Biosynthesis of serotonin in Raphé nuclei of rat brain: effect of p-chlorophenylalanine

The activity of tryptophan hydroxylase (EC 1.99.1.4) in the region of the raphé nuclei of rat brain was higher than that of any other brain area. The content of serotonin and the rate of serotonin synthesis were also highest in the raphé nuclei. Following the administration of p-chlorophenylalanine the injection of tryptophan and pargyline increased the content of serotonin in the region of the raphé nuclei of rat brain. The results suggest that the raphé nuclei retained the capacity to hydroxyl-late tryptophan to some extent after the injection of p-chlorophenylalanine.     

Increase of Brain Tryptophan caused by Drugs which stimulate Serotonin Synthesis

THE concentrations of tryptophan normally present in the mammalian brain are below the Michaelis constant (K_m) of tryptophan hydroxylase^1,2, suggesting that the rate of serotonin synthesis depends more on the concentration of brain tryptophan than on the amount of enzyme. We wish to report that various treatments which have been shown to increase brain serotonin synthesis also increase the concentration of tryptophan in brain. Conversely, p-chlorophenylalanine (PCPA), which inhibits serotonin synthesis³, decreases tryptophan in brain.     

Characterization of experimental phenylketonuria augmentation of hyperphenylalaninemia with α-methylphenylalanine and p-chlorophenylalanine

Phenylalanine in conjuction with p-chlorophenylalanine or α-methylphenylalanine was administered to suckling rats to induce hyperphenylalaninemia reminiscent of untreated phenylketonuria, and developmental parameters were monitored. The experimental model utilizing p-chlorophenylalanine was found to be unsatisfactory, in that the drug had general deleterous effects on growth, numerous side effects including increased mortality, and affected brain levels of biogenic monoamine neurotransmitters. The model utilizing α-methylphenylalalanine was relatively free from nonspecific effects and thus, changes observed in the animals were attributable to expereimental phenylketonuria. The latter animals had slightly decreased body and brain weights, and exhibited grossly elevated serum phenylalanine and urinary excretion of phenylketone metabolites. Hyperphenylalaninemia produced greatly disrupted brain amino acids at 10 days of age (prior to the formalization of the blood-brain barrier and specific transport systems) which was limited by 30 days of age to changes in glycine, γ-aminobutyric acid and the aliphatic and aromatic amino acids which compete for uptake in t he brain by a common carrier. These animals also exhibited a myelin deficit and changes in proteins from isolated nerve cell preparations. Mature animals which had daily treatment up to 60 days of age results obtained with animal models and the clinical findings in untreated phenylketonuric patients.     

The inverse relationship between the activity of pyridoxal kinase and the level of biogenic amines in rabbit brain

In a series of experiments, an inverse relationship was found to exist between the activity of pyridoxal kinase and the concentrations of brain norepinephrine, dopamine, and serotonin. When the biogenic amine content of the brain is lowered, the activity of pyridoxal kinase rises. Conversely, when the concentration of brain biogenic amines is elevated, a depression occurs in the activity of pyridoxal phosphokinase. This relationship, which resembles an in vivo allosteric inhibition phenomenon, not only is highly suggestive of a definite relation between the brain vitamin B₆ content and the metabolism of the biogenic amines, but also suggests that the concentration of brain biogenic amines might control the production of coenzyme pyridoxal phosphate.     

Effects ofp-chlorophenylalanine on cortical monoamines and on the activity of noradrenergic neurons

The catecholamines noradrenaline, dopamine, adrenaline, the indoleamine 5-hydroxy-tryptamine (5-HT; serotonin), and some of their major metabolites were assayed, using high performance liquid chromatography (HPLC), in the neocortex of normal rats as well as in animals in which 5-HT synthesis had been inhibited withp-chlorophenylalanine. Besides important depletions in serotonin and in 5-hydroxyindole-3-acetic acid, noradrenaline levels were significantly reduced, but the content in 3-methoxy-4-hydroxyphenylglycol was increased, indicating an augmented utilization of this amine. The levels of dopamine and 3-methoxytyramine were also reduced, although homovanillic acid and 3,4-dihydroxyphenylacetic acid levels remained constant. The spontaneous unitary activity of identified noradrenergic neurons in the Locus coeruleus was increased, indicating an hyperactivity of this system. These results can be interpreted in relation to functional interactions between the catecholamines and serotonin; i.e.: a decrease in endogenous serotonin results in the loss of a negative feedback control of noradrenaline release.Special Issue dedicated to Prof. Eduardo De Robertis.     

Effect of <Emphasis Type="Italic">p</Emphasis>-Chlorophenylalanine on Tryptophan Hydroxylase in Rat Pineal

KOE and Weissman have demonstrated that p-chlorophenylalanine (pCPA) depletes serotonin in the brain of mammals¹. pCPA induces a variety of behaviour changes in rat, cat and other animals^2,3, presumably because of the depletion of serotonin in the brain. The biochemical mechanism of the depletion remains, however, to be elucidated, although it has been proposed that pCPA might inactivate tryptophan hydroxylase in the brain⁴. We demonstrate here that pCPA does not inactivate tryptophan hydroxylase in rat pineal, although pCPA depletes the serotonin level in the pineal.     

Improved Production of Human Immunoglobulin γ1 Chain Fc Polypeptides in Escherichia coli and Their Properties

Exposure to some xenobiotics (pentobarbital, 3-terf-butyl-4-methoxyphenol (BHA), chloretone (acetone chloroform), 1, l-bis-(p-chlorophenyl)-2,2,2-trichloroethane (DDT) and polychlorinated biphenyls (PCB)) for a 5 hr period increased the concentrations of brain serotonin and 5-hydroxyindole acetic acid (5HIAA). The decrease in the brain serotonin level elicited by /7-chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, was prevented by the concomitant administration of chloretone. The administration of both chloretone and pargyline (an inhibitor of monoamine oxidase) caused significant elevation of the brain 5HIAA level as compared with that in a pargyline control, however, the concentration of brain serotonin was not different between pargyline alone and chloretone plus pargyline. These results show that the increase in the brain serotonin level caused by chloretone is not due to acceleration of brain serotonin synthesis, but to retardation of the degradation of brain serotonin, and the increase in brain 5HIAA caused by chloretone may be due to the reduced removal of 5HIAA from the brain. Chloretone plus pargyline caused significant elevation of hypothalamus catecholamines, as compared to in the pargyline control, so the catecholamine turnover rates may be accelerated by the administration of chloretone.     

INCREASED CEREBRAL EXCITABILITY CAUSED BY p-CHLOROPHENYLALANINE IN YOUNG RATS

—Single intraperitoneal doses of p-chlorophenylalanine (p-CP; 100 mg/kg or more), lowered the threshold of flurothyl-induced seizures in 1- and 2-week old rats after 24 h, but not after 4 or 72 h. In older rats there was no change in cerebral excitability after a single dose, but the seizure threshold was lowered after several daily doses. Rats given p-CP in lower doses daily from birth exhibited lowered seizure thresholds only until 3-4 weeks of age. Depletion of brain serotonin and inhibition of liver phenylalanine hydroxylase by p-CP exhibited time courses and dose responses similar to those reported for older animals. Activity of phenylalanine hydroxylase returned to normal more rapidly than the content of serotonin after a dose of p-CP, and recovery of normal seizure threshold seemed to be more closely associated with recovery of the hepatic enzyme than with restoration of the brain amine. It appears, therefore, that hyperphenylalanemia or some consequence of it may be an important factor contributing to increased cerebral excitability in p-CP intoxication and possibly in clinical phenylketonuria and that depletion of brain serotonin may not be the only or even the predominant cause of the changes in cerebral excitability in these conditions.     

Interaction of diet and drugs in the regulation of brain 5-hydroxyindoles and the response to painful electric shock.

The long-term consumption of a tryptophan-poor, corn diet by rats decreases electroshock response thresholds. This hyperalgesia appears to be related directly to diet-induced reductions in the brain concentrations of the putative neurotransmitter, serotinin. Rehabilitating corn-fed animals by feeding them the corn diets supplemented with tryptophan restores brain serotonin and pain thresholds to normal; similarly, injecting the tryptophan-deficient, corn-fed animals with fluoxetine, a drug that blocks the uptake of serotonin into brain neurons, also restores the electroshock response thresholds to control levels. The tryptophan hydroxylase inhibitor, $\text{p}$-chlorophenylalanine, increases the hyperalgesia to electroshock in corn-fed rats and further reduces brain serotonin concentrations. Injection of the amino acid valine, on the other hand, produces hyperalgesia and decreases brain serotonin in casein-fed rats but not in animals fed the corn diet. These data lend support to the hypothesis that serotonin neurons may mediate the sensitivity or reactivity to painful stimuli.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.     

THE EFFECTS OF CHRONIC REGIMENS OF CLORGYLINE AND PARGYLINE ON MONOAMINE METABOLISM IN THE RAT BRAIN

The effects of chronic administration of clorgyline and pargyline on rat brain monoamine metabolism have been examined. The inhibitory selectivity of these drugs towards serotonin deamina-tion (MAO type A) and phenylethylamine deamination (MAO type B) can be maintained over a 21-day period by proper selection of low doses of these drugs (0.5-1.0 mg/kg/24h). The results are consistent with MAO type A catalyzing the deamination of serotonin and norepinephrine and with MAO type B having little effect on these monoamines. Dopamine appears to be dcaminated in vivo principally by MAO type A. Clorgyline administration during a 3-week period was accompanied by persistent elevations in brain norepinephrine concentrations; serotonin levels were also increased during the first 2 weeks, but returned towards control levels by the third week of treatment. Low doses of pargyline did not increase brain monoamine concentrations, but treatment with higher doses for 3 weeks led to elevations in brain norepinephrine and 5-hydroxytryptamine; at this time significant MAO-A inhibition had developed. The changes in monoamine metabolism seen at the end of the chronic clorgyline regimen are not due to alterations in tryptophan hydroxylase activity. At this time tyrosine hydroxylase activity was also unaffected.     

CNS mediated inhibition of gastric secretion by bombesin: independence from interaction with brain catecholaminergic, and serotoninergic pathways and pituitary hormones

The effects of hypophysectomy and pharmacologic manipulation of brain biogenic amines on gastric secretion (volume and titratable acidity) and on CNS-mediated inhibition of gastric secretion by bombesin were studied in pylorus-ligated rats. Bombesin (100 ng), given intracisternally (i.c.), reduced the gastric secretory volume by 61%, raised pH values to 5 and virtually suppressed the titratable acidity of gastric secretion. Hypophysectomy did not modify the volume of secretion, lowered the gastric acid concentration by 37% and did not alter the magnitude of bombesin's suppressive effect, suggesting that pituitary-derived substances do not participate in the expression of bombesin's action. Depletion of brain catecholamines by combined administration of the neurotoxic agent 6-hydroxydopamine (400 μg twice, i.c.) and the catecholamine synthesis inhibitor α-methyl-p-tyrosine (250 mg/kg) or blockade of dopamine receptors by haloperidol (25 μg, i.c.), which induced a rise in plasma prolactin levels (indirect evidence of suppression of dopaminergic inhibitory tonus) neither modified gastric secretion nor the antisecretory effect of bombesin. Depletion of brain serotonin by the indolamine neurotoxin 5,6-dihydroxytryptamine (50 μg, i.c.) combined with p-chlorophenylalanine (315 mg/kg), an inhibitor of tryptophane-hydroxylase, did not affect gastric secretion or bombesin's action. Administration of dopamine, serotonin or noradrenaline at 10-μg dose levels i.c. had no effect on gastric secretion. The demonstration that pharmacologic measures designed to interfere with the normal functioning of brain catecholaminergic and serotoninergic systems did not modify gastric secretion is not in favor of their involvement in the brain control of gastric secretion. Moreover, the fact that the potent antisecretory action of bombesin is not mimicked by, nor dependent upon, intact biogenic amine pathways further supports the concept that a direct neuropeptidergic pathway may participate in the CNS regulation of gastric secretion.     

α-METHYLTRYPTOPHAN: EFFECTS ON CEREBRAL MONOOXYGENASES IN VITRO AND IN VIVO

Following administration of x-methyltryptophan (AMTP) (50 mg/kg) there was a time dependent decrease of serotonin and a concomitant increase of α-methyl-5-hydroxy-tryptamine (AM-5-HT) in most cerebral areas. AMTP is hydroxylated to α-methyl-5-hydroxytryptophan (AM-5-HTP) by cerebral tryptophan hydroxylase in vitro and in vivo. Hydroxylation of AMTP in vitro and in vivo was markedly inhibited in p-chlorophenylalanine (p-CP) treated rats. After administration of AMTP, the conversion in vivo of tyrosine to norepinephrine was inhibited. This inhibition was not apparent in p-CP pretreated animals. p-Chloroamphetamine (p-CA) (10 mg/kg) lowered serotonin and AM-5-HT levels in some areas of the brain, but unlike p-CP, alone or in combination with AMTP it did not significantly inhibit hydroxylation of tryptophan (Trp). AMTP, as substrate of tryptophan hydroxylase, has a K_m of 1.08 × 10^-4 M (using 6-MPH₄, as cofactor) and as competitive inhibitor, a K₁ of 2.09 × 10^-4M with L-Trp as substrate. AMTP becomes an uncompetitive inhibitor when its concentration is equal to or exceeds that of L-Trp. D-AMTP is neither a substrate nor an inhibitor of tryptophan hydroxylase. DL-AM-5-HTP (K₁, 1.5 × 10^-5 M) and AM-5-HT (K¹ 4.0 × 10^-5 M) are competitive inhibitors.     

Possible role of brain histamine in morphine addiction.

Several biogenic amines have been suggested to play a possible role in opiate addiction. While some reports indicated changes in brain norepinephrine and dopamine concentrations and/or synthesis (1,2,3), others have demonstrated the involvement of serotonin or acetylcholine (4,5,6,7). In view of recent reports suggesting a possible role for histamine in brain function as another putative neurotransmitter (8), we have investigated whether this biogenic amine might also participate in morphine addiction and withdrawal.     

Serotonin in the central nervous system of the cockroach, Periplaneta americana

Studies on serotonin in the insect nervous system has long been neglected, although serotonin is a putative neurotransmitter. During the course of this study the serotonin content was found to be significantly higher than that found in mammalian midbrain. Parachlorophenylalanine was found to inhibit the first step of the biosynthetic pathway by inhibiting tryptophan-hydroxylase enzyme and leading to alterations in the concentrations of metabolites such as 5-hydroxy tryptophan, 5-hydroxy indole acetic acid and tryptophan. Using a dose of 15 μg/g the inhibitory effect was not long lasting and recovery was observed to restore the normal levels. Higher trytophan levels were observed after a certain period of P-chlorophenylalanine treatment because there was a block in the biosynthetic path and tryptophan could not be utilized for 5-HT synthesis. A negative correlation between brain tryptophan and protein content was observed in both the cases of P-chlorophenylalanine and reserpine treatments.     

Effect of Growth Conditions on Serotonin Content of Tetrahymena pyriformis*

SYNOPSIS The serotonin content of Tetrahymena was measured under several growth conditions and after exposure to drugs. Serotonin was maximal during stationary phase, then declined to a level typical of logarithmically growing cells. Addition of 5-hydroxytryptophan to the culture increased cellular serotonin content, but neither L-tryptophan, reserpine, p-chlorophenylalanine, nor desmethylimipramine altered the serotonin content.     

DEVELOPMENTAL CHARACTERISTICS OF PHENYLETHANOLAMINE AND OCTOPAMINE IN THE RAT BRAIN

Abstract— Phenylethanolamine and octopamine have been detected in the developing rat brain. Maximum concentration of these amines occurs early in development (16-17 days of gestation). At this developmental stage, the brain concentration of these amines is higher than that of norepinephrine. There is a sharp decline in the phenylethanolamine and octopamine concentrations on day 18 of gestation to approximately those of the adult. This decrease coincides with an increase in-monoamine oxidase activity of fetal brain, with an increase in the activities of tyrosine hydroxylase and dopamine-β-hydroxylase, and with the appearance of a saturable active uptake mechanism for norepinephrine. The administration of iproniazid, a monoamine oxidase inhibitor, to pregnant rats produced an increase in phenylethanolamine, octopamine and norepinephrine concentrations in the fetal rat brain at 16 days of gestation. p -Chlorophenylalanine, an inhibitor of phenylalanine hydroxylase, decreased fetal brain norepinephrine; this drug increased brain levels of phenylethanolamine and octopamine. The combined administration of iproniazid, p -chlorophenylalanine and phenylalanine to pregnant rats resulted in increased concentrations of octopamine and in a several-fold increase of phenylethanolamine levels; norepinephrine concentrations were sharply reduced. The possible significance of these findings in relation to pathological conditions such as phenylketonuria is discussed.     

Brain biogenic amines and reproductive dominance in bumble bees (Bombus terrestris)

To begin to explore the role of biogenic amines in reproductive division of labor in social insects, brain levels of dopamine, serotonin, and octopamine were measured in bumble bee (Bombus terrestris) workers and queens that differ in behavioral and reproductive state. Levels of all three amines were similar for mated and virgin queens. Young workers that developed with or without a queen had similar amine levels, but in queenright colonies differences in biogenic amine levels were associated with differences in behavior and reproductive physiology. Dominant workers had significantly higher octopamine levels compared with workers of lower dominance status but of similar size, age, and ovary state. High dopamine levels were associated with the last stages of oocyte development irrespective of worker social status and behavior. These results suggest that biogenic amines are involved in behavioral and physiological aspects of regulation of reproduction in bumble bees. Accepted: 10 December 1999     

Quantitative high-performance thin-layer chromatography of dansyl derivatives of biogenic amines

A procedure for the separation, detection, and quantification of picomole levels of dansyl derivatives of the biogenic amines, dopamine, norepinephrine, octopamine, and serotonin, has been developed using high-performance thin-layer chromatography. The detection limit is 1 to 2 pmol. Each of the amine derivatives has been detected in insect brain tissue and a solvent system has been developed for the separation and quantification of octopamine in insect tissue samples.     

Atomoxetine-Induced Increases in Monoamine Release in the Prefrontal Cortex are Similar in Spontaneously Hypertensive Rats and Wistar-Kyoto Rats

Spontaneously hypertensive rats (SHRs) are used as a model for attention-deficit/hyperactivity disorder (ADHD), since SHRs are hyperactive and show defective sustained attention in behavioral tasks. The psychostimulants amphetamine and methylphenidate and the selective norepinephrine reuptake inhibitor atomoxetine are used as ADHD medications. The effects of high K⁺ stimulation or psychostimulants on brain norepinephrine or dopamine release in SHRs have been previously studied both in vitro and in vivo, but the effects of atomoxetine on these neurotransmitters have not. The present study examined the effects of administration of atomoxetine on extracellular norepinephrine, dopamine, and serotonin levels in the prefrontal cortex of juvenile SHRs and Wistar-Kyoto (WKY) rats. Baseline levels of prefrontal norepinephrine, dopamine, and serotonin were similar in SHRs and WKY rats. Systemic administration of atomoxetine (3 mg/kg) induced similar increases in prefrontal norepinephrine and dopamine, but not serotonin, levels in both strains. Furthermore, there was no difference in high K⁺-induced increases in extracellular norepinephrine, dopamine, and serotonin levels in the prefrontal cortex between SHRs and WKY rats. These findings indicate that monoamine systems in the prefrontal cortex are similar between SHRs and WKY rats.