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CHAPTER 2.3 - Tryptophan Hydroxylase and Serotonin Synthesis Regulation
Abstract
Potential determinants of existing tryptophan hydroxylase (TPH) activity in vivo are (1) a supply of tryptophan via the tryptophan transporter, (2) de novo synthesis of tetrahydrobiopterin and efficient regeneration of this coenzyme, and (3) iron metabolism. TPH is an iron protein. Iron binding is relatively strong in TPH2, while it is very weak in TPH1. Owing to the poorly liganded iron of TPH, various complexities arise. Irreversible inactivation of TPH and neural deterioration seem to be caused by an H2O2 reaction with non-liganded or poorly liganded Fe2+. Within the cell, the amount of ferrous iron available to TPH is below saturation. The metabolism of iron may control serotonin production by attenuating actual TPH activity in the cell interior. Phosphorylation of TPH was studied in terms of its functional aspects with the use of brain TPH, while the identification of phosphorylation sites was mainly conducted on the basis of the amino acid sequence of TPH1. Protein turnover is cell-type specific in terms of its machinery, and its regulation is one of the major post-translational modifications of a protein. TPH degradation is likely linked to protein phosphorylation. Functionally, TPH, the rate-limiting enzyme in serotonin biosynthesis, is involved in neural development, which likely affects animal behaviors in the adult stages. Future studies will define the sensitive period during development when TPH activity is required for the fine-tuning of neuronal organization that is responsible for each behavior in the adult.
... Both are important in the rate-limiting of serotonin biosynthesis but have differential expression patterns with TPH2 operating predominantly in the brain whereas TPH1 additionally regulates peripheral actions of serotonin in the body (Lin et al., 2007;Walther and Bader, 2003). TPH has a principal role in the homeostatic regulation of intrasynaptic serotonin levels by maintaining adequate levels of synthesis during baseline and dynamically reacting to heightened neurotransmission (such as during stress and threat) by refilling emptied vesicles (Boadle-Biber et al., 1986;Hasekawa and Nakamura, 2010;Herr et al., 1975;Linthorst and Reul, 2010). Specifically, higher efficiency of stress-induced transcription of genes controlling TPH expression likely results in a faster "vesicular refill" which stabilizes serotonergic homeostasis and attenuates the impact of acute threat and long-term stress on serotonin-mediated physiological processes (Hasekawa and Nakamura, 2010;Linthorst and Reul, 2010). ...
... TPH has a principal role in the homeostatic regulation of intrasynaptic serotonin levels by maintaining adequate levels of synthesis during baseline and dynamically reacting to heightened neurotransmission (such as during stress and threat) by refilling emptied vesicles (Boadle-Biber et al., 1986;Hasekawa and Nakamura, 2010;Herr et al., 1975;Linthorst and Reul, 2010). Specifically, higher efficiency of stress-induced transcription of genes controlling TPH expression likely results in a faster "vesicular refill" which stabilizes serotonergic homeostasis and attenuates the impact of acute threat and long-term stress on serotonin-mediated physiological processes (Hasekawa and Nakamura, 2010;Linthorst and Reul, 2010). Indeed, higher TPH expression has been related to strengthened emotional regulation (i.e. ...
... Higher basal TPH expression throughout neuro-development in utero and early childhood facilitates self-regulation and dampens the impact of stressful experiences on adult physiology and behavior (Chen and Miller, 2012). Conversely, blocking TPH synthesis during critical developmental phases has been demonstrated to result in emotional and behavioral dysregulation in animals when reaching adulthood, such as attenuation of the prepulse inhibition, aggression, muricide in rats, anxiety, and behavioral disinhibition (Hasekawa and Nakamura, 2010). Therefore, higher baseline TPH expression throughout critical phases of socio-emotional development and a higher stress-induced TPH gene expression can effectively strengthen homeostatic regulation of neurophysiological processes and thereby facilitate neuro-development and increase emotional resilience. ...
YILDIRIM, B.O., DERKSEN, J.J.L. Systematic review, structural analysis, and new theoretical perspectives on the role of serotonin and associated genes to the etiology of psychopathy and sociopathy. NEUROSCI BIOBEHAV REV XX(X) XXX-XXX, 2013.- Since its theoretical inception, psychopathy has been considered by philosophers, clinicians, theorists, and empirical researchers to be substantially and critically explained by genetic factors. In this systematic review and structural analysis, new hypotheses will be introduced regarding gene-gene and gene-environment interactions in the etiology of psychopathy and sociopathy. Theory and research from neurobiological and behavioral sciences will be integrated in order to place this work in a broader conceptual framework and promote synergy across fields. First, a between groups comparison between psychopathy and sociopathy is made based on their specific dysfunctions in emotional processing, behavioral profiles, etiological pathways, HPA-axis functioning, and serotonergic profiles. Next, it is examined how various polymorphisms in serotonergic genes (e.g., TPH, 5HTT, HTR1A, HTR2A, HTR2C, and HTR3) might contribute either individually or interactively to the development of these disorders and through which specific biological and behavioral endophenotypes this effect could be mediated. A short introduction is made into mediating variables such as GABAergic functioning and testosterone which could potentially alter the decisive effect of serotonergic genotypes on behavior and physiology. Finally, critical commentary is presented on how to interpret the hypotheses put forward in this review.
... Another collection of evidence that supports a role for 5HT in the pathogenesis of depression are studies of monoamine depletion. The synthesis of serotonin is entirely dependent on availability of its precursor, L-tryptophan (L-Trp) (86,87) and is an essential amino acid, meaning it is only obtained through the diet. Thus, manipulation of this amino acid's concentration through dietary restrictions is an acceptable strategy for monoamine depletion. ...
... in catecholamine synthesis, TPH is not sensitive to end-product inhibition and is entirely dependent on physiologic stimulation and the local concentration of L-Trp (86,87). With the aid of pyridoxal phosphate (Vitamin B6), the aromatic L-amino acid decarboxylase (AADC) then catalyzes 5-HTP into 5-hydroxytyptamine [5HT or serotonin] (173). ...
The projects discussed in this dissertation outline the physiologic consequences of S-palmitoylation, a lipid-based post-translational modification, on the serotonin and norepinephrine transporters (SERT and NET, respectively). SERT and NET are trans-membrane proteins that belong to the SLC6 family of secondary active transporters. During neuro- and neuro-hormonal transmission, SERT and NET utilize the differential ionic concentrations of the extracellular and intracellular spaces to remove their corresponding substrates (serotonin [5HT] and norepinephrine [NE]) from the extracellular space. Each monoamine modulates distinct and overlapping physiologic functions, with dysregulation driving the pathogenesis of neuro-cognitive, neuro-humoral, sympathetic, cardiac, and vascular diseases. Because of this, these transporters are a target in the development of therapeutics to battle human disease associated with monoamine signaling. Prescription therapeutics that target SERT and NET include methylphenidate (Ritalin), bupropion (Wellbutrin), selective-serotonin (Lexapro, Prozac, Lustral), and serotonin-norepinephrine reuptake inhibitors (Pristiq, Cymbalta). SERT and NET are also targets for drugs of abuse like cocaine (COC), amphetamine (AMPH), methamphetamine (METH), and methylenedioxymethamphetamine (MDMA). As a medical student, I approached my graduate research training from the point-of-view that dysregulated physiology precedes pathophysiology, which can be observed through clinical phenotypes and rectified by intelligent chemo-therapeutic design and selection. More clearly, this is the pathogenesis-to-presentation model of medicine. The experimental design of this dissertation seeks to (1) outline the normal physiologic consequences of S-palmitoylation for SERT and NET, (2) describe how these processes become dysregulated and facilitate disease pathogenesis, and (3) use this knowledge to outline how therapeutics can potentially rectify these pathophysiology’s and re-justify monoamine homeostasis. The first project of this dissertation explores the regulation of SERT by S-palmitoylation and how this process is altered by therapeutic manipulation with escitalopram (Lexapro). Our studies revealed that when SERT-expressing cells are acutely challenged with the irreversible palmitoyl acyl-transferase (DHHC) inhibitor, 2-bromopalmitate (2BP), SERT palmitoylation was reduced in a time-wise fashion without changing surface or total SERT expression. Acute inhibition of SERT palmitoylation decreased SERT Vmax without altering surface expression or relative affinity of the transporter for 5HT (Km). This suggests that palmitoylation regulates SERT acutely by adjusting SERT kinetics without altering levels of SERT at the cellular surface. In longer time intervals with higher 2BP concentrations, inhibition of SERT palmitoylation promoted a loss in SERT surface density and total protein, suggesting that palmitoylation is involved in trafficking of SERT through cell surface recruitment or endocytosis, dependent on SERT’s state-of-palmitoylation. Additionally, palmitoylation may prevent a loss of total SERT protein by opposing lysosomal degradation or supporting biogenesis. When treated with escitalopram, SERT palmitoylation was reduced alongside 2BP inhibition. These results correlate with losses in surface SERT and 5HT uptake under the same conditions, suggesting that escitalopram may bind and configure SERT to a conformation that discourages palmitoylation, leading to internalization and downregulation of SERT activity. The second project explored the pathogenesis of autism and how escitalopram may be efficacious in its treatment. In previous research, functionally-rare SERT coding variants associated with autism and obsessive-compulsive disorders (ASD and OCD, respectively) exhibit increased surface expression and transport capacity. ASD is a disorder of developmental delay in cognitive processes characterized by difficulties in communication, interaction in social settings, and obsessive-compulsive patterns in thought and behavior. Here, we reveal that the ASD SERT coding variant, F465L, confers an increase in palmitoylation and confirm from previous studies an increase in F465L cell surface levels and Vmax when compared to WT hSERT. Promising studies from clinical and functional magnetic resonance imaging (fMRI) data describe therapeutic approaches for adults with severe forms of ASD/OCD that consist of SSRIs like escitalopram. Acyl-biotinyl exchange (ABE) and cell surface analysis of WT and F465L hSERTs treated with 2BP or escitalopram reveal reductions in both palmitoylation and SERT surface levels to basal WT conditions. These results suggest dysregulated palmitoylation is a step in the pathogenesis of autism and obsessive-compulsory based cognitive illnesses, and escitalopram may be effective in rectifying this process. The third project investigated the family of enzymes responsible for protein palmitoylation called palmitoyl-acyl transferases or DHHCs. We have previously published that 5 members of this family (DHHC2, 3, 8, 15, and 17) catalyze palmitoylation of the dopamine transporter (DAT). This information, alongside our data from project one, led us to hypothesize that these enzymes may play a role in regulating SERT trafficking, activity, and serotonergic tone. When SERT was co-expressed with 9 different DHHCs, we observed an increase in SERT palmitoylation from DHHC1, 8, 15, and 17. Increased SERT palmitoylation status was consistent with increased levels of SERT surface expression, transport capacity, and total cellular SERT protein. From this group, SERT saturation analysis was performed in the absence or presence of DHHC 1 and 8 with transport capacity normalized to total cellular protein (pmol/min/mg) and SERT surface density. We identified that DHHC 1 and 8 modulate SERT activity differently, with DHHC1 directing a trafficking-dependent increase in transport capacity, while DHHC 8 directly enhanced SERT Vmax independent of cell surface levels. These results outline the diversity of DHHC outcomes for SERT trafficking, activity, and expression. It is possible that DHHCs may operate independently to palmitoylate SERT on different intracellular cysteines, and at different points in SERTs life-cycle, to accomplish the cell’s current physiologic objectives. The fourth project examined palmitoylation of NET and how perturbance of this process controls NET processing, trafficking, and may be involved in the development of a vascular disorder termed orthostatic intolerance (OI). NET is a catecholamine transporter that facilitates the reuptake of epinephrine, norepinephrine, and/or dopamine from pre-synaptically stimulated neurons, controlling cognitive functions and sympathetic tone. In this study, we demonstrate that native rat and heterologous human NETs are palmitoylated. Treatment of heterologous cells expressing NET with 2BP resulted in acute time-dependent decreases in Net palmitoylation, surface density, transport capacity, and total NET protein levels. As inhibition of NET palmitoylation was increased by 2BP concentration and time of exposure, we observed losses of total NET protein without loss of beta-actin, suggesting no changes in cellular cyto-toxicity. Physiologically, OI is a syndrome characterized by hyperadrenergic symptoms involving postural tachycardia, syncope, and excessive plasma NE. We have demonstrated that the OI coding variant, A457P has reduced palmitoylation and total expression compared to WT. These processes were partially recovered upon co-expression of DHHC1, an ER bound DHHC, suggesting that co-translational palmitoylation may facilitate NET biogenesis and that its dysregulation may be a mechanism in the pathogenesis of orthostatic intolerance.
... Serotonin is synthesized in the midbrain in a small population of raphe nucleus neurons where tryptophan hydroxylase is expressed [30]. However, serotonin synthesis is not limited to the central nervous system (CNS), as tryptophan hydroxylase is also found in enterochromaffin cells in the gastrointestinal tract [31]. In fact, it should be noted that most of the serotonin in the human body is produced by this cell type [32]. ...
... There are two isoforms of TPH that can participate in this reaction: TPH1, expressed predominantly peripherally; and TPH2, expressed only in the brain. L-aromatic amino acid decarboxylase (AADC) then converts 5-HTP to serotonin [19,31]. ...
Serotonin modulates several physiological and cognitive pathways throughout the human body that affect emotions, memory, sleep, and thermal regulation. The complex nature of the serotonergic system and interactions with other neurochemical systems indicate that the development of depression may be mediated by various pathomechanisms, the common denominator of which is undoubtedly the disturbed transmission in central 5-HT synapses. Therefore, the deliberate pharmacological modulation of serotonergic transmission in the brain seems to be one of the most appropriate strategies for the search for new antidepressants. As discussed in this review, the serotonergic system offers great potential for the development of new antidepressant therapies based on the combination of SERT inhibition with different pharmacological activity towards the 5-HT system. The aim of this article is to summarize the search for new antidepressants in recent years, focusing primarily on the possibility of benefiting from interactions with various 5-HT receptors in the pharmacotherapy of depression.
... These studies provided the scientists with very important information that the brain independently synthesizes 5-HT from L-tryptophan, and suggested that exogenous 5-HT administration incorporates to 5-HT contents in the nervous system. Next studies resulted in molecular identity of two major enzymes in 5-HT biosynthesis pathway: tryptophan hydroxylase (TPH) and AADC [6,14,65,66] (Figure 1). In the cytosol of the nerve cells, TPH catalyzes hydroxylation of l-tryptophan to produce 5-HTP by incorporation of an atom of atmospheric oxygen into l-tryptophan and the other is reduced to water, in the presence of the cofactor agent, tetrahydrobiopterin. ...
... Molecular sequence of the gene encoding AADC has also been identified and localized in mammals [134,135]. It has a non-specific tissue distribution and is expressed in wide range of cell types [66]. In bivalves, molecular identity, localization, and characterization of TPH and AADC are unknown. ...
... TPH enzymes are notoriously unstable in vitro (Hasegawa and Nakamura 2010). To assess the thermal stability of the recombinant proteins, purified wild-type hTPH2 and S41Y enzymes (purified from bacteria or present in PC12 cell homogenates) were incubated at 37°C or 4°C for varying periods of time. ...
... The TPH enzymes are generally thermally unstable (Hasegawa and Nakamura 2010). A number of reports suggest that particular polymorphisms decrease stability further (Zhang et al. 2006;Winge et al. 2007). ...
Human TPH2 ( hTPH 2) catalyzes the rate‐limiting step in CNS serotonin biosynthesis. We characterized a single‐nucleotide polymorphism (C2755A) in the hTPH 2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased V max with unchanged K m values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37°C) for the S41Y enzyme (as compared to the wild‐type enzyme). The S41Y polymorphism decreased cyclic AMP‐dependent protein kinase A‐mediated phosphorylation ~ 50% relative to wild‐type hTPH 2, suggesting that the S41Y mutation may disrupt the post‐translational regulation of this enzyme. Transfected PC12 cells expressed hTPH 2 mRNA , active protein, and synthesized and released serotonin. Paradoxically, while S41Y‐transfected PC12 cells expressed higher levels of hTPH 2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression.
image We report the functional implications of a polymorphic human tryptophan hydroxylase‐2 gene associated with depression and bipolar disorder. The polymorphic enzyme (serine‐41 converted to tyrosine) has increased activity, but decreased enzyme stability and serotonin production. Moreover, cyclic AMP‐dependent protein kinase (PKA)‐mediated phosphorylation of the mutant enzyme is decreased suggesting modified regulation of the S41Y variant leading to altered serotonin.
... Serotonin is a neurotransmitter that has a broad variety of actions in the body, including the regulation of mood. This compound is produced by the hydroxylation of the amino acid tryptophan to provide 5-hydroxytryptophan, which is then decarboxylated [19]. In humans, serotonin is found mostly in the blood platelets, enterochromaffin cells of the intestinal mucosa, and particular parts of the brain [20,21]. ...
The largest cause of mortality and neurological impairment in the world today is stroke. Survivors of an ischemic stroke often have impairment in motor function, visual field defect, speech difficulties, and depression, among other symptoms. While cerebral ischemia is occurring, the levels of many neurotransmitters in the brain, including dopamine, 5-hydroxytryptamine or serotonin, norepinephrine, and glutamate, change, and these changes contribute to the pathophysiology of cerebral ischemia. Despite the fact that the overall impact of serotonin in cerebral ischemia is still unknown, there is evidence that increased serotonin activity in the hippocampus protects neurons from damage after ischemia. Furthermore, multiple studies have shown that selective serotonin reuptake inhibitors, serotonin/norepinephrine reuptake inhibitors, and serotonin agonists may decrease the size of brain infarcts and enhance functional recovery after stroke. When it comes to cerebral ischemia, there are a variety of mechanisms that might explain the neuroprotective impact of SSRIs such as fluoxetine, sertraline, paroxetine, fluvoxamine, citalopram, and escitalopram, among others. They have antioxidant, anti-apoptotic, and anti-inflammatory properties, which are responsible for their success in the prevention and treatment of stroke. In this review, we will go through the function of serotonin in ischemia as well as the potential mechanisms underlying the neuroprotective effects of selective serotonin reuptake inhibitors (SSRIs) in stroke.
... Serotonin is a neurotransmitter that has a wide range of functions in the body. It is synthesized by hydroxylation of the amino acid tryptophan to 5-hydroxytryptophan followed by decarboxylation [19]. In human, serotonin presents mainly in the blood platelets, enterochromaffin cells of the intestinal mucosa and in specific areas of brain [20]. ...
Stroke is a leading cause of death and neurological disability worldwide. Survivors of ischemic stroke usually suffer from impairment in motor function, visual field defect, speech disorders and depression. Cerebral levels of several neurotransmitters such as dopamine, 5-hydroxytriptamine (5-HT) or serotonin, norepinephrine (NE) and glutamate alter during ischemia and contribute to the pathophysiology of cerebral ischemia. The overall effect of serotonin in cerebral ischemia is still unclear but there are evidences that enhancement of serotonin activity in the hippocampus exerts protection against neuronal damage after ischemia. Besides, several studies showed that selective serotonin reuptake inhibitors (SSRIs), serotonin/norepinephrine reuptake inhibitors (SNRIs) and serotonin agonists reduced cerebral infarct size and improved functional recovery after stroke. There are different mechanisms that may explain the neuroprotective effect of SSRIs such as fluoxetine, sertraline, paroxetine, fluvoxamine, citalopram and escitalopram in cerebral ischemia. They exert antioxidant, antiapoptotic and anti-inflammatory activities which are responsible for their effectiveness in stroke. In this review, we will discuss in detail the role of serotonin in ischemia and the proposed mechanisms of the neuroprotective role of SSRIs in stroke.
... When mammals are under conditions of infection and stress, most L-tryptophan is decomposed into bioactive substances that may interact with the stress response through the kynurenine pathway (Floc'h et al., 2011;O'Farrell and Harkin, 2015;Mahony et al., 2015). Besides, L-tryptophan generates serotonin (5-HT) through two-stage enzymatic reactions: tryptophan hydroxylase and aromatic amino acid decarboxylase (Hasegawa et al., 2010). As a neurotransmitter, 5-HT participates in many physiological functions such as animal emotional and behavioral regulation, harmful free radical elimination (Juan et al., 2006), and regulates the response of behavior and neuroendocrine to stress (Kloete et al., 2005;Moltesen et al., 2016). ...
Evisceration is a peculiar behavior that happens frequently in the processes of aquaculture and transportation of sea cucumbers. This behavior involves a complex physiological process and results in the expulsion of the digestive tract and other viscera. However, studies on evisceration are insufficient, and the identification of the internal regulatory metabolites and potential pathways of evisceration in the sea cucumber are therefore likely to provide a scientific basis for this specific behavior. In this study, ultraperformance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was performed on the coelomic fluids that ejected by evisceration Apostichopus japonicus and in the coeloms of normal A. japonicus to detect changes in metabolites and metabolic pathways in the process of evisceration behavior. In total, nine metabolites were found to have increased and eleven to have decreased in the ejected coelomic fluids of evisceration sea cucumbers. These metabolites included phosphatidylethanolamine, glucosylceramide, L-tryptophan, carbamic acid, and cyclohexylamine. In addition, enrichment of metabolic pathway analyses revealed five significantly changed signaling pathways: Glycosyl-phosphatidyl-inositol (GPI) -anchor biosynthesis, regulation of autophagy, sphingolipid metabolism, nitrogen metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. These results contribute valuable data on the potential physiological mechanisms underlying evisceration behavior of A. japonicus, which could have important implications for the aquaculture and transportation of sea cucumbers.
... The VDRE complex activates transcription of many target genes, among them the tryptophan hydroxylase 2 (TPH2) gene (Demay et al. 1992;Gutknecht et al. 2012;Patrick and Ames 2014). This gene encodes for TPH2 that is part of the metabolic pathway for the neurotransmitter serotonin which regulates mood, so that any upstream or downstream dysfunction along this pathway could lead to an imbalance in serotonin in the serotonergic neurons of the central nervous system, potentially increasing the odds of MDD (Côté et al. 2003;Hasegawa and Nakamura 2010). 5 Partial sequence chromatograms of TaqI polymorphism from study subjects. ...
Heritability of major depressive disorder (MDD) is between 36 and 44%, suggesting that up to nearly half of the phenotypic variability is attributable to genes. A number of genetic polymorphisms have been shown to predispose certain individuals to depression. Of particular interest are the polymorphisms of the vitamin D receptor (VDR) gene. Although the VDR gene has been well characterized and a vast number of polymorphisms have been identified, the association between BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) single-nucleotide polymorphisms (SNPs), together with their haplotypes, and MDD risk have yet to be established. We conducted a matched case-control study with a total of 600 participants comprising 300 major depressive disorder (MDD) cases and 300 controls matched by age, gender and ethnicity in a 1:1 ratio, in four public hospitals in Kuala Lumpur and Selangor. Three adjacent SNPs of the VDR gene-BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236)-were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Odds ratios and 95% confidence intervals (CIs) were obtained from conditional logistic regression using Stata 16. Linkage disequilibrium and haplotype association with MDD were analyzed using the online SNPStats program. None of the genotypes of the three SNPs was significantly associated with risk of developing MDD after adjusting for confounding factors. However, the TAC (BAt) haplotype was associated with increased odds of MDD (adjusted OR = 2.17, 95% CI = 1.30-3.61, p = 0.003) using CCT (baT) as reference haplotype. The findings suggest that the BsmI-ApaI-TaqI TAC (BAt) haplotype of the VDR gene increases susceptibility to MDD.
... This propensity of the A allele to increase risk of depression could possibly play a role in the interaction between ApaI polymorphism and severity. The C>A transversion at the ApaI restriction site is located in the 3' VDR gene transcription initiation site-a ligand binding site of the VDR gene [67]-and may affect the downstream effect of the ligand-binding properties of the Vitamin D receptor, in turn affecting the Vitamin D Response Element (VDRE) complex and activation of the transcription of tryptophan hydroxylase 2 (TPH2) gene, leading to an imbalance of serotonin levels in the brain [68][69][70]. Although several polymorphisms, including ApaI, of the VDR gene have been described, their effects on VDR function and interaction with severity of depression are still poorly understood. ...
A total of 201 patients with major depressive disorder from four hospitals in Malaysia were followed up for 5 years to determine the prognostic factors of recurrent major depressive disorder that could potentially contribute to improving the management of MDD patients. For each individual patient, at the time of recruitment as part of a case-control study, information was collected on recent threatening life events, personality and social and occupational functioning, while blood samples were collected to genotype single nucleotide polymorphisms of vitamin D receptor (VDR), zinc transporter-3 (ZnT3), dopamine transporter-1 (DAT1), brain-derived neurotropic factor (BDNF), serotonin receptor 1A (HT1A) and 2A (HT2A) genes. Kaplan-Meier and Cox-regression were used to estimate hazard functions for recurrence of major depressive disorder. Individuals with severe MDD in previous major depressive episodes had five and a half times higher hazard of developing recurrence compared to mild and moderate MDD (HR = 5.565, 95% CI = 1.631–18.994, p = 0.006). Individuals who scored higher on social avoidance had three and a half times higher hazard of recurrence of MDD (HR = 3.525, 95% CI = 1.349–9.209; p = 0.010). There was significant interaction between ApaI +64978C>A single nucleotide polymorphism and severity. The hazard ratio increased by 6.4 times from mild and moderate to severe MDD for A/A genotype while that for C/A genotype increased by 11.3 times. Social avoidance and severity of depression at first episode were prognostic of recurrence. Screening for personality factors at first encounter with MDD patients needs to be considered as part of the clinical practice. For those at risk of recurrence in relation to social avoidance, the psychological intervention prescribed should be customized to focus on this modifiable factor. Prompt and appropriate management of severe MDD is recommended to reduce risk of recurrence.
... One study suggested that in female rodents, estrogens may facilitate the action of SSRIs (Estrada-Camarena et al., 2004). Although the mechanism of action through which this occurs is unknown, treatment with estradiol in the dorsal raphe nucleus (DRN) has been to shown to increase the expression of tryptophan hydroxylase 2 (TPH2) (Hiroi et al., 2006), the brain-specific serotonin synthesizing enzyme (Hasegawa et al., 2010). This finding suggested that estradiol may have an indirect effect of increasing serotonin synthesis in the DRN through its modulation of TPH2 expression. ...
A roadblock to successful treatment for anxiety and depression is the high proportion of individuals that do not respond to existing treatments. Different underlying neurobiological mechanisms may drive similar symptoms, so a more personalized approach to treatment could be more successful. There is increasing evidence that sex is an important biological variable modulating efficacy of antidepressants and anxiolytics. We review evidence for sex-specific effects of traditional monoamine based antidepressants and newer pharmaceuticals targeting kappa opioid receptors (KOR), oxytocin receptors (OTR), and N-methyl-D-aspartate receptors (ketamine). In some cases, similar behavioral effects are observed in both sexes while in other cases strong sex-specific effects are observed. Most intriguing are cases such as ketamine which has similar behavioral effects in males and females, perhaps through sex-specific neurobiological mechanisms. These results show how essential it is to include both males and females in both clinical and preclinical evaluations of novel antidepressants and anxiolytics.
... 86 Tryptophan hydroxylase 2 (TPH2), the primary isoform present in the central nervous system, mediates the ratelimiting step in central serotonin synthesis. 87,88 The activity of tryptophan hydroxylase is oxygen dependent, and in vivo the enzyme is partially unsaturated with respect to oxygen at physiologic concentrations of tryptophan. 89,90 In animal models, exposure to low atmospheric oxygen reduces tryptophan hydroxylase activity, whereas exposure to supplemental oxygen increases it. ...
Learning objectives:
After participating in this activity, learners should be better able to:• Assess epidemiologic evidence that increased altitude of residence is linked to increased risk of depression and suicide• Evaluate strategies to address hypoxia-related depression and suicidal ideation ABSTRACT: Suicide and major depressive disorder (MDD) are complex conditions that almost certainly arise from the influences of many interrelated factors. There are significant regional variations in the rates of MDD and suicide in the United States, suggesting that sociodemographic and environmental conditions contribute. Here, we review epidemiological evidence that increases in the altitude of residence are linked to the increased risk of depression and suicide. We consider the possibility that chronic hypobaric hypoxia (low blood oxygen related to low atmospheric pressure) contributes to suicide and depression, which is suggested by animal models, short-term studies in humans, and the effects of hypoxic medical conditions on suicide and depression. We argue that hypobaric hypoxia could promote suicide and depression by altering serotonin metabolism and brain bioenergetics; both of these pathways are implicated in depression, and both are affected by hypoxia. Finally, we briefly examine treatment strategies to address hypoxia-related depression and suicidal ideation that are suggested by these findings, including creatine monohydrate and the serotonin precursors tryptophan and 5-hydroxytryptophan.
... Another possibility is that fluvoxamine influences tryptophan hydroxylase activity as a ratelimiting enzyme in 5-HT biosynthesis [34]. Given that raphe nuclei are the serotonergic cell body regions that are very rich in tryptophan hydroxylase [35], the increase or decrease in 5-HT synthesis rate in the raphe nuclei observed after antidepressant administration may be related to an increase or decrease in tryptophan hydroxylase activity, respectively. ...
A considerable body of evidence indicates the involvement of the neurotransmitter serotonin (5-HT) in the pathogenesis and treatment of depression.
The acute effect of fluvoxamine, on 5-HT synthesis rates was investigated in rat brain regions, using α-(14)C-methyl-L-tryptophan as a tracer. Fluvoxamine (25 mg/kg) and saline (control) were injected intraperitoneally, one hour before the injection of the tracer (30 μCi).
There was no significant effect of fluvoxamine on plasma free tryptophan. After Benjamini-Hochberg False Discovery Rate correction, a significant decrease in the 5-HT synthesis rate in the fluvoxamine treated rats, was found in the raphe magnus (-32%), but not in the median (-14%) and dorsal (-3%) raphe nuclei. In the regions with serotonergic axon terminals, significant increases in synthesis rates were observed in the dorsal (+41%) and ventral (+43%) hippocampus, visual (+38%), auditory (+65%) and parietal (+37%) cortex, and the substantia nigra pars compacta (+56%). There were no significant changes in the 5-HT synthesis rates in the median (+11%) and lateral (+24%) part of the caudate-putamen, nucleus accumbens (+5%), VTA (+16%) or frontal cortex (+ 6%).
The data show that the acute administration of fluvoxamine affects 5-HT synthesis rates in a regionally specific pattern, with a general elevation of the synthesis in the terminal regions and a reduction in some cell body structures. The reasons for the regional specific effect of fluvoxamine on 5-HT synthesis are unclear, but may be mediated by the presynaptic serotonergic autoreceptors.
... Serotonin is synthesized from tryptophan by the aromatic acid decarboxylase (AAAD), which is also involved in the biosynthesis of dopamine and norepinephrine. Since the ratelimiting step in serotonin-synthesis, however, is the amount of TPH available (Hasegawa and Nakamura 2010 ), measuring concentrations of TPH offers one possibility to quantify serotonin synthesis. As measured with PET, high serotonin synthesis capacity rates were observed in the putamen, caudate, thalamus and hippocampus. ...
The serotonergic system is one of the most important modulatory neurotransmitter systems in the human brain. It plays a central role in major physiological processes and is implicated in a number of psychiatric disorders. Along with the dopaminergic system, it is also one of the phylogenetically oldest human neurotransmitter systems and one of the most diverse, with 14 different receptors identified up to this day, many of whose function remains to be understood. The system's functioning is even more diverse than the number of its receptors, since each is implicated in a number of different processes. This review aims at illustrating the distribution and summarizing the main functions of the serotonin (5-hydroxytryptamin, 5-HT) receptors as well as the serotonin transporter (SERT, 5-HTT), the vesicular monoamine transporter 2, monoamine oxidase type A and 5-HT synthesis in the human brain. Recent advances in in vivo quantification of these different receptors and enzymes that are part of the serotonergic system using positron emission tomography are described.
Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d 5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g'-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.
This part 2 of 2 review will show the effects of neurosteroids, made in the brain , on affective disorders . There are sufficient good results in influencing affective disorders by administering steroids like estradiol or progesterone/ pregnenolone and DHEA.
This publication gives a review about current effects of neurosteroids on affective disorders . It will allow clinicians to treat patients with biological treatment, involving neurosteroids, neurotransmitter precursor and Vitamins, involved in brain metabolism .
Serotonin is synthesized in a two-step reaction, from tryptophan to 5-hydroxytryptophan (5-HTP) and then to 5-hydroxytryptamine (serotonin, 5-HT). The latter step is catalyzed by aromatic amino acid decarboxylase (AADC; EC 4.1.1.28), while the first step is catalyzed by tryptophan hydroxylase (TPH; EC 1.14.16.4), the rate-limiting enzyme in serotonin synthesis [1] (Fig. 1).
While anxiogenic stimuli activate neurons in the raphe and lead to serotonin release in limbic forebrain targets, panicogenic stimuli do not necessarily do so. For example, escape performance in an elevated T-maze does not increase c-Fos-like immunoreactivity in the raphe. Nonetheless, the observation of a panicolytic role of serotonin in the PAG and an anxiogenic role in the amygdala and hippocampus suggests that the raphe is not a homogeneous structure. In fact, the dorsal raphe can be divided at least into six subregions based on cytoarchitecture and distribution of serotonergic neurons. These comprise the rostral (DRr), dorsal (DRD), ventral (DRV), lateral wing (lwDR), caudal (DRC), and interfascicular (DRI) portions (Fig. 5.1). Among those, the rostral, ventral, and interfascicular subregions play little role in the control of defense responses, and discussion of their functions can be found elsewhere. Here, we will discuss evidence for a role of the DRD, lwDR and DRC in anxiety and fear.
The circadian system in suprachiasmatic nucleus (SCN) involves regulated serotonin levels and coordinated expression of various clock genes. To understand circadian disfunction in the age-related neurodegenerative disorder Parkinson’s disease (PD), the rotenone-induced PD (RIPD) male Wistar rat model was used. The alterations in the rhythmic dynamic equilibrium of interactions between the various components of serotonin metabolism and the molecular clock were measured. There was significant decrease in the mean 24 h levels of tryptophan, 5-hydroxytryptophan (5-HTP), serotonin (5-HT), N-acetyl serotonin (NAS) and melatonin (MEL) by approximately 63, 51, 76 and 96 % respectively (
p ≤ 0.05). However significant increase in 5-methoxy indole acetic acid (5-MIAA), 5-methoxy tryptophol (5-MTOH), 5-hydroxy tryptophol (5-HTOH) indicated increased serotonin catabolism with the abolition of daily rhythms of MEL, 5-HTP and 5-MIAA in RIPD. 24 h mean levels of rPer1, rCry1, rBmal1 reduced by about 0.5, 0.74 and 0.39-fold and increased for rPer2 by about 1.7-fold. The daily pulse of rPer2, rCry1, rCry2 and rBmal1 significantly decreased by 0.36, 0.6, 0.14, 0.1 and 0.2-fold. As melatonin, an antioxidant and an endogenous synchronizer of rhythm declined in RIPD male Wistar rat model, the effects of melatonin-administration on the rhythmic expression of various clock genes were studied. Interestingly, melatonin-administration resulted in restoration of the phase of rPer1 daily rhythm in RIPD indicating differential sensitivity of various clock components towards melatonin. The animals which were administered both rotenone and MEL for 48 days interestingly showed neuroprotective effects in dark phase on correlations between expression of various genes.
In the past decade, there have been increasing concerns over the effects of pharmaceutical compounds in the aquatic environment, however very little is known about the effects of antidepressants such as the selective serotonin re-uptake inhibitors (SSRIs). Many biological functions within invertebrates are under the control of serotonin, such as reproduction, metabolism, moulting and behaviour. The effects of serotonin and fluoxetine have recently been shown to alter the behaviour of the marine amphipod, Echinogammarus marinus (Leach, 1815). The purpose of this study was to observe behavioural and transcriptional modifications in this crustacean exposed to the two most prescribed SSRIs (fluoxetine and sertraline) and to develop biomarkers of neurological endocrine disruption. The animals were exposed to both drugs at environmentally relevant concentrations from 0.001 to 1μg/L during short-term (1h and 1day) and medium-term (8 days) experiments. The movement of the amphipods was tracked using the behavioural analysis software during 12min alternating dark/light conditions. The behavioural analysis revealed a significant effect on velocity which was observed after 1h exposure to sertraline at 0.01μg/L and after 1 day exposure to fluoxetine as low as 0.001μg/L. The most predominant effect of drugs on velocity was recorded after 1 day exposure for the 0.1 and 0.01μg/L concentrations of fluoxetine and sertraline, respectively. Subsequently, the expression (in this article gene expression is taken to represent only transcription, although it is acknowledged that gene expression can also be regulated at translation, mRNA and protein stability levels) of several E. marinus neurological genes, potentially involved in the serotonin metabolic pathway or behaviour regulation, were analysed in animals exposed to various SSRIs concentrations using RT-qPCR. The expression of a tryptophan hydroxylase (Ph), a neurocan core protein (Neuc), a Rhodopsin (Rhod1) and an Arrestin (Arr) were measured following exposure to fluoxetine or sertraline for 8 days. The levels of Neuc, Rhod1 and Arr were significantly down-regulated to approximately 0.5-, 0.29- and 0.46-fold, respectively, for the lower concentrations of fluoxetine suggesting potential changes in the phototransduction pathway. The expression of Rhod1 tended to be up-regulated for the lower concentration of sertraline but not significantly. In summary, fluoxetine and sertraline have a significant impact on the behaviour and neurophysiology of this amphipod at environmentally relevant concentrations with effects observed after relatively short periods of time.
A method for the analysis of serotonin (5-HT) and its precursors, 5-hydroxytryptophan (5-HTP) and l-tryptophan (TP) in chocolate samples by capillary liquid chromatography-mass spectrometry (cLC-MS) has been developed. Optimum chromatographic conditions were established by using a personalized multifactorial experimental design. Finally the cLC separation was achieved through a mixture of acetonitrile and 5mM ammonium formate at pH 4 (3:97, v/v) as mobile phase in gradient elution, setting the injection volume at 10 μL and using pure water as injection solvent for focusing purposes on the head of the capillary column. For extraction of targets in chocolate samples a new, fast and simple procedure based on the use of acidic extraction medium and sonication was developed. Working in selected ion mode (m/z 177 for 5-HT, m/z 205 for l-tryptophan and m/z 221 for 5-HTP) detection limits were between 0.01 and 0.11 μg g(-1) and linearity was in the concentration range of 0.5-25 μg g(-1). Recoveries higher than 76% with RSDs lower than 8% were obtained from spiked samples for all analytes, showing the effectiveness of the proposed method. Serotonin and its precursors were determined in 5 kinds of commonly consumed chocolates with different cocoa contents (70-100%). The highest serotonin content was found in chocolate with a cocoa content of 85% (2.93 μg g(-1)). Regarding l-tryptophan, the highest content of this amino acid (13.27-13.34 μg g(-1)) was found in chocolate samples with the lowest cocoa content (70-85%). 5-Hydroxytryptophan was not detected in any chocolate samples.
New genetic models that target the serotonin system show that transient alterations in serotonin homeostasis cause permanent changes to adult behaviour and modify the fine wiring of brain connections. These findings have revived a long-standing interest in the developmental role of serotonin. Molecular genetic approaches are now showing us that different serotonin receptors, acting at different developmental stages, modulate different developmental processes such as neurogenesis, apoptosis, axon branching and dendritogenesis. Our understanding of the specification of the serotonergic phenotype is improving. In addition, studies have revealed that serotonergic traits are dissociable, as there are populations of neurons that contain serotonin but do not synthesize it.
The 5-HT 1A agonist 8-OH-DPAT has been reported to disrupt prepulse inhibition (PPI) of the acoustic startle reflex after local administration into the raphe nuclei. Because it is likely that 8-OH-DPAT disrupted PPI by activation of somatodendritic inhibitory receptors, and thereby, via a decrease in 5-HT neurotransmission, we tested whether chronic, drug-induced, depletions of 5-HT have similar effects. Rats were drug-treated for three consecutive days and tested in a short PPI paradigm on day 4, and retested 2 h later, after acute saline or drug administration. Repeated treatment with the 5-HT synthesis inhibitor p-chlorophenylalanine methyl ester (PCPA; 160 mg/kg) produced a small, but significant, attenuation of PPI, and a large decrease in extracellular 5-HT levels in the hippocampus, as measured in independent microdialysis experiments. An even larger depletion of 5-HT was obtained by substituting the 3 rd PCPA administration with the 5-HT releaser d-fenfluramine (10 mg/kg); this combined treatment nearly abolished PPI in the majority of animals. The involvement of 5-HT in the latter effects was confirmed by the finding that low doses of the 5-HT precursor 5-hydroxy-L-tryptophan reinstated PPI during retest. These data, together with recently published studies, provide strong evidence that pharmacologically-induced depletion of 5-HT disrupts PPI.
Background:
The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular 'reactive oxygen species' (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation.
Review:
We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation).The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible.This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, since in some circumstances (especially the presence of poorly liganded iron) molecules that are nominally antioxidants can actually act as pro-oxidants. The reduction of redox stress thus requires suitable levels of both antioxidants and effective iron chelators. Some polyphenolic antioxidants may serve both roles.Understanding the exact speciation and liganding of iron in all its states is thus crucial to separating its various pro- and anti-inflammatory activities. Redox stress, innate immunity and pro- (and some anti-)inflammatory cytokines are linked in particular via signalling pathways involving NF-kappaB and p38, with the oxidative roles of iron here seemingly involved upstream of the IkappaB kinase (IKK) reaction. In a number of cases it is possible to identify mechanisms by which ROSs and poorly liganded iron act synergistically and autocatalytically, leading to 'runaway' reactions that are hard to control unless one tackles multiple sites of action simultaneously. Some molecules such as statins and erythropoietin, not traditionally associated with anti-inflammatory activity, do indeed have 'pleiotropic' anti-inflammatory effects that may be of benefit here.
Conclusion:
Overall we argue, by synthesising a widely dispersed literature, that the role of poorly liganded iron has been rather underappreciated in the past, and that in combination with peroxide and superoxide its activity underpins the behaviour of a great many physiological processes that degrade over time. Understanding these requires an integrative, systems-level approach that may lead to novel therapeutic targets.
Gel filtration of bovine liver extract on a Sephadex G-200 column resolved three macromolecular fractions with dihydropteridine reductase-dependent cytochrome c reducing activity. One of the active fractions was purified from the extract through the steps of solvent fractionation, chromatography on DEAE-Sephadex, and gel filtration. Biochemical and microbiological analyses showed that the purified complex consists of a Mr = 70,000 protein and tetrahydropteroyldiglutamate. In contrast to the extreme lability of free tetrahydropteridines the complex was quite stable against autooxidation under aerobic conditions.
P-enolpyruvate carboxykianse ferroactivator was purified from rat liver cytosol to apparent homogeneity. This protein has an S20 of 4.69. High speed sedimentation equilibrium indicated a molecular weight of 82,000; that obtained by gel filtration was 126,000. this discrepancy in molecular weight by the two methods may indicate that this protein has a high axial ratio. A subunit molecular weight of 23,600 was obtained by sodium dodecyl sulfate electrophoresis. Synthesis of P-enolpyruvate is stimulated 3-fold when P-enolpyruvate carboxykinase is incubated in the presence of ferroactivator plus Fe2+; activity with Fe3+, Mn2+, Co2+, Cd2+, Mg2+, or Ca2+ was not affected by the ferroactivator. P-enolpyruvate synthesis by carboxykinase was activated by Mn2+ in the absence of ferroactivator. Quinolinic acid strongly inhibits carboxykinase activated by ferroactivator and Fe2+, but does not inhibit the enzyme activated by Mn2+. In the reverse reaction, ferroactivator and Fe2+ stimulated oxalacetate synthesis only briefly and carboxykinase activity rapidly returned to control rates. Increased oxalacetate synthesis by carboxykinase that had been incubated with ferroactivator and Co2+ was sustained. Stimulation of P-enolpyruvate synthesis by enzyme incubated in the presence of sulfate and Fe2+ rapidly diminished as a function of incubation time. Under similar conditions, P-enolpyruvate carboxykinase ferroactivator maintained the enzyme in the Fe2+-activated state for at least 4 h. The amounts of purified ferroactivator required to stimulate P-enolpyruvate carboxykinase maximally and the amount present in liver cytosol are sufficient to activate the enzyme maximally in vivo if adequate Fe2+ were present. It is possible that the rate of P-enolpyruvate synthesis in gluconeogenic tissues may be regulated by the availability of intracellular Fe2+ to the ferroactivator and P-enolpyruvate carboxykinase.
The principal pathway for the metabolism of phenylalanine in mammals is via conversion to tyrosine in a tetrahydrobiopterin-dependent hydroxylation reaction occurring predominantly in the liver. Recently, the proposal that certain hyperphenylalaninemic children may have a deficiency of carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, has widened the interest in this area of metabolism. Upon cloning and sequencing the dehydratase, we discovered that this protein is identical to DCoH, the cofactor which regulates the dimerization of hepatic nuclear factor 1 alpha, a homeodomain transcription factor. The identity of the nuclear and cytoplasmic proteins is demonstrated by size, immunoblotting, stimulation of phenylalanine hydroxylase, and dehydratase activity. The evolution of the dual functions of regulation of phenylalanine hydroxylation activity and transcription activation in a single polypeptide is unprecedented.
The oxidation of amino acids by Fenton reagent (H2O2 + Fe(II] leads mainly to the formation of NH+4, alpha-ketoacids, CO2, oximes, and aldehydes or carboxylic acids containing one less carbon atom. Oxidation is almost completely dependent on the presence of bicarbonate ion and is stimulated by iron chelators at levels which are substoichiometric with respect to the iron concentration but is inhibited at higher concentrations. The stimulatory effect of chelators is not due merely to solubilization of catalytically inactive polymeric forms of Fe(OH)3 nor to the conversion of Fe(II) to complexes incapable of scavenging hydroxyl radicals. The results suggest that an iron chelate and another as yet unidentified form of iron are both required for maximal rates of amino acid oxidation. The metal ion-catalyzed oxidation of amino acids is likely a "caged" process, since the oxidation is not inhibited by hydroxyl radical scavengers, and the relative rates of oxidation of various amino acids by the Fenton system as well as the distribution of products formed (especially products of aromatic amino acids) are significantly different from those reported for amino acid oxidation by ionizing radiation. Several iron-binding proteins, peptides, and hemoglobin degradation products can replace Fe(II) or Fe(III) in the bicarbonate-dependent oxidation of amino acids. In view of their ability to sequester metal ions and their susceptibility to oxidation by H2O2 in the presence of physiological concentrations of bicarbonate, amino acids may serve an important role in antioxidant defense against tissue damage.
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Selected References
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Darmon MC, Guibert B, Leviel V, Ehret M, Maitre M, Mallet J. Sequence of two mRNAs encoding active rat tryptophan hydroxylase. J Neurochem. 1988 Jul;51(1):312–316. [PubMed]
In the presence of phenylalanine and molecular oxygen, activated phenylalanine hydroxylase catalyzes the oxidation of tetrahydrobiopterin. The oxidation of this tetrahydropterin cofactor also proceeds if the substrate, phenylalanine, is replaced by its product, tyrosine, in the initial reaction mixture. These two reactions have been defined as coupled and uncoupled, respectively, because in the former reaction 1 mol of phenylalanine is hydroxylated for every mole of tetrahydrobiopterin oxidized, whereas in the latter reaction there is no net hydroxylation of tyrosine during the oxidation of the tetrahydropterin.
During the course of the coupled oxidation of tetrahydrobiopterin, a pterin 4a-carbinolamine intermediate can be detected by ultraviolet spectroscopy (Kaufman, S. (1976) in Iron and Copper Proteins (Yasunobu, K. T., Mower, H. F., and Hayaishi, O., eds) pp. 91-102, Plenum Publishing Corp., New York). Dix and Benkovic (Dix, T. A., and Benkovic, S. J. (1985) Biochemistry 24, 5839–5846) have postulated that the formation of this intermediate only occurs when the oxidation of the tetrahydropteridine is tightly coupled to the concomitant hydroxylation of the aromatic amino acid. However, during the tyrosine-dependent uncoupled oxidation of tetrahydrobiopterin by phenylalanine hydroxylase, we have detected the formation of a spectral intermediate with ultraviolet absorbance that is essentially identical to that of the carbinolamine. Furthermore, this absorbance can be eliminated by the addition of 4a-carbinolamine dehydratase, an enzyme which catalyzes the dehydration of the 4a-carbinolamine. Quantitation of this intermediate suggests that there are two pathways for the tyrosine-dependent uncoupled oxidation of tetrahydrobiopterin by phenylalanine hydroxylase because only about 0.3 mol of the intermediate is formed per mol of the cofactor oxidized.
We have examined the mechanism by which hemin regulates the expression of the human transferrin receptor. Previous work led to the suggestion that the regulatory signal is provided by heme (Ward J. H., Jordan, I., Kushner, J. P., and Kaplan, J. (1984) J. Biol. Chem. 259, 13235-13240). We demonstrated that hemin regulates the expression of the receptor via alterations in the rate of receptor biosynthesis. However, this effect can be completely abolished by addition of desferrioxamine, an intracellular iron chelator. Competition curves demonstrate that desferrioxamine and hemin affect the same intracellular iron pool. Since the chelator cannot remove iron from heme, we propose that hemin acts simply by delivering iron to a chelatable iron pool and that levels of chelatable iron provide the regulatory signal for expression of the transferrin receptor gene.
Recent studies on the cellular transport of sepiapterin, dihydrobiopterin (BH2), and tetrahydrobiopterin (BH4) have concentrated on the following topics:
1) the relatively low permeability of BH2 and BH4 across the cell membrane, 2) the rejection of relatively high concentrations of BH2 and BH4, 3) the high affinity active transport of BH4 in maintaining endogenous BH4 levels in the cell, and 4) the bi-directional rap id transport of sepiapterin. The in vitro studies on sepiapterin uptake, its successive conversion to BH2 and BH4 , and the low permeability of BH4 provided a solid basis of understanding as to why sepiapterin was most efficient at elevating tissue BH4 levels in vivo.
we currently believe that by translating attentional theories into testable hypotheses, we will be able to advance our understanding of schizophrenic psychopathology
by using converging operations and new methodologies, we are now in a position to generate and test critical, operationally defined hypotheses
this approach allows us to dissect both the information processing chain of events and the underlying neurobiological mechanisms that are disordered in schizophrenia (PsycINFO Database Record (c) 2012 APA, all rights reserved)
This chapter discusses the aromatic hydroxylations. Most of the intermediary metabolism is concerned with the transformations of chemically reactive compounds. The amino acids, fatty acids, and sugars that are derived from dietary proteins, fats, and carbohydrates, contain at least one functional group per molecule. Because amino, hydroxyl, and carboxylate groups lead to an activation of adjacent hydrogen atoms, the metabolism of these substances begins with an attack at or near the functional group. All the metabolites that are encountered in the cell are not provided with such convenient reactive handles. The carbocyclic compounds, aromatic and nonaromatic, and the paraffin side chains of some compounds cannot be handled by the usual hydrolysis or dehydrogenation reactions. For the metabolism of some of the stable molecules, a reaction is employed by the cell-aerobic hydroxylation. No intermediate been identified in any aerobic hydroxylation reaction. However, there is some evidence that an epoxide may be an intermediate in the conversion of kynurenic acid to dihydroxykynurenic acid.
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.
Tryptophan hydroxylase (TPH) from central serotonergic neurons in the dorsal raphe nucleus (DRN) and that from the endocrine pineal gland (PG) have been shown to exhibit different biochemical characteristics. We further report here that the isoelectric point determined by chromatofocusing differs between TPH from the rat brainstem and PG. In addition, the levels of TPH mRNA are much greater in the PG than the DRN despite a higher enzymatic activity in the DRN. These data raise the question as to whether different forms of TPH may exist in the DRN and the PG. To address this question, we amplified TPH cDNAs by the polymerase chain reaction (PCR) using poly(A)+ RNA purified from both tissues. Several combinations of oligonucleotide primers encompassing different regions of the published coding sequence of rat pineal TPH were employed for this purpose. Subsequent analysis by gel electrophoresis and Southern blotting of PCR products indicated that DNA fragments of identical length were amplified from both sources. Furthermore, the nucleotide sequences of three independent subclones containing the putative full-length coding region of DRN TPH were determined and found to be identical to that of PG. In situ hybridization using the amplified cDNA as a probe demonstrated specific labeling within the DRN of the rat brain. These data support the hypothesis that tissue-specific differences in TPH characteristics result from differential post-translational events and clearly indicate that a TPH mRNA transcript identical in coding sequence to the PG form is expressed in the DRN.
Tryptophan 5-monooxygenase was purified approximately 5500-fold, to apparent homogeneity with a specific activity of 374 nmol min-−1 mg−1 at 30°C, from rat brain-stem using Sepharose CL-6B, DEAE-Sepharose CL-6B and pteridine-agarose chromatography. Two distinct active forms were separable by DEAE-Sepharose CL-6B and designated as form I and form I1 based on their order of elution from the gel column. The apparent molecular weight of form I was determined to be 300000 by gel filtration on Ultrogel AcA 34 and 288000 by gradient polyacrylamide gel electrophoresis. The enzyme gave a single band on sodium dodecylsulfate/poly- acrylamide gel electrophoresis, the molecular weight of which was estimated to be 59000, indicating that the enzyme might be composed of four identical subunits. The tetrameric structure of the enzyme was further suggested by cross-linking studies using dimethyl suberimidate as a bifunctional reagent. The enzyme activity was stimulated approximately 3.5-fold by the addition of Fe2+. Kinetic studies revealed that this activation was associated with an increase of V value. The purified enzyme had an activity of phenylakdnine hydroxylation but not an activity of tyrosine hydroxylation.
Calmodulin-dependent protein kinase (kinase II) [Yamauchi, T. and Fujisawa, H. (1980) FEBS Lett. 116 141–144] which is involed in the activation of tryptophan 5-monoosygenase was purified 720-fold wiht a 36% yield from rat cerebvral cortex using ammonium sulfate and chromatography on Sepharose CL-4B, Calmodulin-Sepharose 4B and phosphocellulose. The purified enzyme showed one major protein band corresponding in to a molecular weight of 55000 and a faint band upo0n sodium dodecyl sufate/polyacrylamide disc gel electophoresis, wherase it gave a single protein band upon polyacrylamide gel electroporesis whithout soudium dodecyl sulfate. The molecualar weight ad th sedimentation coeffieient of the kinase were dtermined to be 540000 by sedimentation equilibrium and 16.5 S by sucrose density gradient cntrifugation.
The Kinase required absolutely calmodulin and Ca2+ for its activity and the apparent Ka values for calmodulin and Ca+2 were 10 nM and 1.6 μM respectively. The Km values for ATP and Mg2+ wer calculated to be 0.06 mM and 1 mM, respectively. The concentration of tryptophan 5-monooxygenase required to produece half-maximal effects on ist activation by the kinase was estimated to be as low as 0.3 nM, on the basis of th finding that the molecular weight and the specific activity of trytophan 5-monooxygenas were 245000 and 374 nmol min−1 mg oprotein−1 respectively [Nakata, H. and Fuisawa, H. (1982) Eur. J. Biochem. 122, 41–47]. The kinase phosphorylated casein, smooth muscle myosin light chain as well some endogenous protiens of brain cytosol. The enzyme did not phoisphorylte significantly histone, protamine, and phsphorlyase. b. Some other proterties of he Kinase were exiamined.
Tryptophan hydroxylase requires Fe2+ for in vitro enzyme activity. In this study, the intracellular activity of tryptophan hydroxylase was assessed by applying 3-hydroxybenzylhydrazine (NSD-1015), an inhibitor of aromatic l-amino acid decarboxylase, to monolayer cultures of RBL2H3 cells, a serotonin producing mast cell line. The effect of manipulating intracellular ‘free’ iron levels on enzyme activity was analyzed by administration of iron chelators. Desferrioxamine (DFO) suppressed the intracellular enzyme activity. Salicylaldehyde isonicotinoyl hydrazone (SIH) also suppressed enzyme activity, but stimulated it when administered in the Fe-bound form. Hemin also stimulated enzyme activity, which progressively increased over several hours to more than sixfold the initial level. DFO and SIH inhibited the hemin stimulatory effect when administered simultaneously with hemin. Both suppression and stimulation with these chelators took place without a significant decrease or increase in the amount of enzyme. These results indicate that there was an inadequate supply of Fe2+ in the cells to support full activity of tryptophan hydroxylase.
The catalytic subunit of protein kinase A increases brain tryptophan hydroxylase activity. The activation is manifested as an increase in Vmax without alterations in the Km for either tetrahydrobiopterin or tryptophan. The activation of tryptophan hydroxylase by protein kinase A is dependent on ATP and an intact kinase and is inhibited specifically by protein kinase A inhibitors. Protein kinase A also catalyzes the phosphorylation of tryptophan hydroxylase. The extent to which tryptophan hydroxylase is phosphorylated by protein kinase A is dependent on the amount of kinase used and is closely related to the degree to which the hydroxylase is activated. These results suggest that a direct relationship exists between phosphorylation and activation of tryptophan hydroxylase by protein kinase A.
Two full-length cDNA clones that encode functional rat tryptophan hydroxylase (EC 1.14.16.4), the key enzyme in serotonin synthesis, have been isolated from a rat pineal gland library. These tv/o clones correspond to the 1.8 and 4-kilobase mRNA species, respectively. They contain the same coding sequence corresponding to a 51,010 dalton protein and differ in the length of their 3’ untranslated regions.
Weak lead stimulation can modify the human startle reflex It is proposed that, with the aid of physiological measures taken in the interval between lead and startle stimuli, this paradigm offers a promising method of investigating different levels of central processing, the operation of a short time-constant as opposed to a long time-constant system, and the operation of an orienting-Attentional as apposed an Intensity dependent activational system.
A method for the determination of [14C]biopterin biosynthesis from [14C]guanosine-5′-triphosphate by a desalted preparation from rat striatum, based on sequential reverse-phase and cation-exchange high performance liquid chromatography, is described. Synthesis of reduced forms of biopterin by this striatal extract was found to be dependent on enzymatic activity, guanosine-5′-triphosphate, magnesium ions, and a reduced pyridine nucleotide. As demonstrated by the technique of isotope dilution, isotope trapping, 6-lactyl-7,8-dihydropterin (sepiapterin) was found to be an intermediate in biopterin biosynthesis that is catalyzed by the striatal extract. Rat brain was also shown to synthesize biopterin in vivo from intraventricularly administered [14C]guanosine or sepiapterin. Intraventricular injection of sepiapterin increased dihydro- and 5,6,7,8-tetrahydrobiopterin levels in rat brain by more than eightfold. The temporal relationship between the appearance of dihydro- and 5,6,7,8-tetrahydrobiopterin following intraventricular injection of sepiapterin suggests that dihydrobiopterin is the immediate product of sepiapterin reduction which is then reduced further to the functional cofactor 5,6,7,8-tetra-hydrobiopterin. Therefore, in contrast to previous reports, the biosynthesis of biopterin by rat brain does not appear to differ from that occurring in other, nonneural tissues.
A cDNA library was constructed from RNA prepared from P815 mouse mastocytoma cells and screened for tryptophan hydroxylase. An essentially full-length clone that recognizes a major mRNA species of 1.9 kb in mastocytoma cell lines and in pineal gland, duodenum, and brainstem of the mouse was obtained. The predicted amino acid sequence of this mouse mastocytoma clone showed 97 and 87% identity, respectively, with tryptophan hydroxylase clones isolated from rat and rabbit pineal glands, but the mouse clone contains an unusual 3-amino-acid duplication near the N-terminus and lacks a phosphorylation site. A fragment of the cDNA produced an enzymatically active protein when expressed in Escherichia coli, thus demonstrating that the catalytic domain is included in the C-terminal 380 amino acids. The mouse tryptophan hydroxylase locus, termed Tph, was mapped by Southern blot analysis of somatic cell hybrids and by an interspecific backcross to a position in the proximal half of chromosome 7. Because TPH has been mapped to human chromosome 11, this assignment further defines regions of homology between these mouse and human chromosomes.
The free-energy minimization method is applied to systems containing liquid solutions with a solvent of unit activity, a gas phase of constant volume, and solid phases of invariant stoichiometry. The equations derived are employed in a computer program, SOLGASWATER.
Neurogenesis of the locus coeruleus (LC), substantia nigra (SN) and raphe nuclei (RN) was analyzed in autoradiograms prepared from postnatal rhesus monkeys that had been exposed to a pulse of [³H]thymidine on selected embryonic (E) days of the 165-day gestational period. Heavily labeled monoamine (MA) neurons were present only in monkeys exposed to the isotope between E27 and E36 with the peak around E30–E33. The majority of neurons generated on E30 eventually become situated in the medial part of the LC, whereas most cells of the lateral portion are generated on E32 and E33, indicating the existence of a mediolateral spatiotemporal gradient. Proliferation of neurons destined for the compact portion of the LC peaks around E32, whereas production of subcoeruleus cells proceeds more evenly throughout the E30–E33 period. SN neurons are generated between E36 and E43, with peak labeling around E38–E40, and no appreciable spatiotemporal gradients. Neurons of the ventral tegmental area are also generated between E38 and E43. Neurogenesis of the RN occurs between E28 and E43 with only a moderate rostrocaudal spatiotemporal gradient. Neurons of raphe dorsalis and centralis superior undergo final mitosis between E28 and E35, with the peak on E30, whereas cells of raphe magnus, pontis, obscurus and pallidus are produced between E35 and E43, with the peak between E38 and E40. In general, MA neurons that project to different targets may be produced simultaneously within each nucleus irrespective of any spatiotemporal gradients.
1. Attempts and apparently successful procedures to obtain reasonable quantities of electrophoretically homogenous mammalian brain-derived tryptophan hydroxylase, (TPH), have been described, starting in the early 1970s. This work has been carried out with the primary objective to obtain specific antisera to this enzyme to map out serotonergic pathways in the nervous system. 2. By using a multitude of techniques, antisera have indeed been fabricated and employed. However, it is doubtful if pure, native TPH has ever been produced. Indeed, there is strong evidence that more than one isoform of TPH exists in the rat brain. Thus, these antisera are probably directed against TPH-derived polypeptides and not the holoenzyme(s). 3. The difficulty in the purification of TPH lies not only in its subjectivity to proteolysis, but more importantly in its probable capacity to produce superoxide leading to hydrogen perioxide formation. This, in turn, may undergo Fenton chemistry with iron at the active site of the protein to produce hydroxyl radicals that directly attack and destroy the enzyme molecule. Evidence for such a mechanism is presented together with possible protocols that might be used to produce pure stable holo TPH(s). 4. It is hypothesized that similar oxidative events may take place in vivo under certain conditions leading to pathological results. Strategies to block these events are suggested.
p-Chlorophenylalanine (pCPA), a potent inhibitor of serotonin synthesis, specifically depletes brain serotonin in a dose-dependent manner. The resulting impairment of serotonergic inhibitory control of the brain is considered responsible for the consequent muricidal aggression that arises in pCPA-treated rats independent of their strain-dependent genetic predisposition and sex propensity to display this behavior. Judging from the data obtained, the minimal impairment of serotonergic inhibitory control required to induce consistent muricidal aggression in rats of both sexes of the strains considered, corresponds to a brain serotonin depletion of about 55-60%.
Classic tetrahydrobiopterin (BH4) deficiencies are characterized by hyperphenylalaninemia and deficiency of monoamine neurotransmitters. In this article, we report two patients with progressive psychomotor retardation, dystonia, severe dopamine and serotonin deficiencies (low levels of 5-hydroxyindoleacetic and homovanillic acids), and abnormal pterin pattern (high levels of biopterin and dihydrobiopterin) in cerebrospinal fluid. Furthermore, they presented with normal urinary pterins and without hyperphenylalaninemia. Investigation of skin fibroblasts revealed inactive sepiapterin reductase (SR), the enzyme catalyzing the final two-step reaction in the biosynthesis of BH4. Mutations in the SPR gene were detected in both patients and their family members. One patient was homozygous for a TC→CT dinucleotide exchange, predicting a truncated SR (Q119X). The other patient was a compound heterozygote for a genomic 5-bp deletion (1397–1401delAGAAC) resulting in abolished SPR-gene expression and an A→G transition leading to an R150G amino acid substitution and to inactive SR as confirmed by recombinant expression. The absence of hyperphenylalaninemia and the presence of normal urinary pterin metabolites and of normal SR-like activity in red blood cells may be explained by alternative pathways for the final two-step reaction of BH4 biosynthesis in peripheral and neuronal tissues. We propose that, for the biosynthesis of BH4 in peripheral tissues, SR activity may be substituted by aldose reductase (AR), carbonyl reductase (CR), and dihydrofolate reductase, whereas, in the brain, only AR and CR are fully present. Thus, autosomal recessive SR deficiency leads to BH4 and to neurotransmitter deficiencies without hyperphenylalaninemia and may not be detected by neonatal screening for phenylketonuria.
The enzymatic conversion of phenylalanine to tyrosine proceeds according to equations 1 and 2 [1].
The hypothesis that serotonin (5-HT) influences the onset of differentiation (cessation of division) of prospective 5-HT target neurons during embryogenesis was tested by administering the 5-HT depleting drug p-chlorophenylalanine (pCPA) to pregnant rats and dating the time of last cell division for fetal neurons using long survival 3H-thymidine autoradiography. PCPA specifically retarded the onset of neuronal differentiation in brain regions known to contain 5-HT terminals or to have a high 5-HT content in the adult (5-HT target cells). Conversely, the 'stress' of daily vehicle injections accelerated onset of differentiation in these regions, possibly due to stimulation of 5-HT synthesis by glucocorticoids. These data support the model of 5-HT as a humoral signal for the differentiation of cells later recognized by 5-HT neurons as appropriate targets for synaptogenesis.
The preincubation of tryptophan hydroxylase extracted from various areas of the central nervous system of the rat with 30 mM dithiothreitol and 50 μM ferrous ammonium sulfate under nitrogen atmosphere resulted in a persistent increase of its activity. Studies on the enzyme characteristics indicated that this activation was associated with a doubling in itsV
max and a shift (from 7.6 to 7.2) of the optimal pH for its activity. In contrast, the molecular weight and the apparent affinities of tryptophan hydroxylase for its pterin cofactor and for tryptophan were not significantly altered by the preincubation with dithiothreitol and ferrous ammonium sulfate. Since this treatment did not prevent the stimulatory effects of various compounds (phosphatidylserine, ATP and Mg2+, Ca2+) on tryptophan hydroxylase activity, this might be a good procedure to activate this enzyme with only minor changes in its regulatory properties.
Bovine pineal tryptophan hydroxylase is a pterin-dependent aromatic amino acid monooxygenase with a broad substrate specificity and with low Km values for the amino acid substrates, L-tryptophan and L-phenylalanine. p-Chlorophenylalanine, an inhibitor of the tryptophan hydroxylation in the brain, also serves as a good substrate of the bovine pineal enzyme. The full activity of this enzyme is detected in vitro only after preincubation with dithiothreitol under reductive conditions. The enzyme is profoundly and more or less specifically inhibited by L-5HTP, the product of tryptophan hydroxylation, suggesting a possible regulatory role of this hydroxylated amino acid. The Km of the enzyme for tetrahydrobiopterin, a presumed natural cofactor, is significantly higher than the expected tissue concentration if the cofactor is assumed to be uniformly distributed in the tissue. These properties of bovine pineal tryptophan hydroxylase are distinguishable from those of the hydroxylase in the brain indicating that the hydroxylation of tryptophan in the brain leading to the synthesis of serotonin and the reaction in the pineal gland leading to the formation of melatonin are catalyzed by different tryptophan hydroxylases and are probably under separate regulatory mechanisms.
A 4a-carbinolamine intermediate is generated stoichiometrically during the tetrahydrobiopterin-dependent phenylalanine hydroxylation reaction catalyzed by phenylalanine hydroxylase. The dehydration of the carbinolamine is catalyzed by the enzyme, 4a-hydroxytetrahydropterin dehydratase. We have now examined the distribution of the dehydratase activity in various rat tissues by activity measurements and by immunoblot analysis to explore the possibility that the dehydratase may also play a role in tyrosine and tryptophan hydroxylation. The only two tissues that express relatively high dehydratase activity are liver and kidney, which are also the only two tissues that express phenylalanine hydroxylase activity. The dehydratase activity was generally very low in those tissues which contain high levels of tyrosine and tryptophan hydroxylase activity, except for the pineal gland. These results suggest that the dehydratase may not play an important role in the regulation of the synthesis of those neurotransmitters which are derived from the hydroxylated aromatic amino acids.
Tryptophan hydroxylase (TPH) from central serotonergic neurons in the dorsal raphe nucleus (DRN) and that from the endocrine pineal gland (PG) have been shown to exhibit difference biochemical characteristics. We further report here that the isoelectric point determined by chromatofocusing differs between TPH from the rat brainstem and PG. In addition, the levels of TPH mRNA are much greater in the PG than the DRN despite a higher enzymatic activity in the DRN. These data raise the question as to whether different forms of TPH may exist in the DRN and the PG. To address this question, we amplified TPH cDNAs by the polymerase chain reaction (PCR) using poly(A)+ RNA purified from both tissues. Several combinations of oligonucleotide primers encompassing different regions of the published coding sequence of rat pineal TPH were employed for this purpose. Subsequent analysis by gel electrophoresis and Southern blotting of PCR products indicated that DNA fragments of identical length were amplified from both sources. Furthermore, the nucleotide sequences of three independent subclones containing the putative full-length coding region of DRN TPH were determined and found to be identical to that of PG. In situ hybridization using the amplified cDNA as a probe demonstrated specific labeling within the DRN of the rat brain. These data support the hypothesis that tissue-specific differences in TPH characteristics result from differential post-translational events and clearly indicate that a TPH mRNA transcript identical in coding sequence to the PG form is expressed in the DRN.
Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.
Eight weeks of latent iron deficiency in weaned rats maintained on an experimental low iron content diet (18–20 mg/kg) did not significantly alter the packed cell volume and hemoglobin concentration; however, the hepatic and brain nonheme iron contents decreased by 66% and 21% (p < 0.001), respectively. The tryptophan concentration decreased by 31% and 34% in liver and brain, respectively, in rats on experimental diet (p < 0.01). The brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid contents were reduced by 21% and 23% (p < 0.01 and p < 0.02), respectively. However, in the brain, weight, protein, DNA, and the activities of monoamine oxidase, aldehyde dehydrogenase, and liver tryptophan oxygenase were found to remain unaltered.
When rehabilitated with a diet containing 390 mg/kg iron, rats previously maintained on the experimental diet for 2 weeks showed partial recovery in tryptophan levels both in liver and brain. However, brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid levels remained unaltered. The hepatic iron content improved without any change in brain iron content. The latent iron deficiency produced significant alterations in the metabolism of 5-hydroxytryptamine and brain iron content that could not be recovered 2 weeks after the iron rehabilitation.
The activity of tryptophan hydroxylase (EC 1.14.16.4) from rat brain was significantly decreased 1 h following a single systemic injection of 3,4-methylenedioxymethamphetamine (MDMA) when assessed ex vivo by radioenzymatic assay or in vivo by the quantitation of 5-hydroxytryptophan accumulation following central L-aromatic amino acid decarboxylase inhibition. Recovery of enzymatic activity in vivo, which occurred within 24 h of low-dose MDMA treatment, appeared not to involve synthesis of new enzyme protein, because the return of enzymatic activity was not prevented by prior cycloheximide. Acutely MDMA-depressed cortical tryptophan hydroxylase activity could be completely restored in vitro by a prolonged (20-24 h) anaerobic incubation in the presence of dithiothreitol and Fe2+ at 25 degrees C; partial reconstitution occurred when 2-mercapto-ethanol was substituted for dithiothreitol. Cortical tryptophan hydroxylase acutely inactivated by methamphetamine or p-chloroamphetamine could be similarly reactivated. MDMA-inactivated cortical tryptophan hydroxylase derived from rats killed later than 3 days after drug treatment could not be significantly reactivated under the conditions described above, indicating the development of irreversible enzymatic damage. Kinetic analysis of enzyme reactivation revealed an approximate doubling of enzyme Vmax with no change in enzyme affinity for either substrate, tryptophan, or pterin cofactor. These studies suggest that MDMA and its congeners inactivate central tryptophan hydroxylase by inducing oxidation of key enzyme sulfhydryl groups. The reactivation capacity of drug-inactivated enzyme at various times after MDMA treatment may provide a means of assessing the development of MDMA-induced neurotoxicity.
The activity of rat hippocampal tryptophan hydroxylase was reduced from 30-60% 3 h after the administration of a 10-15 mg/kg dose of either fenfluramine, methamphetamine or 3,4-methylenedioxymethamphetamine (MDMA). Tryptophan hydroxylase inactivated by these drug treatments could be reconstituted by a prolonged anaerobic incubation in the presence of 5 mM dithiothreitol and 50 microM Fe2+. Drug-inactivated enzyme obtained from rats killed 18 h after multiple doses of either D(+)- or L(-)-MDMA could not be similarly restored. These observations suggest that the rapid decrease in central tryptophan hydroxylase activity induced by amphetamine analogs results from the reversible oxidation of a sulfhydryl site(s) within the enzyme molecule, whereas the irreversible decrease in enzymatic activity measured 18 h after multiple-dose MDMA treatment may reflect serotonergic toxicity.
Evidence is presented indicating that a cAMP-dependent mechanism activates tryptophan hydroxylase (TrpH), the rate-limiting enzyme for serotonin (5-HT) biosynthesis. Forskolin, a selective activator of adenylate cyclase, stimulated 5-HT formation in synaptoneurosome preparations of rat striatum, substantia nigra, hypothalamus, and amygdala. Further studies of striatum revealed that the forskolin-induced activation of serotonin synthesis is readily reversible. Also, it may be self-limited by a mechanism of desensitization, since after an initial exposure to forskolin followed by removal, a re-exposure of synaptoneurosomes to forskolin was no longer stimulatory. In contrast to these results for 5-HT synthesis, forskolin-induced stimulation of dopamine synthesis persisted following removal of forskolin; hence the response was not rapidly reversible or desensitized. In soluble extracts of striatum, 8-thiomethyl-cyclic AMP enhanced TrpH activity, supporting a direct role of cyclic AMP and cyclic AMP-dependent protein kinase in regulating TrpH. In agreement with previous reports, 8-thiomethyl cyclic AMP also stimulated tyrosine hydroxylase activity in soluble striatal extracts. We conclude that cyclic AMP is an important regulator of TrpH, in addition to its known effects on tyrosine hydroxylase.
The psychotomimetic agent, methylenedioxymethamphetamine, produced a rapid, persistent and dose-dependent reduction in cortical tryptophan hydroxylase activity when administered acutely to rats. This effect did not occur in vitro and did not require N-demethylase activity in the whole animal. Kinetic analysis revealed the loss of enzyme activity to be due to an alteration in Vmax with no change in the affinity of the enzyme for either its cofactor or substrate. Coadministration of the serotonin (5-HT) uptake inhibitor, citalopram, only partially antagonized the loss of tryptophan hydroxylase activity 3 hr after methylenedioxymethamphetamine, but completely prevented the loss of cortical 5-HT. Recovery of enzyme activity did occur by 1 week if the neurotoxic effect of methylenedioxymethamphetamine was blocked by fluoxetine. The effect of methylenedioxymethamphetamine on 5-HT synthesis was not affected by pretreatment with alpha-methyl-p-tyrosine, reserpine or yohimbine. Ketanserine and methiothepin, 5-HT receptor antagonists, did partially block the methylenedioxymethamphetamine-induced loss of tryptophan hydroxylase activity, suggesting a possible role for neurotransmitter release in the acute effects of the drug on enzyme activity.
A new procedure that permits large-scale purification of tyrosine 3-monooxygenase (tyrosine hydroxylase) (L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the cytosolic fraction of bovine adrenal medulla is described. The homogenous enzyme revealed a subunit Mr of 60,000 and a specific activity of 425 nmol.min-1.mg-1. The N-terminal amino-acid sequence (27 residues) revealed 89% homology with the human pheochromocytoma enzyme as deduced from its cDNA sequence. The pure enzyme contained 0.66 +/- 0.09 mol iron, 0.13 mol zinc and 0.62 +/- 0.04 mol phosphate per mol subunit of Mr = 60,000. A broad light absorption band with its maximum around 700 nm (epsilon 700 nm = 1.3 (mM monomer)-1.cm-1) explains its blue-green color. EPR spectra at 3.6 K revealed high-spin Fe(III) (S = 5/2) in an environment of nearly axial symmetry (g values at 7.2-6.7, 4.7-5.3 and 1.9-2.0). A close correlation was observed between the absorbance at 700 nm and the intensity of the axial type of EPR spectrum. The absorption peak at 700 nm is compatible with a ligand-to-iron charge-transfer transition as a result of catecholate coordination to the iron. Physicochemical studies suggest that the enzyme does not undergo such major substrate- or cofactor-induced conformational changes as have been reported for the related enzyme, phenylalanine hydroxylase.
Drug treatments which influence brain serotonergic systems were administered to lactating female mice during the early postpartum period, and their effects on aggressive behavior, locomotor activity and brain monoamines were examined. P-chlorophenylalanine (200 and 400 mg/kg) and 5-hydroxytryptophan (100 mg/kg) inhibited fighting behavior of postpartum mice toward unfamiliar male intruder mice. These drug-treated postpartum females showed increased latencies to attack male intruders and also reduced frequencies of attack. In addition, postpartum mice treated with the serotonin receptor antagonists, mianserin (2 and 4 mg/kg), methysergide (4 mg/kg) and methiothepin (0.25 and 0.5 mg/kg), displayed significantly less aggressive behavior than control mice, as measured by reduced number of attacks. Whole brain monoamine and monoamine metabolite levels were measured after drug treatments. The behavioral results are discussed in terms of drug-induced changes in brain chemistry and indicate a possible role for serotonin in the mediation of maternal aggressive behavior of mice.
Two full-length cDNA clones that encode functional rat tryptophan hydroxylase (EC 1.14.16.4), the key enzyme in serotonin synthesis, have been isolated from a rat pineal gland library. These two clones correspond to the 1.8- and 4-kilobase mRNA species, respectively. They contain the same coding sequence corresponding to a 51,010-dalton protein and differ in the length of their 3' untranslated regions.