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The synthesis and solid-state structure of the fumarate salt of the synthetic psychedelic 5-meth­oxy-N,N-di­allyl­tryptamine (5-MeO-DALT) is reported., The title compound, bis­(N,N-diallyl-5-meth­oxy­tryptammonium) (5-MeO-DALT) fumarate (systematic name: bis­{N-[2-(5-meth­oxy-1H-indol-3-yl)eth­yl]- N-(prop-2-en-1-yl)prop-2-en-1-aminium} (E)-but-2-enedioate), 2C17H23N2O+·C4H2O4 2−, has a single tryptammonium cation and half of a fumarate dianion in the asymmetric unit. The tryptammonium and fumarate ions are held together in one-dimensional chains by a series of N—H⋯O hydrogen bonds. These chains are combinations of R 4 4(22) rings, and C 2 2(14) and C 4 4(28) parallel chains along [111].

A reverse melatonin biosynthetic pathway was recently discovered in plants, by which N-acetylserotonin (NAS) is converted into serotonin by N-acetylserotonin deacetylase (ASDAC) rather than into melatonin by N-acetylserotonin O-methyltransferase (ASMT). In this study, we generated transgenic rice plants in which ASDAC was either suppressed or overexpressed to determine whether ASDAC is functionally involved in melatonin biosynthesis. ASDAC-suppressed rice showed increased levels of NAS, 5-methoxytryptamine (5-MT), and melatonin, whereas ASDAC-overexpressed rice exhibited less melatonin synthesis than observed in the wild type. This finding is strong evidence of the role of ASDAC in melatonin biosynthesis in rice. The increased levels of 5-MT, which is produced either by ASDAC from melatonin or by serotonin O-methyltransferase (SOMT) from serotonin in ASDAC-suppressed rice, was ascribed to enhanced SOMT enzyme activity rather than increased transcripts, such as ASMT or caffeic acid O-methyltransferase (COMT) encoding SOMT activity.

New psychoactive substances (NPS) are a new breed of synthetically produced substances designed to mimic the effects of traditional illegal drugs. Synthetic cannabinoids and synthetic cathinones are the two most common groups, which try to mimic the effects of the natural compounds 9Δ-tetrahydrocannabinol and cathinone, respectively. Similarly, synthetic tryptamines are designer compounds which are based on the compounds psilocin, N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine found in some mushrooms. One of the most important tryptamine compounds found in seizures is 5-methoxy-N,N-diisopropyltryptamine, which has been placed as controlled substance in USA and some European countries. The control of this compound has promoted the rising of another tryptamine, the 5-methoxy-N-methyl-N-isopropyltryptamine, which at the time of writing this article has not been banned yet. So, it is undeniable that this new substance should be monitored. 5-methoxy-N-methyl-N-isopropyltryptamine has been reported by the Spanish Early Warning System and detected in our laboratory in two pill samples purchased in a local smart shop. This has promoted the need of stablishing consumption markers for this compound in consumers’ urine. In the present work, the metabolism and pharmacokinetic of 5-methoxy-N-methyl-N-isopropyltryptamine has been studied by an in vivo approach, using adult male mice of the inbred strain C57BLJ/6. The use of ultra-high performance liquid chromatography coupled to high resolution mass spectrometry allowed the identification of four metabolites. After the pharmacokinetic study in serum and urine, the O-demethylated metabolite and the non-metabolised parent compound are proposed as consumption markers in hydrolysed urine. Data reported in this work will help hospitals and forensic laboratories to monitor the consumption and potential intoxication cases related to this tryptamine.

Melatonin (N-acetyl-5-methoxytryptamine) is a highly pleiotropic hormone with antioxidant, antiproliferative, oncolytic and neuroprotective properties. Here, we present evidence that the N-acetyl side chain plays a key role in melatonin's antiproliferative effect in HT22 and sw-1353 cells, but it does so at the expense of antioxidant and neuroprotective properties. Removal of the N-acetyl group enhances the antioxidant and neuroprotective properties of the indole, but it can lead to toxic methamphetamine-like effects in several cell lines. Inhibition of NFkB mimicked melatonin's antiproliferative and antioxidant effects, but not neuroprotection. Our results strongly suggest that neuroprotective and antiproliferative effects of melatonin rely on different parts of the molecule and are likely mediated by different mechanisms. We also predict that melatonin metabolism by target cells could determine whether melatonin inhibits cell proliferation, prevents toxicity or induces cell death (e.g. apoptosis or autophagy). These observations could have important implications for the rational use of melatonin in personalized medicine.

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.

Melatonin is used in the therapy of sleep and mood disorders and as a neuroprotective agent. The aim of our study was to demonstrate that melatonin supported (via its deacetylation to 5-methoxytryptamine) CYP2D-mediated synthesis of serotonin from 5-methoxytryptamine. We measured serotonin tissue content in some brain regions (the cortex, hippocampus, nucleus accumbens, striatum, thalamus, hypothalamus, brain stem, medulla oblongata, and cerebellum) (model A), as well as its extracellular concentration in the striatum using an in vivo microdialysis (model B) after melatonin injection (100 mg/kg i.p.) to male Wistar rats. Melatonin increased the tissue concentration of serotonin in the brain structures studied of naïve, sham-operated, or serotonergic neurotoxin (5,7-dihydroxytryptamine)-lesioned rats (model A). Intracerebroventricular quinine (a CYP2D inhibitor) prevented the melatonin-induced increase in serotonin concentration. In the presence of pargyline (a monoaminoxidase inhibitor), the effect of melatonin was not visible in the majority of the brain structures studied but could be seen in all of them in 5,7-dihydroxytryptamine-lesioned animals when serotonin storage and synthesis via a classic tryptophan pathway was diminished. Melatonin alone did not significantly increase extracellular serotonin concentration in the striatum of naïve rats but raised its content in pargyline-pretreated animals (model B). The CYP2D inhibitor propafenone given intrastructurally prevented the melatonin-induced increase in striatal serotonin in those animals. The obtained results indicate that melatonin supports CYP2D-catalyzed serotonin synthesis from 5-methoxytryptamine in the brain in vivo, which closes the serotonin-melatonin-serotonin biochemical cycle. The metabolism of exogenous melatonin to the neurotransmitter serotonin may be regarded as a newly recognized additional component of its pharmacological action.

Involvement of hormonal response (catecholamine release) to acute hypoxia induced by radioprotectors in modification of their radioprotective properties was studied in experiments on outbred mature female albino mice, female albino rats, and dogs of both sexes. The response intensity was evaluated by the reduction of radioprotective and toxic properties of indralin (a α1-adrenoceptor agonist and a radioprotector). The radioprotective effect of indralin was measured using lethal doses of whole-body γ-irradiation ((60)Co) and its acute toxicity was assessed by LD50. It was found that repeated administration of indralin with 30-60-min intervals was followed by weakening of its radioprotective effect. Similar sensitization effect of indralin was observed after pretreatment with cystamine and epinephrine. Comparison of the severity of sensitization after administration of epinephrine and cystamine in the dose providing radioprotective effect showed that the potential aminothiol-induced release of catecholamines can provide optimal long-term radioprotective effect of epinephrine.

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 (OaSNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing OaSNAT in their cytoplasm. To localize the OaSNAT in chloroplasts, we used a chloroplast targeting sequence (CTS) from tobacco protoporphyrinogen IX oxidase (PPO), which expresses in chloroplasts. The purified recombinant CTS:OaSNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast-targeted CTS:OaSNAT lines and cytoplasm-expressed OaSNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS:OaSNAT lines than in the OaSNAT 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.

Melatonin and its metabolites including 6-hydroxymelatonin (6(OH)M), N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and 5-methoxytryptamine (5MT) are endogenously produced in human epidermis. This production depends on race, gender and age. The highest melatonin levels are in African-Americans. In each racial group they are highest in young African-Americans [30-50 years old (yo)], old Caucasians (60-90 yo) and Caucasian females. AFMK levels are the highest in African-Americans, while 6(OH)M and 5MT levels are similar in all groups. Testing of their phenotypic effects in normal human melanocytes show that melatonin and its metabolites (10(-5) M) inhibit tyrosinase activity and cell growth, and inhibit DNA synthesis in a dose dependent manner with 10(-9) M being the lowest effective concentration. In melanoma cells, they inhibited cell growth but had no effect on melanogenesis, except for 5MT which enhanced L-tyrosine induced melanogenesis. In conclusion, melatonin and its metabolites [6(OH)M, AFMK and 5MT] are produced endogenously in human epidermis and can affect melanocyte and melanoma behavior.

Background According to the European Drug Report, the use of novel psychoactive substances (NPS) is constantly growing. NPS are widely abused by human adolescent subjects. 5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is one of the most frequently used hallucinogenic NPS. 5-MeO-DIPT intoxication results in hallucinations, vomiting, and tachycardia. Long-term exposure to 5-MeO-DIPT was reported to lead to development of post-hallucinogenic perception disorder. The aim of the present study was to determine whether repeated-intermittent administration of 5-MeO-DIPT during adolescence affects learning and memory in adult rats. Methods Rats were treated with 5-MeO-DIPT in a dose of 2.5 mg/kg from 30 to 33 and 37 to 40 Postnatal Day (PND). The experiments were conducted when the animals reached 90 PND. The effect of 5-MeO-DIPT on cognitive functions was assessed using the novel object recognition, open field, and serial pattern learning (SPL) tests. Results Repeated-intermittent exposure to 5-MeO-DIPT during adolescence decreased the number of crossings in the open field test at adulthood. Moreover, 5-MeO-DIPT treatment impaired adult rats’ learning in the SPL test. There was no change in the novel object recognition test. Conclusions The present results show that the performance of adult rats treated with 5-MeO-DIPT during adolescence was impaired in the open field test, which indicates the attenuated exploratory activity. 5-MeO-DIPT treatment undermined adult rats’ performance in the serial pattern learning test, suggesting impairment of long term memory and cognitive flexibility. The present study showed that the exposure to 5-MeO-DIPT during adolescence might lead to long-lasting behavioral changes which persisted long after the exposure period.

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. peerReviewed

This review gives a comparative evaluation of the radioprotective properties and the therapeutic index (TI) of radioprotectors from various pharmacological group in experiments on both small and large animals. It presents a hypothesis explaining the decrease in the TI of cystamine and 5-methoxytryptamine (mexamine), and the retention of that of α1-adrenomimetic indralin, and also compares the effects on large and small animals. The considerable differences in the therapeutic indices of catecholamines, serotonin and cystamine are a consequence of specific features of their mechanisms of radioprotective action. Radioprotectors acting via receptor mediation tend to provide a more expanded window of protection. The reduction in the TI of cystamine in larger animals, such as dogs, may be caused by the greater increase in toxicity of aminothiols in relation to the decrease in their optimal doses for radioprotective effect in going from mice to dogs, which is a consequence of the slower metabolic processes in larger animals. The somatogenic phase of intoxication by cystamine is significantly longer than the duration of its radioprotective effect, and increases with irradiation. The decrease in the radioprotective effect and the TI of mexamine in experiments with dogs may be caused by their lower sensitivity to the acute hypoxia induced by the mexamine. This is because of lower gradient in oxygen tension between tissue cells and blood capillaries under acute hypoxia that is determined by lower initial oxygen consumption in a large animal as compared with a small animal. Indralin likely provides optimal radioprotective effects and a higher TI for large animals via the increased specificity of its adrenergic effect on tissue respiration, which supports the development of acute hypoxia in the radiosensitive tissues of large animals. The stimulatory effect of indralin on early post-irradiation haematopoietic recovery cannot provide a high level of radioprotective action for large animals, but it may promote recovery.

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.

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.—Kim, T.-K., Kleszczyński, K., Janjetovic, Z., Sweatman, T., Lin, Z., Li, W., Reiter, R. J., Fischer, T. W., Slominski, A. T. Metabolism of melatonin and biological activity of intermediates of melatoninergic pathway in human skin cells.

Aging and advanced cancer are characterized by similar neuroendocrine and immune deficiencies; the most important of them consist of diminished nocturnal production of the pineal hormone melatonin (MLT) and decreased production of IL-2. At present, however, it is known that the pineal gland may produce indole hormones other than MLT. The most investigated of them is represented by 5-methoxy-tryptamine (5-MTT), which may exert antitumor, anticachectic, and immunomodulating effects under experimental conditions, in addition to those effects produced by MLT itself. In an attempt to obtain some preliminary data in human subjects about the potential therapeutic properties of 5-MTT, three different studies of 5-MTT have been carried out in advanced solid tumor patients. The first study of MLT plus 5-MTT included 14 thrombocytopenic cancer patients who did not respond to MLT alone. In the second study we have compared the clinical efficacy of MLT plus 5-MTT in a group of 25 untreatable metastatic cancer patients to the results obtained in a control group of 25 cancer patients receiving MLT alone. Finally, the third study of MLT plus 5-MTT included 14 untreatable metastatic cancer patients who did not respond to MLT alone. In all of these studies, MLT and 5-MTT were given orally at the level of 20 mg/day in the evening and at 5 mg/day during the period of maximum light. A normalization of platelet number was achieved by MLT plus 5-MTT in 5 of 14 (36%) thrombocytopenic cancer patients who did not respond to MLT alone. The percentage of disease control obtained by MLT plus 5-MTT in untreatable metastatic cancer patients was significantly higher than that achieved by MLT alone (15/25 [60%] vs. 8/25 [32%], P < 0.05). Finally, the association of 5-MTT with MLT induced disease stabilization in 4 of 14 (29%) untreatable metastatic cancer patients who did not respond to MLT alone.

Brain serotonin has been implicated in the pathophysiology of a wide spectrum of psychiatric disorders, as well as in the mechanism of action of psychotropic drugs. The aim of present study was to identify rat cytochrome P450 (CYP) isoforms which can catalyze the O-demethylation of 5-methoxytryptamine to serotonin, and to find out whether that alternative pathway of serotonin synthesis may take place in the brain. The study was conducted on cDNA-expressed CYPs (rat CYP1A1/2, 2A1/2, 2B1, 2C6/11/13, 2D1/2/4/18, 2E1, 3A2 and human CYP2D6), on rat brain and liver microsomes and on human liver microsomes (the wild-type CYP2D6 or the allelic variant 2D6*4*4). Of the rat CYP isoforms studied, CYP2D isoforms were the most efficient in catalyzing the O-demethylation of 5-methoxytryptamine to serotonin, but they were less effective than the human isoform CYP2D6. Microsomes from different brain regions were capable of metabolizing 5-methoxytryptamine to serotonin. The reaction was inhibited by the specific CYP2D inhibitors quinine and fluoxetine. Human liver microsomes of the wild-type CYP2D6 metabolized 5-methoxytryptamine to serotonin more effectively than did the defective CYP2D6*4*4 ones. The obtained results indicate that rat brain CYP2D isoforms catalyze the formation of serotonin from 5-methoxytryptamine, and that the deficit or genetic defect of CYP2D may affect serotonin metabolism in the brain. The results are discussed in the context of their possible physiological and pharmacological significance in vivo.

Aims: Besides acting as gastrointestinal prokinetic agents, 5-hydroxytryptamine(4) (5-HT4) receptor agonists can induce positive inotropism in human isolated atrium, but not in ventricles. We pharmacologically evaluated the gastroprokinetic 5-HT4 receptor agonists tegaserod, prucalopride, R199715, cisapride, the cisapride metabolite norcisapride, and the 5-HT3 receptor agonist MKC773 on human isolated myocardial trabeculae, and compared their effects with those induced by 5-HT and 5-methoxytryptamine (5-MeOT). Main methods: Atrial and ventricular trabeculae were paced and changes in contractile force were studied in the absence or presence of the 5-HT4 receptor antagonist GR113808. Partial agonism was assessed using 5-HT4 receptor agonists as antagonists against 5-HT. To test the contribution of L-type calcium channels, the inotropic responses to 5-HT and 5-MeOT were studied in the absence or presence of verapamil. Key findings: Like 5-HT and 5-MeOT, cisapride and tegaserod, but not prucalopride, R19971 and MKC-733, induced concentration-dependent positive inotropic responses on atrial trabeculae, which were abolished by GR113808. The L-type calcium channel blocker verapamil attenuated inotropic responses to 5-HT and 5-MeOT. None of the agonists affected the contraction of left ventricular trabeculae. Concentration response curves to 5-HT were shifted to the right in the presence of prucalopride, cisapride Significance: We conclude that (i) inotropic responses to 5-HT and 5-MeOT seem to depend on L-type calcium channels, (ii) tegaserod and cisapride behave as partial 5-HT4 receptor agonists, while prucalopride, norcisapride and MKC-733 cause no significant effects on human atrial trabeculae, (iii) R199715 seems to behave as a 5-HT4 receptor antagonist. (C) 2012 Elsevier Inc. All rights reserved.

Foxy or Methoxy Foxy (5-methoxy- N , N -di(iso)propyltryptamine hydrochloride; 5-MeO-DIPT) is rapidly gaining popularity among recreational users as a hallucinogenic “designer drug.” Unfortunately, much remain unknown about the consequences of its use on neuropsychological development or behavior. During one of two adolescent periods, the rats were given repeated injections of 5 mg/kg or 20 mg/kg of 5-MeO-DIPT or a corresponding volume of isotonic saline. After the animals reached adulthood, they were trained and tested on a number of tasks designed to assess the impact of 5-MeO-DIPT, if any, on spatial memory, presumably involving declarative memory systems as well as a nonspatial task that is considered sensitive to disruptions in nondeclarative memory. Both the 5-MeO-DIPT- and saline-treated rats were able to master spatial navigation tests where the task included a single goal location and all groups performed comparably on these phases of training and testing. Regardless of exposure level during adolescence, the performance of the drug-treated rats was markedly inferior to that of the control animals on a task where the goal was moved to a new location and on a response learning task, suggesting a lack of flexibility in adapting their responses to changing task demands. Detected reductions in serotonin activity in the forebrain similar to the effects of extensively investigated compounds such as methylenedioxymethamphetamine (MDMA), suggest that 5-MeO-DIPT may produce its adverse effects by compromising serotonergic systems in the brain.

Melatonin (Mel) is the major biologically active molecule secreted by the pineal gland. Mel and its metabolites, 6-hydroxymelatonin (6(OH)Mel) and 5-methoxytryptamine (5-MT), possess a variety of functions, including the scavenging of free radicals and the induction of protective or reparative mechanisms in the cell. Their amphiphilic character allows them to cross cellular membranes and reach subcellular organelles, including the mitochondria. Herein, the action of Mel, 6(OH)Mel, and 5-MT in human MNT-1 melanoma cells against ultraviolet B (UVB) radiation was investigated. The dose of 50 mJ/cm2 caused a significant reduction of cell viability up to 48%, while investigated compounds counteracted this deleterious effect. UVB exposure increased catalase activity and led to a simultaneous Ca++ influx (16%), while tested compounds prevented these disturbances. Additional analysis focused on mitochondrial respiration performed in isolated mitochondria from the liver of BALB/cJ mice where Mel, 6(OH)Mel, and 5-MT significantly enhanced the oxidative phosphorylation at the dose of 10&minus, 6 M with lower effects seen at 10&minus, 9 or 10&minus, 4 M. In conclusion, Mel, 6(OH)Mel and 5-MT protect MNT-1 cells, which express melatonin receptors (MT1 and MT2) against UVB-induced oxidative stress and mitochondrial dysfunction, including the uncoupling of oxidative phosphorylation.

The biogenic amines are widespread regulators of physiological processes, and play an important role in regulating heart rate in diverse organisms. Here, we present the first pharmacological evidence for a role of the biogenic amines in the regulation of dorsal blood vessel pulse rate in an aquatic oligochaete, Lumbriculus variegatus (Müller, 1774). Bath application of octopamine to intact worms resulted in an acceleration of pulse rate, but not when co-applied with the adenylyl cyclase inhibitor MDL-12,330a. The phosphodiesterase inhibitor theophylline mimicked the effects of OA, but the polar adenosine receptor antagonist 8(p-sulphophenyl)theophylline was significantly less potent than theophylline. Pharmacologically blocking synaptic reuptake of the biogenic amines using the selective 5-HT reuptake blocker fluoxetine or various tricyclic antidepressants also accelerated heart rate. Depletion of the biogenic amines by treatment with the monoamine vesicular transporter blocker reserpine dramatically depressed pulse rate. Pulse rate was partially restored in amine-depleted worms after treatment with octopamine or dopamine, but fully restored following treatment with serotonin. This effect of 5-HT was weakly mimicked by 5-methoxytryptamine, but not by alpha-methylserotonin; it was completely blocked by clozapine and partially blocked by cyproheptadine. Because they are known to orchestrate a variety of adaptive behaviors in invertebrates, the biogenic amines may coordinate blood flow with behavioral state in L.variegatus.

Environmental day length drives nocturnal pineal melatonin secretion, which in turn generates or entrains seasonal cycles of physiology, reproduction, and behavior. In mammals, melatonin (MEL) binds to a number of receptor subtypes including high-affinity (MT1 and MT2) and low-affinity (MT3, nuclear orphan receptors) binding sites, which are distributed throughout the central nervous system and periphery. The MEL receptors that mediate photoperiodic reproductive and behavioral responses to MEL have not been identified in a reproductively photoperiodic species. Here I tested the hypothesis that MT1 receptors are necessary and sufficient to engage photoperiodic responses by challenging male Siberian hamsters (Phodopus sungorus), a species that does not express functional MT2 receptors, with ramelteon (RAM), a specific MT1/MT2 receptor agonist. In hamsters housed in a long-day photoperiod, late-afternoon RAM treatment inhibited gonadotropin secretion, induced gonadal regression, and suppressed food intake and body mass, mimicking effects of MEL. In addition, chronic (24 h/d) RAM infusions were sufficient to obscure endogenous MEL signaling, and these treatments attenuated gonadal regression in short days. Together, the outcomes indicate that signaling at the MT1 receptor is sufficient and necessary to mediate the effects of photoperiod-driven changes in MEL on behavior and reproductive function in a reproductively photoperiodic mammal.

Polycyclic aromatic hydrocarbons (PAHs) have deleterious effects on neuroendocrine systems in teleost fish affecting, among other processes, reproductive function or stress responses. The hormone melatonin, mainly produced in the pineal organ of vertebrates, is involved in the regulation of biological rhythms as well as other important functions, and may also act as an antioxidant molecule. The effects of environmental pollutants on the endocrine and metabolic activity of the pineal organ have been studied only in mammals. We here evaluate the effects of the PAHs naphthalene (NAP) and benzo(a)pyrene (BaP) and the flavonoid beta-naphthoflavone (BNF) on the pineal organ of rainbow trout by quantifying the diurnal and nocturnal pineal content of some indoles and methoxyindoles, including melatonin. NAP mainly induced diurnal increases in the pineal content of melatonin and other methoxyindoles like 5-methoxytryptamine (5-MT), 5-methoxyindole-3-acetic acid (5-MIAA) or 5-methoxytryptophol (5-MTOL). Those increases did not occur at night, when even occasional decreases were observed compared with controls. NAP also induced some diurnal and nocturnal decreases in the levels of indolic compounds like serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA), while pineal content of 5-hydroxytryptophan (5-HTP) was first decreased (few hours after injection) and then increased (few days after injection) during the day. BaP and BNF induced strong increases in diurnal levels of melatonin, whereas other pineal compounds were unaffected. It seems that an increase of the methylation capacity of the pineal organ takes place during the day, and a decrease occurs at night. Those effects could be mediated by changes in the activity of key enzymes involved in pineal melatonin biosynthesis, maybe as a result of the alteration of the cellular phototransduction mechanisms involved in the light-induced inhibition of melatonin synthesis in the pineal photoreceptor cells. These results demonstrate for the first time that environmental pollutants can disrupt the activity of the pineal organ of teleost fish. This disruption could be a threat for the survival of the animals in their natural environment, although the increases observed in melatonin levels could play a relevant role as a toxicity-protection factor.

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.

Numbers of individuals infected with Human Immunodeficiency Virus (HIV) are increasing in Japan. The majority of them are Men who have sex with men and a part of them take drugs as 'Sex drug' at their sexual intercourse. Especially, Amyl nitrite, Methamphetamine, 5-methoxy-N, N-diisopropyltryptamine (5-MeO-DIPT; Foxy), and 3, 4-methylenedioxy- methamphetamine (MDMA; Ecstasy) are used, and they sometimes cause the physical and mental disorders. However, the actual drug inducing troubles among Japanese HIV-infected drug users had not yet been discussed enough. In this report, we describe three cases with HIV infection; a case developed severe neuroleptic malignant syndrome (NMS) after taking 5-MeO-DIPT, a case with persistent convulsion due to multiple drug intake and a case with rhabdomyolysis due to the non-subjective methamphetamine intake. Through these cases, we raise and discuss several underlying problems associated with drug use among HIV-infected individuals.

A 23-year-old Japanese woman was brought to the emergency department about 6.5 h after taking liquid and later a half tablet purchased on the street. About 4.5 h prior to presentation, she displayed excited and disorganized behavior. On examination, she was not alert or oriented, with a Glasgow Coma Scale score of 13, did not answer any questions from doctors while smirking and looking around restlessly, and sometimes exhibited echolalia, imitating the speech of doctors. She was given intravenous infusion of fluid for 8 h, then discharged. Gas chromatography-mass spectrometry of urine revealed 5-methoxy-diisopropyltryptamine, 5-methoxy-N-methyltryptamine and an unidentified tryptamine. Identifying chemical products based solely on information of users is insufficient, and urinalysis is necessary in cases potentially involving designer drugs.

This work aims at studying the optimization of an on-line capillary electrophoresis (CE)-based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on-line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.

The hallucinogenic “designer drug” known as Foxy or Methoxy Foxy and formally know as 5-Methoxy-N,N-di(iso)propyltryptamine hydrochloride (5-MeO-DIPT) is rapidly gaining popularity among recreational users. However, little is known about the consequences of its use on neuropsychological development or behavior. During one of two adolescent periods, the rats were given repeated injections of either saline or 5 mg/kg of 5-MeO-DIPT. Once the animals reached 80 days of age, they were trained and tested on a number of tasks designed to assess the effects of 5-MeO-DIPT, if any, on memory tasks with spatial components that presumably involve declarative memory systems and on a nonspatial task that is considered sensitive to disruptions in nondeclarative memory. With one exception, both the 5-MeO-DIPT-and saline-treated rats were able to master the spatial navigation tests at comparable rates. However, the performance of the drug-treated rats was markedly inferior to that of the control animals on a response-learning task, suggesting a lack of flexibility in adapting their responses to changing task demands. This could indicate reductions in serotonin activity in the forebrain similar to the effects of studied drugs such as methylenedioxymethamphetamine (MDMA), suggesting 5-MeO-DIPT may act as a toxin compromising serotoninergic systems in the brain.

Lymphocyte number still remains one of the most important immune parameters predicting the prognosis of advanced cancer patients. IL-2 and IL-12 are the main antitumor cytokines in humans, and their effect is modulated by the neuroendocrine system, mainly by the pineal gland through the circadian release of melatonin (MLT) and perhaps that of other indole hormones, such as 5-methoxytryptamine (5-MTT), and 5-methoxytryptophol (5-MTP). MLT has been proven to exert important antitumor immunomodulating effects, whereas the possible immunomodulatory properties of the other pineal indoles are still controversial. In an attempt to better define the pineal neuroendocrine regulation of the anticancer cytokine network, we have evaluated in metastatic solid-tumor patients the effects on lymphocyte number induced by different neuroimmune regimens, consisting of MLT alone (20 mg/day orally in the evening), subcutaneous (s.c.) low-dose IL-2 alone (3 MIU/day in the evening for 6 days/week), s.c. low-dose IL-12 alone (0.5 mcg/kg once/week in the morning), IL-12 plus MLT, IL-2 plus MLT, and IL-2 plus MLT plus 5-MTT (10 mg/day orally in the afternoon) plus 5-MTP (5 mg/day orally at noon). The results showed the following evidence: (1) MLT alone is unable to induce lymphocytosis; (2) MLT significantly enhances IL-2-induced lymphocytosis; (3) IL-12 alone determines lymphocytopenia, which can be reversed by MLT; (4) IL-2 plus IL-12 induces a very pronounced lymphocytosis, which can be further amplified by MLT; (5) a total pineal endocrine replacement therapy with MLT, 5-MTT, and 5-MTP further increases IL-2-induced lymphocytosis with respect to MLT plus IL-2 alone. Therefore, this study confirms that IL-2- and IL-12-dependent anticancer immunity is under a pineal modulation.

Few studies have examined the effects of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in vivo. In these studies, 5-MeO-DIPT was tested in a drug-elicited head twitch assay in mice where it was compared to the structurally similar hallucinogen N,N-dimethyltryptamine (N,N-DMT) and challenged with the selective serotonin (5-HT)2A antagonist M100907, and in a lysergic acid diethylamide (LSD) discrimination assay in rats where its subjective effects were challenged with M100907 or the 5-HT 1A selective antagonist WAY-100635. Finally, the affinity of 5-MeO-DIPT for three distinct 5-HT receptors was determined in rat brain. 5-MeO-DIPT, but not N,N-DMT, induced the head twitch responses in the mouse, and this effect was potently antagonized by prior administration of M100907. In rats trained with LSD as a discriminative stimulus, there was an intermediate degree (75%) of generalization to 5-MeO-DIPT and a dose-dependent suppression of response rates. These interoceptive effects were abolished by M100907, but were not significantly attenuated by WAY-100635. Finally, 5-MeO-DIPT had micromolar affinity for 5-HT 2A and 5-HT 2C receptors, but much higher affinity for 5-HT 1A receptors. 5-MeO-DIPT is thus effective in two rodent models of 5-HT2 agonist activity, and has affinity at receptors relevant to hallucinogen effects. The effectiveness with which M100907 antagonizes the behavioral actions of this compound, coupled with the lack of significant antagonist effects of WAY-100635, strongly suggests that the 5-HT 2A receptor is an important site of action for 5-MeO-DIPT, despite its apparent in vitro selectivity for the 5-HT 1A receptor.

The urinary metabolites of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in humans have been investigated by analyzing urine specimens from its users. For the unequivocal identification and accurate quantification of its major metabolites, careful analyses were conducted by gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and liquid chromatography-tandem mass spectrometry, using authentic standards of each metabolite synthesized. Three major metabolic pathways were revealed as follows: 1) side chain degradation by O-demethylation to form 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT), which would be partly conjugated to its sulfate and glucuronide; 2) direct hydroxylation on position 6 of the aromatic ring of 5-MeO-DIPT, and/or methylation of the hydroxyl group on position 5 after hydroxylation on position 6 of the aromatic ring of 5-OH-DIPT, to produce 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (6-OH-5-MeO-DIPT), followed by conjugation to its sulfate and glucuronide; and 3) side chain degradation by N-deisopropylation, to the corresponding secondary amine 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT). Of these metabolites, which retain structural characteristics of the parent drug, 5-OH-DIPT and 6-OH-5-MeO-DIPT were found to be more abundant than 5-MeO-NIPT. Although the parent drug 5-MeO-DIPT was detectable even 35 h after dosing, no trace of its N-oxide was detected in any of the specimens examined.

Locomotor sensitisation and deficits in prepulse inhibition (PPI) induced by psychostimulants are two paradigms that have been widely studied as animal behavioural models of psychosis. Clozapine is one of the atypical antipsychotic agents which has been widely employed to reverse the aforementioned behavioural changes in these usual models. In this particular study, locomotor sensitisation and prepulse inhibition deficit were induced under the same context by intermittent oral administration of S-(-)-cathinone or Catha edulis extract in rats. The rats were then challenged by administration of the atypical antipsychotic drug, clozapine and were finally challenged with psychostimulants after 2-week of withdrawal. Locomotor activity and PPI were assessed and later analyses of the neurotransmitter levels were made. The results of this experiment show that repeated oral administration of cathinone or C. edulis extract enhanced locomotor and exploratory activity and lead to a gradual deficit in prepulse inhibition. This locomotor sensitisation and PPI deficit could be reversed by administration of clozapine. A challenge with psychostimulant on day 40 (i.e., after 2-week of withdrawal) resulted in a response similar to the initial exposure (day 1). Neurotransmitter level analyses showed a significant increase in the level of dopamine in the prefrontal cortex (p < 0.05). There was also a significant decrease in the level of 5-hydroxytryptamine (5-HT) in the nucleus accumbens (p < 0.05) and its metabolite, 5-hydroxyindole acetic acid (5-HIAA) in the prefrontal cortex (p < 0.01). In the remaining regions (anterior and posterior striatum), there were no significant changes. In conclusion, this is the first study to demonstrate that repeated administration of C. edulis extract, or commercial cathinone, induces prepulse inhibition deficit and clozapine reverses both C. edulis or cathinone-induced sensitised locomotion and prepulse inhibition deficit.

The pharmacological effect of the 5-HT1A receptor ligands, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), indorenate, and buspirone, alone or in combination with the antagonist MM-77, was studied in mice subjected to forced swimming. It was confirmed that this stressful factor produces an anxiolytic-like effect, which is reversed by the mentioned 5-HT1A receptor agonists. Only the 8-OH-DPAT-induced decrease of such an effect could be blocked by the postsynaptic antagonist of the 5-HT1A receptor 1-(2-methoxyphenyl)-4-[(4-succinimido)butyl]-piperazine (MM-77). Stressing by forced swimming seems to induce plastic changes in 5-HT1A receptors, which in turn modify the behavioural actions of 5-HT1A receptor agents.

We have previously determined that melatonin modulates hippocampal synaptic transmission in a biphasic way: an initial depression was followed by a recovery/amplification phase. Here we describe the influence of two novel melatonin receptor ligands, BMNEP (N-bromoacetyl-2-iodo-5-methoxytryptamine) and TMPEI (N-[2-(2-Trimethylammoniumethyleneoxy-7-methoxy)ethyl]propionamide iodide), on the population spike (PS) and excitatory postsynaptic potentials (EPSP) recorded from mouse hippocampal slices. BMNEP, which specifically alkylates and constitutively activates the MT2 melatonin receptor, mimicked the first phase of melatonin's action by irreversibly depressing both the PS and EPSP. TMPEI, a charged ligand of plasma membrane melatonin receptors, amplified those potentials in a manner similar to the effect of melatonin observed during the second, recovery phase. Melatonin had no influence on the potentials amplified by the action of TMPEI. Our results suggest that the biphasic, receptor-dependent action of melatonin and its analogs modulates the efficiency of the hippocampal glutamergic synapse and is most likely mediated through two different, sequentially occurring mechanisms.

Over the past 10 years, there has been a resurgence of interest in examining the role of melatonin in health and disease. While the brunt of research in this area has portrayed melatonin in a favorable light, there is a growing body of evidence suggesting that melatonin may possess adverse effects contributing to the development of various neuropsychiatric disease states. In preclinical models of Parkinson's disease (PD), melatonin has been shown to enhance the severity of this condition while its antagonism, using constant light or pinealectomy, facilitates recovery. To test this hypothesis further, the present study employed the melatonin analogues ML-23 and S-20928 in a post-6-OHDA injection regime to determine whether they may have a favorable effect on the symptoms of this more chronic model of PD. When ML-23 was injected I.P. in a dose of 3 mg/kg twice daily for 3.5 days after 6-OHDA, significant improvement in motor function and regulatory deficits was observed. Similarly, the injection of S-20928 in a 1 mg/kg dose (I.P.), in the same regimen, facilitated modest improvement in motor function and regulatory deficits while the larger dose enhanced the severity of behavioural deficits and produced severe side effects causing deterioration in condition during the course of drug administration. ML-23 administration totally abolished the 6-OHDA-induced mortality, which accompanies dopamine (DA) degeneration, while S-20928 had no effect on this parameter. These results suggest that some melatonin analogues can aid in recovery from DA depleting lesions after DA degeneration has commenced and the recovery is not attributable to the antioxidative properties of this hormone. While the exact mechanism by which ML-23 and S-20928 are exerting their therapeutic effect is unclear, it is possible that antagonism of melatonin receptors may play some role and this should be considered when assessing the potential of melatonin analogues for treatment of human neuropsychiatric disorders.

There is evidence that melatonin and its metabolites could bind to nuclear sites in neurones, suggesting that this hormone is able to exert long-term functional effects in the central nervous system via genomic mechanisms. This study was designed to investigate (i) whether systemically administered melatonin can exert long-term effects on spinal cord windup activity, and (ii) whether blockade of melatonin degradation with eserine could prevent this effect. Rats receiving melatonin (10 mg/kg ip), the same dose of melatonin plus eserine (0.5 mg/kg ip), or saline were studied. Seven days after administration of the drugs or saline, spinal windup of rats was assessed in a C-fiber reflex response paradigm. Results show that rats receiving melatonin exhibited a reduction in spinal windup activity. This was not observed in the animals receiving melatonin plus eserine or saline, suggesting a role for melatonin metabolites in long-term changes of nociceptive transmission in the rat spinal cord.

The overflow of tritium from stimulated rat hypothalamic slices preincubated with [3H]-serotonin (5-HT) was significantly enhanced by reducing the frequency of stimulation from 3 Hz to 1 Hz while keeping the number of impulses constant. The 5-HT receptor agonist 5-methoxytryptamine inhibited in a concentration-dependent manner the electrically-evoked release of [3H]-5-HT with IC50 values of 560 nmol/l and of 34 nmol/l when the stimulations were delivered at 3 Hz and 1 Hz, respectively. The terminal 5-HT autoreceptor antagonist methiothepin enhanced in a concentration-dependent manner the electrically-evoked release of [3H]-5-HT and this effect was greater at a frequency of stimulation of 3 Hz than at 1 Hz. In the same paradigm, the 5-HT reuptake inhibitors citalopram and paroxetine did not alter the overflow of radioactivity elicited by stimulation at 3 Hz but significantly decreased it at 1 Hz. In the presence of 5-HT autoreceptor blockade achieved with methiothepin, citalopram increased the overflow of [3H]-5-HT to the same extent at 1 Hz and at 3 Hz. The IC50 values for inhibition of [3H]-5-HT release by the selective alpha 2-adrenoceptor agonist UK 14.304 were 35 nmol/l at 3 Hz and 30 nmol/l at 1 Hz. It is concluded that modulation of 5-HT release by 5-HT autoreceptors, but not by alpha 2-adrenoceptors is dependent on the synaptic concentration of 5-HT as a function of the frequency of depolarization.

Rationale. Indorenate has been proposed to possess antihypertensive, anorectic, stimulus control and anxiolytic-like actions. This compound has affinity mainly for the serotonergic1A/1B receptors, hence it could possess antidepressant-like activity. Objectives. The general purpose of this study was to explore the possible antidepressant-like effects of the serotonergic compound indorenate in the forced swimming test (FST). Methods. In a first approach, a comparison of the actions of several doses of indorenate (2.5, 5.0, 10 mg/kg) with those of other 5-HT1A agonists, buspirone (5.0, 10.0 mg/kg) and 8-OH-DPAT (0.25, 0.50, 1.0 mg/kg), was performed in the FST. Secondly, in order to determine the serotonergic receptors that are participating in indorenate's action, different doses of serotonergic antagonists were administered. The compounds used were the 5-HT1A/1B and β-adrenergic antagonist pindolol (2.5, 5.0 mg/kg), the 5-HT1B receptor antagonist GR 55562 (0.75, 1.5, 3.0 mg/kg), the 5-HT1A antagonist WAY 100635 (0.25, 0.5, 1.0 mg/kg) and the 5-HT2 antagonist ketanserin (1.0, 2.0, 4.0 mg/kg). Results. Indorenate (10 mg/kg), 8-OH-DPAT (1.0 mg/kg) and buspirone (5.0 and 10.0 mg/kg) reduced immobility behaviour in the FST, considered as an antidepressant-like effect. Both doses of pindolol (2.5 and 5.0 mg/kg) and WAY 100635 (0.5 and 1.0 mg/kg) antagonised the antidepressant-like effect of indorenate. Neither 5-HT1B (GR55562) nor 5-HT2 (ketanserin) antagonists produced changes in the effect of indorenate in the FST. Conclusions. Indorenate produces antidepressant-like actions in the FST that are mediated by the stimulation of 5-HT1A receptors.

Melatonin and 5-methoxytryptamine inhibited forskolin-stimulated cyclic AMP formation in cultured neural cells prepared from embryonic chick retina. Both methoxyindoles exhibited similar potency and efficacy, with EC50 values of 0.8 nM for melatonin and 7.2 nM for 5-methoxytryptamine. Inhibition of cyclic AMP formation by 5-methoxytryptamine or melatonin was prevented by pretreatment with pertussis toxin. Pretreatment of cultures with 5-methoxytryptamine for 24 h reduced the subsequent inhibitory cyclic AMP response to 5-methoxytryptamine but not that to 2-iodomelatonin. Putative melatonin receptors on cultured retinal cells were labeled with 2-[125I]iodomelatonin. Melatonin displaced specific 2-[125I]iodomelatonin with a Ki value (0.8 nM) similar to the EC50 for inhibition of cyclic AMP formation. In contrast, 5-methoxytryptamine only inhibited 2-[125I]iodomelatonin binding at very high concentrations (Ki = 650 nM). Pretreating cultured cells for 24 h with 2-iodomelatonin or melatonin, but not with 5-methoxytryptamine, reduced subsequent 2-[125I]iodomelatonin binding. Thus, 5-methoxytryptamine appears to inhibit forskolin-stimulated cyclic AMP formation at a site distinct from the 2-iodomelatonin binding site.

Exposure of neuronal cells to the Alzheimer's amyloid beta protein (Abeta) results in extensive oxidative damage of bio-molecules that are profoundly harmful to neuronal homeostasis. It has been demonstrated that melatonin protects neurons against Abeta-mediated neurotoxicity, including cell death and a spectrum of oxidative lesions. We undertook the current study to determine whether melatonin membrane receptors are involved in the mechanism of neuroprotection against Abeta neurotoxicity. For this purpose, we characterized the free-radical scavenging potency of several compounds exhibiting various affinities for melatonin membrane receptors (MLT 1a and 1b). Abeta-mediated neurotoxicity was assessed in human neuroblastoma cells and in primary hippocampal neurons. In sharp contrast with melatonin, no neuroprotection against Abeta toxicity was observed when we used melatonin membrane receptor agonists that were devoid of antioxidant activity. In contrast, the cells were fully protected in parallel control experiments when either melatonin, or the structurally unrelated free-radical scavenger phenyl-N-t-butyl nitrone (PBN), were added to Abeta-containing culture media. This study demonstrates that the neuroprotective properties of melatonin against Abeta-mediated toxicity does not require binding of melatonin to a membrane receptor and is likely the result of the antioxidant and antiamyloidogenic features of the agent.

Ventral spinocerebellar tract neurons located in laminae V–VII of cat lumbar spinal cord were tested for the effects of ionophoretically applied monoamines and receptor selective agonists. Extracellularly recorded responses, monosynaptically evoked by group I afferents in a muscle nerve, were compared before, during, and after ionophoresis. They were analyzed with respect to changes in the number of evoked spikes and in the latency. Both serotonin (5-HT) and noradrenaline (NA) were found to facilitate responses of all neurons tested. Ionophoresis of three serotonin subtype receptor agonists (5-carboxamidotryptamine maleate, 5 methoxytryptamine HCl, and alpha-methyl 5-hydroxytryptamine) and of two NA receptor agonists (phenylephrine and isoproterenol) likewise had a facilitatory effect. However, three other 5-HT receptor agonists (8-hydroxy-dipropylaminotetraline hydrobromide), 2-methyl 5-hydroxytryptamine, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl and two NA receptor agonists (tizanidine and clonidine) had the opposite effect because they depressed responses of the tested neurons. These results show that information forwarded by means of the ventral spinocerebellar tract may be modulated by monoamines and that several receptor subtypes, located pre- or postsynaptically, may be involved. The results also demonstrate that transmission by means of group I muscle afferents may not only be facilitated by monoamines but also depressed by selective receptor subtype activation. J. Comp. Neurol. 443:298–309, 2002. © 2002 Wiley-Liss, Inc.

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.

The cytochrome P450 2D (CYP2D) mediates synthesis of serotonin from 5-methoxytryptamine (5-MT), shown in vitro for cDNA-expressed CYP2D-isoforms and liver and brain microsomes. We aimed to demonstrate this synthesis in the brain in vivo. We measured serotonin tissue content in brain regions after 5-MT injection into the raphe nuclei (Model-A), and its extracellular concentration in rat frontal cortex and striatum using an in vivo microdialysis (Model-B) in male Wistar rats. Naive rats served as control animals. 5-MT injection into the raphe nuclei of PCPA-(tryptophan hydroxylase inhibitor)-pretreated rats increased the tissue concentration of serotonin (from 40 to 90% of the control value, respectively, in the striatum), while the CYP2D inhibitor quinine diminished serotonin level in some brain structures of those animals (Model-A). 5-MT given locally through a microdialysis probe markedly increased extracellular serotonin concentration in the frontal cortex and striatum (to 800 and 1000% of the basal level, respectively) and changed dopamine concentration (Model-B). Quinine alone had no effect on serotonin concentration; however, given jointly with 5-MT, it prevented the 5-MT-induced increase in cortical serotonin in naive rats and in striatal serotonin in PCPA-treated animals. These results indicate that the CYP2D-catalyzed alternative pathway of serotonin synthesis from 5-MT is relevant in the brain in vivo, and set a new target for the action of psychotropics.

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.

Recent studies have shown that the hematopoietic system is under neuroendocrine control. In particular, thrombopoiesis has been proven to be stimulated by melatonin, and the pineal indole has been shown to be effective in the treatment of thrombocytopenia resulting from different causes. At present, however, there are no data concerning the possible thrombopoietic activity of pineal indoles other than melatonin. The present study was carried out to evaluate the effect of a concomitant administration of the pineal indole 5-methoxytryptamine in patients with cancer-related thrombocytopenia who did not respond to melatonin alone. The present study included 30 patients, who were randomized to receive melatonin alone (20 mg/day orally in the evening) or melatonin plus 5-methoxytryptamine (1 mg/day orally in the early afternoon). A normalization of platelet count was achieved in 5/14 (36%) patients treated with melatonin plus 5-methoxytryptamine and in none of the patients treated with melatonin alone (P < 0.05). Moreover, mean platelet number significantly increased only in the patients treated with melatonin plus 5-methoxytryptamine. This preliminary clinical study would suggest that 5-methoxytryptamine, a pineal indole, may also exert thrombopoietic activity. Further studies, however, will be required to establish whether 5-methoxytryptamine may play a direct thrombopoietic activity, or whether it may act by improving melatonin's efficacy.

Indorenate (INDO), initially described as an antihypertensive agent, also has some effects on behaviour, with anxiolytic and anorectic actions being reported. The aim of the present experiment was to examine the activity of INDO at the behavioural level at various serotonin (5-hydroxytryptamine, 5-HT) receptor sites by comparing its stimulus properties with those of other 5-HT receptor agonists and by examining its interactions with some 5-HT antagonists. Rats were trained to discriminate between 10.0 mg/kg INDO (administered intraperitoneally (90 min before the start of the session) from saline. A Fixed Ratio 10 (FR10) schedule of reinforcement was in effect in each drug condition. During generalization test sessions, the discrimination index (DI, responses to drug lever/responses to drug + saline lever) was calculated from the responses emitted before the first reinforcer of the session. DI was a function of the dose of INDO employed. Generalization to the discriminative stimulus properties of INDO was observed with the 5-HT1A receptor agonist 8-OH-DPAT (1.0 mg/kg produced 90% generalization) and the 5-HT1B/2C receptor agonist 1-(3-trifluoromethylphenyl) piperazine (TFMPP) (3.0 mg/kg produced up to 75% generalization). Yohimbine (5.6 mg/kg), buspirone (1.0 mg/kg), 6-chloro-2-(1-piperaziny)pyrazine (1.0 mg/kg) and m-chlorophenyl piperazine (mCPP) (1.0 mg/kg) induced a DI of 70%, 50% and 48% and 55%, respectively. In generalization tests, ritanserin (0.01–1.0 mg/kg) induced saline-like responding. NAN-190 (3.0 mg/kg), a 5-HT1A receptor antagonist, was able to reduce the DI of INDO to 50%. Although the 5-HT2C/2A receptor antagonists cinanserin (10.0 mg/kg) and metergoline (0.3 mg/kg) were able to reduce the stimulus properties of INDO to 60% and 30%, respectively, only ritanserin (1.0 mg/kg) reduced the stimulus properties of INDO to 25% with a clear dose–response relationship. The results suggest that INDO acts as an agonist at 5-HT1A receptor sites, but its activity at 5-HT1B/2C receptor sites also contributes to its discriminative function.

It has been suggested that the external carotid vasodilatation produced by serotonin (5-hydroxytryptamine; 5-HT) in anaesthetised dogs with intact vagosympathetic trunks is mediated by sympatho-inhibitory '5-HT1D' receptors and musculotropic '5-HT1-like' receptors. The present study has re-analysed this suggestion with regard to the classification schemes recently proposed by the NC-IUPHAR subcommittee on 5-HT receptors. In pentobarbital-anaesthetised dogs with intact vagosympathetic trunks, 1-min intracarotid (i.c.) infusions of 5-carboxamidotryptamine (5-CT; 0.01-0.3 microg/min), 5-HT (0.3-30 microg/ min), 5-methoxytryptamine (5-MeO-T; 1-100 microg/min) or sumatriptan (1-100 microg/min) dose-dependently increased the external carotid blood flow without affecting blood pressure or heart rate. The selective 5-HT1D receptor agonist, PNU-142633 (1-1000 microg/min), was essentially inactive. After mesulergine (300 microg/kg, i.v.), an antagonist at cardiovascular 5-HT7 receptors, the above responses to 5-HT, 5-CT and 5-MeO-T were blocked, whilst those to sumatriptan remained unaffected. In contrast, after the 5-HT1B/1D receptor antagonist, GR127935 (10 microg/kg, i.v.), the responses to 5-HT, 5-CT and 5-MeO-T were not affected, but those to sumatriptan were abolished. Furthermore, after the selective 5-HT1B receptor antagonist, SB224289 (300 microg/kg, i.v.), the responses to 5-HT, 5-CT and 5-MeO-T were significantly enhanced, whereas those to sumatriptan were abolished. Interestingly, the responses to all these agonists remained unmodified after the selective 5-HT1D receptor antagonist, BRL15572 (300 microg/kg, i.v.). The above results suggest that the '5-HT1-like' receptors, which mediate canine external carotid vasodilatation, display the pharmacological profile of sympatho-inhibitory 5-HT1B receptors and musculotropic 5-HT7 receptors, and confirm the existence of vasoconstrictor 5-HT1B receptors.

In a study on the embryotoxicity of pineal indoles on developing chick embryos in vivo, the pineal indoles—namely, melatonin (MEL), methoxytryptamine (MTA) and methoxytryptophol (MTP)—were injected into the yolk sacs of the chick embryos through the air chambers of the eggs on the 4th day of incubation. The eggs were opened and the embryos examined after 6, 10 or 14/15 days of incubation. Abnormalities were found to occur mainly in the 6- and 10-day-old embryos, which exhibited external malformations such as twisted vertebral column, abdominal hernia, exteriorization of heart and viscera, defects of eye, beak and limb. From the results obtained from embryos on the 14th or 15th day of incubation, MEL was found to be the most toxic indole in regard to the mortality induced, whereas MTA had the highest teratogenicity because of the frequent incidence of abnormal embryos. Effect of MTP treatment on the development of chick embryos varied greatly between doses, and there were no abnormal embryos found on the 14th or l5th day of incubation.

It has been shown that 5-hydroxytryptamine and melatonin, an indoleamine for which 5-hydroxytryptamine is a precursor, influence the release of vasopressin and oxytocin from the rat hypothalamus both in vivo and in vitro. The oral administration of melatonin has been shown to decrease oxytocin release and modulate the nocturnal vasopressin release in humans. 5-hydroxytryptamine and its metabolites, 5-hydroxytryptophol, 5-methoxytryptamine and 5-methoxytryptophol, are detected within the pineal, and there is evidence that 5-methoxytryptamine and 5-methoxytryptophol may have some physiological role. The aim of this study was to evaluate the effects of 5-hydroxytryptamine, 5-hydroxytryptophol, 5-methoxytryptamine and 5-methoxytryptophol on neurohypophysial hormone release from the rat hypothalamus in vitro. It was found that 5-hydroxytryptamine and 5-hydroxytryptophol increased neurohypophysial hormone release, 5-methoxytryptamine decreased the release of vasopressin and oxytocin and 5-methoxytryptophol was found to have no effect, thus providing further evidence for a role of indole compounds in the control of neurohypophysial hormone secretion.