Characterization, development, and localization of the deoxycytidine phosphorylating systems in mammalian brain

The accumulation of deoxycytidine by rabbit and mouse brain was studied in vitro. Brain slices from brain stem, cerebellum, and forebrain of rabbits of various ages (1 day to 2.5 years) and forebrain from adult mice were incubated for various times in artificial CSF containing 6 nM [3H]deoxycytidine at 37 degrees C under 95% O2/5% CO2. Rabbit and mouse brain slices of all ages accumulated [3H]deoxycytidine by a saturable system (IC50 = 4 microM) and converted it to [3H]deoxycytidine phosphates and [3H]DNA. When slices from all brain regions of 1-day-old rabbits were incubated in 6 nM [3H]deoxycytidine for 30 min, tissue-to-medium ratios of 3H were between 1.2 and 2.5 and declined with age, except in cortex; the percentages of total 3H in perchloric acid homogenates of brain slices as [3H]DNA were 10-24% and declined to low levels in middle age. However, at all ages and in all regions tested, 30-85% of the [3H]deoxycytidine within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [3H]deoxycytidine for 30 min, the highest percentage of [3H]deoxycytidine phosphates plus [3H]DNA was present in the nuclear and mitochondrial fractions of all brain regions. Deoxycytidine phosphates were synthesized from deoxycytidine in all brain regions tested into middle age.     

Cerebral Blood Flow and Oxidative Metabolism in Conscious Fischer-344 Rats of Different Ages

Abstract: The cerebral metabolic rates for O₂ and for glucose were measured in conscious, fasted male Fischer-344 rats at the ages of 3, 12, and 24 months, and cerebral blood flow was determined with ¹⁴C-iodoantipyrine. The metabolic rates for oxygen and glucose were obtained by multiplying blood flow by the O₂ and glucose concentration differences, respectively, between blood in the femoral artery and in the superior sagittal sinus. Mean cerebral blood flow and the metabolic rates for oxygen and glucose did not differ significantly (p > 0.05) between 3 and 12 or between 12 and 24 months. Nor did the arteriovenous differences for O₂ and for glucose change significantly with age. Because the superior sagittal sinus drains blood mainly from the cerebral cortex, the results indicate that average cerebral cortical oxidative metabolism, and the coupling ratios between the cerebral metabolic rate for oxygen and cerebral blood flow and between the cerebral metabolic rate for glucose and cerebral blood flow, do not change significantly with age in the Fischer-344 rat.     

A stimulatory subunit in the polypeptide elongation factor-1 of the chick brain

Abstract: The higher-molecular-weight elongation factor-1 (EF-1_H) of the chick brain was observed to contain three subunits (denominated α, β, and γ), contrary to a previous report that the brain EF-1_H consisted of aggregates of low-molecular-weight elongation factor- 1 (EF-1_L). Crude EF-1_H, obtained from 20-day embryonic brain, was treated with 0.4 M ammonium chloride and 0.1 mM GTP, and EF-1_βγ, was obtained using a DEAE-Sephadex column equilibrated in 0.025 mM GTP. Both EF-1_β, and EF-1_γ, were isolated by means of a DE-52 column equilibrated in 6 M urea and were found to have molecular weights of 2.8 and 4.8 × 10⁴, respectively. EF-1_β and EF-1_γ were also obtained from young rat and calf brains by the same procedures. The molecular weight of the isolated EF-1_α was 5 × 10⁴. It was found that EF-1_β stimulated the two EF-1_α-dependent reactions, i.e., phenylalanyl-tRNA binding (reaction 1) and polyphenylalanine synthesis (reaction 2), and also stimulated the nucleotide exchange reaction in the EF- 1_α-guanine nucleotide binary complex (reaction 3). The degrees of stimulation of reactions 1, 2, and 3 by the addition of EF-1_β were 2 to 3 times, about 18 times, and 2 to 3 times as much as with EF-1_α alone, respectively. The amino acid compositions of EF-1_α -1_β, and -1_γ and EF-2 were very similar to those of other eukaryotic tissues. Thus the constituents and properties of EFs of the brain were found to be basically similar to those of other tissues of eukaryotes, although EF-1_β, and EF-1, had not been reported in the brain. A possible physiological significance of EF-1_β during brain development is also discussed.     

Hippocampal electrical activity and gamma-aminobutyrate metabolism in brain tissue following administration of homocysteine

Abstract: The concentration of γ-aminobutyrate (GABA) and the activity of glutamate decarboxylase and GABA-transaminase were measured in extracts of mouse brain before the onset and during the course of generalized seizures induced by systemic administration of homocysteine thiolactone. The results indicate that whole brain GABA metabolism is unaffected by subconvulsive and convulsive doses of homocysteine at all stages of the generalized seizure. Electroencephalographic monitoring of rat brain electrical activity via hippocampal electrode implantation allowed the course of homocysteine-induced seizures to be followed and afforded a means of quantifying such seizures.     

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [3H]Flunitrazepam and [3H]Ethyl-β-carboline-3-carboxylate Binding Sites in Rat Brain Membranes

Abstract: This study was designed to analyze possible differences in the binding of [³H]flunitrazepam ([³H]FNZP) and [³H]ethyl - β - carboline - 3 - carboxylate ([³H]β-CCE), to rat brain membranes, in various experimental conditions. In cerebral cortex, hippocampus, cerebellum, and orain stem the number of binding sites for [³H]β-CCE was higher than for [³H]FNZP; both were displaced by clonazepam. Until the 7th day of postnatal brain development the numbers of [³H]FNZP and [³H]β-CCE sites were equivalent; but later on, the β-carboline sites increased to a higher level. Noradrenergic denervation by 6-hydroxydopamine was followed in the hippocampal formation. Already after 2 days, there was a decrease in [³H]FNZP sites, which reached 70% of control after 14 days. Similar results were obtained with DSP-4 denervation. This change was only in B_max and not in K_D, In contrast, the [³H]β-CCE sites did not change with denervation. Neonatal injection of l - 2,4,5 - trihydroxyphenylalamine or DSP-4 produced in the adult a decrease in [³H]FNZP sites in the cerebral cortex, in parallel with the noradrenergic denervation. On the other hand, there was an increase in the cerebellum and brain stem, in correspondence with the hyperinnervation by sprouting. In these rats, the number of sites for [³H]β-CCE did not change in the different brain regions. With 0.1% Triton X-100, applied to synaptosomal membranes, [³H]FNZP binding was reduced by 35%, while that of [³H]β-CCE was not significantly changed. These results suggest that there is heterogeneity of binding sites for benzodiazepine receptors in rat brain. A tentative interpretation of the experiments involving noradrenergic denervation and hyperinnervation, as well as those with Triton X-100, is that [³H]FNZP binds to pre- and postsynaptic receptors, while [³H]β-CCE binds mainly to postsynaptic benzodiazepine receptors.     

Differential effects of protein malnutrition and ascorbic acid deficiency on histidine metabolism in the brains of infant nonhuman primates

Abstract: Male infant nonhuman primates (M. nemes-trina) born in captivity were used in the study. They were divided into three groups. The first group of three animals was fed a 20% casein diet and the second group of six monkeys received a 2.0% casein diet. The third group of four monkeys received a 20% casein diet totally devoid of ascorbic acid for 3.5 weeks before the diet was supplemented with ascorbic acid (20 mg/kg diet). All the diets were given to the animals in two daily rations of 100 g/animal. The monkeys fed a 2% casein diet failed to grow, and after about 3.5 months showed variable degrees of edema, hypoalbuminemia, evidence of psychomotor disturbance, depressed plasma levels of many essential amino acids, and other features consistent with the diagnosis of protein-energy malnutrition. Examination of the brains revealed significant alterations in the levels of histidine (+ 172%) and homocarnosine (+ 146%) in comparison with the control well-fed monkeys. Associated with the increase in brain histidine was a marked elevation of brain histamine level. Protein deficiency also led to poor brain retention of ascorbic acid but not to the same degree observed in the ascorbic acid-deficient animals. The latter group of animals, after receiving their diet for about 8 months, demonstrated a modest elevation in the plasma levels of most amino acids in comparison with controls. Ascorbic acid deficiency elicited a significant reduction (p < 0.01) in brain level of histidine, with hardly any change in homocarnosine level. In addition, vitamin C deficiency produced elevation of brain histamine level comparable to findings in the protein-energy-deficient monkeys. The results suggested that protein deficiency raised brain histamine level mainly through increased availability of the precursor amino acid histidine, while defective degradation might account for the increased brain level of this amine in ascorbic acid-deficient monkeys. Histamine has been proposed to have a predominantly depressant action on relevant neurons, and has also been shown to participate with other neuro-transmitters in influencing the function of the pituitary gland by regulating release of the hypothalamic hormones into the portal vessels. The relevance of the findings of marked increases in brain histamine in experimental protein and ascorbic acid deficiencies to the behavioral and extensive endocrinological alterations seen in human malnutrition deserves some intensive investigation.     

Depolarization-evoked release of gamma-hydroxybutyrate from rat brain slices

The release of gamma-hydroxybutyrate from preloaded rat brain striatal slices was investigated. K+-induced depolarization caused an efflux of gamma-hydroxybutyrate of about 50 fmol min-1 mg-1 (wet weight), but in a Ca2+-free medium containing Mg2+, the evoked release was reduced by 50-60%. The release was higher when 100 microM veratridine was used as a depolarizing agent. The efflux of gamma-hydroxybutyrate is related to veratridine and K+ concentration, and is strongly inhibited by 10 microM tetrodotoxin. The Ca2+ channel blocker verapamil induces a large decrease in the efflux of gamma-hydroxybutyrate after both K+- and veratridine-induced depolarization. These results are in favour of a possible transmitter function for gamma-hydroxybutyrate in rat striatum.     

Stimulation of Free Fatty Acid and Diacylglycerol Accumulation in Cerebrum and Cerebellum During Bicuculline-Induced Status Epilepticus. Effect of Pretreatment with α-Methyl-p-Tyrosine and p-Chlorophenylalanine

The pool size and composition of free fatty acids (FFA) and diglycerides (DG) from the cerebrum and cerebellum of rats undergoing bicuculline-induced seizures were studied. A fourfold increase in cerebral FFA occurred 3-4 min after bicuculline injection; arachidonic and stearic acids were the principal fatty acids accumulated. Cerebellar FFA also increased, but to a lesser extent. An increased production of arachidonic acid took place in the cerebrum as a function of time after bicuculline injection. Other fatty acids produced were oleic, palmitic, and docosahexaenoic acids. A twofold increase in cerebral arachidonic acid was seen at the time of the first generalized tonic-clonic convulsion. However, a 13- to 17-fold increase in arachidonic acid was seen approximately 5-6 min after bicuculline injection. The rise in other FFA was much smaller. Stearoyl- and arachidonoyl-DG were also accumulated. The drug alpha-methyl-p-tyrosine was found to (a) potentiate the bicuculline-stimulated release of cerebellar FFA, and (b) inhibit by 70% the production of stearoyl- and arachidonoyl-DG in the cerebrum and cerebellum. Basal production of FFA was stimulated by p-chlorophenylalanine, but the drug had no effect on the bicuculline-induced changes. Hydrolysis of phospholipids enriched in stearoyl-arachidonoyl groups, such as phosphatidylinositol of excitable membranes, may be stimulated during seizures.     

On the ultrastructural localization of catecholamines in the beta lobes (corpora pedunculata) of Periplaneta americana

Summary In the brain of the cockroach Periplaneta americana, the beta lobes of the corpora pedunculata respond with an intense positive reaction to a specific fluorescence histochemical method for catecholamines. The fluorescence reaction disappears completely after prolonged treatment of the cockroaches with reserpine. An ultrastructural examination of the beta lobes in formaldehyde-glutaraldehyde-osmium fixed preparations reveals the presence of two types of fibres: 1) Fibres and nerve endings containing small clear vesicles and sligthly larger vesicles with a semi-dense content. The appearance and size distribution of these vesicles ist not affected by treatment with reserpine. 2) Fibres containing larger and denser vesicles, but practically no clear vesicles. The size distribution of these dense vesicles is only slightly affected by treatment of the cockroaches with reserpine.If brain slices are incubated in a medium containing noradrenaline or -methyl-noradrenaline and fixed in permanganate, small vesicles with electron-dense central cores show up, similar to those which have been described in vertebrate adrenergic nerve fibres (small granular vesicles). They are confined to one of the two types of fibres (a and b) visible in these preparations, namely to type b, whose correspondence with type 2 fibres of formaldehyde-glutaraldehyde-osmium fixed preparations is discussed.The authors wish to thank Mr. E. Chessa and Mr. F. Piccirilli for technical assistance in photography.     

2''-Deoxycoformycin Inhibition of Adenosine Deaminase in Rat Brain: In Vivo and In Vitro Analysis of Specificity, Potency, and Enzyme Recovery

2'-Deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase (ADA), is increasingly used as a tool to investigate adenosine metabolism and neuromodulation. To advance further the usefulness of DCF for studies of purines in the CNS, we determined the inhibitory potency of this compound against ADA and adenylate deaminase (AMPDA) in brain, the rate of ADA recovery in various brain regions after single or repeated intraperitoneal DCF administrations, and the effect of DCF on several neurotransmitter synthetic enzymes. In vitro, the Ki values for inhibition of ADA and AMPDA were found to be 23 pM and 233 microM, respectively. In vivo, DCF inhibited ADA with ED50 values ranging from 155 to 280 micrograms/kg at 2 h posttreatment, and 98% inhibition was achieved with 1 mg/kg. AMPDA activity was not affected by doses up to 5.0 mg/kg. In contrast to the greater than 95% inhibition of ADA seen 1 day after DCF at 5 mg/kg, the effectiveness of a second similar DCF treatment on the activity that had recovered by 14 days was dramatically reduced. Eight days after DCF treatment with doses of 5-50 mg/kg, the degree of ADA activity recovery in 10 brain regions examined was similar; it averaged 35% of control values at the low dose but showed some heterogeneity, ranging from 15 to 54% of control values, at the higher doses. Forty days after treatment with a single dose of 5 mg/kg, ADA activity recovered by 68-78% of control values in brain regions with normally high levels of activity and by 44-59% of control values in other regions. The activities of choline acetyltransferase, glutamic acid decarboxylase, and histidine decarboxylase (an enzyme colocalized with ADA in hypothalamic neurons) were unaffected by DCF treatment, a result suggesting the lack of a generalized neurotoxic effect. The very low doses of DCF required for ADA inhibition in vivo are consistent with the high potency of this drug against ADA in vitro, and any physiological effects observed at low doses might therefore be ascribed to inhibition of ADA.