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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.

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.

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.

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.

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.

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

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.

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.

Emerging evidence has implicated a potential role for 5-HT(4) receptors in cognition and anxiolysis. One of the main target structures of 5-HT(4) receptors on 'cognitive and emotional' pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5-HT(4) receptors on GABA(A) receptor channels in PFC pyramidal neurons. Application of 5-HT(4) receptor agonists produced either an enhancement or a reduction of GABA-evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABA(A) receptor beta3 or beta1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that beta3 and beta1 subunits are co-expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5-HT(4) receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5-HT(4) receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5-HT(4) receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network.

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.

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.

The recent cloning of three distinct melatonin receptor subtypes (Mel1a, Mel1b and Mel1c) which are part of a new family of G-protein coupled receptors, and probably mediate the physiological actions of the hormone, has spurred interest in the design of analogues with subtype selectivity. The 5-methoxyl and N-acetyl groups of melatonin are important for binding to and activation of the receptor. The indole nucleus serves to hold these two groups at the correct distance from one another and allows them to adopt the required orientation for interaction with the receptor binding pocket. We have investigated the subtype selectivity of a number of analogues of melatonin in which the structure has systematically been modified in order to probe the similarities and differences in the interaction of ligand and receptor subtype. At all three subtypes 5-methoxyl and N-acetyl groups of melatonin are important for high affinity binding. However, replacing the 5-methoxyl group (eg with 5-H, 5-OH, 5-Me or 5-BzO) reduces affinity much less at the Mel1b receptor subtype than at either Mel1a or Mel1c cloned subtypes. This suggests differences between the Mel1b and Mel1a/1c subtypes in the size and shape of the binding pocket or in the manner in which melatonin interacts with the receptor at this position. Further studies have revealed that analogues with longer N-acyl carbon chains behave similarly at each subtype. These observations suggest that the 'pocket' into which the N-acetyl group fits is very similar for each subtype. Substitutions at the 2-position on the indole ring improved affinity at each receptor subtype but did not give selective analogues. The systematic 'mapping' of the requirements for binding at each receptor subtype should allow the design of more selective agonists and antagonists, which will be valuable tools for the characterization and classification of functional melatonin receptors.

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 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.

1. The aim of this study was to characterize the receptors mediating the atropine-resistant neurogenic contraction to 5-hydroxytryptamine (5-HT) in the longitudinal muscle of the guinea-pig proximal colon and to determine the type of tachykinin receptors involved in the contractile response to 5-HT by the use of selective antagonists. 2. In the presence of atropine (0.3 microM), guanethidine (5 microM), hexamethonium (100 microM), ketanserin (0.1 microM) and indomethacin (3 microM), 5-HT (0.01-3 microM) produced concentration-dependent neurogenic contractions of colonic strips and at 0.3 microM produced a maximal effect (pEC50 = 7.39 +/- 0.09, n = 18). The 5-HT4 receptor stimulant, 5-methoxytryptamine (5-MeOT, 0.03-10 microM) also produced neurogenic contractions with similar maximum effect to those of 5-HT (pEC50 = 6.89 +/- 0.16). 3. The 5-HT4 receptor antagonist, DAU 6285 (3 microM) shifted the concentration-response curves to both 5-HT and 5-MeOT to the right without significant depression of the maximum, but the 5-HT1/5-HT2 receptor antagonist, metitepine (0.1 microM) and the 5-HT3 receptor antagonist, ondansetron (0.3 microM) had no effect on the control curves to 5-HT and 5-MeOT. 4. The selective NK1 receptor antagonist, FK 888 (1 microM) markedly attenuated the contractions to 5-HT and 5-MeOT. In contrast, the selective NK2 receptor antagonist, SR 48968 (10 nM) and the selective NK3 receptor antagonist, SR 142801 (10 nM) had no effect on the contractions to 5-HT and 5-MeOT. 5. These results indicate that the 5-HT-induced atropine-resistant neurogenic contraction of guinea-pig proximal colon is due to activation of 5-HT4 receptors, presumably located on excitatory motor neurones, innervating the longitudinal muscle. The contraction evoked by activation of the 5-HT4 receptors is mediated primarily via NK1 receptors but not NK2 or NK3, suggesting that the 5-HT4 receptor-mediated contraction is evoked indirectly via tachykinin release from tachykinin-releasing excitatory neurones.

The effect of benzodiazepines (BZP) on melatonin release was investigated in the pineal gland of the rainbow trout, Oncorhynchus mykiss, maintained under in vitro perifusion culture conditions. Melatonin and the methoxyindoles 5-methoxytryptophol (5-MTOL), 5-methoxyindoleacetic acid (5-MIAA), and 5-methoxytryptamine (5-MT) were determined directly in samples of the superfusion medium by HPLC with electrochemical detection. Melatonin release was significantly increased by addition of diazepam and clonazepam in a dose-related and reversible manner. The effects of benzodiazepines were more pronounced in light-adapted pineal organs, when melatonin secretion is low, than under scotopic conditions. When the perifusion medium was replaced by a medium containing low calcium, high magnesium concentrations, melatonin release was considerably decreased by 70% in light-adapted and 20% in dark-adapted pineal organs. Addition of diazepam to low Ca2+, high Mg(2+)-medium reversed the decrease of melatonin release and produced a clear rise in its secretion rate. Addition of the BZP antagonist flumazenil to the perifusion medium slightly decreased melatonin release in the light- and dark-adapted state, whereas the peripheral receptor antagonist PK 11195 did not alter melatonin release. The effect of diazepam is reduced by simultaneous addition of flumazenil to the superfusion medium, suggesting that the effects of diazepam are receptor-mediated. The methoxyindoles 5-MTOL, 5-MIAA, and 5-MT showed no significant changes of their release pattern after diazepam application in light- and dark-adapted pineal organs. These results suggest that BZP can influence melatonin production and release by an intrapineal action possibly on the melatonin synthesizing photoreceptor cell.

1. The involvement of neurokinins in the non-cholinergically-mediated contractile response induced by stimulation of 5-HT3 and 5-HT4 receptors has been examined in the longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum. 2. The 5-HT3 receptor agonist, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT), showed a lower potency in this preparation than the more selective 5-HT4 receptor agonist 5-methoxytryptamine. The effect of both drugs was markedly reduced by atropine. 3. Substance P (SP) and neurokinin B (NKB) produced biphasic concentration-response curves in the preparation. Neurokinin A (NKA), the NK1 receptor agonist, [Sar9,Met(O2)11]SP and the NK3 receptor agonist, senktide yielded monophasic concentration-response curves. 4. After desensitization of the NK1 receptor with SP or [Sar9,met(O2)11]SP, in the presence of atropine, the contractile response to 2-methyl-5-HT was entirely blocked. Desensitization of NK3 receptors with NKB, also in the presence of atropine, fully suppressed the 5-HT4 receptor-mediated contraction evoked by 5-methoxytryptamine. 5. In preparations prelabelled with [3H]-choline, SP produced a concentration-dependent increase in tritium overflow, an index of [3H]-acetylcholine release, while an inverse relationship was found with NKB. At low neurokinin concentrations, the releasing effect of NKB was much more marked. 6. It is suggested that in the response to 5-HT3 receptor stimulation, there is a role for SP and acetylcholine. NKB appears to be preferentially involved in the release of acetylcholine elicited by stimulation of 5-HT4 receptors.

1. The 5-HT4 receptor has only recently been identified but has yet to be cloned. This paper describes the pharmacology of a potent and selective 5-HT4 receptor antagonist, GR113808, which will be useful in the further characterization of this receptor. 2. On the guinea-pig ascending colon, GR113808 (1 nM-0.1 microM) behaved as an antagonist of 5-hydroxytryptamine (5-HT)-induced contraction, producing rightward displacements of the concentration-effect curve to 5-HT and a concentration-related depression of the maximum effect. However, the compound had no effect on cholecystokinin (CCK-8)-induced contraction in concentrations up to 1 microM. 3. In the guinea-pig colon preparation, onset and offset of the antagonism by GR113808 of 5-HT-induced contraction was examined. Incubation of the tissues for either 15 min, 30 min or 60 min produced similar rightward displacements of the concentration-effect curves to 5-HT, with no increase in the degree of depression of the maxima with increasing time of incubation. Experiments examining offset of antagonism (0.01 microM) demonstrated that washout for 30 min was required to reverse fully the effects of the antagonist. 4. Potency estimates in the colon for GR113808 were made by determining approximate pA2 values (30 min) using the Gaddum equation. The values obtained were 9.2, 9.7 and 9.2 when tested against the agonists 5-HT, 5-methoxytryptamine and R,S-zacopride respectively. 5. On the carbachol-contracted tunica muscularis mucosae preparation of the rat thoracic oesophagus, GR113808 behaved as an antagonist of 5-HT-induced relaxation, producing no reduction in maximum response. Analysis of these data yielded a pA2 of 9.3. GR1 13808 also antagonised the relaxant effects of 5-methoxytryptamine (pA2 = 9.0) and R,S-zacopride (pA2 = 9.4). The compound had no effect on isoprenaline-induced relaxation of the carbachol-contracted oesophagus at a concentration of 1 MicroM.6. In tests of selectivity, GR113808 had only low affinity for 5-HT3 receptors (pKi = 6.0) and had no functional activity at either 5-HT2 or 5-HT1-like receptors on vascular smooth muscle preparations. In a range of binding assays, GRi 13808 was shown to have no appreciable affinity for any other receptor type investigated.7. In the anaesthetized piglet, GRI13808 was a potent antagonist of 5-methoxytryptamine-induced tachycardia (mean DRo = 97.2 microg kg-1 h-1). The compound was ineffective against isoprenaline-induced tachycardia.8. The present results are discussed in comparison with those for existing antagonists at the 5-HT4receptor. The results of this study indicate that GRI13808 will be a valuable antagonist for studying 5-HT4 receptor mechanisms in vitro and in vivo and validate its use as a radioligand for determining 5-HT4 receptor distribution.

Twenty-six title compounds with the ethylamine part of tryptamine or 5-methoxytryptamine fixed in an anticlinal ecliptic conformation were synthesized for assaying them at the different known serotonin receptors. Several alkylation methods have been improved and adapted for the space consuming norbornane system. The structures were fully elucidated by high-field NMR spectroscopy. All 1H- and 13C-signals could be assigned by means of 1H-1H- and 13C-1H-correlation spectroscopy (COSY). Selektivitat sterisch fixierter Tryptamin- und 5-Methoxytryptamin-derivate an Serotoninrezeptorsubtypen, 1. Mitt: Synthese von N-Alkyl- und N,N-Dialkyl-3-indolylbicyclo[2.2.1]heptan-2-aminen Sechsundzwanzig Titelverbindungen wurden dargestellt, um sie pharmakologisch an verschiedenen Serotoninrezeptortypen zu prufen. In diesen Verbindungen liegt der Ethylaminteil des Tryptamins bzw. 5-Methoxytryptamins in anticlinal ekliptisch fixierter Konformation vor. Hierzu wurden verschiedene Alkylierungsmethoden optimiert und an die Erfordernisse des sterisch anspruchsvollen Norbornansystems angepast. Alle Strukturen wurden durch Hochfeld-NMR-Spektroskopie gesichert. Durch 1H-1H-und 13C-1H-Korrelationsspektroskopie (COSY) konnten alle Protonen- und Kohlenstoffresonanzen des Norbornansystems zweifelsfrei zugeordnet werden.

An efficient synthesis of [fluoroacetyl-18F]fluoromelatonin (Nω-[18F]Fluoroacetyl-5-methoxytryptamine) starting from [18F]fluoride and ethyl p-toluensulfonyloxyacetate is described. The total time required for its synthesis is ca. 90 min. The radiochemical yield, purity and specific activity (end of bombardment) of the desired compound are 14.2%,>98%, and 540 mCi/μmol, respectively.

Melatonin deacetylase, an enzyme activity recently discovered in the Xenopus laevis retina, regulates local melatonin levels. The deacetylase occurs in retina, retinal pigment epithelium, and skin, all sites of melatonin action, and is widely distributed among vertebrates. We have solubilized the enzyme from Xenopus retina and pigment epithelium using nonionic detergents, and have developed a specific enzyme assay. We have characterized the enzyme and now report that the deacetylase is relatively specific for melatonin and is inhibited by the melatonin precursor N-acetylserotonin and the product of the deacetylase, 5-methoxytryptamine. Inhibition of deacetylase activity by eserine (physostigmine) suggests a relationship between deacetylase and cholinesterase activities. However, among a variety of cholinesterase inhibitors tested, only eserine inhibits the deacetylase. Furthermore, eserine is much less potent as an inhibitor of the deacetylase than the cholinesterases, and purified cholinesterases failed to deacetylate melatonin. We also show that melatonin deacetylase and aryl acylamidase (an enzyme related to cholinesterases) activities are differentially extractable from Xenopus ocular tissues, and that they exhibit different pH optima and inhibition profiles. Our results provide an initial characterization of the Xenopus retinal melatonin deacetylase, and indicate that deacetylase activity is distinct from cholinesterase and aryl acylamidase activities.

We have studied the regional distribution of 5-hydroxytryptamine (5-HT) receptor subtypes in fresh circular segments of human cerebral, middle meningeal, and temporal arteries. Vasomotor responses induced by a series of 5-HT agonists and antagonists with some degree of selectivity were studied by using a sensitive in vitro system. Nine 5-HT agonists were examined for contractile effects on the arteries. In cerebral and meningeal arteries 5-carboxamidotryptamine (5-CT) was more potent than 5-HT. The opposite order of potency (5-HT-5-CT) was found in temporal arteries. In the cerebral arteries 5-methoxytryptamin (5-MeOHT) was more potent than sumatriptan while sumatriptan was more potent than 5-MeOHT in meningeal and temporal arteries. The 5-HT1, receptor antagonist, methiothepin, competitively antagonized 5-CT-induced contractions in cerebral arteries, with a PA2, value of 9.05. 5-HT-induced contractions were competitively antagonized by ketanserin (5-HT2) in the temporal arteries PA2, value of 9.06). Methiothepin and ketanserin had non-competitive antagonistic effects in the middle meningeal arteries. The 5-HT3, selective antagonist ondansetron did not cause any shift of the contractions induced by 2-methyl-5-HT in the temporal, cerebral and middle meningeal arteries. These results suggest that the cerebral arteries mainly contain 5-HTid., or 5-HT1,-like receptors, and the temporal artery 5-HT2, receptors; the data further indicate the presence of both receptor subtypes in the middle meningeal artery.

The aim of this study was to determine whether pineal indoles affect cyclic nucleotide levels in mammary gland slices of BALB/c adult mice. Melatonin at 0.1 nM-10 microM concentrations decreased cAMP and augmented cGMP concentration in murine mammary gland slices in the presence of a phosphodiesterase inhibitor (1 mM theophylline), an index of cyclic nucleotide synthesis. Melatonin-induced changes in cyclic nucleotide levels were significantly larger at the end of the light period (2000) than in the morning (at 1000). Indole-induced inhibition of cyclic AMP levels by mammary slices exhibited the following order of potency: 5-methoxytryptamine > melatonin > or = 6-hydroxymelatonin > serotonin, N-acetylserotonin > 5-hydroxytryptophol. The order of potency for indole-induced augmentation of cyclic GMP levels was: 5-methoxytryptamine > melatonin > 6-hydroxymelatonin > serotonin, N-acetylserotonin, 5-hydroxytryptophol. When melatonin or 5-methoxytryptamine (10 nM) were examined for their effects on cAMP and cGMP levels in mammary glands of mice killed at six different time intervals during the 24-hr cycle, the activity was maximal during night. The data demonstrate that 5-methoxytryptamine and melatonin decreased cAMP and increased cGMP levels in mammary gland slices. Methoxyindole-induced changes in cyclic nucleotide synthesis in murine mammary glands exhibit the time-dependency known to occur in several other melatonin-influenced responses.

The possible effect of melatonin, 5-methoxytryptamine, 5-methoxytryptophol, 6-chloromelatonin and 2-iodomelatonin on testosterone production by Leydig cells in vitro was investigated. The ability of individual indoles to inhibit testosterone production was found to depend on the concentration used. The relative inhibitory potency of the compounds tested was: 6-chloromelatonin greater than 2-iodomelatonin greater than melatonin greater than 5-methoxytryptamine greater than 5-methoxytryptophol. The results revealed that natural indoles which are synthesized in the pineal gland and their halogenized derivatives are capable of influencing directly testosterone production by Leydig cells. Also, these results demonstrated that melatonin exerts its remarkable antigonadotrophic effects, at least in part, through the direct decrease of testosterone production. Moreover, 6-chloromelatonin and 2-iodomelatonin, which are reported to inhibit melatonin binding to target tissues, possess properties of biological melatonin analogues under the conditions of the model system used.

The Harderian glands of Syrian hamsters contain melatonin and the enzymes N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) which synthesize melatonin from serotonin. Because the Harderian glands share this metabolic pathway with the pineal gland, we examined the effects of short-day photoperiods, which stimulate pineal-mediated gonadal regression, and N-(2,4-dinitrophenyl)-5-methoxytryptamine (ML-23), which has been described as a melatonin antagonist, on melatonin synthesis in the Harderian glands of the hamster. Harderian glands of male hamsters kept in short days had reduced NAT activity and melatonin concentration, but HIOMT activity was unchanged from that of longday controls. In males kept in short days, ML-23 restored melatonin concentrations to levels seen in long days but did not affect the short-day induced reduction in NAT activity. ML-23 had no effect upon NAT or HIOMT activity or melatonin concentration in male hamsters kept on long days. Harderian glands of female hamsters kept on short days had reduced melatonin concentrations, but NAT and HIOMT activities similar to those of long-day controls. ML-23 had no effect on Harderian NAT or HIOMT activities or melatonin concentration in females kept in short days. However, in females kept in long days, ML-23 treatment led to increased NAT activity and decreased melatonin concentrations. We conclude from these results that short-day photoperiods alter some aspects of melatonin synthesis in hamster Harderian glands and that these effects differ in males and females. ML-23 does not usually prevent the effects of short days on Harderian melatonin synthesis, suggesting that it is not a melatonin antagonist in the Syrian hamster.

"Synaptic" ribbons and spherules are typical organelles of the mammalian pinealocytes, that present quantitative day-night variations parallel to pineal melatonin levels. The European hamster is a wild photoperiodic mammal whose pineal melatonin concentrations do not vary during the 24 hr light-dark cycle. The aim of this study was thus to see if a rhythmic variation in synaptic structures was nevertheless present in this species. It was found that, in contrast to all of the photoperiodic species studied to date, the European hamster pinealocyte contains no ribbons, and virtually no spherules. Moreover, constant administration of melatonin does not result in the occurrence of these organelles; nevertheless, the pinealocytes have the potentiality to produce spherules and, to a lesser extent, ribbons, as they are observed in ganglionectomized animals and in the deep pineal of some hamsters after ablation of the superficial part of the gland. The formation of pineal synaptic structures, normally lacking in the European hamster, thus seems directly related to the noradrenergic innervation of the gland.

Dark-adapted retinas of mice (C57BL/6J) incubated in the dark in media containing 1 mM 3-isobutylmethylxanthine (IBMX) or 5 mM Co2+ accumulate cyclic AMP (cAMP). A portion of this pool is light sensitive, as light can prevent or reverse its accumulation. Similarly, tryptamine, serotonin, 5-methoxytryptamine, bufotenine, and 5-methoxydimethyltryptamine can block the accumulation of the light-sensitive pool of cAMP, whereas tryptophan, melatonin, N-acetylserotonin, 5-methoxytryptophol, and tetrahydro-beta-carbolines are inactive. The phenomenon is not seen with mutant mouse retinas (rd/rd), which lack most photoreceptors, but persists in abnormal retinas containing photoreceptors but with extensive neuronal depletion in the inner retina. Tryptamine also inhibits cAMP accumulation in either dark or light-adapted retinas exposed to forskolin alone but not in media containing high levels of forskolin plus 1 mM IBMX. There is some suggestion that serotonin 5-HT-2 antagonists can partially reverse the action of the tryptamines, but hitherto undescribed receptors may be involved. Current data suggest that photoreceptors are the target for the action of the tryptamines.

The involvement of the Harderian gland, atrophied eyes, and melatonin in the perception of photoperiodic changes has been studied in the mole rat, a fossorial blind mammal the thermoregulatory capacity of which is photoperiod-dependent. When transferred from a long photoperiod to a short one, mole rats increase their resistance to cold, a perfectly reversible phenomenon. After 2 weeks under short photoperiod the thermoregulatory capacities of animals without Harderian glands are less than those of the controls. The Harderian gland appears thus to be implicated in the detection of photoperiodic changes. After 5 weeks, however, the Harderianectomized animals had perfectly integrated the photoperiodic change, so demonstrating that other photoreceptor organs exist. The atrophied eyes, which, under these conditions, do not seem to play an important role, are involved when the animals are transferred from short photoperiod to long photoperiod. Melatonin, but not 5-methoxytryptamine, appears to be a crucial compound in such a phenomenon. These results, which demonstrate that in mammals (at least in the mole rat, as in nonmammalian vertebrates), nonocular photoreceptors exist, suggest that the mechanism by which mammals integrate photoperiodic changes is not the same when the animals are transferred from long to short photoperiod as when transferred from short photoperiod to long photoperiod.

The role of the pineal and of photoperiod in the regulation of gonadal activity has been studied in the European hamster, a wild hamster commonly found in the vicinity of Strasbourg, France. Besides the presence of a probable endogenous circannual rhythm in reproductive capacities, it appears that, as in all other hamster species studied to date, the sexual activity of this animal is directly regulated by changes in the photoperiodic environment. Pinealectomy as well as a constant release of melatonin or of 5-methoxytryptamine has no effect on the gonadal atrophy induced by short photoperiod, but induces testicular regression in animals kept under long photoperiod. The pineal in this species appears thus to be involved in the maintenance of sexual activity in long photoperiod, while it is apparently not required for the gonadal inhibitory effect of short photoperiod. These results are similar to those obtained in the Turkish hamster. 5-Methoxytryptophol is not implicated in the phenomenon of synchronization of the sexual activity with the photoperiodic changes.

In superfused rat hypothalamic slices prelabeled with [3H]-5-hydroxytryptamine [( 3H]-5-HT), the 5-HT autoreceptor agonists 5-methoxytryptamine and RU 24969 inhibited in a concentration-dependent manner the electrically evoked release of [3H]-5-HT. Exposure to phorbol-12,13-dibutyrate increased in a concentration-dependent manner the stimulation-evoked overflow of [3H]-5-HT and shifted to the right the 5-methoxytryptamine inhibition curve. Exposure to forskolin, a potent activator of adenylate cyclase, increased the stimulation-evoked [3H]-5-HT overflow and shifted to the left the 5-methoxytryptamine or RU 24969 inhibitory curves on transmitter release. A similar interaction was observed in the presence of 1-isobutyl,3-methylxanthine (IBMX), a phosphodiesterase inhibitor, or 8-bromo-cyclic AMP and the serotonin autoreceptor agonist 5-methoxytryptamine. In the presence of phorbol-12,13-dibutyrate or forskolin, the enhancing effect of the 5-HT autoreceptor antagonist methiothepin on the stimulation-evoked [3H]-5-HT overflow was significantly less pronounced than in the absence of these compounds. These results indicate that the presynaptic 5-HT autoreceptors that modulate the release of [3H]-5-HT in rat hypothalamic slices may be coupled to the phosphoinositide cycle and protein kinase C-dependent mechanisms. In addition, the increase in intracellular level of cyclic AMP by forskolin, IBMX, and 8-bromo-cyclic AMP potentiates the inhibitory effects of the autoreceptor agonist 5-methoxytryptamine on [3H]-5-HT release, although the mechanism of the interaction remains to be elucidated.

Antisera were raised against tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-methoxytryptophan, and 5-methoxytryptamine, by conjugating each molecule to bovine serum albumin and to human serum albumin via glutaraldehyde, in such a way as to preserve the original part. Antibody specificity was tested with the enzyme-linked immunosorbent assay method. The specificity of each anti-indolealkylamine-glutaraldehyde antibody was established with competition experiments by using an adsorbed immunogenic conjugate and indolealkylamines either free or conjugated with poly-L-lysine. The nonconjugated compounds were poorly recognized. In the same way, the nonreduced conjugates always appeared less immunoreactive than the reduced ones. Calculated from the specificity study of each antiserum, the cross-reactivity ratios were found to be smallest for the most immunoreactive conjugates. Thus, a specific immune response was defined for each compound belonging to the same metabolic pathway.

Though melatonin is primarily metabolised to 6-hydroxy-melatonin, we have recently shown that it can also be demethylated to form N-acetyl-serotonin. The question therefore arises as to whether demethylation is a general metabolic pathway that can apply to other pineal methoxyindoles. To investigate this possibility we administered deuterated methoxy-tryptophol (dML) and deuterated methoxy-tryptamine (dMT) to rats and analysed the urine for the presence of deuterated methoxyindole acetic acid (dMIAA) and deuterated hydroxyindole acetic acid (dHIAA). The method of analysis was gas chromatography mass spectrometry (GCMS), where the relevant molecular ion and fragment ions were monitored. The results showed that the major metabolite in all cases was dMIAA. There was no evidence to suggest that the compounds had been demethylated to form dHIAA. The study therefore indicates that the demethylation of melatonin is a specific metabolic pathway that does not apply to other methoxyindoles.

A quantitative gas chromatographic mass spectrometric method using a deuterated analogue as internal standard were developed for the analysis of 5-methoxytryptamine in brain. The internal standard was synthesized in two steps and possessed high isotopic purity. The analytical procedure involves the addition of the internal standard to the homogenizing medium containing the tissue, followed by homogenization, extraction with chloroform and derivatization with pentafluoropropionic anhydride. The identification and quantitation of 5-methoxytryptamine is based on monitoring the ions at m/z 306, 319 and 482 for the pentafluoropropionyl derivative of 5-methoxytryptamine and m/z 308, 322 and 486 for the internal standard. Using this procedure 5-methoxytryptamine was identified and quantitated in the sheep pineal gland. The mean 5-methoxytryptamine level (N = 12) was 545 +/- 180 pmol g-1.

The effect of acute and chronic lithium treatments on 5-hydroxytryptamine (5-HT, serotonin) release and on its regulation by presynaptic 5-HT autoreceptors was studied in [3H]5-HT preloaded superfused rat brain slices. The [3H]5-HT overflow evoked by a 30-s exposure to 65 mM K+ was increased after 3 weeks of ingestion of lithium-containing diet in the three brain areas examined. Acute injection of 4 mEq/kg lithium chloride did not affect 5-HT release. The K+-induced release observed in both control and chronically lithium-treated animals was Ca2+-dependent. Chronic lithium treatment was also found to be associated with a decrease in basal [3H]5-HT overflow in the cortex and hypothalamus and with an increase in spontaneous hippocampal 5-HT overflow. The Ca2+-independent overflow induced by fenfluramine was also decreased in cortical slices from lithium-treated animals. The sensitivity of the inhibitory 5-HT autoreceptors was assessed by the response to the 5-HT agonist 5-methoxytryptamine. The results indicate a marked reduction in the maximal inhibition of [3H]5-HT release induced by 5-methoxytryptamine in slices obtained from animals which have been treated with lithium for 3 weeks. These data suggest that the functional down regulation of the prejunctional 5-HT sites may be responsible for the increase in K+-stimulated 5-HT overflow in brain slices of animals treated chronically with lithium.

Serotonin (5-hydroxytryptamine, 5-HT; 0.5 micro M and above) stimulated the release of [3H]dopamine ([3H]DA) from particulate fractions of the carp (Cyprinus carpio) retina. The 5-HT effect was dose- and Ca2+-dependent, and was structurally specific. A similar response was not elicited by the other indoles (5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine, 5-hydroxytryptophan, or 5-hydroxyindoleacetic acid) examined. An increase in [3H]DA release was elicited by addition of 5-HT agonists (5-methoxytryptamine, 5-methoxy-N,N-dimethyltryptamine, and tryptamine), but not antagonized by three 5-HT antagonists (metergolin, methysergide, and spiperone). Either DA alone or noradrenaline (0.5 mM) produced a large increase in [3H]DA release from the particulate fractions, but this action was Ca2+-independent. Further, no significant release of [3H] gamma-aminobutyric acid could be evoked by 5-HT (0.5 mM) under similar experimental conditions. Taken together, the present data suggest that 5-HT stimulates [3H]DA release from the fish retina through a specific receptor-mediated mechanism on dopaminergic terminals, but not through an exchange or nonspecific phenomenon.

Serotonin (50 microM) or its agonist 5-methoxytryptamine (5 microM) stimulated the acrosome reactions of golden hamster sperm within 15 min after addition to sperm capacitated in vitro for 4.5 h. The stimulation was inhibited by the serotonin receptor antagonists quipazine or cyproheptadine. Epinephrine (70 microM), norepinephrine (50 microM), and dopamine (25 microM) were unable to stimulate acrosome reactions even at 30 min under the same conditions, even though previous studies had demonstrated stimulation by these catecholamines at the same concentrations when present from the start of the capacitation time course. Epinephrine (5 microM) also was unable to stimulate at 30 min. These results demonstrate that serotonin and its agonist have a more direct effect on the hamster sperm acrosome reaction than other biogenic amines and that the effect is receptor-mediated.