above proteins can be purified by tyrosine-affinity chromatography. They have molecular weights different from proteins staining for dopa oxidase and proteins that bind anti-tyrosinase antibody in Western blots. It is suggested that they may be a hithergo unrecognized part of the cellular apparatus governing melanogenesis.     

Effect of aluminum on the blood-brain barrier permeability during nitric oxide-blockade-induced chronic hypertension in rats

We examined the effect of aluminum on the permeability of the blood-brain barrier (BBB) during nitric oxide-blockade-induced chronic hypertension in rats. Animals were given the inhibitor of nitric oxide synthase, l-NAME (N ^ω-nitro-l-arginine methyl ester), for 4 wk to induce chronic hypertension. Two groups of rats were given an intraperitoneal injection of aluminum chloride. The integrity of the BBB was assessed by a quantitative measurement for Evans blue (EB) dye. The arterial blood pressure in l-NAME- and l-NAME plus aluminum-treated animals was significantly elevated from 115±2.8 and 110±1.7 mm Hg to 174±5.2 and 175±4.8 mm Hg, respectively (p<0.01). The EB dye content in the brain regions of the rats in the l-NAME group was increased, but there was no statistical significance compared to the saline group. The extravasation of EB dye was significantly increased in the brain regions of the animals treated with aluminum compared to the rats treated with saline (p<0.05). A significantly higher EB dye content in the brain regions was observed in the l-NAME plus aluminium group compared to l-NAME, aluminum, and saline groups (p<0.01). These findings indicate that exposure to a high level of aluminum leads to an additional increase in BBB permeability where nitric oxide-blockade-induced chronic hypertension potentiates the effect of aluminum to enhance BBB permeability to EB dye.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.