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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.

Purpose The dried urine spots (DUSs) technique is increasing continuously as an easy sampling method for monitoring substance abuse due to its advantages of stability and convenience regarding transport and storage. 5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a new type of tryptamine hallucinogen, the use of which has been banned in many countries. And according to the previous research, 5-MeO-DIPT is not stable in urine. In order to improve its stability, an LC-MS/MS method for determining 5-MeO-DIPT in DUSs was developed. Method 10 μl urine was spotted on Whatman FTATM classic card, then extracted with 200 μl methanol, and liquid chromatography-tandem mass spectrometry in positive ion multiple reaction monitoring mode was utilized for analysis. Results The LOD and LLOQ of the method were 0.1 ng/ml and 0.2 ng/ml, respectively. The accuracy and precision were 98.2%–103.9% and 2.7%–8.5%, respectively. It was found that the stability of 5-MeO-DIPT in DUSs was better than the stability of 5-MeO-DIPT in urine stored at 25 °C. Moreover, this method was also applied to detect 5-MeO-DIPT in the urine of individuals known to have used 5-MeO-DIPT. It was found that the concentrations of 5-MeO-DIPT were 0.3–2.3 ng/ml, which were lower than those obtained via GC–Orbitrap-MS. The small volume of urine required (10 μl), combined with the simplicity of the analytical technique, makes this an useful procedure for the screening of drug of abuse.

BACKGROUND AND OBJECTIVES: The popularity of tryptamines such as N,N-dimethyltryptamine (DMT) appears to be increasing in the United States (US), but epidemiologic literature on prevalence of use is scant. This paper aims to determine trends in prevalence and correlates of past-year tryptamine use among a nationally representative sample of young adults in the US. METHODS: Participants in the National Survey on Drug Use and Health survey were queried about past-year use of tryptamines—specifically DMT, α-methyltryptamine (AMT), and 5-MeO-DIPT (“Foxy”). Data were examined from young adults (ages 18–25), years 2007–2014 (N = 144,787). Linear trends in prevalence of past-year tryptamine use were examined in the full sample and stratified by specific demographic and drug use characteristics. RESULTS: Tryptamine use is rare, but increased from .2% in 2007/08 to .7% in 2013/14, a 273% relative increase (p < .001). While prevalence increased among all demographic groups, prevalence was substantially higher among individuals who use other drugs. In particular, between 2007/08 and 2013/14, prevalence of tryptamine use increased among past-year ecstasy users (from 2.1% to 10.0%) and LSD users (from 7.0% to 15.5%) (ps < .01). Prevalence of tryptamine use tended to be higher among lifetime and past-year users of psychedelic drugs compared to users of non-psychedelic drugs. CONCLUSION: While tryptamine use is not prevalent in the general young adult population, prevalence is increasing. Users of various other drugs—particularly drugs with psychedelic effects—report higher prevalence of tryptamine use. SCIENTIFIC SIGNIFICANCE: Users of other drugs can be targeted when disseminating information about tryptamines to ensure user safety. (Am J Addict 2018;27:578–585)

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.

A series of N-benzylated-5-methoxytryptamine analogues was prepared and investigated, with special emphasis on substituents in the meta position of the benzyl group. A parallel series of several N-benzylated analogues of 2,5-dimethoxy-4-iodophenethylamine (2C-I) also was included for comparison of the two major templates (i.e., tryptamine and phenethylamine). A broad affinity screen at serotonin receptors showed that most of the compounds had the highest affinity at the 5-HT2 family receptors. Substitution at the para position of the benzyl group resulted in reduced affinity, whereas substitution in either the ortho or the meta position enhanced affinity. In general, introduction of a large lipophilic group improved affinity, whereas functional activity often followed the opposite trend. Tests of the compounds for functional activity utilized intracellular Ca(2+) mobilization. Function was measured at the human 5-HT2A, 5-HT2B, and 5-HT2C receptors, as well as at the rat 5-HT2A and 5-HT2C receptors. There was no general correlation between affinity and function. Several of the tryptamine congeners were very potent functionally (EC50 values from 7.6 to 63 nM), but most were partial agonists. Tests in the mouse head twitch assay revealed that many of the compounds induced the head twitch and that there was a significant correlation between this behavior and functional potency at the rat 5-HT2A receptor.

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.

Many N,N-dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5-methoxy-2-methyl-N,N-diallyltryptamine (5-MeO-2-Me-DALT), 5-methoxy-2-methyl-N-allyl-N-cyclohexyltryptamine (5-MeO-2-Me-ALCHT), and 5-methoxy-2-methyl-N,N-diisopropyltryptamine (5-MeO-2-Me-DIPT) using gas chromatography-mass spectrometry (GC-MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC-MS

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.

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.

5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DiPT) is a designer hallucinogen that is a synthetic tryptamine derivative. It is highly abused and is involved in criminal activities because of its psychotropic properties. Herein, we presented an UHPLC-MS/MS method allowing for the qualitative and quantitative determination of 5-MeO-DiPT in human hair. The hair was first decontaminated and then cut into pieces. Thirty milligrams of hair samples was pulverized below 4°C in the presence of 0.5mL deionized water containing 0.1% formic acid. After centrifuging twice, 5μL of supernatant was injected into the LC-MS/MS system. A T3 column (100mm×2.1mm, 1.8μm) was used, and mobile phases consisted of 20mmol/L ammonium acetate, 5% acetonitrile and 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). The gradient elution was used at a flow rate of 0.3mL/min. The resulting calibration curve for 5-MeO-DiPT was y=281.50213x+0.00231 (R2=0.992), the limit of detection (LOD) was 0.05pg/mg, and the lower limit of quantification (LLOQ) was 0.1pg/mg. The accuracy was between 92.1% and 105.6%, and the intra- and interday precision, recovery and matrix effect were acceptable. The validated method was successfully used in 106 real cases, and the concentration of 5-MeO-DiPT in hair samples of these suspected users was 0.2-7532.5pg/mg. These cases present data to document illegal 5-MeO-DiPT use.

The last fifteen years have seen the emergence and overflow into the drug scene of “superpotent” N-benzylated phenethylamines belonging to the “NBOMe” series, accompanied by numerous research articles. Although N-benzyl substitution of 5-methoxytryptamine is known to increase its affinity and potency at 5-HT2 receptors associated with psychedelic activity, N-benzylated tryptamines have been studied much less than their phenethylamine analogs. To further our knowledge of the activity of N-benzyltryptamines, we have synthesized a family of tryptamine derivatives and, for comparison, a few 5-methoxytryptamine analogs with many different substitution patterns on the benzyl moiety, and subjected them to in vitro affinity and functional activity assays vs. the human 5-HT2 receptor subtypes. In the binding (radioligand displacement) studies some of these compounds exhibited only modest selectivity for either 5-HT2A or 5-HT2C receptors suggesting that a few of them, with affinities in the 10–100 nanomolar range for 5-HT2A receptors, might presumably be psychedelic. Unexpectedly, their functional (calcium mobilization) assays reflected very different trends. All of these compounds proved to be 5-HT2C receptor full agonists while most of them showed low efficacy at the 5-HT2A subtype. Furthermore, several showed moderateto-strong preferences for activation of the 5-HT2C subtype at nanomolar concentrations. Thus, although some N-benzyltryptamines might be abuse-liable, others might represent new leads for the development of therapeutics for weight loss, erectile dysfunction, drug abuse, or schizophrenia This work was supported by FONDECYT (Chile) regular research grants 1110146 and 1150868 to BKC and CONICYT doctoral grant 21140358 to MT-S SI

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.

Antioxidants are prospective radioprotectors because of their ability to scavenge radiation-induced reactive oxygen species (ROS). The hematopoietic system is widely studied in radiation research because of its high radiosensitivity. In the present study, we describe the beneficial effects of 5-methoxytryptamine-α-lipoic acid (MLA), which was synthesized from melatonin and α-lipoic acid, against radiation-induced hematopoietic injury. MLA administration significantly enhanced the survival rate of mice after 7.2 Gy total body irradiation. The results showed that MLA not only markedly increased the numbers and clonogenic potential of hematopoietic cells but also decreased DNA damage, as determined by flow cytometric analysis of histone H2AX phosphorylation. In addition, MLA decreased the levels of ROS in hematopoietic cells by inhibiting NOX4 expression. These data demonstrate that MLA prevents radiation-induced hematopoietic syndrome by increasing the number and function of and by inhibiting DNA damage and ROS production in hematopoietic cells. These data suggest MLA is beneficial for the protection of radiation injuries.

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.

Recent advances in the knowledge of the mechanisms responsible for antitumor immunity have stimulated the elaboration of new cancer immunotherapeutic strategies. Moreover, more recent discoveries have demonstrated that immune responses are under a physiological modulatory control played by several neuroendocrine pathways, which explain the differences between the in vivo and in vitro immune responses. While until a few years ago the evaluation of the immune status of cancer patients was substantially established on the basis of clinical empirical criteria, recent discoveries of the antitumor cytokine network have allowed the biochemical bases of anticancer immunity to be defined, leading to new anticancer immunotherapeutic strategies, on the basis of patient neuroendocrine and neuroimmune status, in an attempt to correct the great number of cancer-related alterations on the basis of knowledge of the physiopathology of anticancer immunity. The rationale for cancer neuroimmunotherapy consists of the possibility to enhance the efficacy of the various immunotherapeutic strategies by a concomitant administration of antitumor cytokines (namely IL-2), in addition to neuroendocrine endogenous molecules (namely the pineal indole hormones), able to stimulate the anticancer immunoresponse by amplifying the anticancer reaction and/or by counteracting the generation of immunosuppressive events.

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.

Characterization of the melatonin (MLT) biosynthesis pathway in plants is still limited. Additionally, a metabolomic analysis of MLT biosynthesis in plants is still a challenge due to analyte structural and chemical diversity, low analyte abundances, and plant matrix complexities. Herein, a sensitive liquid chromatography-mass spectrometry (LC-MS) method enabling the simultaneous determination of seven plant MLT biosynthetic metabolites was developed. In the proposed strategy, the targeted metabolites, which included tryptophan (Trp), tryptamine (TAM), 5-hydroxytryptophan (5HTP), serotonin (5HT), N-acetylserotonin (NAS), 5-methoxytryptamine (5MT), and MLT, were purified from plant extracts using a one-step dispersive solid-phase extraction (DSPE). The samples were then chemically labeled with dansyl chloride (DNS-Cl), followed by analysis using LC-MS. The limit of detection (LOD) values ranged from 0.03 to 1.36 pg/mL and presented a 22- to 469-fold decrease when compared to the unlabeled metabolites. Due to the high sensitivity of the proposed method, the consumption of plant materials was reduced to 10 mg FW. Ultimately, the established method was utilized to examine the distributions of MLT and its intermediates in rice shoots and roots with or without cadmium (Cd) stress. The results suggested that under normal condition, MLT may also be generated via a Trp/TAM/5HT/5MT/MLT path (Pathway II) in addition to the previously reported Trp/TAM/5HT/NAS/MLT path (Pathway I), although Pathway I was shown to be dominant. During Cd stress, MLT was also shown to be produced through these two pathways, with Pathway II shown to be dominant in rice shoots and roots.

For the development of novel 5-HT(4) receptor ligands we have designed and synthesized two series of 5-methoxytryptamine derivatives varying the substitution on the primary amine. Their biological activities were evaluated in a receptor binding assay where a subset of compounds showed comparable potency to the agonists serotonin and 5-methoxytryptamine. Structure-activity analyses have highlighted promising avenues for further synthetic work and binding modes were proposed by docking these compounds into a homology model of the 5-HT(4) receptor.

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.

Density functional theory has been applied to elucidate the mechanism of the O-demethylation reaction that generates serotonin from 5-methoxytryptamine (5-MT); a process that is efficiently catalyzed by P450 CYP2D6. Two substrates, the neutral 5-MT and the protonated 5-MTH(+), were used to probe the reactivity of CYP2D6 compound I. Notably, the H-abstraction process is found to be slightly more facile for 5-MT. However, our DFT augmented by docking results show that the amino acid Glu216 in the active site holds the NH(3)(+) tail of the 5-MTH(+) substrate in an upright conformation and thereby controls the regioselectivity of the bond activation. Thus, the substrate protonation serves an important function in maximizing the yield of serotonin. This finding is in accord with experimental conclusions that 5-MTH(+) serves as the substrate for the CYP2D6 enzyme. The study further shows that the H-abstraction follows two-state reactivity (TSR), whereas the rebound path may involve more states due to the appearance of both Fe(IV) and Fe(III) electromers during the reaction of 5-MTH(+).

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.

Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT(6) receptor (5-HT(6)R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT(6)R are poorly understood due to the lack of selective 5-HT(6)R ligands. In the present study, we examined functional coupling of the human 5-HT(6)R, 5-HT(7A)R, or 5-HT(7B)R with various Galpha-proteins (Galpha(15), Galpha(qs5), or Galpha(qG66Ds5)) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT(6)R with Galpha(qG66Ds5) produced the highest levels of Ca(2+) signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT(6)R HTS system (Z'-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT(6)R, we screened approximately 500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC(50) value of 6.3 microM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT(6)R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT(6)R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT(6)R interacting protein. Taken together, these results suggest that the 5-HT(6)R/Galpha(qG66Ds5) FDSS6000 system can be utilized to screen for selective 5-HT(6)R ligands and to examine any functional relationships between 5-HT(6)R and its binding proteins.

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.

We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.

To understand the change in environmental conditions when a probe is incorporated in micelles, an absorption and fluorescence spectral study on the solubilization behaviour of 5-methoxy tryptamine (5-MT) and N,N-dimethyl-5-methoxy tryptamine (5-NMT) has been made in CTAB and SDS for their neutral and cationic forms. The blue shift in emission wavelength is accompanied by increase in intensity with increasing surfactant concentration for almost all the cases except for the cationic probe in CTAB surfactant. This exceptional behaviour can be attributed to the quenching of emission intensity caused by Br(-) ions. Spectral correlations with solvent polarity parameters indicate relatively less polar binding sites in CTAB and SDS as compared to water. The binding constant and cmc values have been determined for CTAB and SDS using both the neutral and the cationic probes, which are in good agreement with the literature values. Higher value of binding constant and lower polarity of the binding sites justify 5-NMT as a better binding probe as the two methyl groups make the molecule more hydrophobic and drag it to the interior of both the micelles.