Tryptophan Hydroxylation In Brain Research Articles

Measurement of tryptophan hydroxylase mRNA levels by competitive RT-PCR

Tryptophan hydroxylase (TPH) is the rate limiting enzyme in serotonin biosynthesis [D.G. Grahame-Smith, Tryptophan hydroxylation in brain, Biochem. Biophys. Res. Commun. 16 (1964) 586–592 [19]]. As such, the TPH gene is a likely target for modulation of serotonergic function, which has been associated with several psychiatric disorders [E.C. Azmitia, P.M. Whitaker-Azmitia, Awakening the sleeping giant: anatomy and plasticity of the brain serotonergic system, J. Clin. Psychiatry 52 (12, Suppl.) (1991) 4–16 [1]; R.P. Hart, R. Yang, L.A. Riley., T.L. Green, Post-transcriptional control of tryptophan hydroxylase gene expression in rat brain stem and pineal gland, Mol. Cell. Neurosci. 2 (1991) 71–77 [20]; M.J. Owens, C.B. Numeroff, Role of serotonin in the pathophysiology of depression: focus on the serotonin transporter, Clin. Chem. 40 (1994) 288–295 [24]]. Unfortunately, it has been technically difficult to measure TPH mRNA levels in central serotonergic neurons due to its low levels. For example, detection with ribonuclease protection assays requires pooling of 5–10 dissected brainstems [M.C. Darmon, B. Guibert, V. Leviel, M. Ehret, M. Maitre, J. Mallet, Sequence of two mRNAs encoding active rat tryptophan hydroxylase, J. Neurochem. 51 (1988) 312–316 [15]; B.L. Jacobs, E.C. Azmitia, Structure and function of the brain serotonin system, Physiol. Rev. 72 (1992) 165–229 [21]]. This protocol describes the use of competitive RT-PCR to measure TPH mRNA levels from rat brain. First described in 1988, competitive RT-PCR has become an accepted method of measuring RNA abundance [M. Clementi, S. Menzo, P. Bagnarelli, A. Manzin, A. Valenza, P.E. Varaldo, Quantitative PCR and RT-PCR in virology, PCR Methods Appl. 2 (1994) 191–196 [12]; N.C.P. Cross, Quantitative PCR techniques and applications, Br. J. Haematol. 89 (1995) 693–697 [14]; K.P. Foley, M.W. Leonard, J.D. Engel, Quantitation of RNA using the polymerase chain reaction, Trends Genet. 9 (1993) 380–385 [17]; P.D. Siebert, J.W. Larrick, Competitive PCR, Nature 359 (1992) 558 [27]]. Competitive RT-PCR uses co-amplification with a known quantity of an in vitro transcribed RNA which amplifies using the same primers and thus competes for reactants with the product of interest. As the two products amplify with the same efficiency, the relative abundance of the two amplification products remains constant, and thus can be used to determine initial tissue TPH mRNA levels [G. Gilliland, S. Perrin, K. Blanchard, H.F. Bunn, Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 2725–2729 [18]; A.M. Wang, M.V. Doyle, D.F. Mark, Quantitation of mRNA by the polymerase chain reaction, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 9717–9721 [31]]. We first demonstrate equivalent results between RNA slot blots and competitive RT-PCR using the CA77 thyroid C cell line [M.S. Clark, A.F. Russo, Tissue-specific glucocorticoid regulation of tryptophan hydroxylase mRNA levels, Mol. Brain Res. 48 (1997) 346–354 [9]]. We then describe the use of competitive RT-PCR to measure TPH mRNA levels in RNA isolated from rat brain poly-A + RNA. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Serotonin; Signal transduction: gene expression

Daily variations in in vivo tryptophan hydroxylation and in the contents of serotonin and 5-hydroxyindoleacetic acid in discrete brain areas of the rat.

The in vivo rate of brain tryptophan hydroxylation was determined through 5-hydroxytryptophan accumulation (5-HTPacc) following the administration of NSD 1015, a L-aromatic amino-acid decarboxylase inhibitor. This measurement was performed every 4 h throughout a 24 h hour period in 10 discrete brain areas of rats maintained on a regular 12 h/12 h light-dark cycle. The concentrations of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were also determined in untreated rats. Daily variations in 5-HTPacc were found in all the areas studied, the 5-HTPacc being higher during the dark period in most structures. These results strongly suggest that tryptophan hydroxylation is involved in the control of the 5-HT biosynthesis circadian rhythm. However, various patterns of 5-HT and 5-HIAA daily variations were observed, suggesting that the circadian factors affecting serotonin metabolism can be different among brain areas.

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.

Effects of disulfiram and coprine on rat brain tryptophan hydroxylation in vivo

The effects of disulfiram and coprine on brain tryptophan hydroxylation, and on the brain-levels of serotonin and 5-hydroxyindole-3-acetic acid, were studied in 45 and 235 days old rats. Both drugs were found to affect the parameters measured. Disulfiram increased the rate of tryptophan hydroxylation and the serotonin level in young rats, while these parameters appeared to be unaffected in old disulfiram-treated rats. In contrast, coprine increased the rate of tryptophan hydroxylation and possibly also the serotonin level in old rats while no significant effects were seen in young coprine-treated rats. Regarding the 5-hydroxyindole-3-acetic acid concentration, this appeared to be increased by disulfiram in both age-groups, while no significant effects were found with coprine. The lack of similarity in the action of disulfiram and coprine, which are both potent aldehyde dehydrogenase inhibitors, suggests that the effects found were not caused by an impaired metabolism of monoamine-derived biogenic aldehydes.

Effects of aspartame ingestion on the carbohydrate-induced rise in tryptophan hydroxylation rate in rat brain

Effects of aspartame (aspartyl-phenylalanine-methylester) on increases in brain-tryptophan level and hydroxylation rate following a high-carbohydrate, protein-free meal were tested. After an overnight fast, rats consumed a protein-free meal containing one of several levels of aspartame. Blood and brain amino acid levels and the in vivo rate of tryptophan hydroxylation in brain were estimated at intervals thereafter. Ingestion of the meal alone increased brain-tryptophan level and hydroxylation rate. Aspartame did not modify these effects, except at doses of 530 mg/kg body weight or more. Results suggest a threshold dose of aspartame can be identified for the rat in single-meal studies above which suppression of carbohydrate-induced increases in brain-tryptophan level and serotonin synthesis occurs. This dose, however, is large and, when corrected for species differences in metabolic rate, is unlikely to be ingested by a human subject as a single load.

Brain tyrosine hydroxylation: alteration of oxygen affinity in vivo by immobilization or electroshock in the rat.

Abstract— The rates of brain tyrosine and tryptophan hydroxylation, estimated in vivo from the accumulation of DOPA and 5‐hydroxytryptophan after the administration of a decarboxylase inhibitor, appear dependent on the availability of oxygen as a substrate. During two types of physical stress, electroshock and curare‐immobilization, the rate of brain tyrosine hydroxylation was greater than in unstressed controls and was not significantly decreased when the stresssed animals were made hypoxic. The loss of oxygen dependence by brain tyrosine hydroxylation during stress was observed in several brain regions and was not associated with alterations in the concentrations of brain tyrosine. tryptophan, serotonin, dopamine or norepinephrine. The rate of brain tryptophan hydroxylation was not affected by stress and remained oxygen dependent. The increase in catecholamine synthesis during stress appears to be the result of increased catecholaminergic nerve impulse flow. These experiments are consistent with the hypothesis that during neuronal stimulation an allosteric change in tyrosine hydroxylase increases the affinity of the enzyme for oxygen allowing greater catecholamine synthesis despite limiting concentrations of this substrate.

Inhibition of rat brain tryptophan hydroxylation with p-chloroamphetamine

p-Chloroamphetamine (PCA) at high concentrations (0.0025 to 0.02 M) inhibited rat brainstem tryptophan hydroxylase and, to a lesser extent, rat corpus striatum tyrosine hydroxylase. Hog kidney aromatic l-amino acid decarboxylase was not affected. Inhibition of tryptophan hydroxylase by p-chloroamphetamine was competitive with tryptophan and noncompetitive with 6,7-dimethyl-5,6,7,8-tetrahydropterine (DMPH 4). In rats given p-chloroamphetamine, brainstem tryptophan-hydroxylating activity was only slightly reduced, whereas striatal tyrosine-hydroxylating activity was not altered. Synthesis rates of brain serotonin and dopamine in rats treated with p-chloroamphetamine, estimated by measuring the accumulation of 5-hydroxytryptophan and dopa, respectively, after blockade of decarboxylase, were both decreased. In view of the high concentrations of p-chloroamphetamine required to inhibit tryptophan and tyrosine hydroxylases in vitro, mechanisms other than inhibition of enzyme by PCA might be responsible for the decelerated synthesis of serotonin and dopamine in brain (e.g. blockade of monoamine re-uptake).

Brain tryptophan hydroxylation: dependence on arterial oxygen tension.

The accumulation of cerebral 5-hydroxytryptophan after decarboxylase inhibition was decreased in rats maintained at arterial O(2) tensions below 60 mm-Hg. In contrast, brain lactate was stable above 40 mm-Hg and brain adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate were unchanged above 30 mm-Hg. There was a linear correlation of brain 5-hydroxytryptophan accumulation to cerebral venous O(2) tension. Cerebral tryptophan hydroxylase appears to have a poor affinity for oxygen and to be affected by slight hypoxia. The resultant decreases in monoamine neurotransmitter metabolism may explain the behavioral changes of mild oxygen deprivation.

Tryptophan hydroxylation in brain

There are several facts suggesting that the first step in the biosynthesis of 5-hydroxytryptamine, the formation of 5-hydroxytryptophan by tryptophan hydroxylation, may occur in brain: 1. 1. There is a rapid turnover of 5-hydroxytryptamine in brain ( Udenfriend and Weissbach, 1958); 2. 2. 5-hydroxytryptamine does not freely enter the brain from the circulation ( Udenfriend, Weissbach and Bogdanski, 1957); 3. 3. Intracerebral but not intraperitoneal injections of DL -[3- 14C] tryptophan produces labelling of brain 5-hydroxytryptamine ( Gal, Marshall and Poczik, 1963). It has now been possible to demonstrate enzymic tryptophan hydroxylation in isolated brain tissue.