UPTAKE OF GLUCOSE ANALOGUES BY RAT BRAIN CORTEX SLICES: Na+-INDEPENDENT MEMBRANE TRANSPORT

Abstract— The glucose analogues 3-O-methyl-D-glucose and α-methyl-D-glucoside were not metabolized in brain tissue.
The uptake of these two sugars into the intracellular compartment of brain cortex slices was investigated using media with normal and low Na⁺ concentration (replacement of all NaCl with choline Cl). The cellular transport was not Na⁺-dependent. The transport mechanism clearly distinguished between the two sugars in both normal and low Na⁺ media.     

UPTAKE OF GLUCOSE ANALOGUES BY RAT BRAIN CORTEX SLICES: A KINETIC ANALYSIS BASED UPON A MODEL

Abstract— In slice preparations the exchange of dissolved substances between cells and incubation medium is delayed by diffusion through the extracellular space. The delay may seriously interfere with the study of membrane transport in terms of unidirectional fluxes across the cell membranes. A three-compartment serial model has been developed to describe exchange between slice and incubation medium. By aid of this model it is shown that the diffusion delay prevents determination of unidirectional fluxes for the two non-metabolizable glucose analogues 3-O-methylglucose and α-methyl-glucosidc. The membrane transport of the slowly transported α-methylglucoside can however be examined by aid of the model whereas the transport of 3-O-methylglucose is so rapid that it can not be examined with respect to V_max K_m and K_r. An attempt to determine these parameters will result in falsely large values which reflect extracellular diffusion and not membrane transport.     

UPTAKE AND METABOLISM OF 5-METHYL-TETRAHYDROFOLIC ACID BY RAT BRAIN SLICES

DNA-dependent DNA polymerases were partially purified from nuclei of cells from the occipital lobe of human brain. The purification procedure included successive DEAE-cellulose and phosphocellulose column chromatography, gel filtration and sucrose density gradient centrifugation steps. Four enzymes corresponding to DNA polymerases-α, β, γ, and terminal deoxynucleotidyl transferase were found. Brain DNA polymerases could be differentiated from one another by size, template preferences and sensitivity to sulfhydryl blocking agents.     

GABA UPTAKE IN RAT BRAIN SLICES: INHIBITION BY GABA ANALOGUES AND BY VARIOUS DRUGS

Abstract— 2-Hydroxy-, 2-chloro-, 2- and Cmethyl-GABA are linear competitive inhibitors of GABA uptake in rat brain slices. These analogues are thus potential substrates for the GABA transport system and possible'false transmitters'. 2-Hydroxy-GABA is the most potent inhibitor of GABA uptake yet described. No specific inhibitor of GABA uptake was revealed amongst the drugs tested.     

COMPETITIVE INHIBITION OF GABA UPTAKE IN RAT BRAIN SLICES BY SOME GABA ANALOGUES OF RESTRICTED CONFORMATION

Abstract— trans -4-aminocrotonic acid, dl - cis -aminocyclohexane-ltarboxylic acid and 4-aminotetrolic acid were found to be competitive inhibitors of GABA uptake in rat brain slices. These inhibitors are analogues of extended conformations of GABA, which indicates that these conformations are important in the initial binding of this inhibitory transmitter to its transport carrier.     

UPTAKE AND RELEASE OF NIPECOTIC ACID BY RAT BRAIN SLICES

—Nipecotic acid, a potent inhibitor of GABA uptake, is taken up by slices of rat cerebral cortex by a sodium-dependent, ‘high affinity’ system (K_m 11 μM), and can be released from these slices by an increased potassium ion concentration in a calcium-dependent manner. Nipecotic acid and GABA appear to be taken up by the same osmotically-sensitive structures. GABA and substances which inhibit GABA uptake also inhibit the uptake of nipecotic acid. GABA can release preloaded nipecotic acid from brain slices, and nipecotic acid can release preloaded GABA. This indicates that GABA and nipecotic acid can be counter-transported using the same mobile carrier. Nipecotic acid appears to have a higher affinity than GABA for this carrier.     

EFFECTS OF HISTIDINE ON THE TRANSPORT OF L-DOPA BY SLICES OF BRAIN CORTEX AND STRIATUM OF THE RAT

Abstract— L-Histidine inhibits the uptake of L-DOPA by brain cortex or striatum slices in short-term experiments, but increases it in long-term experiments. The potentiation of the steady-state accumulation of L-DOPA by histidine is a result of the cis- and trans -effects of histidine on the transport of L-DOPA at both sides of the cell membrane. The results demonstrate the importance of hetero-exchange phenomena in determining the cellular level attained for a given amino acid at the steady-state.     

UPTAKE AND RELEASE OF D- AND L-ASPARTATE BY RAT BRAIN SLICES

D-Aspartate is accumulated by slices of adult rat cortex by a high affinity uptake which is abolished if the sodium ions in the incubation medium are replaced by choline. A small uptake of D-aspartate takes place if the sodium ions are replaced by lithium ions. It appears likely that D-aspartate shares the same transport system with L-aspartate, and that the uptake of D-aspartate is into the same osmotically-sensitive particles as those which accumulate L-aspartate. D-Aspartate is released from cerebral cortex slices by raised potassium concentrations, provided calcium is present in the perfusing buffer. Both D- and L-aspartate produce gross hyperactivity when injected intraperitoneally into immature rats. Radioactive D-aspartate may be very useful in examining the neurotransmitter role of the naturally- occurring L-aspartate e.g. in studies of the autoradiographic localization of high affinity L-aspartate accumulation, its main advantage being that, unlike L-aspartate, D-aspartate does not undergo rapid metabolism.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

ALTERATION OF TRICARBOXYLIC ACID CYCLE METABOLISM IN RAT BRAIN SLICES BY HALOTHANE

Abstract—

• 1 Metabolism of [2-¹⁴C]pyruvate, [1-¹⁴C]acetate and [5-¹⁴C]citrate in the rat cerebral cortex slices was studied in the presence of halothane. Metabolites assayed include acetylcholine (ACh), citrate, glutamate, glutamine, γ-aminobutyrate (GABA) and aspartate. The trichloroacetic acid soluble extract, the trichloroacetic acid insoluble precipitate and its lipid extract were also studied.
• 2 In control experiments, pyruvate preferentially labelled ACh, citrate, glutamate, GABA and aspartate. Acetate labeled ACh, but to a lesser extent than pyruvate. Acetate also labeled lipids and glutamine. Citrate labeled lipids but not ACh and served as a preferential precursor for glutamine. These data support a three-compartment model for cerebral tricarboxylic acid cycle metabolism.
• 3 Halothane caused increases in GABA and aspartate contents and a decrease in ACh content. It has no effect on the contents of citrate, glutamate and glutamine.
• 4 Halothane preferentially inhibited the metabolic transfer of radioactivity from pyruvate into almost all metabolites, an effect probably not related to pyruvate permeability. This is interpreted as halothane depression of the‘large metabolic compartment’ which includes the nerve endings.
• 5 Halothane increased the metabolic transfer of radioactivity from acetate into lipids but did not alter such a transfer into the trichloracetic acid extract.
• 6 Halothane increased the metabolic transfer of radioactivity from citrate into the trichloroacetic acid precipitate, lipids and especially glutamine. Transfer of citrate radioactivity into GABA was somewhat decreased.
• 7 The differential effects of halothane on acetate and citrate utilization suggest that the ‘small metabolic compartment’ should be subdivided. Therefore, at least three metabolic compartments are demonstrated.
• 8 Halothane did not interfere with the dicarboxylic acid portion of the tricarboxylic acid cycle.

     

HIGH AFFINITY UPTAKE OF l-GLUTAMINE IN RAT BRAIN SLICES

—l -Glutamine is taken up into rat brain slices by a specific‘high affinity’uptake system (K_m 52 μm ) which is not influenced by high concentrations of l -glutamate and l -asparagine. The uptake system appears to be associated with cellular structures that do not survive homogenization under conditions which yield synaptosomes. The‘high affinity’uptake of glutamine is dependent on the external sodium ion concentration and can be inhibited by p-chloromercuriphenylsulphonate, amino-oxyacetic acid, ouabain, dibenamine and allylglycine. The effects of several inhibitors indicate that l -asparagine uptake is mediated by a system different from the‘high affinity’system mediating l -glutamine uptake.     

UPTAKE AND RELEASE OF TAURINE FROM RAT BRAIN SLICES

Abstract— Rapid efflux of [³⁵S]taurine from rat brain slices was observed on electrical stimulation. Slower release resulted when the Ca²⁺ content of the perfusion medium was replaced with Mg²⁺. Uptake of [³⁵S]taurine into rat cortical slices was unaffected by GABA, glutamic acid, glycine and leucine but was inhibited by alanine, ouabain, KCN and 2,4-dinitrophenol. Of a number of analogues of taurine, 2-aminoethylsulphinic acid was the most potent in inhibiting the uptake of [³⁵S]taurine. The rate of uptake was found to be decreased by lowering the incubation temperature. The possibility that taurine may be a neurotransmitter is discussed.     

THE EFFECT OF PHOSPHOLIPASES ON THE UPTAKE OF ATROPINE AND ACETYLCHOLINE BY SLICES OF MOUSE BRAIN CORTEX

The uptake of [³H]atropine, [³H]acetylcholine and [¹⁴C]inulin in mouse brain cortex slices was studied in slices treated with phospholipases A or C. In control experiments the slices took up atropine and acetylcholine against a concentration gradient, indicating active uptake. This uptake was decreased by ouabain, by anaerobic conditions and by an increase in the potassium ion concentration. The phospholipases were found to decrease the uptake of atropine and particularly that of acetylcholine in the slices. The uptake of labelled inulin in enzyme-treated slices, as compared to untreated slices, was not decreased, indicating no change in the inulin space. The effect of the phospholipases was time dependent and, up to a certainlimit, concentration dependent. The effect of ouabain in decreasing the uptake of atropine was not eliminated by the enzyme treatment. The effect of anaerobic conditions in decreasing the uptake was weak after treatment with phospholipases. The effect of higher concentrations of potassium ions was abolished by treatment with phospholipase A. The results emphasize the importance of phospholipids as substances controlling structural order in membranes and suggest their participation in active transport.