Biosynthesis of N-Acetyldopamine and N-Acetyloctopamine by Schistocerca gregaria Nervous Tissue

N-Acetyltyramine, N-acetyldopamine and N-acetyloctopamine were the major products when either L-[3H]tyrosine or [3H]tyramine were incubated with thoracic ganglia of the desert locust, Schistocerca gregaria. No label was incorporated into L-DOPA under these conditions, although 2-3% of the radioactivity could be recovered in dopamine and octopamine. Addition of the aromatic amino acid decarboxylase inhibitor, 3-hydroxybenzylhydrazine (NSD 1015), prevented the formation of N-acetylcompounds from L-[3H]tyrosine, without resulting in an accumulation of label in L-DOPA. In contrast, incubation of samples of haemolymph with L-[3H]tyrosine resulted in the recovery of 7% of label in L-DOPA, which was increased to 17% in the presence of NSD 1015. These results provide evidence that the initial step in the synthesis of dopamine and octopamine by S. gregaria nervous tissue is the conversion of L-tyrosine to tyramine, which is subsequently metabolised to N-acetyltyramine, N-acetyldopamine or N-acetyloctopamine.     

A comparison of the signalling properties of two tyramine receptors from Drosophila

In invertebrates, the phenolamines, tyramine and octopamine, mediate many functional roles usually associated with the catecholamines, noradrenaline and adrenaline, in vertebrates. The α‐ and β‐adrenergic classes of insect octopamine receptor are better activated by octopamine than tyramine. Similarly, the Tyramine 1 subgroup of receptors (or Octopamine/Tyramine receptors) are better activated by tyramine than octopamine. However, recently, a new Tyramine 2 subgroup of receptors was identified, which appears to be activated highly preferentially by tyramine. We examined immunocytochemically the ability of CG7431, the founding member of this subgroup from Drosophila melanogaster, to be internalized in transfected Chinese hamster ovary (CHO) cells by different agonists. It was only internalized after activation by tyramine. Conversely, the structurally related receptor, CG16766, was internalized by a number of biogenic amines, including octopamine, dopamine, noradrenaline, adrenaline, which also were able to elevate cyclic AMP levels. Studies with synthetic agonists and antagonists confirm that CG16766 has a different pharmacological profile to that of CG7431. Species orthologues of CG16766 were only found in Drosophila species, whereas orthologues of CG7431 could be identified in the genomes of a number of insect species. We propose that CG16766 represents a new group of tyramine receptors, which we have designated the Tyramine 3 receptors.     

Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust

The metabolic transformation of tyrosine (TYR) by the decarboxylase and hydroxylase enzymes was investigated in the central nervous system of the locust, Locusta migratoria. It has been demonstrated that the key amino acids, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan (5HTP) and tyrosine are decarboxylated in all part of central nervous system. DOPA and 5HTP decarboxylase activities show parallel changes in the different ganglia, but the rank order of the activity of TYR decarboxylase is different. Enzyme purification has revealed that the molecular weights of TYR decarboxylase and DOPA/5HTP decarboxylase are 370,000 and 112,000, respectively. The decarboxylation of DOPA by DOPA/5HTP decarboxylase is stimulated, whereas the decarboxylation of DOPA by TYR decarboxylase is inhibited in the presence of the cofactor pyridoxal-5'-phosphate. TYR hydroxylase could not be detected and 3H-TYR is found to be metabolised to tyramine (TA), but not to DOPA. The haemolymph contains a significant concentration of DOPA (120 pmol/100 microl haemolymph), and the ganglia incorporates DOPA from the haemolymph by a high affinity uptake process (K(M)=12 microM and V(max)=24 pmol per ganglion/10 min). Our results suggest that no tyrosine hydroxylase is present in the locust CNS and the DOPA uptake into the ganglia by a high affinity uptake process as well as the DOPA decarboxylase enzyme may be responsible for the regulation of the ganglionic dopamine (DA) level. Two types of decarboxylases exist, one of them decarboxylating DOPA and 5HTP (DOPA/5HTP decarboxylase), other decarboxylating TYR (TYR decarboxylase). The DOPA/5HTP decarboxylase enzyme present in the insect brain may correspond to the 5HTP/DOPA decarboxylase in vertebrate brain, whereas TYR decarboxylase is characteristic only for the insect brain.     

Trace amines differentially regulate adult locomotor activity, cocaine sensitivity, and female fertility in Drosophila melanogaster

The trace biogenic amines tyramine and octopamine are found in the nervous systems of animals ranging in complexity from nematodes to mammals. In insects such as Drosophila melanogaster, the trace amine octopamine is a well-established neuromodulator that mediates a diverse range of physiological processes, but an independent role for tyramine is less clear. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC). We previously reported the identification of two Tdc genes in Drosophila: the peripherally-expressed Tdc1 and the neurally-expressed Tdc2. To further clarify the neural functions of the trace amines in Drosophila, we examined normal and cocaine-induced locomotor activity in flies that lack both neural tyramine and octopamine because of mutation in Tdc2 (Tdc2(RO54)). Tdc2(RO54) flies have dramatically reduced basal locomotor activity levels and are hypersensitive to an initial dose of cocaine. Tdc2-targeted expression of the constitutively active inward rectifying potassium channel Kir2.1 replicates these phenotypes, and Tdc2-driven expression of Tdc1 rescues the phenotypes. However, flies that contain no measurable neural octopamine and an excess of tyramine due to a null mutation in the tyramine beta-hydroxylase gene (TbetaH(nM18)) exhibit normal locomotor activity and cocaine responses in spite of showing female sterility due to loss of octopamine. The ability of elevated levels of neural tyramine in TbetaH(nM18) flies to supplant the role of octopamine in adult locomotor and cocaine-induced behaviors, but not in functions related to female fertility, indicates mechanistic differences in the roles of trace amines in these processes.     

Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility

The trace biogenic amine tyramine is present in the nervous systems of animals ranging in complexity from nematodes to mammals. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC), a member of the aromatic amino acid family, but this enzyme has not been identified in Drosophila or in higher animals. To further clarify the roles of tyramine and its metabolite octopamine, we have cloned two TDC genes from Drosophila melanogaster, dTdc1 and dTdc2. Although both gene products have TDC activity in vivo, dTdc1 is expressed nonneurally, whereas dTdc2 is expressed neurally. Flies with a mutation in dTdc2 lack neural tyramine and octopamine and are female sterile due to egg retention. Although other Drosophila mutants that lack octopamine retain eggs completely within the ovaries, dTdc2 mutants release eggs into the oviducts but are unable to deposit them. This specific sterility phenotype can be partially rescued by driving the expression of dTdc2 in a dTdc2-specific pattern, whereas driving the expression of dTdc1 in the same pattern results in a complete rescue. The disparity in rescue efficiencies between the ectopically expressed Tdc genes may reflect the differential activities of these gene products. The egg retention phenotype of the dTdc2 mutant and the phenotypes associated with ectopic dTdc expression contribute to a model in which octopamine and tyramine have distinct and separable neural activities.     

Tyramine Functions independently of octopamine in the Caenorhabditis elegans nervous system

Octopamine biosynthesis requires tyrosine decarboxylase to convert tyrosine into tyramine and tyramine beta-hydroxylase to convert tyramine into octopamine. We identified and characterized a Caenorhabditis elegans tyrosine decarboxylase gene, tdc-1, and a tyramine beta-hydroxylase gene, tbh-1. The TBH-1 protein is expressed in a subset of TDC-1-expressing cells, indicating that C. elegans has tyraminergic cells that are distinct from its octopaminergic cells. tdc-1 mutants have behavioral defects not shared by tbh-1 mutants. We show that tyramine plays a specific role in the inhibition of egg laying, the modulation of reversal behavior, and the suppression of head oscillations in response to anterior touch. We propose a model for the neural circuit that coordinates locomotion and head oscillations in response to anterior touch. Our findings establish tyramine as a neurotransmitter in C. elegans, and we suggest that tyramine is a genuine neurotransmitter in other invertebrates and possibly in vertebrates as well.     

Evidence for a possible neurotransmitter/neuromodulator role of tyramine on the locust oviducts

Visualization of the tyraminergic innervation of the oviducts was demonstrated by immunohistochemistry, and the presence of tyramine was confirmed using high-performance liquid chromatography coupled to electrochemical detection. Oviducts incubated in high-potassium saline released tyramine in a calcium-dependent manner. Stimulation of the oviducal nerves also resulted in tyramine release, suggesting that tyramine might function as a neurotransmitter/neuromodulator at the locust oviducts. Tyramine decreased the basal tension, and also attenuated proctolin-induced contractions in a dose-dependent manner over a range of doses between 10(-7) and 10(-4) M. Low concentrations of tyramine attenuated forskolin-stimulated cyclic AMP levels in a dose-dependent manner. This effect was not blocked by yohimbine. High concentrations of tyramine increased basal cyclic AMP levels of locust oviducts in a dose-dependent manner; however, the increases in cyclic AMP were only evident at the highest concentrations tested, 5 x 10(-5) and 10(-4) M tyramine. The tyramine-induced increase in cyclic AMP shared a similar pharmacological profile with the octopamine-induced increase in cyclic AMP. Tyramine increased the amplitude of excitatory junction potentials at low concentrations while hyperpolarizing the membrane potential by 2-5 mV. A further increase in the amplitude of the excitatory junction potentials and the occurrence of an active response was seen upon washing tyramine from the preparation. These results suggest that tyramine can activate at least three different endogenous receptors on the locust oviducts a putative tyramine receptor at low concentrations, a different tyramine receptor to inhibit muscle contraction, and an octopamine receptor at high concentrations.     

THE BIOSYNTHESIS OF OCTOPAMINE-CHARACTERIZATION OF LOBSTER TYRAMINE β-HYDROXYLASE

—Tyramine β-hydroxylase catalyzes the biosynthesis of octopamine in the lobster nervous system. This enzyme has been characterized and a rapid microassay, based on the enzymic release of tritiated water from [1,2-(side chain) ³H] tyramine, has been developed. Lobster tyramine β-hydroxylase resembled mammalian dopamine β-hydroxylase. The most conspicuous differences were that the lobster enzyme was inhibited by anions, particularly fumarate, and had a higher affinity for substrates. Tyramine β-hydroxylase activity was present in both particulate and soluble fractions of homogenates of the lobster nervous system. Bound activity, extracted by repeated freezing and thawing, was partially purified. The enzyme had the following properties: (1) The optimum pH for the conversion of tyramine to octopamine was 7·4. (2) The apparent Michaelis constant for tyramine was 0·15 mm and for ascorbic acid was 0·2 mm at pH 6·6. (3) The purified enzyme was inhibited by salts; the degree of inhibition was sensitive to the anion and decreased in the order chloride ? fumarate > sulphate > acetate. (4) Tyramine β-hydroxylase was inhibited by metal chelating agents and by cupric sulphate at concentrations greater than 10^?4m ; N-ethylmaleimide had no significant effect on activity in concentrations up to 3 mm . (5) The purified enzyme also β-hydroxylated dopamine to form norepinephrine, with an apparent Michaelis constant of 0·24 mm . This activity co-purified with tyramine β-hydroxylase, suggesting that a single enzyme catalyzed both reactions.     

Induced tyramine overproduction in transgenic rice plants expressing a rice tyrosine decarboxylase under the control of methanol-inducible rice tryptophan decarboxylase promoter

Tyramine, one of the various biogenic amines found in plants, is derived from the aromatic L-amino acid tyrosine through the catalytic reaction of tyrosine decarboxylase (TYDC). Tyramine overproduction by constitutive expression of TYDC in rice plants leads to stunted growth, but an increased number of tillers. To regulate tyramine production in rice plants, we expressed TYDC under the control of a methanol-inducible plant tryptophan decarboxylase (TDC) promoter and generated transgenic T(2) homozygous rice plants. The transgenic rice plants showed normal growth phenotypes with slightly increased levels of tyramine in seeds relative to wild type. Upon treatment with 1% methanol, the transgenic rice leaves produced large amounts of tyramine, whereas no increase in tyramine production was observed in wild-type plants. The methanol-induced accumulation of tyramine in the transgenic rice leaves was inversely correlated with the tyrosine level. These data indicate that tyramine production in rice plants can be artificially controlled using the methanol-inducible TDC promoter, suggesting that this promoter could be used to selectively induce the expression of other proteins or metabolites in rice plants.     

Isolation, cloning, and tissue expression of a putative octopamine/tyramine receptor from locust visceral muscle tissues

Octopamine has been shown to play major roles in invertebrate nervous systems as a neurotransmitter, neuromodulator, and neurohormone. Tyramine is the biochemical precursor of octopamine and its neuromodulatory role is now being investigated and clarified in invertebrates, particularly in insects. Both octopamine and tyramine mediate their actions via G protein-coupled receptors (GPCRs) and are believed to play important functions in the regulation of physiological processes in locust oviduct. Here we report the isolation, cloning, and tissue expression of a putative octopamine/tyramine receptor from the locust, Locusta migratoria. Degenerate oligonucleotides in PCR reactions were first used to obtain partial cDNA sequences and then these partial sequences were used in screens to obtain a full-length cDNA. The cloned cDNA is about 3.1 kb long and encodes a protein of 484 amino acid residues with typical characteristics of GPCRs including seven transmembrane domains and many signature residues. The amino acid sequence of the cloned cDNA displays sequence similarities with known GPCRs, particularly octopamine/tyramine receptors. Screening of the locust genomic DNA library resulted in isolation of a genomic DNA with the same size as the cDNA, indicating that the gene is intron-less. RT-PCR and Northern blot analyses revealed the expression of the receptor mRNA in brain, ventral nerve cord, oviduct, and midgut tissues. Southern blot analyses using EcoRI and HindIII restriction endonucleases recognized at least two distinct gene bands.     

Insulin‐like receptor substrate gene chico regulates octopamine metabolism in Drosophila melanogaster

Octopamine, one of the main insect biogenic amines, plays an important role in the control of fitness in Drosophila melanogaster Meigen. The present study examines the effects of a null mutation of the gene of the insulin‐like receptor substrate (chico), in the heterozygous state, on octopamine metabolism, heat stress resistance and fecundity of D. melanogaster. A rise in the activity of one of the key enzymes of octopamine synthesis, tyrosine decarboxylase, as well as that of an enzyme of its degradation, octopamine‐dependent N‐acetyl transferase, is observed in chico^1/+ females. It is also found that the resistance to heat stress is decreased and fecundity is reduced dramatically in chico^1/+ flies. Such changes in these parameters in D. melanogaster females result from a rise in octopamine titre, which suggests that chico affects the octopamine level by regulating the activity of tyrosine decarboxylase.     

Possible role of octopamine and tyramine in the antihypertensive and antidepressant effects of tyrosine

The administration of a dose of 200 mg/kg of tyrosine (as either the free amino acid or the ethyl ester) increased the 24-hour excretion of p-hydroxyphenethyleneglycol (p-HPG) and p-hydroxyphenylethanol, metabolites of octopamine and tyramine, by 147 and 50%, respectively. One hour after this dose of tyrosine, brain levels of p-HPG and p-hydroxyphenylacetic acid (p-HPA), another metabolite of tyramine, were increased by 82 and 196%, respectively. Pretreatment with Ro4-4602, a peripheral decarboxylase inhibitor, reduced by 50% the tyrosine-induced increases in brain p-HPA levels, suggesting that tyramine was partially formed in the brain parenchyma. Tyrosine caused only slight, but non-significant increases in brain levels of catecholamine metabolites. These results suggest that tyrosine-induced increases in the production of tyramine and octopamine in brain may account for some of the effects of tyrosine, such as its antihypertensive and reported antidepressant properties.     

Genetic Control of Biogenic-Amine Systems in Drosophila Under Normal and Stress Conditions

The contents of octopamine and its precursors (tyrosine and tyramine) were studied in adults of two lines of Drosophila virilis with contrasting stress responses. It was demonstrated that in individuals responding to stress by a hormonal stress reaction (line 101), the contents of octopamine and tyrosine are lower than in nonresponding flies (line 147). It was found that there is no difference between the lines in the level of tyramine under normal conditions. The dopamine response to stressor was also studied. Genetic analysis of these differences revealed that they are controlled by a single gene and that the gene is not sex-linked. The gene controlling the response was found to be linked to chromosome 6 of D. virilis.     

Tyramine receptor (SER-2) isoforms are involved in the regulation of pharyngeal pumping and foraging behavior in Caenorhabditis elegans

Octopamine regulates essential processes in nematodes; however, little is known about the physiological role of its precursor, tyramine. In the present study, we have characterized alternatively spliced Caenorhabditis elegans tyramine receptor isoforms (SER-2 and SER-2A) that differ by 23 amino acids within the mid-region of the third intracellular loop. Membranes prepared from cells expressing either SER-2 or SER-2A bind [3H]lysergic acid diethylamide (LSD) in the low nanomolar range and exhibit highest affinity for tyramine. Similarly, both isoforms exhibit nearly identical Ki values for a number of antagonists. In contrast, SER-2A exhibits a significantly lower affinity than SER-2 for other physiologically relevant biogenic amines, including octopamine. Pertussis toxin treatment reduces affinity for both tyramine and octopamine, especially for octopamine in membranes from cells expressing SER-2, suggesting that the conformation of the mid-region of the third intracellular loop is dictated by G-protein interactions and is responsible for the differential tyramine/octopamine affinities of the two isoforms. Tyramine reduces forskolin-stimulated cAMP levels in HEK293 cells expressing either isoform with nearly identical IC50 values. Tyramine, but not octopamine, also elevates Ca2+ levels in cells expressing SER-2 and to a lesser extent SER-2A. Most importantly, ser-2 null mutants (pk1357) fail to suppress head movements while reversing in response to nose-touch, suggesting a role for SER-2 in the regulation of foraging behavior, and fail to respond to tyramine in assays measuring serotonin-dependent pharyngeal pumping. These are the first reported functions for SER-2. These results suggest that C. elegans contains tyramine receptors, that individual SER-2 isoforms may differ significantly in their sensitivity to other physiologically relevant biogenic amines, such as octopamine (OA), and that tyraminergic signaling may be important in the regulation of key processes in nematodes.     

Neurochemistry of GABAergic activities in the central nervous system ofLocusta migratoria

Summary The biochemical elements of GABA-ergic synapses in the central nervous tissue were examined by a comparative neurochemical approach. The high concentration of GABA as well as the activities of glutamate decarboxylase and GABA-transaminase suppose a high content of GABAergic elements in the nervous system of the locust.Nerve endings isolated from the ganglia of locusts accumulated exogenous GABA in a carriermediated, sodium dependent process into compartments from where it could partially be released under depolarizing conditions. The transport was stimulated by extracellular chloride, was modulated by specific ionophores (enhanced by valinomycin, inhibited by CCCP) and could effectively be blocked by GABAergic ligands (DABA, muscimol). Binding studies revealed the existence of multiple binding sites for GABA which differ in number, affinity, pharmacology and ion dependency. The putative receptors for GABA (Na⁺-independent binding sites) in locust nervous tissue exceeded the concentrations found in vertebrate brain tissue and showed different binding pharmacology.Abbreviations GABA -amino butyric acid - GAD glutamate decarboxylase - GABA-T GABA-transaminase - DABA diamino butyric acid     

Factors affecting tyramine production in <Emphasis Type="Italic">Enterococcus durans</Emphasis> IPLA 655

The decarboxylation of tyrosine by certain lactic acid bacteria leads to the undesirable presence of tyramine in fermented foods. Tyramine is the most frequent biogenic amine found in cheese and is also commonly found in other fermented foods and beverages. The tyramine-producing strain Enterococcus durans IPLA 655 was grown in a bioreactor under different conditions to determine the influence of carbon source, tyrosine and tyramine concentrations, and pH on tyramine production. The carbon source appeared to have no significant effect on the production of tyramine. In contrast, tyrosine was necessary for tyramine production, while the presence of tyramine itself in the growth medium inhibited such production. pH showed by far the greatest influence on tyramine synthesis; tyramine was produced in the greatest quantities at pH 5.0, although this was accompanied by a reduced growth rate.     

The uptake and subcellular distribution of aromatic amines in the brain of the rat

The hydroxylated phenylethylamines p-tyramine, m-tyramine, octopamine, metaraminol and norepinephrine were accumulated by homogenates of rat brain much more vigorously than β-phenethylamine or amphetamine. The affinity concentrations (K_m) for initial (5-min) uptake by homogenates of whole brain were 0.5, 3 and 6 μM for DL-norepine-phrine, p-tyramine and DL-octopamine, respectively. The uptake of these three hydroxylated compounds was much more vigorous in striatal tissue than in cortical tissue, and in both tissues the rate of uptake decreased in the sequence: norepinephrine > tyramine > octopamine. The uptake of these three substances was inhibited by reduced temperature, by lack of glucose, by CN- and DNP, and by desmethylimipramine, cocaine and ouabain. The uptake of norepinephrine and octopamine appeared to require Na⁺. Pretreatment of rats with reserpine or 6-hydroxydopamine decreased the ability of brain to take up norepinephrine or octopamine. Previously accumulated labelled phenylethylamines migrated in sucrose density gradients with a peak of radioactivity corresponding to an equilibrium position of catecholamine-containing nerve endings. The magnitude of the retention of [³H]amine in this synaptosornal peak decreased in the order: norepinephrine > octopamine > tyramine. The accumulated amines were released by sonic, osmotic and thermal stresses which disrupt neuronal membranes. The presence of a β-hydroxyl group appeared to protect amines from destruction by monoamine oxidase, presumably by virtue of uptake in presynaptic storage vesicles. During superfusion, tyramine and metaraminol appeared to displace [³H]norepinephrine from binding sites in brain slices.     

Characterization of a <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strain Able to Produce Tyramine and Partial Cloning of a Putative Tyrosine Decarboxylase Gene

The aim of this article was to analyze the ability of wine Lactobacillus plantarum strains to form tyramine. Preliminary identification of L. plantarum strains was performed by amplification of the recA gene. Primers pREV and PlanF, ParaF and PentF were used respectively as reverse and forward primers in the polymerase chain reaction tests as previously reported. Furthermore, the gene encoding for the tyrosine decarboxylase (TDC) was partially cloned from one strain identified as L. plantarum. The strain was further analyzed by 16S rDNA sequence and confirmed as belonging to L. plantarum species. The tyrosine decarboxylase activity was investigated and tyramine was determined by the high-performance liquid chromatography method. Moreover, a negative effect of sugars such as glucose and fructose and L-malic acid on tyrosine decarboxylase activity was observed. The results suggest that, occasionally, L. plantarum is able to produce tyramine in wine and this ability is apparently confined only to L. plantarum strains harboring the tdc gene.