Review article: the many potential roles of intestinal serotonin (5-hydroxytryptamine, 5-HT) signalling in inflammatory bowel disease.

BACKGROUND: Serotonin (5-hydroxytryptamine, 5-HT) is an important mediator of every major gut-related function. Recent investigations also suggest that 5-HT can influence the development and severity of inflammation within the gut, particularly in the setting of inflammatory bowel disease (IBD). AIM: To review the roles that the intestinal serotonin signalling system plays in gut function, with a specific focus on IBD. METHODS: We reviewed manuscripts from 1952 to 2017 that investigated and discussed roles for 5-HT signalling in gastrointestinal function and IBD, as well as the influence of inflammation on 5-HT signalling elements within the gut. RESULTS: Inflammation appears to affect every major element of intestinal 5-HT signalling, including 5-HT synthesis, release, receptor expression and reuptake capacity. Importantly, many studies (most utilising animal models) also demonstrate that modulation of selective serotonergic receptors (via agonism of 5-HT4 R and antagonism of 5-HT3 R) or 5-HT signal termination (via serotonin reuptake inhibitors) can alter the likelihood and severity of intestinal inflammation and/or its complicating symptoms. However, there are few human studies that have studied these relationships in a targeted manner. CONCLUSIONS: Insights discussed in this review have strong potential to lead to new diagnostic and therapeutic tools to improve the management of IBD and other related disorders. Specifically, strategies that focus on modifying the activity of selective serotonin receptors and reuptake transporters in the gut could be effective for controlling disease activity and/or its associated symptoms. Further studies in humans are required, however, to more completely understand the pathophysiological mechanisms underlying the roles of 5-HT in this setting.

Gender-specific regulation of tyrosine hydroxylase in thymocyte differentiation antigen-1 knockout mice.

Thymocyte differentiation antigen-1 (Thy-1) is a cell surface glycoprotein found on T cells and neurons and is involved in cell-to-cell interactions. In addition, Thy-1 knockouts (KO) are a potential mouse model of restless legs syndrome (RLS) based on clinical observations and the role of dopamine in the disease. In this study, we analyzed the activity and quantity of tyrosine hydroxylase (TH; the rate-limiting enzyme in dopamine production) and determined phosphorylation levels for the enzyme phosphoserine-40 (pSer-40). There was no significant difference in the total TH activity and pSer-40 TH levels between Thy-1 KO and control groups; however, TH specific activity was significantly lower (by 26%) in Thy-1 KO mice. This difference is due in part to increased TH protein levels in this group (increased by 29%). When analyzed by gender, Thy-1 KO female mouse striata contained less TH specific activity compared with control females (decreased by 41%) and male control or Thy-1 KO animals (decreased by 30%). TH specific activity and pSer-40 TH levels in male Thy-1 KO and control displayed no differences. However, pSer-40 TH was significantly higher in control females (38%) compared with control or Thy-1 KO males. The Thy-1 KO females exhibited significantly lower (28%) pSer-40 TH (normalized to GAPDH or TH) than control females. Indeed, the Thy-1 KO females had 50% of the pSer-40 TH found in controls. Our results suggest a gender effect on TH specific activity, TH protein levels, and serine-40 phosphorylation of TH in Thy-1 KO female mice.

A threshold neurotoxic amphetamine exposure inhibits parietal cortex expression of synaptic plasticity-related genes.

Compulsive drug abuse has been conceptualized as a behavioral state where behavioral stimuli override normal decision making. Clinical studies of methamphetamine users have detailed decision making changes and imaging studies have found altered metabolism and activation in the parietal cortex. To examine the molecular effects of amphetamine (AMPH) on the parietal cortex, gene expression responses to amphetamine challenge (7.5 mg/kg) were examined in the parietal cortex of rats pretreated for nine days with either saline, non-neurotoxic amphetamine, or neurotoxic AMPH dosing regimens. The neurotoxic AMPH exposure [three doses of 7.5 mg/kg/day AMPH (6 h between doses), for nine days] produced histological signs of neurotoxicity in the parietal cortex while a non-neurotoxic dosing regimen (2.0 mg/kg/day x 3) did not. Neurotoxic AMPH pretreatment resulted in significantly diminished AMPH challenge-induced mRNA increases of activity-regulated cytoskeletal protein (ARC), nerve growth-factor inducible protein A (NGFI-A), and nerve growth-factor inducible protein B (NGFI-B) in the parietal cortex while neither saline pretreatment nor non-neurotoxic AMPH pretreatment did. This effect was specific to these genes as tissue plasminogen activator (t-PA), neuropeptide Y (NPY) and c-jun expression in response to AMPH challenge was unaltered or enhanced by amphetamine pretreatments. In the striatum, there were no differences between saline, neurotoxic AMPH, and non-neurotoxic AMPH pretreatments on ARC, NGFI-A or NGFI-B expression elicited by the AMPH challenge. These data indicate that the responsiveness of synaptic plasticity-related genes is sensitive to disruption specifically in the parietal cortex by threshold neurotoxic AMPH exposures.

Cocaine-responsive gene expression changes in rat hippocampus.

Chronic cocaine use is known to elicit changes in the pattern of gene expression within the brain. The hippocampus plays a critical role in learning and memory and may also play a role in mediating behaviors associated with cocaine abuse. To profile the gene expression response of the hippocampus to chronic cocaine treatment, cDNA hybridization arrays were used to illuminate cocaine-regulated genes in rats treated non-contingently with a binge model of cocaine (45 mg/kg/day, i.p.) for 14 days. Validation of mRNA changes illuminated by hybridization array analysis was accomplished by measuring immunoreactive protein (via specific immunoblots). The induction of protein kinase Calpha, potassium channel 1.1, and metabotropic glutamate receptor 5 seen by hybridization arrays was confirmed at the level of protein. Immunoblot screening of previously described cocaine-responsive genes demonstrated increased levels of protein tyrosine kinase 2, beta-catenin, and protein kinase Cepsilon. While some of these changes exist in previously described cocaine-responsive models, others are novel to any model of cocaine use. The inductions of potassium channel 1.1, protein tyrosine kinase 2 and metabotropic glutamate receptor 5 are novel findings to hippocampal cocaine-responsive gene expression. These proteins have been shown to subserve learning and memory and/or long-term potentiation functions within the hippocampus. Additionally, these genes are known to interact with one another, forming a more complex pattern of gene expression changes. The findings suggest altered expression of genes with a number of different functions in the rat hippocampus after a 'binge' style of non-contingent cocaine administration. These changes in gene expression may play roles in neuronal plasticity and the behavioral phenomena associated with cocaine abuse.

Examination of a CYP2E1 repeat polymorphism in a monkey model of alcohol abuse.

BACKGROUND: Cytochrome P-450 2E1 (CYP2E1) is involved in alcohol metabolism, and the expression of this enzyme displays wide phenotypic variability in humans. It has been proposed that some of this variability in expression may be a consequence of the size of a repeat polymorphism in the 5" regulatory region of the gene and that the polymorphism may segregate with alcoholism. This study examined whether the repeat polymorphism exists in macaque monkeys and whether it associates with excessive alcohol consumption in this animal model. METHODS: Ten outbred cynomolgus monkeys (Macaca fascicularis) that displayed a voluntary alcohol consumption ranging from 1.0 to 3.6 g/kg/day were genotyped for a CYP2E1 repeat polymorphism. This polymorphism has been documented in the region from -2519 base pair (bp) to -1953 bp of the human CYP2E1 gene 5" distal promoter. RESULTS: Individual polymerase chain reaction amplification of genomic DNA from each of the 10 monkey samples revealed a single band of approximately 400 bp in the region corresponding to the human CYP2E1 polymorphism. Polymerase chain reaction amplicons from the 10 individuals were sequenced, and each one generated a 370 bp sequence that is 90% identical to the human gene sequence. However, unlike human alleles that contain five to eight repeats, the cynomolgus monkey is homozygous for a single copy of the repeat most closely resembling repeat 8 (88% identical) in the human gene. CONCLUSIONS: These data demonstrate that the CYP2E1 distal promoter region in monkeys is very similar to the human sequence yet lacks the extensive repeated DNA found in humans. This includes the rare repeats 3 and 4, which have been postulated to play a role in transcription regulation and to associate with alcohol abuse liability in humans. These data suggest that the CYP2E1 polymorphism arose late in evolution and that the regulation of the gene by this genetic region is not associated with a heavy alcohol drinking phenotype in the cynomolgus monkey.

Chronic cocaine-mediated changes in non-human primate nucleus accumbens gene expression.

Chronic cocaine use elicits changes in the pattern of gene expression within reinforcement-related, dopaminergic regions. cDNA hybridization arrays were used to illuminate cocaine-regulated genes in the nucleus accumbens (NAcc) of non-human primates (Macaca fascicularis; cynomolgus macaque), treated daily with escalating doses of cocaine over one year. Changes seen in mRNA levels by hybridization array analysis were confirmed at the level of protein (via specific immunoblots). Significantly up-regulated genes included: protein kinase A alpha catalytic subunit (PKA(calpha)); cell adhesion tyrosine kinase beta (PYK2); mitogen activated protein kinase kinase 1 (MEK1); and beta-catenin. While some of these changes exist in previously described cocaine-responsive models, others are novel to any model of cocaine use. All of these adaptive responses coexist within a signaling scheme that could account for known inductions of genes(e.g. fos and jun proteins, and cyclic AMP response element binding protein) previously shown to be relevant to cocaine's behavioral actions. The complete data set from this experiment has been posted to the newly created Drug and Alcohol Abuse Array Data Consortium (http://www.arraydata.org) for mining by the general research community.

A cocaine analog, 2beta-propanoyl-3beta-(4-tolyl)-tropane (PTT), reduces tyrosine hydroxylase in the mesolimbic dopamine pathway.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Previously published results have established that chronic cocaine administration (30-45 mg/kg per day, 10-14 days) resulted in an upregulation of TH gene expression in dopaminergic pathways of rats. The present studies tested the effects of a tropane analog, PTT (2beta-propanoyl-3beta-(4-tolyl)-tropane), on TH expression. This drug has similar actions to cocaine, but possesses markedly different pharmacokinetics (20 times more potent at binding the dopamine transporter, markedly increased metabolic stability, and 10-20 times more potent in behavioral measures). Moreover, PTT demonstrates an increased selectivity for the dopamine (DA) and norepinephrine (NE) transporters compared with cocaine. In direct contrast to the previously reported effects of cocaine, 10 days of PTT administration (3.0 mg/kg per day, i.p.) produced a uniform downregulation of TH protein and activity gene expression. TH activity and immunoreactive protein where decreased by 54 and 69%, respectively in the nucleus accumbens. Within the ventral tegmental area, TH activity and protein were decreased by 33 and 19%, respectively. The underlying mechanisms for these fundamental differences are unclear, but likely reflect varying and selective affinities and lengths of occupancy at biogenic amine transporters.

Fundamentals of DNA hybridization arrays for gene expression analysis.

DNA hybridization arrays [also known as macroarrays, microarrays and/or high-density oligonucleotide arrays (Gene Chips)] bring gene expression analysis to a genomic scale by permitting investigators to simultaneously examine changes in the expression of literally thousands of genes. For hybridization arrays, the general approach is to immobilize gene-specific sequences (probes) on a solid state matrix (nylon membranes, glass microscope slides, silicon/ceramic chips). These sequences are then queried with labeled copies of nucleic acids from biological samples (targets). The underlying theory is that the greater the expression of a gene, the greater the amount of labeled target, and hence, the greater output signal. In spite of the simplicity of the experimental design, there are at least four different platforms and several different approaches to processing and labeling the biological samples. Moreover, investigators must also determine whether they will utilize commercially available arrays or generate their own. This review will cover the status of the hybridization array field with an eye toward underlying principles and available technologies. Future developments and technological trends will also be evaluated.

Identification of substrate orienting and phosphorylation sites within tryptophan hydroxylase using homology-based molecular modeling.

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in the biosynthesis of serotonin. The inherent instability of TPH has prevented a crystallographic structure from being resolved. For this reason, multiple sequence alignment-based molecular modeling was utilized to generate a full-length model of human TPH. Previously determined crystal coordinates of two highly homologous proteins, phenylalanine hydroxylase and tyrosine hydroxylase, were used as templates. Analysis of the model aided rational mutagenesis studies to further dissect the regulation and catalysis of TPH. Using rational site-directed mutagenesis, it was determined that Tyr235 (Y235), within the active site of TPH, appears to be involved as a tryptophan substrate orienting residue. The mutants Y235A and Y235L displayed reduced specific activity compared to wild-type TPH ( approximately 5 % residual activity). The K(m) of tryptophan for the Y235A (564 microM) and Y235L (96 microM) mutant was significantly increased compared to wild-type TPH (42 microM). In addition, kinetic analyses were performed on wild-type TPH and a deletion construct that lacks the amino terminal autoregulatory sequence (TPH NDelta15). This sequence in phenylalanine hydroxylase (residues 19 to 33) has previously been proposed to act as a steric regulator of substrate accessibility to the active site. Changes in the steady-state kinetics for tetrahydrobiopterin (BH(4)) and tryptophan for TPH NDelta15 were not observed. Finally, it was demonstrated that both Ser58 and Ser260 are substrates for Ca(2+)/calmodulin-dependent protein kinase II. Additional analysis of this model will aid in deciphering the regulation and substrate specificity of TPH, as well as providing a basis to understand as yet to be identified polymorphisms.

Intersubunit binding domains within tyrosine hydroxylase and tryptophan hydroxylase.

Tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT) belongs to the aromatic amino acid hydroxylase superfamily, which includes phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH). The crystal structures for both PAH and TH have been reported, but a crystallographic model of TPH remains elusive. For this reason, we have utilized the information presented in the TH crystal structure in combination with primary sequence alignments to design point mutations in potential structural domains of the TPH protein. Mutation of a TH salt bridge (K170E) was sufficient to alter enzyme macromolecular assembly. We found that the disruption of the cognate intersubunit dimerization salt bridge (K111-E223) in TPH, however, did not affect the macromolecular assembly of TPH. Enzyme peaks representing only tetramers were observed with size exclusion chromatography. By contrast, a single-point mutation within the tetramerization domain of TPH (L435A) was sufficient to disrupt the normal homotetrameric assembly of TPH. These studies indicate that, although the proposed salt bridge dimerization interface of TH is conserved in TPH, this hypothetical TPH intersubunit binding domain, K111-E223, is not required for the proper macromolecular assembly of the protein. However, leucine 435 within the tetramerization domain is necessary for the proper macromolecular assembly of TPH.

p-ethynylphenylalanine: a potent inhibitor of tryptophan hydroxylase.

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in serotonin biosynthesis. The enzyme activity is dependent on molecular oxygen, a tetrahydropterin cosubstrate, and ferrous iron. The present study demonstrates that TPH is inhibited by a novel compound, p-ethynylphenylalanine (pEPA), produced by the Heck reaction of trimethylsilylacetylene with N-tertbutyloxycarbonyl-4-iodo-L-phenylalanine methyl ester. pEPA is a more potent and specific inhibitor of TPH than p-chlorophenylalanine (pCPA). In the present study, pEPA was demonstrated to inhibit competitively and reversibly TPH in vitro (Ki = 32.6 +/- 6.2 microM vs. tryptophan). pEPA displayed little inhibitory activity toward tyrosine hydroxylase (EC 1.14.16.2), the initial and rate-limiting enzyme for catecholamine biosynthesis, and no inhibition of phenylalanine hydroxylase or tyrosinase. In addition, pEPA was a poor ligand for the serotonin transporter and several serotonin receptors. Administration of pEPA (30 mg/kg) to rats produced a 95 +/- 5% decrease in TPH activity in brain homogenates and a concomitant decrease in serotonin and 5-hydroxyindole-3-acetic acid levels (85%) at 24 h after injection. In contrast, pCPA produced a similar effect (87 +/- 5% decrease in TPH activity) only at 10 times the concentration (300 mg/kg). These results suggest that pEPA is a selective, reversible, and potent inhibitor of TPH both in vitro and in vivo. The potential for pEPA to inhibit selectively and reversibly the biosynthesis of serotonin may contribute to the characterization of the role of serotonin in behavioral and physiological activities.

Dopamine toxicity in neuroblastoma cells: role of glutathione depletion by L-BSO and apoptosis.

Dopamine (DA), while an essential neurotransmitter, is also a known neurotoxin that potentially plays an etiologic role in several neurodegenerative diseases. DA metabolism and oxidation readily produce reactive oxygen species (ROS) and DA can also be oxidized to a reactive quinone via spontaneous, enzyme-catalyzed or metal-enhanced reactions. A number of these reactions are cytotoxic, yet the precise mechanisms by which DA leads to cell death remain unknown. In this study, the neuroblastoma cell line, SK-N-SH, was utilized to examine DA toxicity under varying oxidant states. Cells pretreated with the glutathione (GSH)-depleting compound, L-buthionine sulfoximine (L-BSO), exhibited enhanced sensitivity to DA compared to controls (non-GSH-depleted cells). Furthermore, in cells pretreated with L-BSO, the addition of ascorbate (250 microM) afforded significant protection against DA-induced toxicity, while pyruvate (500 microM) had no protective effect. To further characterize the possibility that DA is associated with oxidative stress, additional studies were carried out with manganese (30 microM) as a pro-oxidant. Manganese and DA (200 microM), although not cytotoxic when individually administered to SK-N-SH cells, had a synergistic action on cytotoxicity. Finally, morphological and molecular markers of programmed cell death (apoptosis) were observed in cells treated with DA and L-BSO. These markers included membrane blebbing and internucleosomal DNA fragmentation. These results suggest that DA toxicity is tightly linked to intracellular oxidant/antioxidant levels, and that environmental factors, such as excessive Mn exposure, may modulate cellular sensitivity to DA.

Regulation of rat dopamine transporter mRNA and protein by chronic cocaine administration.

This study describes a direct comparison of dopamine transporter (DAT) mRNA and protein, as well as its binding sites, in tissue from the same animals after chronic cocaine administration. Rats were treated twice daily with 25 mg/kg cocaine or with saline. After 8 days of cocaine administration, changes in DAT mRNA levels in the substantia nigra pars compacta and ventral tegmental area were measured by in situ hybridization, and DAT protein in the striatum was quantified by immunoblotting. Whereas chronic cocaine treatment significantly reduced levels of DAT mRNA in the substantia nigra pars compacta and ventral tegmental area as compared with vehicle-treated controls, cocaine treatment did not alter DAT protein levels in the striatum. Furthermore, the density of DAT binding sites was also measured in the striatum by quantitative autoradiography using two DAT radioligands, 33-(4-[125I]iodophenyl)tropane-2-carboxylic acid methyl ester ([125I]RTI-55) and [3H]propanoyl-3beta-(4-tolyl)tropane ([3H]PTT). Similar to the results of immunoblotting of DAT protein, [1251]RTI-55 and [3H]PTT binding site levels also remained unaltered. These results indicate a dissociation in the regulation of DAT mRNA and its protein levels as a result of cocaine administration in rats. This study also indicates that the DAT ligands [3H]PTT and [125I]RTI-55 provide an accurate assessment of DAT protein levels.

Manganese uptake and distribution in the central nervous system (CNS).

Information about the nature of manganese (Mn)-binding ligands in plasma and serum, and its transport mechanism across the blood-brain barrier (BBB) is sparse. Most studies to date have focused on distribution, excretion, and accumulation of intravenous and intraperitoneal solutions of soluble divalent salts of Mn. Mn is transported in the blood primarily in the divalent oxidation state (Mn2+) and crosses the BBB via specific carriers at a rate far slower than in other tissues. Mn transport across the BBB occurs both in the 2+ and 3+ oxidation state. Within the CNS, Mn accumulates primarily within astrocytes, presumably because the astrocyte-specific enzyme, glutamine synthetase (GS), represents an important regulatory target of Mn. Compared to Mn2+, Mn3+ has a slower elimination rate and therefore, may have a greater tendency to accumulate in tissues. Furthermore, in view of the dependence of Mn accumulation within the CNS on iron (Fe) homeostasis, the oxidation state of Mn may represent a key determinant in the differential distribution, accumulation and secretion profiles of Mn, a fact that has received little attention in experimental biology toxicology. Accordingly, the distribution and membrane transport of Mn emphasizes the importance of: 1) the oxidation state of Mn, as it governs the affinity of Mn to endogenous ligands, and 2) the reaction of Mn3+ with transferrin, the plasma iron-carrying protein. This review will focus on transport kinetics of Mn across the BBB (both in the 2+ and 3+ oxidation state), the putative role of transferrin in the transport of Mn across the BBB, the transport of Mn by astrocytes, as well as the physiological significance of Mn to the function GS.

Cytotoxic and genotoxic potential of dopamine.

A variety of in vitro and in vivo studies demonstrate that dopamine is a toxic molecule that may contribute to neurodegenerative disorders such as Parkinson's disease and ischemia-induced striatal damage. While much attention has focused on the fact that the metabolism of dopamine produces reactive oxygen species (peroxide, superoxide, and hydroxyl radical), growing evidence suggests that the neurotransmitter itself may play a direct role in the neurodegenerative process. Oxidation of the dopamine molecule produces a reactive quinone moiety that is capable of covalently modifying and damaging cellular macromolecules. This quinone formation occurs spontaneously, can be accelerated by metal ions (manganese or iron), and also arises from selected enzyme-catalyzed reactions. Macromolecular damage, combined with increased oxidant stress, may trigger cellular responses that eventually lead to cell death. Reactive quinones have long been known to represent environmental toxicants and, within the context of dopamine metabolism, may also play a role in pathological processes associated with neurodegeneration. The present discussion will review the oxidative metabolism of dopamine and describe experimental evidence suggesting that dopamine quinone may contribute to the cytotoxic and genotoxic potential of this essential neurotransmitter.

Quantitative RT-PCR: pitfalls and potential.

Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field.

Identification of amino-terminal sequences contributing to tryptophan hydroxylase tetramer formation.

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin. In the rabbit, TPH exists as a tetramer of four identical 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Previous studies demonstrated that within the carboxyl-terminus of TPH, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hydrophobic repeat identified within the regulatory domain of TPH (residues 21-41) may also be involved in macromolecular assembly. To test this hypothesis, a series of amino-terminal deletions (Ndelta15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chromatography. The amino-terminal deletion Ndelta15, upstream from the 4,3-hydrophobic repeat, was capable of forming tetramers. However, when a portion of the 4,3-hydrophobic repeat was deleted (Ndelta30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete removal of the 4,3-hydrophobic repeat (Ndelta41) rendered the enzyme incapable of forming tetramers; a monomeric form predominated. In addition, a double-point mutation (V28R-L31R) was created in the hydrophobic region of the enzyme. The introduction of two arginines (R) at positions 28 and 31 respectively, in the helix disrupted the native tetrameric state of TPH. According to size-exclusion chromatography analysis, the double-point mutant (V28R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is information within the amino-terminus that is necessary for tetramer formation of TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus.

Dopamine, in the presence of tyrosinase, covalently modifies and inactivates tyrosine hydroxylase.

Dopamine has been implicated as a potential mediating factor in a variety of neurodegenerative disorders. Dopamine can be oxidized to form a reactive dopamine quinone that can covalently modify cellular macromolecules including protein and DNA. This oxidation can be enhanced through various enzymes including tyrosinase and/or prostaglandin H synthase. One of the potential targets in brain for dopamine quinone damage is tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. The present studies demonstrated that dopamine quinone, the formation of which was enhanced through the activity of the melanin biosynthetic enzyme, tyrosinase, covalently modified and inactivated tyrosine hydroxylase. Dihydroxyphenylalanine (DOPA; the catechol-containing precursor of dopamine) also inactivated tyrosine hydroxylase under these conditions. Catecholamine-mediated inactivation occurred with both purified tyrosine hydroxylase as well as enzyme present in crude pheochromocytoma homogenates. Inactivation was associated with covalent incorporation of radiolabelled dopamine into the enzyme as assessed by immunoprecipitation, size exclusion chromatography, and denaturing sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Furthermore, the covalent modification and inactivation of tyrosine hydroxylase was blocked by antioxidant compounds (dithiothreitol, reduced glutathione, or NADH). In addition to kinetic feedback inhibition and the formation of an inhibitory dopamine/Fe+3 complex, these findings suggest that a third mechanism exists by which dopamine (or DOPA) can inhibit tyrosine hydroxylase, adding further complexity to the regulation of catecholamine biosynthesis.

Advances in the molecular characterization of tryptophan hydroxylase.

The neurotransmitter serotonin has been implicated in numerous physiological functions and pathophysiological disorders. The hydroxylation of the aromatic amino acid tryptophan is rate-limiting in the synthesis of serotonin. Tryptophan hydroxylase (TPH), as the rate-limiting enzyme, determines the concentrations of serotonin in vivo. Relative serotonin concentrations are clearly important in neural transmission, but serotonin has also been reported to function as a local antioxidant. Identification of the mechanisms regulating TPH activity has been hindered by its low levels in tissues and the instability of the enzyme. Several TPH expression systems have been developed to circumvent these problems. In addition, eukaryotic expressions systems are currently being developed and represent a new avenue of research for identifying TPH regulatory mechanisms. Recombinant DNA technology has enabled the synthesis of TPH deletions, chimeras, and point mutations that have served as tools for identifying structural and functional domains within TPH. Notably, the experiments have proven long-held hypotheses that TPH is organized into N-terminal regulatory and C-terminal catalytic domains, that serine-58 is a site for PKA-mediated phosphorylation, and that a C-terminal leucine zipper is involved in formation of the tetrameric holoenzyme. Several new findings have also emerged regarding regulation of TPH activity by posttranslational phosphorylation, kinetic inhibition, and covalent modification. Inhibition of TPH by L-DOPA may have implications for depression in Parkinson's disease (PD) patients. In addition, TPH inactivation by nitric oxide may be involved in amphetamine-induced toxicity. These regulatory concepts, in conjunction with new systems for studying TPH activity, are the focus of this article.