Stimulation by Dibutyryl Cyclic AMP of Serotonin Synthesis and Tryptophan Transport in Brain Slices

N⁶,O²-′Dibutyryl cyclic AMP (dibutyryl cyclic AMP), a derivative of 3′,5′-adenosine monophosphate (cyclic AMP) resistant to phosphodiesterase inactivation, has been reported to stimulate serotonin and melatonin synthesis in the pineal gland in vitro^1–3. In brain adenyl cyclase and phosphodiesterase, which catalyse the formation and the inactivation of cyclic AMP, are found chiefly in the synaptosomal fraction of the tissue homogenates⁴, where vesicles containing monoamine are also present⁵. These factors prompted us to study the effects of cyclic AMP and its dibutyryl derivative on the synthesis of brain monoamines.     

Comparative Ontogenesis of Brain Tryptamine, Serotonin, and Tryptophan

The pattern of ontogenetic development of tryptophan (TP), tryptamine (T), indole-3-acetic acid (IAA), 5-hydroxytryptamine (5-HT; serotonin), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the brains of rats aged 1-45 days is presented. Analysis of the five components in each brain allows the calculation of the acid/amine and amine/amino acid ratios. These metabolic indexes are a useful tool to study and compare the metabolic origins and fates of both amines. The ontogenetic patterns of TP, T, and IAA are very similar, especially during the first week postpartum. The highest and lowest levels found for T were 2.2 ng/g and 0.1 ng/g at the 1st and 5th day, respectively. The temporal relationship between the T/TP and IAA/T ratios suggests the existence of mechanisms protecting T against monoamine oxidase (MAO) which develop in parallel to synaptogenesis. Significant correlations were found between TP and IAA during the whole period studied and between TP and T during the first week after birth. The 5-HT peak found during the first postpartum week could be due to a non-neuronal pool of 5-HT protected against MAO and possibly contained in mast cells. Preliminary determinations on leptomeningeal membranes suggest the existence of such a pool.     

Synthesis of Serotonin in Traumatized Rat Brain

Abstract: Previous studies have demonstrated that focal freezing lesions in rats cause a widespread decrease of cortical glucose use in the lesioned hemisphere and this was interpreted as a reflection of depression of cortical activity. The serotonergic neurotransmitter system was implicated in these alterations when it was shown that (1) cortical serotonin metabolism was increased widely in focally injured brain and (2) inhibition of serotonin synthesis prevented the development of cortical hypometabolism. In the present studies we applied an autoradiographic method that uses the accumulation of the ¹⁴C-labeled analogue of serotonin α-methylserotonin to assess changes in the rate of serotonin synthesis in injured brain. The results confirmed that 3 days after the lesion was made, at the time of greatest depression of glucose use, serotonin synthesis was significantly increased in cortical areas throughout the injured hemisphere. The increase was also seen in the dorsal hippocampus and area CA3, as well as in the medial geniculate and dorsal raphe, but not in any other subcortical structures including median raphe. Present results suggest that the functional changes in the cortex of the lesioned hemisphere are associated with an increased rate of serotonin synthesis mediated by activation of the dorsal raphe. We also documented by α-[¹⁴C]aminoisobutyric acid autoradiography that there was increased permeability of the blood-brain barrier, but this was restricted to the rim of the lesion.     

Streptozotocin-Induced Diabetes Reduces Brain Serotonin Synthesis in Rats

The rate of brain 5-hydroxytryptamine (serotonin) synthesis and turnover in streptozotocin-diabetic rats was assessed using three separate methods: the rate of 5-hydroxytryptophan accumulation following decarboxylase inhibition with Ro 4-4602; the decline in 5-hydroxyindoleacetic acid levels following monoamine oxidase inhibition with pargyline; and the rate of 5-hydroxyindoleacetic acid accumulation following blockade of acid transport with probenecid. Each of the three methods revealed that 5-hydroxytryptamine synthesis and turnover is decreased by 44-71% in diabetic rats with plasma glucose levels of between 500 and 600 mg%. In addition, the levels of free and bound plasma tryptophan were measured and the levels of the free amino acid were found to be the same in control and diabetic rats. Since diabetic rats exhibit a 40% decrease in brain tryptophan, the free tryptophan level in plasma does not predict brain tryptophan levels in diabetic rats. These data are discussed within the context of psychiatric disturbances experienced by diabetic patients.     

Postmortem and Regional Changes of Serotonin, 5-Hydroxyindoleacetic Acid, and Tryptophan in Brain

Using a specific and sensitive high pressure liquid chromatographic technique for the measurement of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and tryptophan (TRP), we found that there were no changes in 5-HT or 5-HIAA in the rat cortex when left in situ for 6 h at room temperature or 24 h at 4 degrees C. Only a minimal 14% increase in 5-HT was observed after 24 h at 4 degrees C in the striatum of the same animals. Concentrations of TRP, however, were increased significantly in both brain regions by these postmortem delay procedures. A second study revealed that there were significant regional 5-HT and 5-HIAA concentration differences within the cerebral cortex. The frontal cortex was shown to have the highest concentrations of 5-HT and 5-HIAA. Further, within the frontal cortex, 5-HIAA levels varied, showing apparent progressive rostral to caudal increases. 5-HT concentrations, however, remained constant within the frontal cortex. These results are discussed in reference to the conflicting reports of the previous human suicide and postmortem studies.     

Serotonin Synthesis Rate Measured in Living Dog Brain by Positron Emission Tomography

In vivo measurements by positron emission tomography of the brain serotonin synthesis rates in the normal dog, in the dog with increased plasma tryptophan concentration, and in the dog under different arterial oxygen tensions are described. The method described here permits repeated measurements in the same brain for the first time. An increase in the plasma tryptophan concentration from 16.6 to 191.5 and then to 381 microM resulted in close to a linear increase in the brain serotonin synthesis rate. When PaO2 was raised from 76 +/- 2 to 106 +/- 1 mm Hg, the rate of serotonin synthesis in the dog brain increased from 39 +/- 8 to 54 +/- 10 pmol g-1 min-1. The estimates of the Michaelis-Menten constants, Kappm and Vmax, for the transport of tryptophan through the blood-brain barrier are 303 +/- 54 microM and 63 +/- 10 nmol g-1 min-1, respectively.     

Stress-and Endotoxin-Induced Increases in Brain Tryptophan and Serotonin Metabolism Depend on Sympathetic Nervous System Activity

Stressful treatments and immune challenges have been shown previously to elevate brain concentrations of tryptophan. The role of the autonomic nervous system in this neurochemical change was investigated using pharmacological treatments that inhibit autonomic effects. Pretreatment with the ganglionic blocker chlorisondamine did not alter the normal increases in catecholamine metabolites, but prevented the increase in brain tryptophan normally observed after footshock or restraint, except when the duration of the footshock period was extended to 60 min. The footshock- and restraint-related increases in 5-hydroxyindoleacetic acid (5-HIAA) were also prevented by chlorisondamine. The increases in brain tryptophan caused by intraperitoneal injection of endotoxin or interleukin-1 (IL-1) were also prevented by chlorisondamine pretreatment. The footshock-induced increases in brain tryptophan and 5-HIAA were attenuated by the beta-adrenergic antagonist propranolol but not by the alpha-adrenergic antagonist phenoxybenzamine or the muscarinic cholinergic antagonist atropine. Thus the autonomic nervous system appears to be involved in the stress-related changes in brain tryptophan, and this effect is due to the sympathetic rather than the parasympathetic limb of the system. Moreover, the main effect of the sympathetic nervous system is exerted on beta- as opposed to alpha-adrenergic receptors. We conclude that activation of the sympathetic nervous system is responsible for the stress-related increases in brain tryptophan, probably by enabling increased brain tryptophan uptake. Endotoxin and IL-1 also elevate brain tryptophan, presumably by a similar mechanism. The increase in brain tryptophan appears to be necessary to sustain the increased serotonin catabolism to 5-HIAA that occurs in stressed animals, and which may reflect increased serotonin release.     

Amino Acid Analysis Demonstrates that Increased Plasma Free Tryptophan Causes the Increase of Brain Tryptophan During Exercise in the Rat

Rats were trained to run on a horizontal treadmill for 2 h at 20 m/min. This activity considerably increased plasma free tryptophan (TRP) (+70%) but did not alter plasma total TRP levels and had little or no effect on plasma concentrations of the other large neutral amino acids (LNAAs) that compete with TRP for entry into the brain. Brain TRP levels increased by 80%. The only other brain LNAA to be affected by exercise was threonine, which rose moderately. The results indicate that increased plasma free TRP was specifically responsible for the increase of brain TRP after 2 h of exercise. Brain lysine was also increased whereas glycine, alanine, and gamma-aminobutyric acid were decreased. The differences between the present findings and those previously obtained following 2 h immobilization stress are discussed.     

Inactivation of Brain Tryptophan Hydroxylase by Nitric Oxide

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by nitric oxide (NO) and by the NO generators sodium nitroprusside, diethylamine/NO, S -nitroso- N -acetylpenicillamine, and S -nitrosocysteine. The inactivation occurs in an oxygen-free environment and is enhanced by dithiothreitol and ascorbic acid. Protection against the effect of NO on tryptophan hydroxylase is afforded by oxyhemoglobin, reduced glutathione, and exogenous Fe(II). Catalase partially protects the enzyme from NO-induced inactivation, whereas both superoxide dismutase and uric acid are without effect. These findings indicate that tryptophan hydroxylase is a target for NO and suggest that critical iron-sulfur groups in this enzyme serve as the substrate for NO-induced nitrosylation of the protein, resulting in enzyme inactivation.     

Long-Term L-DOPA Treatment Causes Indiscriminate Increase in Dopamine Levels at the Cost of Serotonin Synthesis in Discrete Brain Regions of Rats

(1) The treatment of choice for Parkinson’s disease (PD) is 3,4-dihydroxyphenylalanine (L-DOPA) with peripheral decarboxylase inhibitor, but long-term therapy leads to motor and psychiatric complications. In the present study we investigated 5-hydroxytryptamine (5-HT) and dopamine concentrations in serotonergic and dopaminergic nuclei following chronic administration of L-DOPA to find whether the neurotransmitter synthesis in these brain areas are compensated. (2) Rats were administered L-DOPA (250 mg/kg) and carbidopa (25 mg/kg) daily for 59 and 60 days, and killed on the 60th day, respectively at 24 h and 30 min after the last dose. L-DOPA, norepinephrine, 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), dopamine, homovanillic acid (HVA), and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in striatum, nucleus raphe dorsalis (NRD), nucleus accumbens (NAc), substantia nigra, cerebellum, and cortex employing HPLC-electrochemical procedure. (3) Prolonged treatment of L-DOPA caused depression in the animals as revealed in a forced swim test. Serotonin content was significantly decreased in all brain regions studied 30 min after long-term L-DOPA, except in NAc. The cortex and striatum showed lowered levels of this indoleamine 24 h after 59 doses of L-DOPA. Dopamine, HVA, and DOPAC concentrations were significantly higher in all the regions studied after 30 min, and in the cerebellum after 24 h of L-DOPA. The levels of DOPAC were elevated in all the brain areas studied 24 h after prolonged L-DOPA treatment. (4) The present results suggest that long-term L-DOPA treatment results in significant loss of 5-HT in serotonergic and dopaminergic regions of the brain. Furthermore, while L-DOPA metabolism per se was uninfluenced, dopamine synthesis was severely impaired in all the regions. The imbalance of serotonin and dopamine formation may be the cause of overt cognitive, motor, and psychological functional aberrations seen in parkinsonian patients following prolonged L-DOPA treatment.     

Increase in Survival of Subcultured Fibroblasts mediated by Serotonin

WE have already reported a cytospecific effect of serotonin in enhancing the growth of fibroblasts in vitro¹, apparently by shortening the lag phase. We have now investigated this effect and the kinetics of cell growth in order to provide information about the mechanism of action of serotonin.     

Influence of Fluoxetine on Regional Serotonin Synthesis in the Rat Brain

Abstract: The aim of the present study was to test the hypothesis that there should be a difference between the effects of an acute and an 8-day (chronic) administration of fluoxetine (10 mg/kg) on the rate of serotonin [5-hydroxytryptamine (5-HT)] synthesis. The 5-HT synthesis rate was measured in discrete regions of the rat brain using the α-[¹⁴C]methyl- l -tryptophan autoradiographic method. The results show that the acute and chronic fluoxetine treatments influence the 5-HT synthesis rate in different ways. A single dose of fluoxetine induced a significant increase in 5-HT synthesis in the visual, auditory, and parietal cortices, substantia nigra, hypothalamus, ventral thalamus, and dorsal hippocampus. In contrast, after a chronic treatment a decrease was observed in the substantia nigra, caudate, and nucleus accumbens, the auditory, parietal, sensorimotor, and frontal cortices, and ventral tegmental area. A significant decrease in the rate of 5-HT synthesis was observed in the dorsal raphe after both the single and chronic treatments. The results suggest that extracellular 5-HT has a delayed influence on the brain 5-HT synthesis rate in structures with serotonergic terminals. The findings from the acute study could be important for patients who have just started receiving fluoxetine treatment, as an increase in the 5-HT synthesis rate might occur in the acute phase of their treatment. In addition, the findings from the chronic treatment study might give us a better understanding of how the brain serotonergic system adapts during a prolonged exposure to extracellular 5-HT.     

Brain Tryptophan Hydroxylation in the Portacaval Shunted Rat: A Hypothesis for the Regulation of Serotonin Turnover In Vivo

Regional and whole-brain tryptophan-hydroxylating activity and serotonin turnover were investigated in portacaval shunted (PCS) rats using an in vivo decarboxylase inhibition assay. To saturate tryptophan hydroxylation with amino acid substrate, rats were administered a high dose of tryptophan 1 h prior to analysis of brain tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid. The analysis revealed, as expected, higher brain concentrations of tryptophan and 5-hydroxyindoles and increased serotonin synthesis rate in PCS rats as compared with shamoperated controls. Saturating levels of brain tryptophan were achieved in both PCS and sham animals after exogenous tryptophan administration. The tryptophan load resulted in increased brain serotonin turnover in all regions and in whole brain compared with rats that did not receive a tryptophan load. Tryptophan-loaded PCS rats showed increased brain serotonin turnover compared with tryptophan-loaded sham rats. Regionally, this supranormal tryptophan-hydroxylating activity was most pronounced in the mesencephalon-pons followed by the cortex. It is concluded that, at least in the PCS rat, brain tryptophan hydroxylation is an inducible process. Since it is known that brain tissue from PCS rats undergoes a redox shift toward a reduced state and that the essential cofactor tetrahydrobiopterin is active in tryptophan hydroxylation only when present in its reduced form, it is hypothesized that this is the reason for the supranormal tryptophan-hydroxylating activity displayed by the PCS rats. The hypothesis further suggests that alterations in tetrahydrobiopterin availability may serve as a mechanism by which brain tryptophan hydroxylation, and therefore serotonin turnover, can be regulated with high sensitivity in vivo.     

Adrenalectomy-Induced Increase of Brain Protein Synthesis Is Antagonized by Corticosterone Replacements in Free-Moving Rats

Abstract: The autoradiographic method with l [³⁵S]-methionine was used to determine whether changes in glucocorticoid circulating levels were associated with changes in local rates of protein synthesis in rat brain. Chronic bilateral adrenalectomy induced an increase of me-thionine incorporation rates into proteins in 60 of the 62 brain regions examined (mean effect, +50%). This effect was confirmed biochemically and quantified by correcting for the relative contribution of methionine derived from protein degradation to the precursor pool for protein synthesis in the whole brain. Acute or chronic administration of corticosterone, at doses that normalize basal levels of adrenocorticotrophic hormone, reversed or prevented the adrenalectomy-induced increase of protein synthesis in most regions. However, in nearly all the regions studied (59 of 62), acute corticosterone administration to sham-operated rats did not change the apparent rate of protein synthesis. These results demonstrate that glucocorticoids exert a generalized inhibitory action on brain protein synthesis, because the stimulatory and persistent effect of adrenalectomy on protein synthesis was antagonized by corticosterone replacements at physiological doses. Thus, the regulation of overall brain protein synthesis by glucocorticoids emphasizes the role of neuroendocrine events on long-term neurochemical processes.     

Modulation of Neurosteroid Synthesis/Accumulation by l-Ascorbic Acid in Rat Brain Tissue: Inhibition by Selected Serotonin Antagonists

Abstract: We have investigated the possibility that the synthesis/accumulation of neurosteroids, i.e., brain-produced steroids putatively endowed with modulatory actions in the CNS, is regulated by monoaminergic receptor-mediated mechanisms. In minces of rat brain cortex, l -ascorbic acid concentration-dependently (0.07–1.0 m M ) increases the levels of pregnenolone, allotetrahydrodeoxycorticosterone, and dehydroepiandrosterone. This effect of l -ascorbic acid is region-dependent: in hippocampus, progesterone and allopregnanolone are also increased, whereas dehydroepiandrosterone is unchanged, and in corpus striatum only progesterone is increased significantly. 5-Hydroxytryptamine (10 µ M ), 1-(3-chlorophenyl)piperazine (1.0 µ M ), and 5-methoxytryptamine (0.4 µ M ) mimic the effect of l -ascorbic acid, whereas a pretreatment with p -chlorophenylalanine (400 mg/kg i.p., 2 days) reduces the amplitude of the l -ascorbic acid effect on brain cortical neurosteroids. The effect of l -ascorbic acid is blocked by the nonselective serotonin antagonists methiothepin, clozapine, methysergide, and pizotifen, but not mesulergine, spiperone, MDL 72222, and dl -propranolol, nor by the catecholaminergic receptor antagonists prazosin and S (−)-sulpiride. l -Ascorbic acid is not additive with dibutyryl-cyclic AMP and, furthermore, the inhibition of adenylate cyclase by MDL 12330A, but not of phospholipase C by U-73122, markedly attenuates the l -ascorbic acid-induced increase of pregnenolone in rat brain cortical minces. Together these data suggest that l -ascorbic acid plays a role in the modulation of neurosteroidogenesis, presumably by favoring the activation of the purported serotonin type 6 receptor by endogenous serotonin.     

Tryptophan boost caused by senescence occurred independently of cytoplasmic glutamine synthetase

We examined to determine whether senescence-induced tryptophan levels are positively associated with levels of glutamine synthetase (GS1), the initial enzyme in tryptophan biosynthesis. We generated transgenic rice plants in which GS1 was suppressed by RNA interference technology. The transgenic line showed a dramatic decrease in GS1 protein and glutamine content, but the levels of tryptophan and mRNA of the key tryptophan biosynthetic genes upon senescence were comparable to those of the wild type.