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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.

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

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

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

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

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

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

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.

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

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

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

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.

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The aim of this study was to identify the receptor type(s) by which 5-hydroxytryptamine applied to the intestinal mucosa excites the terminals of myenteric AH neurons. The AH neurons have been identified as the intrinsic primary afferent (sensory) neurons in guinea-pig small intestine and 5-hydroxytryptamine has been identified as a possible intermediate in the sensory transduction process. Intracellular recordings were taken from AH neurons located within 1mm of intact mucosa to which 5-hydroxytryptamine was applied. Trains of action potentials and/or slow depolarizing responses were recorded in AH neurons in response to mucosal application of 5-hydroxytryptamine (10 or 20microM) or the 5-hydroxytryptamine-3 receptor agonist, 2-methyl-5-hydroxytryptamine (1 or 3mM), and to electrical stimulation of the mucosa. The 5-hydroxytryptamine-2 receptor agonist, alpha-methyl-5-hydroxytryptamine (100microM), and the 5-hydroxytryptamine-1,2,4 receptor agonist, 5-methoxytryptamine (10microM), did not elicit such responses. The 5-hydroxytryptamine-3 receptor-selective antagonist, granisetron (typically 1microM), and the 5-hydroxytryptamine-3,4 receptor antagonist, tropisetron (typically 1microM), each reduced or abolished the responses to 5-hydroxytryptamine, while the selective 5-hydroxytryptamine-4 receptor antagonist, SB 204070 (1microM), did not. It is concluded that application of 5-hydroxytryptamine to the mucosa activates a 5-hydroxytryptamine-3 receptor that triggers action potential generation in the mucosal nerve terminals of myenteric AH neurons.

Stimulation of the serotoninergic innervation of the leech pharynx or application of serotonin to the isolated pharynx induced four distinct types of contractile activity: an increase in basal tonus, large phasic contractions of 10‐15 s in duration, smaller phasic contractions occurring at approximately 1 Hz, and a relaxation after washout of serotonin. Application to the isolated pharynx of the selective serotonin agonists (6)-8-hydroxy-2-(di-n-propylamino)tetralin, N-(3-trifluoromethylphenyl)piperazine, 1-(m-chlorophenyl)-piperazine, (6)-2,5-dimethoxy-4-iodoamphetamine, 2-methyl-5-hydroxytrypamine, a-methyl-5-hydroxytryptamine, and 5-methoxytryptamine induced distinct types of pharyngeal contractile activity. The results of this study suggest that the leech pharynx possesses more than one type of serotonin receptor. © 1999 Elsevier Science Inc. All rights reserved.

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

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

Intestinal ischemia/reperfusion (I/R) causes serious systemic injury, mainly from a variety of bioactive substances released from the injured intestine. To assess the possible roles of serotonin (5-hydroxytryptamine, 5-HT), a bioactive amine mainly stored in the intestine, in I/R injury, we assayed the levels of tryptophan, 5-HT, and 5-hydroxyindole acetic acid (5-HIAA) in the blood and intestine in a rat I/R model. Plasma 5-HT increased significantly over time after reperfusion; the plateau level was obtained 4 h after reperfusion and was associated with an increase in 5-HIAA. Plasma tryptophan levels declined gradually after reperfusion. The ratio of 5-HIAA/5-HT was significantly higher in I/R rats than in control rats, suggesting that elevated 5-HT was quickly metabolized in the systemic circulation. In the intestine, 5-HT decreased dramatically, whereas tryptophan increased. This phenomenon was prominent in the severely damaged intestine. These findings suggest that the injured intestine released large amounts of 5-HT, whereas its synthesis in the injured intestine was suppressed. An increase in 5-HT in the circulation may be related to various circulatory disturbances observed in humans after intestinal ischemia.

Many cnidarians exhibit a diurnal rhythm of expansion and contraction. Previous studies correlated this behavior to various environmental factors, including prey-catching, the photosynthetic requirement of the zooxanthellae, and tidal movement in the intertidal zone. Whether intrinsic factors affect the diurnal rhythm of expansion/contraction were not addressed. The present study investigated the effect of the photoperiod hormone melatonin (N-acetyl-5-methoxytryptamine) and its homologue on the expansion and contraction of a sea anemone. Addition of exogenous melatonin at 10–4 M induced the expansion of the oral disk. At an exogenous concentration of 10–3 M, protrusion of the actinopharynx was induced. Melatonin thus exerted its effect in the sea anemone in a dose-dependent manner. Addition of another indoleamine, 5-methoxytryptamine, at a concentration of 10–4 M resulted in the thythmic contraction of the body column, resembling the respiratory rhythm of coelenteric flushing. Serotonin (5-hydroxytryptamine), however, did not induce any apparent effect at an exogenous concentration of 10–3 M. Simultaneous addition of melatonin and 5-methoxytryptamine induced both types of responses, suggesting that their effects are not antagonistic with each other. This and their different responses suggested that the two indoleamines acted on two different sites in the sea anemone. At a concentration of 10–4 M, melatonin was also able to modulate the effect of Antho-RF-amide in the contraction of the oral disk. Eserine, an inhibitor of melatonin deacetylation, also induced protrusion of the actinopharynx. This strongly supported the concept that melatonin was produced endogenously and probably at or near the site of action. J. Exp. Zool. 279:201–207, 1997. © 1997 Wiley-Liss, Inc.

The influence of serotonin (5-hydroxytryptamine, 5-HT) and serotonin analogues on the direct current electroretinogram (d.c. ERG) and the standing potential of the albino rabbit eye (SP) was studied. After unilateral vitrectomy, corneal recordings were obtained during simultaneous intravitreal perfusion with a control solution alternating with 5-HT at concentrations of 25, 120 and 200 μM. The c-wave increased at 25 and 120 μM when changing from control solution to test solution (P < 0.05) but did not decrease significantly when changing back to control solution (P > 0.05). The c-wave was reversibly elevated at 200 μM (PHS-5-HT, P < 0.01; 5-HT-PHS, P < 0.05). To analyse further the influence on the c-wave, in vivo intraretinal microelectrode recordings were obtained during intravitreal perfusion with 5-HT. The transepithelial potential (TEP) increased (P < 0.01), while the slow PIII was not significantly affected (P > 0.05). The serotonin receptor agonists 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, 5-methoxytryptamine, α-methyl-5-hydroxytryptamine and 2-methyl-5-hydroxytryptamine, caused a significant reversible elevation of the c-wave, whereas 5-carboxyamidotryptamine did not. Tropisetron did not block the serotonin effect and LY53857 had an effect of its own on the c-wave. The results seem to indicate that the influence of serotonin on the c-wave is mainly due to an effect on the retinal pigment epithelium (RPE) and that more than one type of serotonin receptor may be involved.

It has been suggested that the late hypotensive response to serotonin (5-hydroxytryptamine; 5-HT) in vagosympathectomized rats is mediated by '5-HT1-like' receptors since this effect is mimicked by 5-carboxamidotryptamine (5-CT), is not modified by cyproheptadine, ketanserin or MDL 72222, but it is blocked by methysergide. The present study was set out to reanalyze this suggestion in terms of the classification schemes proposed in 1994 and 1996 by the NC-IUPHAR subcommittee on the classification and nomenclature of 5-HT receptors. I.v. bolus injections of 5-CT (0.01-0.3 microg x kg(-1)), 5-HT (1-30 microg x kg(-1)) and 5-methoxytryptamine (5-MeO-T; 1-30 microg x kg(-1)) produced dose-dependent hypotensive responses with a rank order of agonist potency: 5-CT5-HT5-methoxytryptamine with sumatriptan (30-1000 microg x kg(-1)) inactive. The depressor responses to 5-HT and 5-CT were not attenuated by i.v. GR127935 (300-3000 microg x kg(-1)) or equivalent volumes of saline. In contrast, lisuride, methiothepin, mesulergine, metergoline and clozapine dose-dependently antagonized the responses to 5-HT and 5-CT; the rank order of apparent pA2 values against 5-HT and 5-CT, respectively, was: lisuride (7.7; 7.8)methiothepin (6.8; 7.0)or = mesulergine (6.4; 6.6)clozapine (5.7; 5.8); metergoline displayed variable potencies (5.6; 6.4). Except for lisuride, which also affected isoprenaline-induced hypotension, the antagonism by the other drugs was selective. Based upon the above rank order of agonist potency, the blockade by a series of drugs showing high affinity for the cloned 5-ht7 receptor and the lack of blockade by GR127935, our results indicate that the 5-HT receptor mediating hypotension in vagosympathectomized rats is operationally similar to other putative 5-ht7 receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine coronary and external carotid arteries and guinea-pig ileum as well as feline tachycardia).

Picomole to femtomole per gland/ml levels of 5-methoxytryptamine (5MT) in the pineal gland and serum of the rat and quail were determined, in the absence of a monoamine oxidase inhibitor, by using the ultra-sensitive technique of capillary column gas chromatography/electron-capture negative ion chemical ionization mass spectrometry. Diurnal rhythms of pineal 5MT with high levels at mid-light and low levels at mid-dark were found in both species, while serum 5MT levels showed less or no diurnal variations. The definitive presence of 5MT in the pineal gland and blood circulation provides further evidence that it is potentially a neurohormone. Whether 5MT is implicated in the photoperiodic responses and/or other physiological functions of animals, however, remains to be investigated.

The effects of the 5-HT receptor agonist, 5-methoxytryptamine, on plasma glucose levels were investigated in rats. 5-Methoxytryptamine induced a significant hyperglycemia above the dosage of 1 mg/kg. 5-Methoxytryptamine-induced hyperglycemia was antagonized by pretreatment with the 5-HT1 and 5-HT2 receptor antagonist, methysergide, or the 5-HT2A receptor antagonist, ketanserin, whereas the 5-HT3 and 5-HT4 receptor antagonist, tropisetron, and the 5-HT4 receptor antagonist, SDZ 205-557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester), showed no effect. In addition, the peripheral 5-HT2 receptor antagonist, xylamidine, reduced 5-methoxytryptamine-induced hyperglycemia. These results suggest that 5-methoxytryptamine-induced hyperglycemia is mediated by the peripheral 5-HT2A receptor, although it has a high affinity for the 5-HT4 receptor. Adrenodemedullation abolished the 5-methoxytryptamine-induced hyperglycemia. 5-Methoxytryptamine did not affect the blood levels of the pancreatic hormones, insulin and glucagon. The hyperglycemia induced by 5-methoxytryptamine was not affected by pretreatment with dexamethasone which inhibits corticosterone release. These results indicate that 5-methoxytryptamine-induced hyperglycemia is elicited by a facilitated adrenaline release from the adrenal gland. Therefore, it is suggested that the 5-HT2A receptor may be partly involved in the pharmacological effects induced by the 5-HT4 receptor agonist, 5-methoxytryptamine. © 1997 Elsevier Science B.V. All rights reserved.

A series of tryptamine analogues has been prepared and tested for their 5-HT1 receptor agonist properties. The incorporation of an alkoxy group at the C-5 position of the indole nucleus resulted in a short-lived and dose-dependent immediate antihypertensive and bradycardic response in anaesthetized spontaneously hypertensive rats (SHR). In addition, a carbomethoxy function at the β-position of the side-chain of the tryptamines significantly increased the mean resting arterial blood pressure (MAP) in pithed rats and also produced contraction of the canine basilar artery in a dose-dependent fashion. Structure-activity relationships (SAR) suggest that the 5-alkoxy group is an important pharmacophore in the production of the antihypertensive effect and that the introduction of a hydroxymethylene group on the side-chain, instead of the carbomethoxy group, changed the receptor affinity profile.

The dinoflagellate Gonyaulax polyedra was previously shown to undergo asexual encystment in response to decreased temperature (15° instead of 20°C rearing temperature) in combination with short-days, a response which can be mimicked by melatonin and, much more efficiently, by 5-methoxytryptamine (= 5-MT). We demonstrate that these cyst-inducing conditions lead to enormous accumulations of the two methoxyindoleamines. The circadian rhythmicity of melatonin is maintained for the two days usually preceding cyst formation, though, at an elevated level. Transiently, very high concentrations of melatonin can occur, eventually exceeding 1 millimolar. These extreme concentrations decay rapidly; during this decline, 5-MT and 5-methoxytryptophol appear in large amounts. The concentrations of 5-MT which are measured during this process are higher than those required for cyst induction by the exogenous indoleamine.

In an attempt to define the role of the pineal hormone melatonin and two analogues (5-methoxytryptamine, 5MT, and 6-hydroxymelatonin, 6HM) in limiting oxidative stress, the present study investigated the changes in glutathione, lipid peroxidation, and the activity of the antioxidant enzyme glutathione peroxidase after exercise (swimming for 60 min) with or without treatment with the indolamines mentioned. Lipid peroxidation was measured by estimating tissue levels of malondialdehyde and 4-hydroxyalkenals; the experimental animals in these studies were male Sprague-Dawley rats. In the liver, swimming exercise increased the levels of reduced glutathione (GSH) and also significantly increasing oxidized glutathione (GSSG), while decreasing the GSH/GSSG ratio, an index directly related to oxidative stress. When the animals were treated with melatonin, the concentrations of GSH and GSSG were also increased after swimming; however, no reduction in the GSH/GSSG ratio appeared. In the animals treated with 6HM the changes were the same as in those treated with melatonin. In muscle as well, the concentration of GSH and the GSH/GSSG ratio were decreased following 60 min of swimming. Pretreatment of the rats with melatonin prevented these effects. Pretreatment of the rats with both 5MT and 6HM also prevented the changes. Brain GSH/GSSG ratio was not affected by either exercise or indolamine administration. Swimming enhanced lipid peroxidation in the liver, muscle and brain; however, this was prevented in animals treated with melatonin or 6HM before swimming. Glutathione peroxidase was significantly elevated after exercise in the brain but not in the liver and muscle. It is concluded that swimming imposes a severe oxidative stress and suggests that melatonin and, to a lesser degree, 5MT and 6HM confer protection against the oxidative damage associated with swimming for 60 min. This mechanism may be reasonably attributed to their indole structure, which possibly allows these molecules to act as free-radical scavengers.

We have investigated the effects of 5-hydroxytryptamine (5-HT) on electrically induced contractions of rabbit isolated bladder. Electrical field stimulation evoked twitch contractions which were potentiated by 5-HT (0.3-10 microM). The potentiating effect of 5-HT was inhibited by ondansetron (pA2 9.2) and granisetron (pA2 9.1) but not by methysergide or SB 204070 ((1-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1,4-benzodioxan-5-carboxylate hydrochloride). This suggests that the potentiating effect of micromolar concentrations of 5-HT on neuromuscular transmission in rabbit isolated bladder is mediated by 5-HT3 receptors. The receptors involved in the response to lower concentrations of 5-HT, observed in some tissues, remain to be characterised.

This study examined effects of 5-methoxytryptamine (5-MOT), an agonist at 5-HT4 and 5-HT2B receptors, on the contractile response and acetylcholine release in rat stomach fundus strips. 5-MOT (10−9 − 10−5 M) produced a concentration-dependent increase in the contraction, while it evoked acetylcholine release in a ‘bell-shaped’ concentration-dependent manner. Atropine reduced 5-MOT (10−8 − 10−6 M)-induced contractions, but it had little effect on the contractions evoked by higher concentrations. 5-MOT-induced contraction and acetylcholine release were inhibited by SDZ 205–557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-[diethylamino] ethyl ester), a 5-HT4 receptor antagonist. In the presence of atropine, both SDZ 205–557 and yohimbine, a 5-HT2B receptor antagonist, inhibited the contraction. In the presence of tetrodotoxin, the contraction was inhibited by yohimbine, but not by SDZ –557. These results suggest that the contractile action of 5-MOT in rat stomach fundus involves atropine-sensitive and atropine-resistant components. The sensitive contraction appears to be mediated through 5-HT4 receptors located on cholinergic neurons, whereas the resistant contraction is mediated through 5-HT4 receptors located on non-cholinergic neurons and through 5-HT2B receptors.

The effects of GYKI-46 903 ((+)endo-4-propionyloxy-6-(4-fluorophenyl)-1-azabicyclo [3.3.1]non-6-ene HCl), on 5-HT3 receptors have been studied and compared with ondansetron in peripheral organs in vitro and in vivo, and in a receptor binding assay in membranes prepared from rat cerebral cortex. GYKI-46 903 was found to be a non-competitive antagonist at 5-HT3 receptors present in non-stimulated longitudinal muscle strip of guinea-pig ileum (pD2' against serotonin = 5.54), and also in 5-methoxytryptamine-pretreated electrically stimulated ileal preparations (pD2' against serotonin = 5.26). On the contrary, ondansetron was found to be a competitive antagonist for 5-HT3 receptors; the pA2 value against serotonin was 7.40 in non-stimulated ileum, and it was 7.08 in electrically stimulated ileal preparation pretreated with 5-methoxytryptamine. In displacement studies, the pIC50 values of GYKI-46 903 and ondansetron against [3H]granisetron binding to rat cerebral cortex membranes were 6.91 and 8.58 respectively. GYKI-46 903, when administered by intravenous infusion, antagonized the decrease in heart rate evoked by serotonin (Bezold-Jarisch reflex) in anaesthetized rats, and the maximal reversal was less than 50%. This was in striking contrast with ondansetron, which, after intravenous injection, completely antagonized the serotonin-induced bradycardia with an ID50 value of 3.28 ug/kg. These data classify GYKI-46 903 as a non-competitive antagonist for 5-HT3 receptors.