THE EFFECT OF SULFHYDRYL REAGENTS ON THE TITYUSTOXIN-INDUCED ACETYLCHOLINE RELEASE IN RAT BRAIN SLICES1

Abstract— Pre-treatment of rat brain slices with organic mercurials prevents the increased acetylcholine release induced by tityustoxin. This inhibition is reversed by dithiothreitol. N-Ethylmaleimide blocks the tityustoxin effect irreversibly. Simultaneous incubation with mercurials and toxin reveals a competition of both ligands for a membrane sulfhydryl group apparently required for tityustoxin activity.     

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.     

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.     

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.     

UPTAKE AND RELEASE OF EXOGENOUS [1-14C]ACETYLCHOLINE BY BRAIN CORTEX SLICES FROM THE RAT

Abstract— Radioactive acetylcholine ([¹⁴C]ACh) that is taken up by rat cerebral cortex slices, incubated aerobically in a physiological saline-glucose paraoxon-[¹⁴C]ACh medium, apparently by a passive diffusion process at concentrations > 1 mm consists essentially of two forms, a readily exchangeable and releaseable or mobile form, and a bound or retained form, poorly (or not) exchangeable. The quantity of retained ACh consists of a considerable fraction of that taken up amounting to 54% with external 0.1 mm -[¹⁴C]ACh and about constant, 27%, for the range 5-50mm -[¹⁴C]ACh. All its ACh is released on homogenization with 0.1 n -perchloric acid or on tissue disintegration in distilled water. The cerebral uptake of ACh differs basically from that of urea as there is no retention of the latter following its uptake. Cerebral cortex slices are superior to those of cerebellar cortex, subcortical white matter, kidney cortex, liver and spleen in taking up and retaining [¹⁴C]ACh. Deprivation in the incubation media of glucose or Na⁺ or Ca²⁺. or the presence of dinitrophenol, whilst causing little change in ACh uptake, induces considerable changes in swelling and ACh retention; the greater the amount of swelling the smaller is that of retention. It seems that the latter is segregated in compartments characterized by a low permeability to exogenous ACh. About half of it is independent of changes in incubation conditions whilst the other half enters the compartment by an Na⁺, Ca²⁺ and energy-dependent process. At least part of the retention is neuronal as it is diminished by protovera-trine, the diminution being blocked by tetrodotoxin. Mobile ACh (i.e. total uptake minus retained ACh) is largely unaffected by protoveratrine, ouabain, etc. It seems that the retained ACh is directly proportional to the amount of mobile ACh minus the amount that enters with swelling. If the latter is largely glial in location, then the retained ACh is simply proportional to the mobile neuronal ACh. Suggestions are made as to the location of the retained ACh in the brain cells and to the processes involved in its segregation there. Release of retained ACh occurs on change of the Na⁺ gradient. Atropine and d-tubocurarine also diminish the amount of retained ACh but the percentage diminution falls with increase of the concentration of exogenous ACh.     

ANALYSIS OF THE PREFERENTIAL RELEASE OF NEWLY SYNTHESIZED ACETYLCHOLINE BY CORTICAL SLICES FROM RAT BRAIN WITH THE AID OF TWO DIFFERENT LABELLED PRECURSORS

—The release of newly synthesized acetylcholine (ACh) by cortical slices from rat brain in the presence of 25 mm -KCl was studied. The slices were incubated for 5 min in a medium containing both [2-¹⁴C]pyruvate and choline labelled with 3 deuterium atoms (choline-d₃) in order to label at the same time the acetyl moiety and the choline moiety of ACh. The non-labelled ACh and the ACh-d₃ were measured by pyrolysis-gas chromatography/mass spectrometry, and the [^I4C]ACh by liquid scintillation counting. It was found that the newly formed [⁴C]ACh as well as the newly formed ACh-d₃ had a more than 2.5 times greater probability of being released than the preformed non-labelled ACh. These findings strongly suggest that it is not simply the ACh synthesized immediately inside the nerve ending membrane from incoming undiluted labelled choline, which is preferentially released, but that all newly formed ACh has a greater probability of being released than preformed ACh. No preferential release of newly formed ACh was observed when the incubation medium contained 5.6 mm -pyruvate instead of 10 mm -glucose + 0.6 mm -pyruvate. The cause of this difference remains unexplained.     

THE RELATIONSHIP BETWEEN ACETYLCHOLINE RELEASE FROM BRAIN SLICES AND THE ACETYLCHOLINE CONTENT OF SUBCELLULAR FRACTIONS PREPARED FROM BRAIN

Abstract— Acetylcholine (ACh) release from sliced cerebral cortex of rats was measured when the tissue was incubated in a high K⁺ (46 m m ) medium containing eserine. In the absence of hemicholinium (HC-3), ACh release was well maintained, but in the presence of HC-3, ACh release declined within 15–20 min. Subcellular fractions representing nerve-ending free (cytoplasmic) ACh and nerve-ending bound ACh were prepared from slices that had been stimulated to release ACh in the presence of HC-3. Both nerve-ending stores of ACh were depleted when their content was compared to tissue that had not been stimulated and there was no demonstrable difference in the rate of depletion of either of the two fractions. Stimulating slices with K⁺ in the absence of HC-3 also depleted cytoplasmic and vesicle-bound ACh. It is concluded that, under these experimental conditions, both nerve ending stores of ACh are available for release and that, in the absence of HC-3, ACh synthesis can maintain ACh release, but cannot maintain tissue ACh content.     

HYPEROSMOLALITY-INDUCED GABA RELEASE FROM RAT BRAIN SLICES: STUDIES OF CALCIUM DEPENDENCY AND SOURCES OF RELEASE

Abstract— The effects of hyperosmolal superfusion upon the release of preloaded, radio-labeled GABA has been studied, using both first cortical and first pontine brain slices. GABA release was stimulated with either hyperosmolal Na⁺ or sucrose superfusion in cortical slices. This stimulated release of radio-labeled GABA was partially Ca²⁺-dependent in cortical slices. When barium ions replaced Ca²⁺ in hyperosmolal medium, a similar effect was seen. High concentration of magnesium in Ca²⁺ -free hyperosmolal medium did not induce stimulation. The increased release of α-aminoisobutyric acid (AIBA), a non-metabolized amino acid induced by hyperosmolality, was not Ca²⁺-dependent.
GABA release was also stimulated with hyperosmolal sucrose superfusion in pontine slices. The effect of pre-treatment of cortical and pontine slices with β-alanine or L-2,4-diaminobutyric acid (DABA) was used to study the source of exogenous GABA release induced by hyperosmolality. In cortical slices, β-alanine blocked the hyperosmolal release of GABA and also slightly inhibited GABA uptake. DABA did not change hyperosmolal GABA release, although it inhibited GABA uptake. In pontine slices, both DABA and β-alanine inhibited GABA uptake, but were unable to inhibit the hyperosmolal release of GABA.
The data suggest that hyperosmolality causes increased release of GABA from neurons, analogous to that seen with K⁺-depolarization. AIBA, unlike GABA, is released from brain cells as a non-Ca²⁺ -dependent response to osmotic equilibration. The observation that pre-treatment with β-alanine inhibits the hyperosmolal release of GABA suggests that hyperosmolality alters glial cell function.     

MUSCIMOL UPTAKE, RELEASE AND BINDING IN RAT BRAIN SLICES

Abstract— The GABA analogue, muscimol, was taken up relatively inefficiently compared to GABA by slices of rat cerebral cortex at 37 C. Muscimol uptake followed saturation kinetics (K_m ImM. V_m 0.1 μmol g mini and showed an absolute dependence on sodium ions. The relative susceptibilities of muscimol uptake and GABA high affinity uptake to a variety of inhibitors, including (-)-nipecotic acid. (+)-2.4-diaminobutyric acid and arecaidine, and the stimulation of muscimol efflux by 50μM-GABA, suggest that muscimol and GABA share some common transport carriers. Since L-histidine inhibited muscimol uptake hut not GABA high affinity uptake, at least part of the observed muscimol uptake may be mediated by the 'small basic'amino acid transport system. Muscimol appeared to he taken up into nerve terminals, since uptake was inhibited by the neuronal uptake inhibitor cis -3-aminocyclohexanecarboxylic acid but not by the glial uptake inhibitor β-alanine. Muscimol efflux was stimulated in a calcium-dependent manner by an increased potassium ion concentration.
Sodium-independent binding of muscimol was observed in slices of rat cerebral cortex at 4 C. Binding could be inhibited by a variety of substances. including GABA, isoguvacine and (+)-bicuculline methochloride, which are known to inhibit the binding of muscimol to putative GABA receptors associated with synaptic membranes purified from rat brain.     

DEMONSTRATION OF ACETYLCHOLINE RELEASE BY MEASURING EFFLUX OF LABELLED CHOLINE FROM CEREBRAL CORTICAL SLICES

Abstract— To demonstrate release of ACh in the absence of inhibition of cholinesterase, slices of cerebral cortex were incubated with [³H]choline, after which they were placed in a tissue bath for superfusion. Hemicholinium (HC-3) increased the spontaneous efflux of [³H]choline. Electrical stimulation at 4/s increased the efflux of [³H]choline to the same extent whether the slices were stimulated early or late during superfusion. The effect of stimulation on efflux of [³H]choline was abolished by tetrodotoxin and by the absence of calcium. The extent of choline efflux resulting from stimulation, as calculated from the specific radioactivity of the incubation medium, was the same when the slices were incubated with 0.1 or 1.0mM choline, but was less with lower concentrations of choline. We conclude that the increased efflux of [³H]choline evoked by stimulation probably originates from stores of [³H]ACh synthetized during incubation.     

ACCELERATED EFFLUX OF [14C] AND [3H] AMINO ACIDS FROM SUPERFUSED SLICES OF RAT BRAIN1

Abstract— The rate of spontaneous efflux of several isotopically-labelled amino acids was measured in superfused slices of rat brain. The group specificity of amino acid efflux was studied by examining the pattern of accelerated efflux in the presence of extracellular unlabeled amino acid. The mediated efflux of γ-aminobutyric acid and l -glutamic acid was highly specific. Less specificity was found for amino acids in the small neutral, large neutral and basic groups. The efflux of γ-aminobutyric acid and l -glutamic acid was accelerated by structurally similar amino acids which are known to depress or excite motor neurons. This finding raises the possibility that the physiological effects of non-specific amino acids during local iontophoresis may be secondary to an accelerated release of more specific transmitter amino acids from neighbouring cells.     

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.     

CHARACTERISTICS OF D-GLUCOSAMINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The uptake of D-glucosamine by rat brain synaptosomes is studied as a function of time, temperature and synaptosomal protein and substrate concentrations. The rate of D-glucosamine uptake, after correcting for simple diffusion, obeys Michaelis-Menten kinetics. The apparent kinetic constants for the uptake process are K_m = 2.5 0.8 m m , V_max = 3.7 ± 1.2 nmol/mg protein/min. D-Glucose, D-mannose, 2-deoxy-D-glucose and 3-0-methyl-o-glucose are potent inhibitors of D-glucosamine uptake. 2-Deoxy-D-glucose and D-glucosamine inhibit the uptake of one another in a simple competitive manner, indicating their sharing of a common transport system. Cytochalasin B, phloretin and phloridzin are powerful competitive inhibitors of D-glucosamine uptake with apparent inhibitor constants ( K₁ ) of 7.0 × 10^-5, 2.3 × 10^-3 and 0.4 mM, respectively. The uptake is unaffected by Na⁺, Li⁺ and Mg²⁺, partially inhibited by NH₄⁺, Mn²⁺ and Ca²⁺, and slightly stimulated by PO₄-ions. D-Glucosamine uptake is also sensitive to inhibition by several sulfhydryl reagents, thus implying the involvement of sulfhydryl groups in the transport process. The apparent affinity constants for synaptosomal transport for both D-glucosamine and 2-deoxy-D-glucose are about 4 times greater in 7-day-old than in the adult rat brains.     

FURTHER EXPERIMENTS ON THE UPTAKE OF ACETYLCHOLINE AND ATROPINE AND THE RELEASE OF ACETYLCHOLINE FROM MOUSE BRAIN CORTEX SLICES AFTER TREATMENT WITH PHOSPHOLIPASES

—Uptake of acetylcholine (ACh) in mouse brain cortex slices, previously shown with ACh synthesized from tritiated choline is confirmed with acetyl[1-¹⁴C]choline. Radioactivity from tritiated sodium acetate also accumulates in slices, but forms hardly any ACh. Uptake of ACh increases in a Ca²⁺-free medium, decreases again upon addition of a 3 × 10⁵ molar concentration of an anticholinergic benzilate compound and is completely blocked by the same compound at 3 × 10³ m. Slices preloaded with labelled ACh release, after extensive washing, some of their radioactivity into an outer medium free from ACh. Phospholipase, A or C, increases the release of radioactivity from the slices. An equilibrium is reached both with controls and phospholipase-treated slices. Remaining radioactivity seems to be due to bound ACh. Calcium and magnesium ions have no effect on the uptake of tritiated atropine, although low concentrations of Ca²⁺ decrease the effects of phospholipase C on atropine uptake. The inhibitory effect of K⁺ on atropine uptake disappears completely after treatment with small amounts of phospholipase A, but even high concentrations of phospholipase C have no effect.     

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.

     

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.     

大白鼠脑M胆碱受体的溶脱

 本文报告膜蛋白溶脱剂溶脱大鼠脑M胆碱受体的结果,其中0.5％CHAPS,0.35％洋地黄皂苷和10％甘油的混合液效果较好,可溶脱30％的受体,并得到22％有活性的受体。溶脱的受体有较好的稳定性,与膜结合受体有同样的配体结合特异性,可饱和性及可逆性。平衡结合及动力学研究表明溶脱受体和膜结合受体对[~3H]QNB有类似的亲和性。     

BIOSYNTHESIS OF RIBONUCLEIC ACID IN RAT BRAIN SLICES

The incorporation of uridine into RNA in brain slices was studied. Optimal conditions for uridine incorporation were determined. The characteristics of the product suggest that de novo DNA-directcd synthesis of fairly high molecular weight material takes place. Incorporation into RNA of several areas of brain was studied. The incorporation was also studied as a function of the age of the animal. Finally, an apparent correlation was observed between the decrease in uridine incorporation with age and the increase of the enzyme uridine nucleosidase which hydrolyses uridine to uracil, a material which cannot be incorporated into RNA.