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Serotonin N-acetyltransferase (SNAT) functions as the penultimate or final enzyme in melatonin biosynthesis, depending on the substrate. The Escherichia coli orthologue of archaeal SNAT from Thermoplasma volcanium was identified as RimI (EcRimI), with 42% amino acid similarity to archaeal SNAT. EcRimI has been reported to be an N-acetyltransferase enzyme. Here, we investigated whether EcRimI also exhibits SNAT enzyme activity. To achieve this goal, we purified recombinant EcRimI and examined its SNAT enzyme kinetics. As expected, EcRimI showed SNAT activity toward various amine substrates including serotonin and 5-methoxytryptamine, with Km and Vmax values of 531 μM and 528 pmol/min/mg protein toward serotonin and 201 μM and 587 pmol/min/mg protein toward 5-methoxytryptamine, respectively. In contrast to the rimI mutant E. coli strain that showed no growth defect, the EcRimI overexpression strain exhibited a 2-fold higher growth rate than the control strain after 24 h incubation in nutrient-rich medium. The EcRimI overexpression strain produced more melatonin than the control strain in the presence of 5-methoxytryptamine. The enhanced growth effect of EcRimI overexpression was also observed under cadmium stress. The higher growth rate associated with EcRimI expression was attributed to increased protein N-acetyltransferase activity, increased synthesis of melatonin, or the combined effects of both. [ABSTRACT FROM AUTHOR]

Recently, the presence of melatonin in fermented beverages has been correlated with yeast metabolism during alcoholic fermentation. Melatonin, originally considered a unique product of the pineal gland of vertebrates, has been also identified in a wide range of invertebrates, plants, bacteria, and fungi in the last two decades. These findings bring the challenge of studying the function of melatonin in yeasts and the mechanisms underlying its synthesis. However, the necessary information to improve the selection and production of this interesting molecule in fermented beverages is to disclose the genes involved in the metabolic pathway. So far, only one gene has been proposed as involved in melatonin production in Saccharomyces cerevisiae, PAA1, a polyamine acetyltransferase, a homolog of the vertebrate's aralkylamine N-acetyltransferase (AANAT). In this study, we assessed the in vivo function of PAA1 by evaluating the bioconversion of the different possible substrates, such as 5-methoxytryptamine, tryptamine, and serotonin, using different protein expression platforms. Moreover, we expanded the search for new N-acetyltransferase candidates by combining a global transcriptome analysis and the use of powerful bioinformatic tools to predict similar domains to AANAT in S. cerevisiae. The AANAT activity of the candidate genes was validated by their overexpression in E. coli because, curiously, this system evidenced higher differences than the overexpression in their own host S. cerevisiae. Our results confirm that PAA1 possesses the ability to acetylate different aralkylamines, but AANAT activity does not seem to be the main acetylation activity. Moreover, we also prove that Paa1p is not the only enzyme with this AANAT activity. Our search of new genes detected HPA2 as a new arylalkylamine N-acetyltransferase in S. cerevisiae. This is the first report that clearly proves the involvement of this enzyme in AANAT activity. [ABSTRACT FROM AUTHOR]

Melatonin is catabolized both enzymatically and nonenzymatically. Nonenzymatic processes mediated by free radicals, singlet oxygen, other reactive intermediates such as HOCl and peroxynitrite, or pseudoenzymatic mechanisms are not species- or tissue-specific, but vary considerably in their extent. Higher rates of nonenzymatic melatonin metabolism can be expected upon UV exposure, e.g., in plants and in the human skin. Additionally, melatonin is more strongly nonenzymatically degraded at sites of inflammation. Typical products are several hydroxylated derivatives of melatonin and N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK). Most of these products are also formed by enzymatic catalysis. Considerable taxon- and site-specific differences are observed in the main enzymatic routes of catabolism. Formation of 6-hydroxymelatonin by cytochrome P450 subforms are prevailing in vertebrates, predominantly in the liver, but also in the brain. In pineal gland and non-mammalian retina, deacetylation to 5-methoxytryptamine (5-MT) plays a certain role. This pathway is quantitatively prevalent in dinoflagellates, in which 5-MT induces cyst formation and is further converted to 5-methoxyindole-3-acetic acid, an end product released to the water. In plants, the major route is catalyzed by melatonin 2-hydroxylase, whose product is tautomerized to 3-acetamidoethyl-3-hydroxy-5-methoxyindolin-2-one (AMIO), which exceeds the levels of melatonin. Formation and properties of various secondary products are discussed. [ABSTRACT FROM AUTHOR]

Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5-methoxytryptamine (5- MT) pathway. 5- MT is known to be synthesized in vitro from serotonin by the enzymatic action of O-methyltransferases, including N-acetylserotonin methyltransferase ( ASMT) and caffeic acid O-methyltransferase ( COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N-acetyltransferase ( SNAT). Here, we show that 5- MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5- MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild-type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5- MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5- MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5- MT pathway with the ectopic overexpression of COMT in chloroplasts in plants. [ABSTRACT FROM AUTHOR]

Manipulations of the serotonin transmission during early development induce long-lasting changes in the serotonergic circuitry throughout the brain. However, little is known on the developmental consequences in the female progeny. Therefore, this study aimed at exploring the behavioural effects of pre- and postnatal stimulation of the serotonergic system by 5-methoxytryptamine in adolescent female rats on behavioural reactivity and anxiety-like phenotype. Our results show that perinatal 5-methoxythyptamine decreased total distance travelled and rearing frequency in the novel enviroment, and increased the preference for the centre of the arena in the open field test. Moreover, perinatal 5-methoxytryptamine increased the percentages of entries and time spent on the open arms of the elevated plus maze, with respect to perinatally vehicle-exposed rats. Thus, perinatal stimulation of serotonin receptors does not impair the functional response to the emotional challenges in female rats, favouring the occurrence of a stress-resilient phenotype. [ABSTRACT FROM AUTHOR]

Melatonin is an animal hormone as well as a signaling molecule in plants. It was first identified in plants in 1995, and almost all enzymes responsible for melatonin biosynthesis had already been characterized in these species. Melatonin biosynthesis from tryptophan requires four-step reactions. However, six genes, that is, TDC, TPH, T5H, SNAT, ASMT, and COMT, have been implicated in the synthesis of melatonin in plants, suggesting the presence of multiple pathways. Two major pathways have been proposed based on the enzyme kinetics: One is the tryptophan/tryptamine/serotonin/ N-acetylserotonin/melatonin pathway, which may occur under normal growth conditions; the other is the tryptophan/tryptamine/serotonin/5-methoxytryptamine/melatonin pathway, which may occur when plants produce large amounts of serotonin, for example, upon senescence. The melatonin biosynthetic capacity associated with conversion of tryptophan to serotonin is much higher than that associated with conversion of serotonin to melatonin, which yields a low level of melatonin synthesis in plants. Many melatonin intermediates are produced in various subcellular compartments, such as the cytoplasm, endoplasmic reticulum, and chloroplasts, which either facilitates or impedes the subsequent enzymatic steps. Depending on the pathways, the final subcellular sites of melatonin synthesis vary at either the cytoplasm or chloroplasts, which may differentially affect the mode of action of melatonin in plants. [ABSTRACT FROM AUTHOR]

Melatonin is a phylogenetically ancient molecule. It is ubiquitously present in almost all organisms from primitive photosynthetic bacteria to humans. Its original primary function is presumable to be that of an antioxidant with other functions of this molecule having been acquired during evolution. The synthetic pathway of melatonin in vertebrates has been extensively studied. It is common knowledge that serotonin is acetylated to form N-acetylserotonin by arylalkylamine N-acetyltransferase ( AANAT) or arylamine N-acetyltransferase ( SNAT or NAT) and N-acetylserotonin is, subsequently, methylated to melatonin by N-acetylserotonin O-methyltransferase ( ASMT; also known as hydroxyindole-O-methyltransferase, HIOMT). This is referred to as a classic melatonin synthetic pathway. Based on new evidence, we feel that this classic melatonin pathway is not generally the prevailing route of melatonin production. An alternate pathway is known to exist, in which serotonin is first O-methylated to 5-methoxytryptamine (5- MT) and, thereafter, 5- MT is N-acetylated to melatonin. Here, we hypothesize that the alternate melatonin synthetic pathway may be more important in certain organisms and under certain conditions. Evidence strongly supports that this alternate pathway prevails in some plants, bacteria, and, perhaps, yeast and may also occur in animals. [ABSTRACT FROM AUTHOR]

Serotonin N-acetyltransferase ( SNAT), the penultimate enzyme in melatonin biosynthesis, catalyzes the conversion of serotonin into N-acetylserotonin. Plant SNAT is localized in chloroplasts. To test SNAT localization effects on melatonin synthesis, we generated transgenic rice plants overexpressing a sheep ( Ovis aries) SNAT ( Oa SNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing Oa SNAT in their cytoplasm. To localize the Oa SNAT in chloroplasts, we used a chloroplast targeting sequence ( CTS) from tobacco protoporphyrinogen IX oxidase ( PPO), which expresses in chloroplasts. The purified recombinant CTS: Oa SNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast-targeted CTS: Oa SNAT lines and cytoplasm-expressed Oa SNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS: Oa SNAT lines than in the Oa SNAT lines. Notably, enhanced SNAT enzyme activity was not directly proportional to the production of N-acetylserotonin, melatonin, or 2-hydroxymelatonin, suggesting that plant SNAT has a role in the homeostatic regulation of melatonin rather than in accelerating melatonin synthesis. [ABSTRACT FROM AUTHOR]

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45°C (Km, 309 μM; Vmax, 1400 pmol/min/mg protein). AtSNAT also catalyzed 5-methoxytryptamine (5-MT) into melatonin with high catalytic activity (Km, 51 μM; Vmax, 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O-methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5-MT with low catalytic activity (Km, 3.396 mM; Vmax, 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N-acetylserotonin by SNAT or into 5-MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N-acetylserotonin was predominantly produced at high temperatures (45°C), while low temperatures (37°C) favored the production of 5-MT. Our results provide biochemical evidence for the presence of a serotonin O-methylation pathway in plant melatonin biosynthesis. [ABSTRACT FROM AUTHOR]

We investigated the protective effects of melatonin and its metabolites: 6-hydroxymelatonin (6-OHM), N1-acetyl- N2-formyl-5-methoxykynuramine (AFMK), N-acetylserotonin (NAS), and 5-methoxytryptamine (5-MT) in human keratinocytes against a range of doses (25, 50, and 75 mJ/cm2) of ultraviolet B (UVB) radiation. There was significant reduction in the generation of reactive oxygen species (50-60%) when UVB-exposed keratinocytes were treated with melatonin or its derivatives. Similarly, melatonin and its metabolites reduced the nitrite and hydrogen peroxide levels that were induced by UVB as early as 30 min after the exposure. Moreover, melatonin and its metabolites enhanced levels of reduced glutathione in keratinocytes within 1 hr after UVB exposure in comparison with control cells. Using proliferation assay, we observed a dose-dependent increase in viability of UVB-irradiated keratinocytes that were treated with melatonin or its derivatives after 48 hr. Using the dot-blot technique and immunofluorescent staining we also observed that melatonin and its metabolites enhanced the DNA repair capacity of UVB-induced pyrimidine photoproducts (6-4)or cyclobutane pyrimidine dimers generation in human keratinocytes. Additional evidence for induction of DNA repair in cells exposed to UVB and treated with the indole compounds was shown using the Comet assay. Finally, melatonin and its metabolites further enhanced expression of p53 phosphorylated at Ser-15 but not at Ser-46 or its nonphosphorylated form. In conclusion, melatonin, its precursor NAS, and its metabolites 6-OHM, AFMK, 5-MT, which are endogenously produced in keratinocytes, protect these cells against UVB-induced oxidative stress and DNA damage. [ABSTRACT FROM AUTHOR]

Indolic and kynuric pathways of skin melatonin metabolism were monitored by liquid chromatography mass spectrometry in human keratinocytes, melanocytes, dermal fibroblasts, and melanoma cells. Production of 6-hydroxymelatonin [6(OH)M], N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and 5-methoxytryptamine (5-MT) was detected in a cell type-dependent fashion. The major metabolites, 6(OH)M and AFMK, were produced in all cells. Thus, in immortalized epidermal (HaCaT) keratinocytes, 6(OH)M was the major product with Vmax = 63.7 ng/106 cells and Km = 10.2 µM, with lower production of AFMK and 5-MT. Melanocytes, keratinocytes, and fibroblasts transformed melatonin primarily into 6(OH)M and AFMK. In melanoma cells, 6(OH)M and AFMK were produced endogenously, a process accelerated by exogenous melatonin in the case of AFMK. In addition, N-acetylserotonin was endogenously produced by normal and malignant melanocytes. Metabolites showed selective antiproliferative effects on human primary epidermal keratinocytes in vitro. In ex vivo human skin, both melatonin and AFMK-stimulated expression of involucrin and keratins-10 and keratins-14 in the epidermis, indicating their stimulatory role in building and maintaining the epidermal barrier. In summary, the metabolism of melatonin and its endogenous production is cell type-dependent and expressed in all three main cell populations of human skin. Furthermore, melatonin and its metabolite AFMK stimulate differentiation in human epidermis, indicating their key role in building the skin barrier. [ABSTRACT FROM AUTHOR]

In its role as a pineal hormone, melatonin is a pleiotropic, nocturnally peaking and systemically acting chronobiotic. These effects are largely explained by actions via G protein-coupled membrane receptors found in the suprachiasmatic nucleus, but also in numerous other sites. Nuclear (ROR/RZR), cytoplasmic (quinone reductase-2, calmodulin, calreticulin) and mitochondrial binding sites and radical-scavenging properties contribute to the actions of melatonin. Regulation of pineal melatonin biosynthesis is largely explained by control mechanisms acting on arylalkylamine N-acetyltransferase, at the levels of gene expression and/or enzyme stability influenced by phosphorylation and interaction with 14-3-3 proteins. Melatonin is not only a hormone but is also synthesized in numerous extrapineal sites, in which it sometimes attains much higher quantities than in the pineal and the circulation. It is also present in many taxonomically distant groups of organisms, including bacteria, fungi, and plants. Moreover, melatonin is a source of bioactive metabolites, such as 5-methoxytryptamine, N 1-acetyl- N 2-formyl-5-methoxykynuramine and N 1-acetyl-5-methoxykynuramine. [ABSTRACT FROM AUTHOR]

Integration of genomic data from pharmacokinetic pathways and drug targets is an emerging trend in bioinformatics, but is there a clear separation of pharmacokinetic pathways and drug targets? Should we also consider the potential interactions of endogenous substrates of drug-metabolizing enzymes with receptors and other molecular drug targets as we combine pharmacogenomic datasets? We discuss these overarching questions through a specific pharmacogenomic case study of the cytochrome P450 (CYP)2D6, serotonin and dopamine triad. Importantly, CYP2D6 may contribute to the regeneration of serotonin from 5-methoxytryptamine by virtue of its catalytic function as a 5-methoxyindolethylamine O-demethylase. Furthermore, serotonergic neurons provide a regulatory feedback on dopaminergic neurotransmission. Hence, we hypothesize that independent of its role as a pharmacokinetic gene, CYP2D6 may nuance the regulation of serotonergic and dopaminergic neurophysiology. Additionally, we reflect upon the contribution of hyperspecialization in biomedicine to the present disconnect between research on pharmacokinetics and drug targets, and the potential for remedying this important gap through informed dialogue among clinical pharmacologists, human geneticists, bioethicists and applied social scientists. [ABSTRACT FROM AUTHOR]

Proton and carbon‐13 NMR data are presented for 5‐methoxytryptamine, 1; 6‐methoxytryptamine, 2; N,N ‐diisopropyl‐5‐methoxytryptamine, 3, (5‐MeO‐DIPT); and N,N ‐diisopropyl‐5‐methoxyindole‐3‐glyoxylamide, 4, at 300 MHz ( 1 H) and 75 MHz ( 13 C) in CDCl 3 at ambient temperature. Compound 3, considered a potential hallucinogen, had been placed into Schedule I of the Controlled Substances Act, effective April 4, 2003, by the U.S. Drug Enforcement Administration. Compound 4 can serve as a possible precursor to 3. We believe that these are the first proton NMR assignments obtained at medium field (7 tesla) using selective homodecoupling and two‐dimensional homonuclear chemical shift correlation spectra (using one or more of the COSY45, COSY90, and COSYLR experiments) for rigorous aryl proton assignments in this group of compounds. Significant observed differences in the proton and carbon‐13 NMR spectra should allow facile distinction of the 5‐methoxy series, 1 and 3, from the 6‐methoxy series, 2. Energy minimizations to obtain optimized structures for each compound were performed at the Hartree–Fock level with the 6‐31G* basis set, and the resulting geometries are discussed. The presented geometry calculations appear to be the most accurate reported to date for 1 based on the basis set employed, and the first HF/6‐31G* structures for compounds 2, 3, or 4. Appreciable geometry differences in 3 and 4 for the pendant sidechain containing the N[CH(CH 3 ) 2 ] 2 moiety are noteworthy. Proximity of the carbonyl oxygens in 4 to H2 and H4 is suggested as a possible contributing factor in the deshielding of these protons in the NMR spectrum. [ABSTRACT FROM AUTHOR]

The 5-hydroxytryptamine (5HT) receptor agonist, 5-methoxytryptamine, inhibited in a concentration-dependent manner the electrically-evoked release of H-5HT from superfused rat hypothalamic slices, with an IC of 560 nmol/l, without affecting the spontaneous outflow of radioactivity. In the presence of the selective monoamine oxidase B (MAO B) inhibitor, (−)-deprenyl (1 μmol/l), the concentration-effect curve for 5-methoxytryptamine was shifted significantly to the left, and the IC was decreased to 25 nmol/l. Under the same experimental conditions, the potency of the 5HT receptor agonist lysergic acid diethylamide (LSD) at inhibiting the electrically-evoked release of H-5HT was the same in the presence as well as in the absence of (−)-deprenyl. The IC values for LSD were 34 nmol/l in the absence of deprenyl, and 31 nmol/l in the presence of the MAO B inhibitor. It is concluded that deprenyl potentiates the inhibition by 5-methoxytryptamine of H-5HT release, by preventing its inactivation through MAO B. Since 5-methoxytryptamine may be present in the pineal gland of some species, the potent effects of this 5-HT receptor agonist on seretoninergic neutrotransmission may be of physiological relevance. [ABSTRACT FROM AUTHOR]

The effects of different 5-methoxyindoles on the process of protein/peptide secretion characterized by the formation of granular vesicles (GV) have been studied in mouse pinealocytes maintained in explant culture. All 5-methoxyindoles studied clearly influence the number of granular vesicles in the pinealocytes. Comparing all present results it appeared that, in this system, melatonin was the least effective of all 5-methoxyindoles tested in stimulating secretion. 5-Methoxyindole-3-acetic acid, irrespective of the duration of the experiment and of the presence of noradrenaline, increased the number of GV. For all other 5-methoxyindoles, 5-methoxytryptamine, 5-methoxytryptophan, 5-methoxytryptophol and melatonin, it appeared that the effects depend on the duration of application and on the presence or absence of noradrenaline in the medium. Moreover, depending on the experimental conditions and the 5-methoxyindole tested, antagonistic as well as synergistic effects between 5-methoxyindoles and noradrenaline were observed. The present results, which suggest that the 5-methoxyindoles are also active in the pineal gland itself, demonstrate that, as far as the formation of granular vesicles is concerned, there exists a very complex mechanism of regulation, involving the sympathetic innervation and the 5-methoxyindoles (which are themselves under the influence of this innervation). The physiological significance of this system is discussed in relation to a proposed working hypothesis. [ABSTRACT FROM AUTHOR]

5-Hydroxytryptamine (5-HT) stimulated an increase in short-circuit current (I) in guinea-pig isolated ileal mucosa over a wide concentration range (0.1 nM-0.1 mM). The concentration-response relationship was biphasic, consisting of a high potency phase (0.1 nM-1 μM) and a low potency phase (3-10 μM). Stimulation of Isc observed at the high potency phase tended to be sustained while responses at the low potency phase (3-10 μM) contained two components, an initial 'transient' response followed by a 'maintained' response. Both the high potency phase (maximum stimulation ∼30 μA cm) and the low potency phase (maximum stimulation ∼80 μA cm ) 5-HT response were antagonized by tetrodotoxin (TTX, 0.3 μM) and atropine (1 μM). However, another low potency (3 μM-0.1 mM, maximum stimulation ∼30 μA cm) component of the 5-HT response was revealed in the presence of TTX or atropine. In the presence of methysergide (1 μM), the concentration-response relationship of 5-HT was still biphasic and tropisetron (0.1 and 10 μM) antagonized both phases of the 5-HT response. In the presence of methysergide, the high potency phase 5-HT response was mimicked by 5-methoxytryptamine (5-MeOT) and the selective 5-HT agonist SC-53116 but not by BIMU 8. The potent 5-HT antagonist GR 113808 antagonized the response to 5-MeOT in a surmountable manner with an affinity estimate of 9.6 ± 0.3 ( n = 4). The 5-MeOT stimulated increase in I was also antagonized in an unsurmountable manner by granisetron (1 μM). In the presence of methysergide, desensitization of 5-HT receptors with 2-methyl-5-hydroxytryptamine (10 μM) abolished both phases of the 5-HT response. Under the same condition, desensitization of 5-HT receptors with 5-MeOT (10 μM) abolished only the high potency 5-HT response and dextrally shifted the low potency 5-HT response. These data show that neuronal and non-neuronal 5-HT receptors are involved in the regulation of secretion in ileal mucosa. We propose the presence of a neuronal 5-HT receptor located upstream of the well characterized neuronal 5-HT receptors to be responsible for the high potency 5-HT response. A schematic model is proposed to explain our findings and the relationship between this 5-HT receptor and other 5-HT receptor subtypes regulating secretion that have been described in the literature. [ABSTRACT FROM AUTHOR]

The response of a cell line of Xenopus laevis melanophores to serotonin was examined. Serotonin increased intracellular levels of cAMP and induced pigment dispersion in the cells. The responses depended on both the concentration of serotonin applied and on the time for which the cells were exposed to serotonin. Using a recently described, microtiter-plate-based bioassay, a series of serotonin receptor ligands were evaluated as agonists or antagonists at the melanophore serotonin receptor. The pharmacological profile suggests the presence of a receptor which shares some properties with but appears different from other previously described serotonin receptors. [ABSTRACT FROM AUTHOR]

The effects of 5-methoxytryptamine and 5-hydroxytryptamine (5-HT) on both basal and electrically evoked outflow of tritium were studied in guinea-pig myenteric plexus preparations preincubated with [H]-choline. Basal outflow. 5-Methoxytryptamine caused a transient and calcium-dependent increase in basal outflow of [H]acetylcholine that was abolished by tetrodotoxin. Ondansetron (1 μmol/1) did not affect the stimulatory response of 5-methoxytryptamine but ICS 205-930 (1 and 3 μmol/1) produced parallel rightward displacements of the concentration-response curve to 5-methoxytryptamine. The PK value for ICS 205-930 was 6.6 suggesting an involvement of 5-HT receptors. 5-HT caused an increase in basal outflow of [H]acetylcholine and a biphasic concentration-response curve was obtained. The maximal response of the first phase to 5-HT (release of 0.98% of tissue tritium) and the maximal response to 5-methoxytryptamine (0.94% of tissue tritium) were similar but 5-methoxytryptamine (-log EC: 6.9) was less potent than 5-HT (-log EC of the high affinity component: 7.9). ICS 205-930 (0.01-1.0 μmol/1) acted as a competitive antagonist against the low affinity component of the 5-HT concentration-response curve with a pA value of 8.0. It is concluded that stimulation of both 5-HT receptors (by 5-methoxytryptamine and submicromolar concentrations of 5-HT) and 5-HT receptors (by micromolar concentrations of 5-HT) causes a release of acetylcholine which in turn leads to smooth muscle contraction. Electrically evoked outflow. This outflow of [H]acetylcholine was concentration-dependently inhibited by both 5-methoxytryptamine and 5-HT. ICS 205-930 (1 μmol/1) reinforced the inhibitory effect of 5-methoxytryptamine but not that of 5-HT. In the presence of methiothepine (0.1 μmol/1) 5-methoxytryptamine enhanced the evoked outflow of [H]acetylcholine, an effect which was attenuated by 3 μmol/1 ICS 205-930. These results suggest that 5-methoxytryptamine may both inhibit (via 5-HT receptors) and facilitate (via 5-HT receptors) the evoked release of acetylcholine from guinea-pig myenteric neurones. The facilitatory action is unmasked when the 5-HT receptor is blocked by methiothepine. [ABSTRACT FROM AUTHOR]

Agonist-induced desensitization has been utilized to discriminate and independently 'isolate' the neuronal excitatory receptors to 5-hydroxytryptamine (5-HT) in the guinea pig ileum (5-HT and putative 5-HT receptors). Electrically stimulated longitudinal muscle myenteric plexus preparations, and non-stimulated segments of whole ileum were used. Exposure to 5-methoxytryptamine (10 μmol/l) inhibited completely responses to 5-HT at the putative 5-HT receptor without affecting 5-HT-mediated responses. Conversely, exposure to 2-methyl-5-HT (10 μmol/l) inhibited completely responses to 5-HT at the 5-HT receptor without affecting putative 5-HT-mediated responses. The inhibition with 5-methoxytryptamine and 2-methyl-5-HT, either alone or in combination, appeared selective as responses to KCI, DMPP, carbachol, histamine, and substance P were unaffected or only very slightly modified. Furthermore, the pA values for ICS 205-930 at the putative 5-HT (pA = 6.2 to 6.5) and 5-HT (pA = 7.6 to 8.1) receptors (estimated in the presence of 2-methyl-5HT and 5-methoxytryptamine, respectively) were consistent with those estimated in the absence of desensitization. 5-Methoxytryptamine, but not 2-methyl-5-HT, suppressed completely but reversibly the concentration-effect curve to renzapride, suggesting that responses to this agent are mediated exclusively via agonism at the putative 5-HT receptor. It is concluded that 5-methoxytryptamine and 2-methyl-5-HT can be utilized as selective probes to discriminate the putative 5-HT receptor from the 5-HT receptor in guinea pig ileum. This finding is of importance as no selective antagonist exists for the putative 5-HT receptor. Furthermore, the presently described method of agonist-induced desensitization and 5-HT receptor discrimination may be useful for the identification and characterization of the putative 5-HT receptor in other tissues and species. [ABSTRACT FROM AUTHOR]

5-Methoxytryptamine (5-MT), 5-methoxytryptophol (5-ML) and melatonin (Mel) were measured in the plasma after 2, 5, and 8 weeks administration of 25 Μg 5-MT to golden hamsters kept under long photoperiod. 5-MT showed a one compartment kinetic profile in the plasma with half lives of 14.8 min after 2 weeks, 15 min after 5 weeks and 19.1 min after 8 weeks. A rapid metabolism of 5-MT was shown, Mel and 5-ML being detected in the plasma following 5-MT administration. However it was also shown that the gonadal atrophy observed after 5-MT administration cannot be due to its metabolism into these 2 compounds. Indeed when exogenously administered at a dose generating the same plasma concentration as that observed after 5-MT, the gonadal regression observed after the association of 5-ML and Mel is much less than that observed after 5-MT. 5-MT is thus a compound of great physiological interest. [ABSTRACT FROM AUTHOR]

This study tested the photo-dependency of the rhythmic synthesis of 5-methoxytryptamine (5-MT) in the pineal gland of golden hamsters. After pargyline administration, pineal 5-methoxytryptamine and melatonin were measured by HPLC in male golden hamsters kept under short and long photoperiod. In both photoperiodic regimes, a clear 5-MT rhythm was observed which fitted a sinusoidal function with high values occuring during the day-time and low values occuring during the night-time. The duration of the low nighttime levels was clearly proportional to the length of the dark phase. A marked rhythm of melatonin synthesis was also seen with low daytime levels and high night-time values. An inverse relationship between 5-MT and melatonin levels was observed. Thus, after pargyline administration, the rhythms of 5-MT and melatonin in the pineal gland of golden hamsters are photoperiod-dependent and show a reciprocal relationship. [ABSTRACT FROM AUTHOR]

In the European hamster (Cricetus cricetus, L.) pinealectomy as well as a constant high level of either circulating melatonin or 5-methoxytryptamine induce marked changes in the immunocytochemically demonstrable central vasopressinergic innervation. When compared to control animals, a drastic decrease of AVP-immunoreaction was observed in the diagonal band of Broca, the lateral septum, the medial amygdala and the ventral hypothalamus. The results obtained suggest that part of the central vasopressinergic innervation is involved in pineal dependent seasonal functioning of the animal. [ABSTRACT FROM AUTHOR]

Subcutaneous implantation of 5-methoxytryptamine-filled silastic capsules overcomes, in natural as well as in experimental conditions, the inhibitory effect of short photoperiod on the gonadal axis. The results obtained are similar to those obtained after pinealectomy. They indicate that 5-methoxytryptamine when constantly available presents, like melatonin, a progonadal (or counter-antigonadal) effect. Evidently, 5-methoxytryptamine, like melatonin, is involved in the control of the reproductive function. [ABSTRACT FROM AUTHOR]

Late-afternoon-administered 5-methoxytryptamine (5-MT) induces gonadal atrophy in male hamsters kept under long photoperiods (14 hL/ 10 hD). Morning-injection of 1mg 5-MT does not show antigonadal effects. However, 1 mg melatonin (MEL) administered in the morning completely prevents the antigonadotropic effect of 5-MT administered in the late afternoon. This result is specific of MEL since morning injections of 1mg 5-MT do not present this counterantigonadotropic property. When interpreted according to Reiter's concept of down-regulation of MEL receptors, these results suggest that the physiological effects of 5-MT are indirectly due to MEL. [ABSTRACT FROM AUTHOR]

Daily (6 days on 7) subcutaneous injections of 25 μg of 5-methoxytryptamine in saline 0.9% into intact male hamsters given between 9.00 and 9.30 (lights on from 8.00 to 18.00; 10hL/14 hD) during 8 weeks block short photoperiodically induced testicular atrophy. Under these experimental conditions, however, this blockage is not total since the weight of the testes and of the accessory sex organs of the pinealectomized hamsters is higher than that of treated animals. The results indicate that 5-MT, like melatonin, is a physiologically active compound implicated in the control of the reproductive function. [ABSTRACT FROM AUTHOR]

The bioluminescent dinoflagellate Gonyaulax polyedra contains various indoleamines, in particular, melatonin and 5-methoxytryptamine, as well as enzymes of their biosynthetic pathway. Melatonin exhibits a high-amplitude circadian rhythm characterized by a dramatic increase shortly after the onset of darkness. The maximum of melatonin is followed by a peak of 5-methoxytryptamine. These 5-methoxylated indoleamines seem to be involved in the mediation of the information 'darkness'. G. polyedra shows a short-day response, which consists in the formation of asexual cysts. Light break experiments demonstrate the photoperiodic nature of this reaction. Cells become sensitive to short days only upon exposure to a lowered temperature (<16°C). Melatonin mimics the short-day effect, but only at decreased temperature. 5-Methoxytryptamine is even a better inducer of cyst formation, acting also at 20°C and in any lighting schedule, including LL. Cyst induction is associated with stimulation of bioluminescence and cytoplasmic acidification. A model on the intracellular pathway of photoperiodic information transduction assumes increased deacetylation of melatonin under cyst-inducing conditions, binding of 5-methoxytryptamine to the membrane of an acidic vacuole, proton transfer to the cytoplasm, and decreased intracellular pH as the stimulus for encystment. Melatonin shows the property of a scavenger of superoxide anions. This reaction, which is efficiently catalyzed by hemin, leads to the formation of a substituted kynuramine (AFMK). Destruction of melatonin by light-induced superoxide anions in the presence of cellular hemin may represent a property which, during evolution, has made this molecule suitable as an indicator of darkness. On the other hand, AFMK, which is formed under illumination, might have become a mediator of the information 'light'. Photoperiodism in Gonyaulax shows surprising parallels to that in mammals, but allows the analysis of this phenomenon at an entirely cellular level. [ABSTRACT FROM AUTHOR]

5-Methoxytryptamine (5-MT) is taken up rapidly by rat tissues after IP injection (50 mg/kg) and the following peak levels ( μg/g or ml of sample) were observed after 5 min: plasma, 5.3; liver, 126; skeletal muscle, 2.4; brain 0.43. Over 90% of the compound disappeared from the plasma and the tissues studied within 60 min. The compound impaired rope climbing performance of trained rats. Pretreatment of the animals with iproniazid enhanced the effect of 5-MT whereas its main (and probably only) metabolite, 5-methoxyindoleacetic acid, did not interfere with rope climbing at a dose of 75 mg/kg. 5-MT also produced marked cutaneous vasodilatation, analgesia and hindleg paralysis. All pharmacological effects lasted approximately 75 min. It is suggested that 5-MT is the pharmacological active agent and that behavioral changes observed after IP injection of the compound are mainly caused by the peripheral and little, if any, by a central action of this compound. [ABSTRACT FROM AUTHOR]

Encystment, which at a temperature of 15°C is photoperiodically controlled in Gonyaulax polyedra, can also be induced by a decrease of temperature, from 20 to 10 or 8°C in the absence of photoperiodic signals. The cyst-inducing capacity of the decrease in temperature depends on the circadian phase: in constant light, the maximum of sensitivity was found at the beginning of subjective night. In a light/dark cycle, however, cyst formation was reduced during dark phase, indicating that light is required for the process of encystment. A similar light dependence was seen in the effect of the physiologically occurring cyst inducer 5-methoxytryptamine, but not in the encystment response to the protonophores monensin and nigericin. [ABSTRACT FROM AUTHOR]

The article presents the study on the reactivity of ethyl 2-R-4-(2-dimethylaminovinyl)pyrimidine-5-carboxylates with primary amines [1, 2]. The study unexpectedly found the presence of 2-R-10-methoxy-8,13,13b,14-tetrahydroindolo-[2,3-a]pyrimido-[5,4-g]quinolizin-5(7H)-ones rather than the expected pyridopyrimidinones. Presence of tetrahydroquinolizinone rings were recorded on spectrometer and elemental analysis was carried which determined the melting points on a Koffler block.

Pretreatment with the MAO-inhibitors iproniazid, clorgyline, or deprenyl abolishes the effects of LSD on the conditioned avoidance response (CAR) in rats. The effects of serotonin (5-HT) and 5-methoxytryptamine (5-MT) are greatly potentiated by these substances. Brain levels of LSD are not affected by MAO inhibition whereas levels of 5-HT and 5-MT are significantly elevated. It is postulated that the decreased behavioral response to LSD is the result of 'MAO inhibitor-induced' changes whereas the increased response of 5-HT and 5-MT results from increased brain levels of these compounds. [ABSTRACT FROM AUTHOR]