Gamma-hydroxybutyrate increases tryptophan availability and potentiates serotonin turnover in rat brain

Gamma-hydroxybutyrate (GHB) is both a therapeutic agent and a recreative drug. It has sedative, anxiolytic and euphoric effects. These effects are believed to be due to GHB-induced potentiation of cerebral GABAergic and dopaminergic activities, but the serotonergic system might also be involved. In this study, we examine the effects of pharmacological doses of GHB on the serotonergic activity in rat brain. Administration of 4.0 mmol/kg i.p. GHB to rats induces an accumulation of tryptophan and 5-HIAA (5-hydroxyindole acetic acid) in the frontal cortex, striatum and hippocampus without causing significant change in the tissue serotonin content. In the extracellular space, GHB induced a slight decrease in serotonin release. The tryptophan and 5-HIAA accumulation induced by GHB is mimicked by the GHB receptor agonist para-chlorophenyl-transhydroxycrotonate (NCS-356) and blocked by NCS-382 (6,7,8,9-tetrahydro-5-[H]-benzocycloheptene-5-ol-4-ylidene acetic acid) a selective GHB receptor antagonist. GHB induces the accumulation of either a derivative of or [3H]-tryptophan itself in the extracellular space, possibly by increasing tryptophan transport across the blood-brain barrier. The blood content of certain neutral amino-acids, including tryptophan, is also increased by peripheral GHB administration. Some of the effect of GHB could be reproduced by baclofen and reduced by the GABAB antagonist CGP 35348. Taken together, these results indicate that the GHB-induced stimulation of tissue serotonin turnover may be due to an increase in tryptophan transport to the brain and in its uptake by serotonergic cells. As the serotonergic system may be involved in the regulation of sleep, mood and anxiety, the stimulation of this system by high doses of GHB may be involved in certain neuropharmacological events induced by GHB administration.     

Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat

It has been proposed that serotonin (5-HT) facilitates the chemosensory activity of the carotid body (CB). In the present study, we investigated mRNA expression and immunohistochemical localization of the 5-HT synthetic enzyme isoforms, tryptophan hydroxylase 1 (TPH1) and TPH2, and the 5-HT plasma membrane transport protein, 5-HT transporter (SERT), in the CB of the rat. RT-PCR analysis detected the expression of mRNA for TPH1 and SERT in extracts of the CB. Using immunohistochemistry, 5-HT immunoreactivity was observed in a few glomus cells. TPH1 and SERT immunoreactivities were observed in almost all glomus cells. SERT immunoreactivity was seen on nerve fibers with TPH1 immunoreactivity. SERT immunoreactivity was also observed in varicose nerve fibers immunoreactive for dopamine beta-hydroxylase, but not in nerve fibers immunoreactive for vesicular acetylcholine transporters or nerve terminals immunoreactive for P2X₃ purinoreceptors. These results suggest that 5-HT is synthesized and released from glomus cells and sympathetic nerve fibers in the CB of the rat, and that the chemosensory activity of the CB is regulated by 5-HT from glomus cells and sympathetic nerve fibers.     

Calcium activation of brain tryptophan hydroxylase.

Using both radioisotopic and fluorometric techniques to measure the activity of midbrain soluble enzyme, we have demonstrated that calcium activates tryptophan hydroxylase. The observed activation apparently results from an increased affinity of the enzyme for both its substrate, tryptophan, and the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6-MPH₄). The calcium activation of tryptophan hydroxylase appears to be specific for both enzyme and effector: other brain neurotransmitter biosynthetic enzymes, such as aromatic amino acid decarboxylase(s) and tyrosine hydroxylase, are not affected by calcium (at concentrations ranging from 0.01 mM to 2.0 mM); other divalent cations, such as Ba⁺⁺, Mg⁺⁺, and Mn⁺⁺, have no activating effect on tryptophan hydroxylase. This work suggests that increases in brain serotonin biosynthesis induced by neural activation may be due to influx of Ca⁺⁺ associated with membrane depolarization and resulting activation of nerve ending tryptophan hydroxylase.     

Effect of restraint stress on rat brain serotonin

Restraint-induced stress in rats was found to enhance steady state concentrations of whole brain and hypothalamic serotonin, at 1,2 and 4 h after immobilization. The increase was maximal at 1 h and tended to decline thereafter. The rate of accumulation of rat brain serotonin, in pargyline pretreated animals, was significantly enhanced after restraint stress. Bilateral adrenalectomy and metyrapone, an endogenous corticoid synthesis inhibitor, failed to affect restraint stress (1h)-induced increase in rat brain serotonin levels. Thus restraint stress-induced autoanalgesia and potentiation of the pharmacological actions of several centrally acting drugs, in rats, are serotonin-mediated responses. The results also indicate that restraint stress-induced effects on rat brain serotonin are not dependent on endogenous corticoid activity.     

Effects of clofibrate on plasma tryptophan, growth hormone, and prolactin and on brain tryptophan and serotonin in prepubertal rats

The effects of clofibrate administration (200 mg/kg, po) on somatic growth, plasma levels of lipids, tryptophan, growth hormone (GH), and prolactin (PRL), as well as on brain concentrations of tryptophan and 5-hydroxytryptamine (5-HT) were studied in prepubertal male rats. The drug did not significantly alter ponderal growth, but an appreciable reduction of tail length was observed in rats treated for 30 days. Triglyceride concentrations in plasma showed a 43% diminution after 30 days of treatment, whereas free fatty acid (FFA) levels were not modified. Clofibrate administration for 7, 15, or 30 days caused a fall in total tryptophan and a significant increase of the free fraction in plasma with no change in brain tryptophan levels. Brain 5-HT was generally unaffected but a marked elevation of this parameter was noted in rats treated for 15 days. Plasma GH and PRL concentrations remained unaltered. It may be concluded from these findings that the slight reduction of somatic growth, the diminution of triglycerides, and the increase of free tryptophan in plasma, induced by chronic clofibrate treatment, are not associated with variations in brain tryptophan and 5-HT levels or with modifications of plasma GH and PRL titers.     

Effect of food restriction on serotonin metabolism in rat brain

The brain concentration of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) increased in rats maintained on restricted volume of low-protein or normal-protein diet, whereas these two agents decreased in rats fed low-protein diet ad libitum. In these two food-restricted groups brain 5-HT and 5-HIAA concentrations were not correlated with brain tryptophan hydroxylase activity, but the concentrations correlated closely with cerebral tryptophan concentrations. The cerebral tryptophan concentration in the two food-restricted groups was not consistent with the total or free tryptophan concentration in plasma. In these restricted rats cerebral tryptophan concentration was elevated, and, unlike the plasma tryptophan, it showed no diurnal variation. These results suggested that tryptophan uptake into the brain from plasma was enhanced by limiting food volume intake. Tryptophan uptake was increased by glucagon injection without changing the plasma tryptophan level, but injection of hydrocortisone or insulin had little or no effect on tryptophan concentration in either the plasma or brain.d-Glucose injection elevated plasma tryptophan concentration but decreased brain tryptophan concentration.     

Effect of hypoxia on tyrosine and tryptophan hydroxylation in unanaesthetized rat brain

T he T yrosine and tryptophan hydroxylase enzymes have been proposed as rate-limiting steps in the biosynthesis of catecholamines and 5-hydroxytryptamine (5-HT), respectively; thus under normal physiological conditions the rate of amine synthesis appears to be controlled by the activity of these hydroxylase enzymes (see U denfriend , 1966; L ovenberg , J equier and S joerdsma , 1968). Subtle changes in neuronal activity may result not in changes in the levels of the amine neurotransmitters, but rather in alteration in their production and metabolism without measurable change in their levels. Previous studies of the effect of hypoxia on monoamines have dealt with amine levels, but there have been no studies of the effect of lowered oxygen on the synthesis of these substances.     

Depletion of tryptophan hydroxylase by 5,6-dihydroxytryptamine in rat brain. Time course and regional differences

—Wistar rats were injected intraventricularly with 75 μg 5,6-dihydroxytryptamine. Tryptophan hydroxylase was assayed in seven regions of the brain as well as spinal cord at intervals following injection. Spinal cord was depleted to 1 per cent of control by 12 days. Tectum was depleted to 32 per cent of control by 9 days. The time course of depletion in most regions was biphasic, with an early reduction of activity 1 h after injection with continued reduction of activity 1-2 days following injection, and a recovery of activity at 4-6 days. The activity again drops at 9-12 days, and this reduction of activity persists to varying degrees to 60 days, with slight recovery at this time in certain regions.     

Genetic control of tryptophan hydroxylase activity in the mouse brain

Interstrain differences in tryptophan hydroxylase activity have been studied in the brain stem of 12 inbred strains of mice. The Mendelian analysis has been performed using BALB/c and C57BL/6J mice. The activity of tryptophan hydroxylase was shown to be controlled by a single gene having two alleles with additive effect. It was suggested that this gene controls either the structure of enzyme's molecule, or its amount in a nervous tissue.     

Effect of butaclamol enantiomers on hypothalamic tyrosine hydroxylase in the rat brain

The authors demonstrate stereospecificity of the action of butaclamol enantiomers on substrate inhibition of hypothalamic tyrosine hydroxylase (TH) and regulation of the tyrosine hydroxylase response by the presynaptic membrane (presynaptic receptors) of rat hypothalamus synaptosomes under membrane activation with dopamine. The effect of (+)-butaclamol on the substrate inhibition of TH was noticeable at a concentration of 10(-8)M, reaching a maximum at 10(-5)M. (-)-Butaclamol administered at the same concentrations did not influence the substrate inhibition of the enzyme. (+)-Butaclamol added to the incubation medium containing hypothalamic synaptosomes concurrently with dopamine (10(-5)M) completely blocked the regulatory action of the latter on TH, with this action mediated via presynaptic receptors. (-)-Butaclamol (10(-5)M) antagonized the action of dopamine under the same conditions. The data obtained indicate high stereo-specificity of butaclamol enantiomers as regards their effect on presynaptic regulation of TH, suggesting that elimination of the substrate inhibition of hypothalamic TH is a stereoselective effect of neuroleptics and can be a prognostically important criterion in the appraisal of compounds with potential neuroleptic activity.     

Failure of tryptophan load-induced increases in brain serotonin to alter food intake in the rat

The effects on food consumption of 50 and 100 mg/kg $\text{1}$-tryptophan injections, versus control saline treatment, were compared in 24-hour food-deprived rats at two time points in the rats' daily light-dark cycle. No effect of the two tryptophan doses, relative to the saline treatment, on food intake was observed, although tryptophan loading significantly raised brain tryptophan, serotonin, and 5-hydroxyindoleacetic acid levels, in a dose-dependent manner, over baseline concentrations. Implications of these data for serotonergic modulation of food intake regulation are considered.     

Rapid depletion of serum tryptophan, brain tryptophan, serotonin and 5-hydroxyindoleacetic acid by a tryptophan-free diet

Rats were trained for 20 days to eat their normal daily meal in a period of 2 hours. On the twentyfirst day they received a diet in which tryptophan was omitted instead of the usual balanced diet. The ingestion of the tryptophan-free diet produced a marked depletion of free serum tryptophan (90%), brain tryptophan (85%), brain 5-HT (58%) and brain 5-HIAA (76%). These changes were almost maximal within 2 hours after food presentation and persisted for more than 24 hours. The mechanism of these changes is discussed.     

Relationship between tryptophan and serotonin concentrations in postmortem human brain

The present study was planned to test a recent observation of positive correlation between tryptophan and 5-hydroxyindole concentrations in postmortem human hypothalamic samples. Four other brain areas were studied, but no significant correlations were observed between tryptophan and serotonin or 5-hydroxyindoleacetic acid concentrations, except in the nucleus accumbens samples of a suicide victim group. A possible in vivo correlation may have been obscured by postmortem changes. The use of tryptophan concentrations as an index for normalising postmortem brain serotonin data is not supported by the present results.     

Chronic amphetamine administration decreases brain tryptophan hydroxylase activity in cats

Chronic administration of d-amphetamine sulfate (7.5 mg/kg, i.p. every 12 hrs. for 6 days) to cats produced significant decreases in the Vmax of brain-stem and forebrain tryptophan hydroxylase when measured 1 day (?34 and ?46%) and 10 days (?17 and ?30%) after the final amphetamine injection. Serotonin and 5-hydroxyindoleacetic acid (5HIAA) levels were decreased by a similar magnitude. A single injection of amphetamine (7.5 mg/kg) produced no significant changes in tryptophan hydroxylase activity, serotonin, or 5HIAA when measured 1 day after the injection. Neither acute nor chronic amphetamine treatment produced any significant changes in the Km of tryptophan hydroxylase for either tryptophan or the natural co-factor, tetrahydrobiopterin. These data suggest that chronic amphetamine treatment decreases central serotonergic neurotransmission by an action on the rate-limiting enzyme in serotonin biosynthesis.