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Serotonin N -acetyltransferase (SNAT) is the key rate-limiting enzyme in melatonin biosynthesis. SNAT mediates dual pathways of melatonin biosynthesis in plants by using serotonin and 5-methoxytryptamine (5-MT) as substrates, and a high reaction pH and temperature are essential to its activity. However, little is known of its underlying mechanisms. Herein, we present a detailed reaction mechanism of a SNAT from Oryza sativa through combined structural and molecular dynamics (MD) analysis. We report for the first time the crystal structures of plant SNAT in the apo and binary/ternary complex forms with acetyl-CoA (AcCoA), serotonin, and 5-MT. These structures reveal that Os SNAT exhibits a unique enzymatically active dimeric fold that is not found in all the known structures of arylalkylamine N-acetyltransferase (AANAT) family. The key residues W188, D189, D226, N220, and Y233 located around the active pocket have important role in catalysis which is subsequently confirmed by site-directed mutagenesis. Combined with MD simulations, we hypothesize a novel plausible catalytic mechanism in which D226 and Y233 function as catalytic base and acid during the acetyl-transfer reaction. This work provides a molecular framework for understanding the catalytic mechanisms of plant SNAT and has implications for future protein engineering and biocatalytic applications.

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.

5-HT inhibits cardiac sympathetic neurotransmission in normoglycaemic rats, via 5-HT1B, 5-HT1D and 5-HT5A receptor activation. Since type 1 diabetes impairs the cardiac sympathetic innervation leading to cardiopathies, this study aimed to investigate whether the serotonergic influence on cardiac noradrenergic control is altered in type 1 diabetic rats. Diabetes was induced in male Wistar rats by streptozotocin (50 mg/kg, i.p.). Four weeks later, the rats were anaesthetized, pithed and prepared for producing tachycardic responses by electrical preganglionic stimulation (C7-T1) of the cardioaccelerator sympathetic outflow or i.v. noradrenaline bolus injections. Immunohistochemistry was performed to study 5-HT1B, 5-HT1D and 5-HT5A receptor expression in the stellate ganglion from normoglycaemic and diabetic rats. In the diabetic group, i) i.v. continuous infusions of 5-HT induced a cardiac sympatho-inhibition that was mimicked by the 5-HT1/5A agonist 5-carboxamidotryptamine (without modifying noradrenaline-induced tachycardia), but not by the agonists indorenate (5-HT1A), CP 93,129 (5-HT1B), PNU 142633 (5-HT1D), or LY344864 (5-HT1F); ii) SB 699551 (5-HT5A antagonist; i.v.) completely reversed 5-CT-induced cardiac sympatho-inhibition; and iii) 5-HT5A receptors were more expressed in the stellate ganglion compared to normoglycaemic rats. These results show the prominent role of the peripheral 5-HT5A receptors prejunctionally inhibiting the cardiac sympathetic drive in type 1 diabetic rats.

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.

5-Methoxy-N,N-Diisopropyltryptamine (5-MeO-DIPT) is a designer hallucinogen derived from tryptamine and its use has been banned by many countries. In this study, a qualitative and quantitative method was developed for determining 5-MeO-DIPT in urine by gas chromatography high-resolution mass spectrometry. 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT) and 5-methoxy-N-isopropyltryptamine (5-MeO-IPT) were identified as 5-MeO-DIPT metabolites in abusers’ urine. 5-MeO-DIPT was extracted from urine by liquid–liquid extraction with ethyl acetate under alkaline conditions. The extract was analyzed by GC-Orbitrap-MS in full scan mode with a resolution of 60,000 full width at half maxima (FWHM). The linear range of this method was 2–300 ng/mL with r > 0.99, and the limit of detection was 1 ng/mL. The accuracy and precision were 93–108.7% and 3.1–10.3%, respectively. This method is simple and sensitive. It has been successfully used to detect 5-MeO-DIPT in drug abusers’ urine, which showed that the concentrations of 5-MeO-DIPT were between 1 and 2.8 ng/mL. 5-OH-DIPT and 5-MeO-IPT, two urinary major metabolites of 5-MeO-DIPT, were identified in urine samples from 5-MeO-DIPT users. Furthermore, the stability of 5-MeO-DIPT in human urine was investigated. It was discovered that the concentration of 5-MeO-DIPT in urine decreased by 22.8, 33.2 and 38.2% after samples were stored for 24 h at 25°C, 5 days at 4°C and 7 days at 4°C, respectively. And 5-MeO-DIPT in urine were stable after they were stored for 30 days at −20°C. Therefore, it is recommended that urine should be stored under freezing conditions before performing 5-MeO-DIPT analysis.

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.

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.

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.

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

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.

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.

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.

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.

Antagonists, but not agonists, of the 5-HT3 receptor are useful therapeutic agents, and it is possible that partial agonists may also be potentially useful in the clinic. Here we show that 5-fluorotryptamine (5-FT) is a partial agonist at both 5-HT3A and 5-HT3AB receptors with an Rmax (Imax/Imax 5-HT) of 0.64 and 0.45 respectively. It is about 10 fold less potent than 5-HT: EC50=16 and 27 microM, and Ki for displacement of [3H]granisetron binding=0.8 and 1.8 microM for 5-HT3A and 5-HT3AB receptors respectively. We have also explored the potencies and efficacies of tryptamine and a range of 5-substituted tryptamine derivatives. At 5-HT3A receptors tryptamine is a weak (Rmax=0.15), low affinity (EC50=113 microM; Ki=4.8 microM) partial agonist, while 5-chlorotryptamine has a similar affinity to 5-FT (EC50=8.1 microM; Ki=2.7 microM) but is a very weak partial agonist (Rmax=0. 0037). These, and data from 5-methyltryptamine and 5-methoxytryptamine, reveal the importance of size and electronegativity at this location for efficient channel opening.

The lipid peroxidation of triglycerides enriched in polyunsaturated fatty acids was investigated by photoemission techniques and the TBARS assay. Butylated hydroxytoluene, 5-OH-tryptophan and N-acetylserotonin inhibited light emission and TBARS formation in a concentration dependent manner. However, it was enhanced in the presence of melatonin and 5-methoxytryptamine and was dependent on its concentration. The total relative luminic units were found to be lower in those systems incubated in the presence of butylated hydroxytoluene, N-acetylserotonin or 5-OH-tryptophan; this decreased proportionally to the concentration of the compound tested. The order of inhibition was 5-OH-tryptophan>N-acetylserotonin>butylated hydroxytoluene with the following IC50 values: 0.65, 6.5 and 9.0 mM respectively. The free-radical scavenging activity of the indole derivatives was also analyzed by the DPPH method, and the results indicate that 5-OH-tryptophan, and N-acetylserotonin exhibited a dose-dependent free-radical scavenging ability at all of the tested concentrations. Thus, at 10 microM concentration a decrease of 84.71% and 73.50% of initial DPPH was observed, compared to 51.00% of BHT. Melatonin and 5-methoxytriptamine decreased the initial concentration of DPPH only 1.85% and 5.0%, respectively. The possible formation of N(1)-acetyl-N(2) formyl-5-methoxykynuramine (AFMK) during lipid peroxidation of triglycerides enriched in PUFAs with cumene hydroperoxide in the presence of melatonin was also analyzed.

We have tested by [(35)S]-GTPgammaS binding the intrinsic activity of three full agonists (serotonin, 5-methoxytryptamine and 5-methoxy-2-methyl-N,N-dimethyltryptamine) on rat 5-HT(6) receptors cloned in HEK-293 cells, using the scintillation proximity assay. Serotonin and 5-methoxytryptamine are soluble in water, while the agonist 5-methoxy-2-methyl-N,N-dimethyltryptamine is soluble in dimethyl sulfoxide (DMSO). In [(35)S]-GTPgammaS binding 5-HT and 5-methoxytryptamine were able to increase basal binding, while 5-methoxy-2-methyl-N,N-dimethyltryptamine surprisingly showed an inverse agonist activity. So we have tested 5-HT and 5-methoxytryptamine in the presence of DMSO: in this condition the two agonists behaved as antagonists. This interfering effect of DMSO was not observed when GTP-europium filtration binding was used in place of scintillation proximity assay using [(35)S]-GTPgammaS. In addition, DMSO did not affect [(3)H]-5HT binding or cAMP accumulation in cloned HEK-293 cells expressing rat 5-HT(6) receptors. In conclusion, we demonstrated that DMSO, the most common solvent used to dissolve compounds insoluble in water, interferes with the method of scintillation proximity assay using [(35)S]-GTPgammaS. DMSO does not affect basal signal, nor the GTPgammaS binding itself, as indicated by the experiments with GTP-europium. Therefore its interfering effect is likely to occur at the binding of antibodies in the scintillation proximity assay.

Peroxynitrite, a potent oxidative stress inducer, inhibits the mitochondrial electron transfer, induces cell death, and is considered to be involved in the pathology of various diseases. However, the intracellular mechanisms involved in the cell death process are not fully understood. Here we demonstrate that the enhanced nitration of specific tyrosine residues of cytochrome c, which are induced by continuous peroxynitrite exposure, attenuates cytochrome c-induced caspase-9 activation in vitro. Interestingly, cytochrome c nitrated with a single high dose of peroxynitrite preserved its potency, while this did not occur when cytochrome c was treated with continuous peroxynitrite exposure. Although both of these experiments resulted in cytochrome c nitration at the tyrosine residues, it was found that nitration at specific residues was enhanced only when cytochrome c was exposed to continuous peroxynitrite. This is the first report to demonstrate that cytochrome c nitration affects the apoptotic pathway by means of enhancement of nitration at specific tyrosine residues. This result implies that the nitration pattern of cytochrome c may affect the efficacy of the mitochondrial pathway in apoptotic cell death.

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.

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.

In neurons of dorsal motor nucleus of the vagus that is involved in the gastric motility and possibly emesis, application of 5-hydroxytryptamine produces membrane depolarization, and suppresses spike-repolarization and spike-afterhyperpolarization, suggesting divergent effects of 5-hydroxytryptamine through activating multiple subtypes of 5-hydroxytryptamine receptors. However, only the role of 5-hydroxytryptamine 2A receptors has been established to be responsible for the depolarization, and the mechanisms underlying the modulation of spikes remain unknown although a role of 5-hydroxytryptamine 4 receptors was implicated in modulations of spikes. There is now increasing evidence for the role of 5-hydroxytryptamine receptors in neurons involved in generating emesis following administration of anticancer drug. Since antagonists of 5-hydroxytryptamine 3/4 receptors are widely used as anti-emetic drugs, we have reevaluated the functional roles of 5-hydroxytryptamine 3/4 receptors of dorsal motor nucleus of the vagus neurons, especially in modulating transient outward currents that are presumed to be involved in spike-repolarization and spike-afterhyperpolarization. Whole-cell patch-clamp recordings were made from the dorsal motor nucleus of the vagus neurons, which were identified by a retrograde tracing method with dextran-tetramethylrhodamine-lysine injected into a bundle of abdominal vagus nerves. Under a voltage-clamp condition, dorsal motor nucleus of the vagus neurons expressed a prominent A-like current. The activation of 5-hydroxytryptamine 3 receptors reversibly increased the resting membrane conductance while the activation of 5-hydroxytryptamine 4 receptors led to an almost irreversible decrease in the A-like current. A long-lasting suppression of A-like current by transient activation of 5-hydroxytryptamine 4 receptors would result in a long-lasting increase in the excitability of dorsal motor nucleus of the vagus neurons, which might be involved in generation of the long-lasting facilitation of gastric motility or in generation of the long-lasting gastric relaxation through the activation of enteric non-adrenergic non-cholinergic neurons as implicated in the delayed emesis induced by anticancer drugs.

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.

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.

We have developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) system capable of achieving better than 2% accuracy, routinely over a wide concentration range of 1–800 ng mL−1. We demonstrate that the necessary high precision, high accuracy and rapid analysis can be achieved using LC-MS/MS technology. Automated nanoelectrospray ionisation tandem mass spectrometry (nanoESI-MS/MS) technology can be employed to eliminate the chromatographic step completely. In this paper, nanoESI-MS/MS is evaluated and compared directly with LC-MS/MS for the quantitative analysis of two-test analytes, amitriptyline (ATT) and 5-methoxytryptamine (5-MTT), in aqueous/organic mixture. Calibration curves were found to be linear over a wide concentration range of 1–800 ng mL−1 for both analytes using LC-MS/MS. Using nanoESI-MS/MS ATT gave a linear response while 5-MTT gave a non-linear response using nanoESI-MS/MS over the same concentration range as in LC-MS/MS. Accuracy and precision values of quality control samples (QCs) at four concentration levels were analysed in replicates of six at each level using 5-MTT and ATT as test analytes for both techniques. The LC-MS/MS system was capable of achieving accuracy levels of 99.50–101.96% for ATT and 100.17–100.40% for 5-MTT. Accuracy levels using nanoESI-MS/MS were not comparable to LC-MS/MS, they ranged from 90.09–100.18% for ATT and 95.95–113.55% for 5-MTT. The precision values obtained for nanoESI-MS/MS were in good agreement with those obtained by LC-MS/MS.

Foxy is the colloquial name for the hallucinogen 5-ethoxy-diisopropyltryptamine (5-MeO-DIPT). A non-fatality involving a 23-year-old Caucasian man who ingested a capsule containing 5-MeO-DIPT is described. He presented to the Emergency Department, not with visual nor auditory hallucinations but with sensory hallucinations, that of formication and paranoia. He was observed and given supportive care for 4 h, then discharged without any known sequelae. Blood and urine were collected for laboratory analyses. Foxy and its metabolites were identified in urine by gas chromatography‐mass spectrometry. The concentrations of 5-MeO-DIPT in the serum and urine were 0.14 and 1.6 mg/mL, respectively. The drug undergoes oxidative deamination to form 5-methoxy-indole acetic acid. The urinary concentration of this metabolite was 0.17 mg/mL. Also, the urine contained three other related compounds. Two of them have been described in a previous case of 5-MeO-DIPT ingestion as 5methoxy-isopropyltryptamine (5-MeO-IPT) and 5-methoxy-diisopropyltryptamine-N 0 -oxide (5-MeO-DIPT-N 0 -oxide). The third compound was substantially present in the urine and was tentatively identified as 5-hydroxy-diisopropyltryptamine (5-OH-DIPT). Only the parent drug, 5-MeO-DIPT was detected in the serum sample. # 2004 Elsevier Ireland Ltd. All rights reserved.

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.

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.