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.     

METHYL MERCURY INHIBITION OF SYNAPTOSOME AND BRAIN SLICE PROTEIN SYNTHESIS: IN VIVO AND IN VITRO STUDIES

Subacute methyl mercury (MeHg) intoxication was induced in adult rats following the daily intragastric administration of 1 mg MeHg/100 g body weight. Decreased [¹⁴C]leucine incorporation into cerebral and cerebellar slice protein was found. Weight loss occurred during the latent and neurotoxic phases but pair feeding did not reveal a significant defect in amino acid incorporation into slice protein. There was no decline in synaptosome protein synthesis in vitro during the latent phase but a significant decline in cerebellar and cerebral synaptosome synthesis was found during the neurotoxic phase. MeHg in vitro inhibited cerebral slice and synaptosome protein synthesis at half maximal concentrations of 7.5 and 12.5 μM respectively. Inhibition of synthesis in synaptosomes was non-competitive with K₁ of 4 × 10^?6M. MeHg had no effect on [¹⁴C]leucine or [¹⁴C]proline uptake into synaptosomes. There was no significant inhibition of synaptosome basal ATPase or Na + K ATPase at concentrations of MeHg (12 μM) giving half maximal inhibition of protein synthesis. No preferential inhibition of the chloramphenicol (55S) or cycloheximide sensitive components of synaptosome fraction protein synthesis was found, suggesting that the inhibition is common to both mitochondrial and extramitochondrial protein synthesizing systems. Addition of nucleotides and/or atractylate failed to influence protein synthesis and did not reverse the MeHg inhibition. Mannitol, as a replacement for the predominant cation species of the incubation medium, gave 40% inhibition of protein synthesis in the control but protected against further inhibition by MeHg.     

MEASUREMENTS OF RATES OF PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The use of tracer concentrations of labelled amino acids to measure incorporation in incubated slices of brain results in wide fluctuations with time in the specific activity of the precursor. Using concentrations of about 1 mm of labelled amino acid facilitates the accurate measurement of rates of synthesis. These higher precursor levels in the medium decrease the fluctuations in free amino acid specific activity due to dilution by endogenous amino acid and the production of amino acid by protein degradation, and decrease the lag in incorporation due to transport phenomena. Concentrations of 1 mm amino acid in the medium did not inhibit protein synthesis; with valine, leucine, phenylalanine, lysine and histidine, incorporation rates were similar when measured at trace concentrations and at 1 mm medium levels. The source of amino acid for protein synthesis appears to be intracellular. No evidence could be found for the preferential use of extracellular medium amino acid. The rate of incorporation of amino acids in incubated slices of rat brain was 0.087 per cent of the protein amino acid/h.     

INHIBITION OF 5''-NUCLEOTIDASE IN RAT BRAIN BY METHYLXANTHINES

—Rat brain 5′-nucleotidase (EC 3.1.3.5) is inhibited by methylxanthines such as theophylline. Inhibition of the 5′-nucleotidase by theophylline appears more efficient than the inhibition of cAMP phosphodiesterase by this drug. A similar inhibition is observed with caffeine, theobromine, 7′-methyl-xanthine and 1-methylxanthine.     

INHIBITION BY APOMORPHINE OF DOPAMINE DEAMINATION IN THE RAT BRAIN

Abstract— Apomorphine (A) inhibited dopamine deamination by rat brain mitochondria, but did not influence catechol- O -methyltransferase (COMT) activity by brain homogenates. The administration of apomorphine (10mg/kg i.p.) to normal rats increased brain dopamine (DA) by 34 per cent and decreased homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) by 60 per cent. In rats treated with reserpine 15 min prior to A, the latter prevented the rise of cerebral HVA and DOPAC and the depletion of DA produced by the former. Finally, A decreased the L-DOPA-induced accumulation of HVA and DOPAC in the rat basal ganglia. These results indicate that A inhibits DA deamination by monoamine oxidase.
This inhibition seems to be specific since apomorphine did not influence 5-HIAA levels in normal rats and prevented neither central 5-HT depletion nor 5-HIAA rise induced by reserpine.     

ANALYSIS OF THE MAJOR AMINO ACIDS OF RAT BRAIN AFTER IN VIVO INHIBITION OF GABA TRANSAMINASE BY ETHANOLAMINE O-SULPHATE

The effect of in vivo inhibition of GABA transaminase by ethanolamine O-sulphate on the content of the free amino acids in rat brain has been studied. Intracisternal injection of 2.0 mg/kg resulted in a progressive increase in GABA levels with time, to reach after 8 h a 100 per cent increase over saline-injected control animals. The effect of injection of 0.5, 1.0 and 2.0 mg/kg was studied 24 h after injection and the results showed that the increased GABA levels were dependent on the dose of inhibitor employed. Apart from the substantial increase in the GABA concentration of the brain there were no significant changes in the content of the other amino acids except for a small but significant decrease in aspartic acid in one experiment. When the extent of inhibition of the transaminase was correlated with the rise in GABA concentration it was shown that no elevation occurred until more than half of the enzymic activity had been inhibited.     

THE UPTAKE OF PURINES BY RAT BRAIN IN VIVO AND IN VITRO

Abstract— The uptake of [¹⁴C]guanine and some of its [¹⁴C]-labelled derivatives into rat brain was studied in vivo and in vitro. In vivo guanine, guanosine, and hypoxanthine penetrated the brain of adult rats to a very small extent. Inosine was taken up somewhat better. In young animals, also, guanosine was taken up poorly, but guanine was taken up fairly well. When guanine was administered to adult animals, only guanine was found in the brain. In young animals, by contrast, radioactivity from guanine appeared in guanosine and in guanine nucleotides, but no free guanine was found. In vitro guanine was taken up much better and, in fact, remained mostly as guanine in slices from 10-day-old rats. The in vitro conversion of guanine to GMP and its incorporation into RNA was unimpaired by the addition of unlabelled guanosine, an indication that guanine was converted directly to GMP. The uptake of guanine in vitro was not subject to competitive inhibition or influenced by the presence of dinitrophenol. This finding suggested that guanine entered the slice by simple diffusion.     

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 ACUTE HYPERTHERMIA ON POLYRIBOSOMES, IN VIVO PROTEIN SYNTHESIS AND ORNITHINE DECARBOXYLASE ACTIVITY IN THE NEONATAL RAT BRAIN

—Acute hyperthermia produces in situ disaggregation of brain polyribosomes in infant rats, as determined by electron microscopy. Protein synthesis is inhibited in infant, but not weanling, rat brain by 45 min of hyperthermia; this inhibition is reversed during a 2 h recovery period at normothermic conditions. Hepatic protein synthesis was inhibited less than that of brain. Acute hyperthermia also leads to a profound loss of ornithine decarboxylase activity in brain; during recovery the activity of this enzyme overshoots to values greater than those of normothermic control rats. This increase is blocked by cycloheximide administration. In testis, a tissue with high ornithine decarboxylase activity, enzyme activity was not affected by hyperthermia and recovery, indicating tissue specificity for these effects.     

PROTEIN SYNTHESIS IN ISOLATED NUCLEI FROM ADULT RAT BRAIN

Nuclei from adult rat brains isolated with isotonic sucrose were incubated with [³H]leucine and later purified by centrifugation through hypertonic sucrose solutions. It was found that under these conditions, tritiated leucine was incorporated into TCA precipitable material. Protein synthesis was impaired if the nuclei were treated with the nonionic detergent Triton X-100 or hypertonic sucrose. The presence of puromycin or cycloheximide markedly inhibited the incorporation of the radioactive amino acid. Actinomycin D and RNase did not have any effect on the incorporation. Autoradiography indicated the presence of labelled material within the nuclei and not in cytoplasmic contaminants. Glial nuclei were more actively involved in protein synthesis than neuronal nuclei.     

IN VIVO RELEASE OF ENDOGENOUSLY SYNTHESIZED CATECHOLAMINES FROM THE CAT BRAIN EVOKED BY ELECTRICAL STIMULATION AND BY d-AMPHETAMINE

Abstract— The cerebral ventricles of spinal-sectioned cats were perfused with artificial cerebrospinal fluid after the intraventricular administration of [³H]DOPA or [³H]tyrosine. Endogenously synthesized [³H]dopamine or [³H]norepinephrine were identified in the perfusate. Electrical stimulation of catecholaminergic nerve tracts in the hypothalamus increased the efflux of both catecholamines. The addition of d -amphetamine to the perfusing cerebrospinal fluid caused a large increase in [³H]dopamine and a small increase in [³H]norepinephrine appearing in the perfusate. Most of the endogenously synthesized [³H]catecholamines detected in the perfusate following stimuli originated from structures bordering the lateral cerebral ventricle. Thus, norepinephrine and dopamine can be synthesized in and released from catecholaminergic nerve terminals in structures bordering the cerebral ventricles.     

PROTEIN SYNTHESIS IN VITRO IN RAT BRAIN AFTER SHORT-TERM PROTEIN STARVATION AND REFEEDING

Rats were fed a protein-free diet for 4 or 6 days. They were compared with rats kept on the same diet for 3 or 5 days and on adequate protein for one additional day. The incorporation of ¹⁴C-labelled amino acid into protein was studied in systems containing ATP, GTP, phosphoenolpyruvate, pyruvate kinase and if required, a mixture of unlabelled amino acids and either the 6000 g supernatant fraction of a brain homogenate or microsomes and soluble enzymes. The 6000 g supernatant fraction showed variation in amino acid incorporating activity as well as in RNase activity as measured by breakdown of labelled polyuridylic acid. There was no difference in RNase activity in isolated microsomes, but the amino acid incorporating activity was significantly higher in preparations obtained from rats fed one meal of protein after 5 days of protein-starvation.     

EFFECTS OF AMPHETAMINE ADMINISTRATION IN VIVO ON IN VITRO PROTEIN SYNTHESIZING SYSTEM FROM RAT BRAIN 1

Abstract— A highly active in vitro protein synthesizing system (S-28) has been prepared from rat brain. Poly (U)-dependent [³H] phenylalanine incorporation by brain S-28 system is significantly inhibited by D-amphetamine. The extent of inhibition by amphetamine is significantly higher than by other biogenic amines such as dopamine and serotonin. At the 100°g level of amphetamine, the inhibition is about 70°. Experiments with ribosomes and soluble enzymes from control and amphetamine-treated systems indicate that the observed inhibition may be due to the effect of the drug on the ribosomes. Kinetic analysis of the reaction mixture in the presence as well as absence of D-amphetamine indicate that this sympathomimetic drug inhibits polysome formation in vitro.     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

PROTEIN SYNTHESIS IN THE MEDULLA OF THE RAT BRAIN. REPRODUCIBILITY OF THE RESULTS

Abstract— [¹⁴C]Leucine was injected intracranially into the brainstem reticular formation at the level of the upper medulla by the stereotaxic method. Subcellular fractions prepared 3 hr after injection showed that the specific activities of leucine-incorporated proteins decreased in the order soluble, microsomal, nuclear and mitochondrial fractions. Specific activities of proteins in the sera were 2·2 per cent of whole homogenate proteins.
The results from 27 experiments showed that 66·6 per cent of the mean specific activities of proteins extracted from whole homogenates fell within ·1 s.d . and 100 per cent within ± 2 s.d . (close to a normal distribution). The coefficients of variation were between 40 and 50 per cent for whole homogenates, sera and all subcellular fractions. Reproducibility of results and factors concerned with possible errors in the technique are discussed.     

THE EFFECT OF ISCHAEMIA AND RECIRCULATION ON PROTEIN SYNTHESIS IN THE RAT BRAIN

Abstract— Rats were subjected to cerebral compression ischaemia for 15min and were subsequently recirculated with blood for periods up to 3 h. In vivo incorporation of intravenously administered L-[1–¹⁴C]valine into total brain proteins was found to be severely inhibited (about 20% of controls) after 45 min of recirculation. After 3 h, protein synthesis had increased, the specific radioactivity of proteins then being about 40% of controls. The post-ischaemic inhibition of protein synthesis was accompanied by a breakdown in polyribosomes and a concomitant increase in ribosomal subunits. In vitro incorporation of L-[1–¹⁴C]phenylalanine by a postmitochondrial supernatant system derived from animals subjected to 15 min ischaemia and 15 min recirculation was also severely reduced and showed, in contrast to control animals, no response to the addition of a specific inhibitor of polypeptide chain initiation (Poly(I)). Together with the in vivo accumulation of ribosomal subunits this indicates a block in peptide chain initiation during the early stages of recirculation.
Polyribosomes from animals subjected to 15 min ischaemia without recirculation showed a normal rate of in vitro protein synthesis which was inhibited by Poly(I) to a similar extent as polyribosomes from control animals. These results suggest that the post-ischaemic inhibition in chain initiation develops during the early stages of recirculation rather than during the ischaemic period itself.     

INHIBITION OF THE BIOSYNTHESIS OF ISOPRENOID COMPOUNDS BY PHENOLIC ACIDS IN THE RAT BRAIN

—The incorporation of [2-¹⁴C]mevalonate into nonsaponifiable lipids by rat brain homogenates is inhibited by phenolic acids derived from tyrosine. The phenyl acids derived from phenylalanine are inhibitory only at very high concentrations compared with phenolic acids. The brain is more sensitive to inhibition by the phenolic acids than the liver. These studies indicate a possible role for phenolic acids in the impairment of cerebral sterol metabolism in phenylketonuria.     

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.     

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.