Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.     

Dose and time dependent effects of morphine on the incorporation of [3H]valine into soluble brain and liver proteins

Morphine (10^?6–10^?5M) causes an increase in incorporation of [³H]valine into soluble proteins during 4 hr in rat brain cortical slices, liver slices and cultivated astroglial cells. The effects are dose-dependent. They are neither cell specific nor strictly related to classical opiate receptors. Pulse-labeling with [³H]valine for 60 min after incubation in 10^?6–10^?5M morphine, resolves time-dependent changes in incorporation, with both increases and decreases in protein metabolism.     

A Quantitative Regional Analysis of Amino Acids Involved in Rat Brain Protein Synthesis by High Performance Liquid Chromatography

A biochemical method is described for the simultaneous quantitative estimation of unidirectional blood-brain amino acid influx and protein biosynthesis in individual structures of the rat brain. The method involved a double labeling experiment started by the administration of [14C]carboxyl-labeled amino acids and terminated 2 min after infusion of 3H-labeled amino acids, each at tracer quantities, the total labeling period being 45 min. Specific radioactivities of 14C- or 3H-labeled phenylalanine, tyrosine, leucine, isoleucine, and valine were determined in plasma and in small brain tissue samples for free amino acids, aminoacyl-tRNAs, and proteins. Amino acids were converted to their corresponding 5-dimethylamino-naphthalenesulfonyl (Dns, dansyl) derivatives and separated on HPLC C18 reversed-phase columns isocratically according to a newly developed optimizing procedure. The order of influx values between the neutral amino acids in relation to each other was Leu greater than Tyr greater than Ile greater than Phe greater than Val in every structure examined. Although aminoacylation of tRNAs was found to proceed to a comparable degree for neutral amino acids in all regions investigated, the specific radioactivity of amino acids attached to tRNAs differed substantially from that in the free amino acid pool, especially for leucine and valine. The results indicate the necessity of aminoacyl-tRNA determinations for tracer incorporation studies in protein synthesis analysis. Relative protein synthesis rates in the halothane-anesthetized rat were determined to be 30 and 67-91 pmol total amino acid incorporation/min/mg tissue for white and gray matter, respectively.     

Measurement of local rates of brain protein synthesis by quantitative autoradiography: Validation with L-[3H]valine

Following the injection of 4-day old rats with 150 mMl-[3,4-³H]valine (10mol/g, IP) the incorporation of³H into protein was linear 2 hours. Valine specific activity in the brain acid-soluble fraction was constant between 30 and 120 min after injection with a mean value of 82.3% of the injectate. Significant amounts of tritated metabolites accumulated in the brain acid-soluble fraction (41.4% of radioactivity at 120 min) but do not prove an impediment to measuring rates of protein synthesis. The rate of protein synthesis in cerebral cortex of the 4-day old rat was measured by quantitative autoradiography using [³H]valine and³H-sensitive film. The measured rate shows excellent agreement with that found previously usingl-[1-¹⁴C]valine. Our results suggest that [³H]valine can be a useful precursor to measure local rates of brain protein synthesis by quantitative autoradiography.     

Effects of acute and chronic nicotine on elevated plus maze in mice: involvement of calcium channels

The current experiments examined the anxiety-related effects of acute and repeated nicotine administration using the elevated plus maze test in mice. Nicotine (0.1 mg/kg s.c., 5 and 30 min after injection; 0.5 mg/kg, s.c., 5 min after injection) had an anxiogenic effect, shown by specific decreases in the percentage of time spent on the open arms and in the percentage of open arm entries. Tolerance developed to this anxiogenic action after 6 days of daily nicotine administration (0.1 mg/kg, s.c.). Five minutes after the seventh injection, an anxiolytic effect was observed, i.e., specific increases in the percentage of time spent on the open arms and in the percentage of open arm entries. L-type voltage-dependent calcium channel antagonists nimodipine (5 and 10 mg/kg, i.p.), flunarizine (5 and 10 mg/kg, i.p.), verapamil (5, 10, 20 mg/kg) and diltiazem (5, 10, 20 mg/kg, i.p.) were also injected prior to an acute low dose of nicotine or to each injection of chronic nicotine. Our results revealed that calcium channel blockers dose-dependently attenuated both an anxiogenic effect of nicotine as well as the development of tolerance to this effect. Our results suggest that neural calcium-dependent mechanisms are involved in the anxiety-related responses to acute and chronic nicotine injection that may ultimately lead to addiction and smoking relapse in human smokers.     

The program of protein synthesis during the development of the micromere-primary mesenchyme cell line in the sea urchin embryo

Changes in the pattern of protein synthesis were analyzed during the in vitro development of the micromere-primary mesenchyme cell line of the sea urchin embryo. Micromeres were isolated and cultured from 16-cell stage embryos, and primary mesenchyme cells were isolated and cultured from early gastrulae. Both cell isolates developed normally in culture with about the same timing as their in situ counterparts in control embryos. Newly synthesized proteins were labeled with [³H]valine at several stages of development and were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorgraphy. The electrophoretic pattern of labeled proteins changed dramatically during development. More than half of the analyzed proteins underwent qualitative or quantitative changes in their relative rates of valine incorporation and these changes were highly specific to this cell line. Almost all of the changes were initiated prior to gastrulation and many prior to hatching. The highest frequency of changes in the micromere pattern of protein synthesis occurred between hatching and the start of gastrulation. This peak of activity coincided with the normal time of ingression of the primary mesenchyme and preceded the differentiation of spicules by more than 30 hr. Most of the observed changes were characterized as either decreases in the synthesis of proteins that showed maximum incorporation at the 16-cell stage or increases in the synthesis of proteins that showed maxima in the fully differentiated cells. Very few proteins exhibited transient synthetic maxima at intermediate stages. Thus, the program of protein synthesis associated with the development of micromeres consists largely of a switch in emphasis from early to late proteins, with the primary time of switching being between hatching and the onset of gastrulation.     

β-Adrenergic Effects on Plasma and Brain Large Neutral Amino Acids Are Unaltered by Chronic Administration of Antidepressants

Effects of isoproterenol (3 mg kg-1, i.p. for 60 min) and salbutamol (3, 10 mg kg-1, i.p. for 60 min) on large neutral amino acid concentrations in rat plasma and brain were assessed. Phenylalanine, leucine, isoleucine, and valine were measured by gas chromatography with electron-capture detection; tyrosine and tryptophan were measured by HPLC with electrochemical detection. These drugs induced increases in brain tryptophan, tyrosine, phenylalanine, and valine and decreases in plasma tryptophan, tyrosine, leucine, isoleucine, and valine. Effects of salbutamol (3 mg kg-1, i.p. for 60 min) were assessed following chronic administration of phenelzine sulfate and desipramine.HCl (each drug 10 mg kg-1 per day, s.c. via Alzet 2ML4 osmotic minipumps for 28 days). There were no effects of these antidepressants on basal levels of large neutral amino acids in brain and plasma. In both brain and plasma, salbutamol-induced changes in large neutral amino acids were unaffected by these antidepressants. The results indicate that beta-adrenoceptor-regulated availability of plasma and brain large neutral amino acids is unaffected by chronic administration of tricyclic or monoamine oxidase inhibitor antidepressants.     

Macromolecular changes in brain stem of morphinized rats

Incorporation of [3H]valine into trichloroacetic acid-(TCA)-precipitable, water-soluble or membrane-bound material of whole brain and brain-stem did not differ significantly in morphine-intoxicated, morphine abstinent and control rats. The animals were intoxicated with morphine (final dose 340 mg/kg b.w.) for 15 days, using an ingestion method with no impairment of the caloric intake compared to controls. Abstinent rats were withdrawn from morphine for 2 days after 13 days of intoxication. Measurements of [3H]valine or [14C]valine incorporated into soluble or membrane-bound brain stem proteins failed to demonstrate any significant changes in specific protein bands from morphinized rats. Separation was achieved by polyacrylamide gel electrophoresis with or without sodium-dodecyl sulphate (SDS) or by isoelectric focusing. After immunoabsorption chromatography to remove those proteins antigenically similar to serum proteins, an increase in the staining intensity and in incorporation of [3H]valine into two protein bands (with isoelectric points (Ip:s) 5.75 and 7.7) was seen in brain stem from long-term morphine-intoxicated rats. The results show that macromolecular interactions are involved in long-term morphine actions.     

Stimulation of Fructose-1,6-Biphosphatase Activity and Synthesis in the Cerebral Cortex of Rats Submitted to the Convulsant Methionine Sulfoximine

Purification of rat cerebral cortex fructose-1,6-biphosphatase (FBPase) was performed by substrate elution from phosphocellulose, followed by Sephadex G-200 column filtration. The purified enzyme exhibited an optimum at pH 7.5, and its catalytic properties were very similar to those of the purified whole-brain enzyme previously prepared by Majumder and Eisenberg in 1977. The isolated preparation was electrophoretically homogeneous. The molecular weight of the enzyme subunit was 40,000; the hydrophobic amino acids predominated with 592 residues, and tryptophan was not detected. Expressed as mumol fructose-1,6-biphosphate hydrolysed per g brain tissue wet weight per min, FBPase activity increased twofold 24 h after an intraperitoneal injection of 100 mg per kg body weight of the convulsant methionine sulfoximine (MSO); the increase of the rate of incorporation of [1-14C]valine into brain FBPase was 2.8-fold under the same experimental conditions. A rabbit specific antiserum against rat cerebral cortex FBPase was prepared, and immunotitration studies confirmed both an increase in the number of molecules and the activation of brain FBPase, 24 h after administration of MSO. The increase of the number of brain FBPase molecules, induced by MSO, was due to an increase in synthesis of the enzyme, as shown by a double-label valine incorporation study.     

THE NATURE OF THE AMINO ACID POOL USED FOR PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The incorporation into brain slice protein of externally provided [1-¹⁴C]valine was measured at varying levels of valine in the medium, under conditions of constant protein synthesis and equilibration of intracellular valine specific activity. The results indicate that the valine pool used for protein synthesis is not identical to the pool of total free valine. Neither does the incorporation solely occur from an extracellular pool which is in equilibrium with the incubation medium. The data are compatible with a two-site activation model in which aminoacylation of tRNA occurs at both an internal site utilizing amino acid from the intracellular pool and an external (possibly membranous) site converting extracellular valine directly to valyl-tRNA. A good fit to the experimental observations is also provided by a compartmented intracellular valine pool model.     

Rates of protein synthesis by hepatocytes isolated from rats of various ages

The rate of total protein synthesis in isolated hepatocytes was determined. The incorporation of L-[³H]valine into protein is linear for at least two hours of incubation and is affected by the concentration of amino acids in the medium. Uptake of valine by hepatocytes from 1.5- and 18-month-old rats was identical and appears to occur by simple passive diffusion. Within five minutes, the specific activities of the intracellular and extracellular valine pools are equivalent. The specific activities of these pools are saturated by 1.6 mM valine and remain constant for 60 minutes of incubation. The rates of protein synthesis by hepatocytes from 1- to 2-month-old rats is 96.8 pmoles of valine per minute per milligram protein. This is comparable to rates of protein synthesis reported for perfused liver and liver in vivo and is approximately 64% higher than the rate of protein synthesis by hepatocytes from 18-month-old rats.     

Incorporation of [3H]Leucine and [3H]Valine into Protein of Freshwater Bacteria: Uptake Kinetics and Intracellular Isotope Dilution

Incorporation of [³H]leucine and [³H]valine into proteins of freshwater bacteria was studied in two eutrophic lakes. Incorporation of both amino acids had a saturation level of about 50 nM external concentration. Only a fraction of the two amino acids taken up was used in protein synthesis. At 100 nM, the bacteria respired 91 and 78% of leucine and valine taken up, respectively. Respiration of ³H and ¹⁴C isotopes of leucine gave similar results. Most of the nonrespired leucine was recovered in bacterial proteins, while only up to one-half of the nonrespired valine occurred in proteins. In intracellular pools of the bacteria, [³H]leucine reached an isotope saturation of 88 to 100% at concentrations of >40 nM. For [³H]valine, an isotope equilibrium of about 90% was obtained at concentrations of >80 nM. Within an incubation period of typically 1 h, tritiated leucine and valine incorporated into proteins of the bacteria reached an isotope saturation of 2 to 6%. In a 99-h batch experiment, bacterial protein synthesis calculated from incorporation of leucine and valine corresponded to 31 and 51% (10 nM) and 89 and 97% (100 nM), respectively, of the chemically determined protein production. Measured conversion factors of 100 nM leucine and valine were 6.4 × 10¹⁶ and 6.6 × 10¹⁶ cells per mol, respectively, and fell within the expected theoretical values. The present study demonstrates that incorporation of both valine and leucine produces realistic measurements of protein synthesis in freshwater bacteria and that the incorporation can be used as a measure of bacterial production.     

In vitro synthesis of myelin proteolipid protein from rat brain using a total homogenate system

In vitro synthesis of myelin proteolipid protein (PLP) was explored at different ages using rat brain total homogenates, incubated for 30 min with [³H]glycine. Total proteolipids, extracted from the incubated samples, were separated by SDSPAGE and the radioactivity was measured in the band corresponding to myelin PLP. The incorporation into PLP in relation to the incorporation into brain total proteins increased from 0.04% at 10 days of age to 0.63% at 20 days, and declined slowly thereafter. Time course experiments were carried out using brain homogenates obtained from rats of 20 days of age (i.e. at the period of maximal synthesis of PLP). Labeled PLP molecules were already found at 2.5 min of incubation and the incorporation of the label into this protein, relative to the incorporation into total proteins, did not vary throughout the entire incubation time (30 min). Pulsechase experiments using a similar system and adding cycloheximide at different incubation times showed that the appearance of label into mature PLP was immediately blocked by the inhibitor of protein synthesis. These data suggest that PLP is synthesized as such and not as a pre-protein which is subsequently processed to render mature PLP.     

Effect of L-DOPA pretreatment on invivo protein synthesis in various rat brain regions

A single dose of L-dihydroxyphenylalanine (L-DOPA, 500 mg/kg, i.p.) that caused massive disaggregation of brain polysomes also suppressed the incorporation of [³H] lysine into trichloracetic acid (TCA)-precipitable proteins of cortex, cerebellum, hypothalamus, brainstem and striatum. The magnitude of this inhibition of [³H] protein synthesis was similar in all brain regions studied, and was not related to changes in the specific activity of the precursor amino acid.     

反复摄取烟碱对脑肌醇含量的影响

急性实验中,间隔５ｍｉｎ反复注射烟碱０．５,１．０,１．０,２．０,２．０ｍｇ／ｋｇｉｐ,３０ｍｉｎ后大鼠大脑皮层及海马中肌醇含量升高,但纹状体中肌醇含量无显著变化;相同条件下,氯化锂１０ｍｍｏｌ／ｋｇｉｐ３０ｍｉｎ后大脑皮层和海马中肌醇含量显著降低;慢性实验中,烟碱２．０－１０．０ｍｇ／ｋｇｓｃｂｉｄ１４ｄ后,大鼠大脑皮层中肌醇含量显著增高;烟碱２．６９－１１．５３ｍｇ／ｋｇ／ｄｐｏ６４ｄ后,大鼠大脑皮层中肌醇含量也显著增高。表明烟碱的作用不同于氯化锂,反复给予烟碱可使大鼠大脑皮层中肌醇含量增加。     

Protein metabolism and electrophoretic profiles in astroglial primary cultures from different regions of newborn rat brain

Primary astroglial cultures (14 days of age) from cerebral cortex, striatum, and hippocampus of newborn rat brain contained similar amounts of soluble proteins. Uptake and incorporation of [³H]valine into soluble protein measured after 30 and 60 min of incubation, respectively, was on a similar level in the various cultures. [³H]valine labeling of protein bands from the cell soluble fractions and incubation media of hemisphere cultures, and which were separated by isoelectric focusing (IEF) or sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), indicated that proteins are released to the extracellular medium after being synthesized within cultivated cells. Acidic and high molecular weight proteins were more heavily labelled in the incubation media than in the cell soluble fractions. Two-dimensional electrophoresis (IEF×SDS-PAGE) of soluble proteins from the different cultures showed similar patterns, which were quite different from the serum-free extracellular protein patterns. Both fractions were different from the pattern obtained from fetal calf-serum. In striatum and hippocapus culture media a spot was seen with Ip 6.0–6.8 and m.w. 105,000, and in the media from cerebellar cultures another spot was observed with Ip 5.2–5.6 and m.w. 55,000. The results show that the different cultures are similar in their protein synthetic capacity and protein composition. The specific differences observed in proteins obtained from the serum-free incubation media might indicate specific properties among astroglial cells from various brain regions.     

EFFECTS OF ADDED SUBSTANCES ON RNA AND PROTEIN SYNTHESIS IN IMMATURE NEURONS

Abstract— A newly described method for the isolation of morphologically intact neurons from newborn rat brain was used to study the influence of inhibitors and neuroactive substances on RNA and protein synthesis in these cells in vitro . Incorporation of [¹⁴C]-uridine into RNA and [³H]leucine into protein proceeded rapidly and continued up to 3 h. When the incorporation mixture was chased at 20 min with an excess of nonradioactive uridine and leucine, hardly any degradation of labelled RNA was noted during the following 2 h 40 min. In contrast, the specific radioactivity of proteins decreased by 22 per cent indicating turnover of cellular proteins.
Incorporation of labelled leucine into protein was markedly inhibited in the presence of NaF and cycloheximide but not affected in the presence of chloramphenicol or pancreatic RNase. A mixture of ATP + GTP depressed the incorporation by 38 per cent. The responses to ATP + GTP and RNase indicated that the incorporation system was typically cellular. Acetylcholine, γ-aminobutyrate, noradrenaline and phenylalanine in the incubation medium depressed the incorporation of labelled uridine into RNA by 10–30 per cent and 5-hydroxytryptamine by 75 per cent. Acetylcholine, γ-aminobutyrate and noradrenaline had no effect on protein synthesis, while 5-hydroxytryptamine and phenylalanine inhibited the incorporation by 60–80 per cent. Testosterone and prednisolone depressed both RNA and protein synthesis while thyroxine caused slight but non-significant stimulation.     

Brief fasting decreases protein synthesis in the brain of adult rats

The influence of starvation on protein synthesis in the adult rat brain was studied in vivo by an intravenous injection of a flooding dose of unlabeled valine including a tracer dose ofL-[3,4(n)-³H]valine. Brief starvation (24 hours) induced a 20% decline in fractional and absolute rates of brain protein synthesis. This decline resulted from a 20% decrease in the efficiency of protein synthesis (g protein synthesized per day per g RNA) whereas the capacity for protein synthesis (g RNA per mg protein) was maintained. Prolonged starvation (5 days) was marked by no further significant changes in the fractional rate, absolute rate and efficiency of protein synthesis, whereas the capacity for protein synthesis cecreased slightly. The relative contribution of brain to wholebody body protein synthesis increased during fasting, and neither the protein nor the RNA brain content did change during the experiment. These results clearly indicate that brain proteins are spared in response to brief and prolonged food deprivation, and that brain protein synthesis is very sensitive to short-term fasting.     

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.