RATE OF STEROL FORMATION BY RAT BRAIN GLIA AND NEURONS IN VITRO AND IN VIVO

The ability of 11-day-old rat glial and neuronal cells to biosynthesize sterol was studied as a function of time in vivo and in vitro. The in vitro experiments utilized [2-¹⁴C]mevalonic acid as precursor. Glial-enriched cell preparations demonstrated a greater ability to incorporate [2-¹⁴C]mevalonic acid into isoprenoid material than did neuronal-enriched preparations. Approximately 4 h were required for maximal uptake of labelled mevalonate by the glial preparations. Further metabolism of the isoprenoid material, involving squalene turnover and sterol demethylation, was still evident even after 15 h of incubation. In vivo, sterol biosynthesis was studied by intraperitoneal injection of sodium [2-¹⁴C]acetate and [U-¹⁴C]glucose, sacrifice of the animals at 2 or 24 h, subsequent isolation of glial- and neuronal-cell enriched fractions and analysis of labelled isoprenoid material. Glial-enriched fractions again contained the bulk of the labelled isoprenoid material.     

MEASUREMENT OF THE RATE OF GLUCOSE UTILIZATION BY RAT BRAIN IN VIVO

Abstract— A method is described by which the rate of glucose utilization by whole brain of conscious rats may be measured. The basis is the uptake of ¹⁴C derived front [2-¹⁴C] glucose into the acid-soluble metabolite pool of brain. Catheters are placed in the femoral artery and vein under light ether anesthesia. After full recovery of consciousness a single intravenous injection of [2-¹⁴C] glucose is given and arterial blood samples taken at intervals. Simultaneous with the last sample the brain is removed and frozen within 1 s. The accumulation of ¹⁴C into the acid-soluble metabilite pool is measured and the rate of glucose utilization is calculated according to the equation:

The integral is calculated from the plasma glucose specific activity curve and evidence is presented to justify this procedure. The rate of glucose utilization measured by this method was 0·62 μmol/min per g in conscious rats and 0·28 μmol/min per g in sodium pentobarbital anesthetized rats.     

EFFECTS OF LITHIUM TREATMENT IN VITRO AND IN VIVO ON ACETYLCHOLINE METABOLISM IN RAT BRAIN

Abstract— The effects of LiCl on cholinergic function in rat brain in vitro and in vivo have been investigated. The high affinity transport of choline and the synthesis of acetylcholine in synaptosomes were reduced when part (25-75%) of the NaCl in the buffer was replaced with LiCl or sucrose. This appeared to be due to lack of Na⁺ rather than to Li⁺, as addition of LiCl to normal buffer had little effect. Following an injection of LiCl (10mmol/kg, i.p.) into rats the concentration of a pulsed dose of [²H₄]choline (20 μmol/kg, i.v., 1 min) and its conversion to [²H₄]acetylcholine, and the concentrations of [²H₂]acetylcholine and [²H₀]choline were measured in the striatum, cortex, hippocampus and cerebellum. The [²H₄]choline and [²H₄]acetylcholine were initially (15 min after LiCl) reduced (to ?30% in the cortex) and later (24 h after LiCl) increased (to + 50% in the striatum). There was a corresponding initial increase (to +50% in the cerebellum) and later decrease (to ?30% in the hippocampus) of the endogenous acetylcholine and choline. These results indicate an initial decrease and later increase in the utilization of acetylcholine after acute treatment with LiCl. Following 10 days of treatment with LiCl there was an increased rate of synthesis of [²H₄]acetylcholine from pulsed [²H₄]choline in the striatum, hippocampus and cortex (P < 0.05). The high affinity transport of [²H₄]choline and its conversion to [²H₄]acetylcholine was activated (131% of control; P < 0.01) in synaptosomes isolated from brains of 10-day treated rats. Investigation of synaptosomes isolated from striatum, hippocampus and cortex revealed that only striatal [²H₄]acetylcholine synthesis was significantly stimulated. Kinetic analysis demonstrated that the apparent K_T for choline was decreased by 30% in striatal synaptosomes isolated from rats treated for 10 days with LiCl. Striatal synaptosomes from 10-day treated rats compared to striatal synaptosomes from untreated rats also released acetylcholine at a stimulated rate in a medium containing 35 mM-KCl. These results indicate that LiCl treatment stimulates cholinergic activity in certain brain regions and this may play a significant role in the therapeutic effect of LiCl in neuropsychiatric disorders.     

ON THE UPTAKE OF INOSITOL BY RAT BRAIN SYNAPTOSOMES

The uptake of inositol by rat brain synaptosomes occurs via an unsaturable process that even at substrate concentrations as low as 1 μM is unable to achieve a concentration gradient indicative of active transport. Dinitrophenol, ouabain and cytochalasin B did not affect uptake of the cyclitol. The data indicate that inositol uptake by rat synaptosomes occurs by diffusion or by a system with an affinity so low it is difficult to discern. The low capacity, saturable inositol uptake system observed in rabbit brain slices may reflect a species difference or uptake by elements of the slice other than neuronal membranes.     

IN VIVO INHIBITION OF RAT BRAIN PROTEIN SYNTHESIS BY l-DOPA

Abstract— A study has been made of the effect of a single intraperitoneal dose of l -DOPA on the in vivo metabolism of [¹⁴C]leucine and [¹⁴C]lysine by the brain, and on their uptake into brain protein. Administration of 500 mg DOPA/kg to 40-g rats raised the concentrations of several free amino acids; the only amino acid which underwent a statistically significant increment was alanine. Intracisternally-injected [U-¹⁴C]leucine was rapidly metabolized to other labelled compounds; DOPA administration did not influence significantly the rate of its metabolism. No similar metabolic change was observed after administering [U-¹⁴C]lysine intracisternally.
Incorporation of [¹⁴C]leucine and [¹⁴C]lysine into total brain protein was significantly reduced 45 min after DOPA administration. There was also depression of the uptake of labelled amino acid into a supernatant fraction, obtained by high speed centrifugation of the brain homogenate, and into brain microtubular protein (tubulin). Reduced amino-acid incorporation into brain proteins observed 45 min after l -DOPA injection coincided with extensive disaggregation of brain polyribosomes. At 120 min after DOPA treatment, disaggregation was no longer significant and there was a smaller depression in labelled amino aicd incorporation, which disappeared completely 240 min after l -DOPA injection. It is concluded that disaggregation of brain polysomes following DOPA treatment is an accurate reflection of a change in the intensity of brain protein synthesis in vivo.     

IN VIVO INHIBITION OF RAT BRAIN PROTEIN SYNTHESIS BY d-AMPHETAMINE

Abstract— Between 1 and 4 h after rats received a single injection of d-amphetamine (15 mg/kg)(when brain polysomes are known to be disaggregated), the in vivo incorporation of [¹⁴C]lysine into trichloroacetic acid-precipitable brain protein was reduced by 28–48%. Incorporation of the ¹⁴C label into the protein present in a 100,000 g supernatant extract of whole brain was similarly reduced (by 44%). Amphetamine administration suppressed protein synthesis in rat cerebral cortex, cerebellum, hypothalamus, striatum, and brainstem to an equivalent extent. The drug did not significantly affect lysine pool sizes measured in these brain regions; thus the reduced incorporation of labeled lysine was not the result of an isotope dilution effect. We therefore conclude that the brain polysome disaggregation resulting from amphetamine administration is associated with decreased in vivo synthesis of some brain proteins.     

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 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.     

A COMPARISON OF AXONALLY TRANSPORTED PROTEINS IN THE RAT SCIATIC NERVE BY IN VITRO AND IN VIVO TECHNIQUES

An in vitro system for studying fast axonal transport in mammalian nerves has been developed. The viability of in vitro nerve preparations was established on the basis of three criteria: electron microscopy, electrical properties, and the activities of two marker enzymes, 5'-nucleotidase and total ATPase. The specific activity of transported proteins was greater using the in vitro procedure, and the level of locally incorporated radioactivity lower, when compared to in vivo transport experiments. Separation of solubilized transported proteins on polyacrylamide gels in the presence of sodium dodecyl sulfate showed that a large number of polypeptides are transported. Using a double label procedure which employed L-[³H]methionine and L-[³⁵S]methionine, proteins transported in vitro and in vivo were compared. No differences in the electrophoretic distribution of transported proteins from the two systems was seen. The major component of transported proteins electrophoresed with an apparent molecular weight of 105,000 ± 24,000. Using the in vitro system, transported proteins were compared to those labelled locally in either Schwann cells or cells of the dorsal root ganglion. Large differences in the labelling patterns were observed in both comparisons. We conclude that in vitro procedures provide a valid means of studying rapid axoplasmic transport. The proteins carried by rapid axoplasmic transport differ from those synthesized in either the Schwann cells of the sciatic nerve or the cells of the dorsal root ganglion.     

IN VIVO METHYLATION AND TURNOVER OF RAT BRAIN HISTONES

Abstract— The turnover of the different histone components from brain nuclei was studied after the administration of l -[³H]lysine and l -[¹⁴C-methyl]methionine to newborn rats. The radioactivities of the different histone subfractions as well as other proteins were determined over a 280-day period. Biphasic type decay curves (³H and ¹⁴C) were obtained for total brain histones and all the subfractions. From 6 to 40 days of age the half life of total brain histones was 19 days. After reaching brain maturity the half life was 132 days. The lysine rich histone (F₁) was found to turnover the fastest of all the histones, having half lives of 13 and 112 days, respectively. The decay curve for the slightly lysine rich histones (F_2a2, F_2b) gave half lives of 25 days up to 40 days of age and 189 days after reaching brain maturity. The arginine rich histones (F_2a1, F₃) gave a half life of 32 days up to 40 days of age, while no turnover was observed after maturity. The turnover rates of the methyl groups and/or methionyl residues did not vary significantly from the turnover rates of the lysyl residues in the F₂ and F₃ histones. The lysine-rich histones did not contain significant amounts of methionyl residues or methyl groups.
Amino acid analysis of the brain histones revealed that about 3·6 per cent of the lysyl residues in the slightly lysine rich histones were methylated, mainly as ε-N-dimethyllysine. About 13 per cent of the lysyl residues in the arginine rich histones were methylated, mainly as ε-N-monomethyllysine and ε-N-dimethyllysine.     

OBSERVATIONS ON THE BIOSYNTHESIS OF RAT BRAIN CERAMIDE AND CEREBROSIDE IN VIVO

Abstract— The incorporation in vivo of l -[¹⁴C]serine into ceramide and cerebroside of young rat brain has been studied. Acid hydrolysis of labelled ceramide and galactosyl-ceramide followed by selective partitioning of the resulting components indicated that 88 per cent of the radioactivity was present in the long-chain base portion. At early time points (10 min, 20 min) the precursor was incorporated into ceramide and to a lesser degree into glucosyl-ceramide. During time intervals of 5 and 10 h, the specific activity values (d.p.m./μmol) for ceramide and glucosyl-ceramide decreased, while values for galactosyl-ceramide, containing either unsubstituted fatty acids (NFA) or α-hydroxy fatty acids (HFA), increased 50 and 30 per cent, respectively. Analysis of labelled ceramide at all time points studied (10 min-10 h) indicated that l -[¹⁴C]serine was incorporated onto the NFA type. This observation suggests that HFA-ceramide may not be the physiological precursor of HFA-galactosyl-ceramide. In this context, the postulated precursor roles of both ceramide and psychosine in the biosynthesis of brain cerebrosides are discussed.     

大鼠颌下腺及其离体培养细胞脱氢表雄酮（DHEA）的免疫组织化学定位

用免疫组织化学ABC法,研究了颌下腺及无血清培养的颌下腺上皮细胞DHEA的定位,结果显示,大鼠颌下腺的浆液性腺泡的上皮细胞及各级导管上皮细胞均呈DHEA免疫反应阳性,无血清培养腺上皮细胞也呈DHEA免疫反应阳性,阳性物质分布于胞质,胞核呈阴性反应,此结果提示：大鼠颌下腺能自身合成DHEA,DHEA对消化功能可能具有重要的调节作用。     

DISTRIBUTION AND UPTAKE OF AMINO ACIDS IN VARIOUS REGIONS OF THE CAT BRAIN IN VITRO

—(1) The levels of the free amino acids were determined in five areas of the cat brain. The regional pattern was heterogeneous and fairly characteristic for each compound. (2) The uptakes of α-aminoisobutyric acid, taurine, d -aspartic acid, and l -histidine were measured in incubated slices from 31 regions of the cat CNS. Differences in uptake were found among the various areas; the regional pattern of uptake was different for each amino acid. The initial rate of uptake (5 min incubation) very often paralleled the rate at equilibrium (90 min incubation). (3) The regional correlation between distribution in vivo and uptake in vitro was good for aspartate, less so for histidine, and poor for taurine. (4) It is concluded that regional heterogeneity in exit processes, available energy, cell density, or protein content is unlikely to have decisive influence in determining regional differences in distribution and transport of metabolites; it seems that influx is the most important factor.     

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.     

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.     

THE STRUCTURAL SPECIFICITY OF THE HIGH AFFINITY UPTAKE OF l-GLUTAMATE AND l-ASPARTATE BY RAT BRAIN SLICES

Abstract— The high affinity uptake system for l -glutamate and l -aspartate in rat cerebral cortex may not be specific for these likely excitatory synaptic transmitters, as threo-3-hydroxy- dl -aspartate, l -cysteinesulphinate, l -cysteate and d -aspartate strongly inhibit the observed high affinity uptake of l -[³H]glutamate by rat brain slices in a manner consistent with linear competitive inhibition. These substances should therefore be considered as possible substrates for the transport system. Each of these four acidic amino acids excites central neurones in a manner similar to excitation induced by l -glutamate, and as each might occur in brain tissue, their possible synaptic role should be investigated.
l -Glutamate high affinity uptake was shown to be sodium-dependent, but under certain conditions appeared to be less sensitive than GABA uptake to changes in the external sodium ion concentration, and to drugs which modify sodium ion movements. This may be relevant to the efficiency of the glutamate uptake process during synaptic depolarization induced by glutamate.
l -Glutamate high affinity uptake was inhibited in a relatively nonspecific manner by a variety of drugs including mercurials and some electron transport inhibitors.     

THE FORMATION OF CHOLINE AND OF ACETYLCHOLTNE BY BRAIN IN VITRO

Abstract— Free choline and acetylcholine (ACh) in mouse or rat brain were assayed biologically. The subcellular distribution of ACh in brain slices that had been incubated in the presence of eserine was compared to that in control brain; during incubation, the ACh outside nerve endings increased four-fold, the ACh released from synaptosomes by osmotic shock doubled but the ACh bound firmly within nerve endings did not increase. The two nerve ending stores of ACh were labelled to similar specific radioactivities when slices were incubated with [³H]choline, but the specific radioactivity of the ACh formed was much lower than that of the added choline. Tissue incubated in the presence of eserine released choline and ACh into the medium and the tissue levels of both substances increased. Brain tissue exposed to Na⁺-free medium lost 84 per cent of its ACh and 66 per cent of its free choline; the amounts of both substances returned towards control values during subsequent incubation in a normal-Na⁺ medium (choline-free). Both the ACh outside nerve endings and the ACh associated with synaptosomes were depleted when tissue was incubated in Na⁺-free medium.