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

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

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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

Recent additions of designer tryptamines and phenethylamines to the Drug Enforcement Administration's schedule of controlled substances necessitate analytical procedures for their detection and quantitation. As specific immunoassays are not currently available and cross-reactivities with existing assays are unknown, a screening method based on gas chromatography-mass spectrometry was developed. The method was capable of measuring the pentafluoropropionic derivatives of a-methyltryptamine (AMT), N,N-dimethyltryptamine (DMT), 4-bromo-2,5-dimethoxy-beta-phenethylamine (2CB), N,N-dipropyltryptamine (DPT), 2,5-dimethyl-4-N-propylthio-beta-phenethylamine (2C-T-7), and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DiPT). Separation was optimized to allow tentative identification of metabolites, which display common electron impact ionization fragmentation patterns. The screening method gave limits of detection between 5 and 10 ng/mL and demonstrated linearity between 50 and 1000 ng/mL. The method was successfully applied to blood and urine samples in suspected AMT intoxications. Confirmation of 5-MeO-DiPT in one of the subjects' urine was achieved using liquid chromatography-mass spectrometry (LC-MS). Quantitation by selected ion monitoring (SIM) yielded a urinary concentration of 229 ng/mL. The method was linear from 25 to 1500 ng/mL with a correlation coefficient of 0.995. The limit of detection was 5 ng/mL in urine on the LC-MS. Two additional peaks were observed and presumed to be metabolic products reported previously as 5-methoxy-N-isopropyltryptamine (5-MeO-iPT) and 5-methoxy-N,N-diisopropyltryptamine-N'-oxide (5-MeO-DiPT-N-oxide).

Although the serotonergic system plays an important role in various neurological disorders, the role of early serotonergic projections to the developing cerebral cortex is not well understood. Because serotonergic fibers enter the marginal zone (MZ) before birth, it has been suggested that they may influence cortical development through synaptic contacts with Cajal-Retzius (CR) cells. We used immunohistochemistry combined with confocal and electron microscopy to show that the earliest serotonergic projections to the MZ form synaptic contacts with the somata and proximal dendrites of CR cells as early as embryonic day 17. To elucidate the functional significance of these early serotonergic contacts with CR cells, we perturbed their normal development by injecting pregnant mice with 5-methoxytryptamine. Lower reelin levels were detected in the brains of newborn pups from the exposed animals. Because reelin plays an important role in the cortical laminar and columnar organization during development, we killed some pups from the same litters on postnatal day 7 and analyzed their presubicular cortex. We found that the supragranular layers of the presubicular cortex (which normally display a visible columnar deployment of neurons) were altered in the treated animals. Our results suggest a mechanism of how serotonergic abnormalities during cortical development may disturb the normal cortical organization; and, therefore, may be relevant for understanding neurological disorders in which abnormalities of the serotonergic system are accompanied by cortical pathology (such as autism).

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.

Foxy is slang for 5-methoxy-N,N-diisopropyltryptamine. It has hallucinogenic properties, similar to other tryptamine compounds, and is mildly euphoric. This case report describes a 21-year-old Caucasian man who ingested a pill called Foxy containing an unknown amount of drug. He was observed in hospital for 2 h, during which time he had mild hallucinations and could not move his limbs. A urine sample was collected approximately 4 h after drug ingestion. The patient was then discharged with no follow up assessment. The 5-methoxy-N,N-diisopropyltryptamine was identified in the urine by gas chromatography-mass spectrometry. Standards prepared from the pure material were used in the identification. Quantitative analysis using the same analytical technique resulted in a urinary concentration of 1.7 micro g/mL. Through oxidative deamination, the metabolite, 5-methoxy-indole acetic acid, was formed. It was identified in the urine, and the concentration was determined to be 1.3 micro g/mL using gas chromatography-mass spectrometry. Two other compounds were discovered in the urine sample as a result of a routine drug screen. From their mass spectra, they were tentatively identified as 5-methoxy-N-isopropyltryptamine and 5-methoxy-N,N-diisopropyltryptamine-N'-oxide.

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.

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.

Autism is classified as a pervasive developmental disorder, with several cardinal features including sensory disturbances, obsessive-compulsive-like behavior, lack of bonding to caregivers and motor disturbances. To date, there is a lack of an animal model of the disease. The current work is aimed at producing such a model by treating developing rat pups with a serotonergic agonist, 5-methoxytryptamine (5-MT; 1 mg/kg) during development (from gestational age 12 days to postnatal day 20), thus mimicking one of the hallmark neurochemical features of the illness-increases in the neurotransmitter, serotonin. Animals were then tested in behavioral paradigms that may resemble the human illness. Treated rat pups were found to be overreactive to auditory or tactile sensory stimuli, to display changes in the negative geotaxic test of motor development, to show lack of separation-induced vocalizations when their dam was removed and to show decreased alternation in the spontaneous alternation task. As well, the animals showed metabolic abnormalities in the brain using in vivo proton magnetic resonance spectroscopy, which are consistent with those observed in autistic children. In summary, the model we are proposing shows some of the behavioral and metabolic features of autism, as well as being produced through alteration of a neurochemical system known to be altered in autism.

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

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

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

Substances based on the N-(2-methoxybenzyl)phenethylamine template ('NBOMe' derivatives) play an important role in medicinal research but some of these derivatives have also appeared as 'research chemicals' for recreational use which has attracted attention worldwide. A major challenge associated with newly emerging substances includes the lack of analytical data and the ability to correctly identify positional isomers.Six N-benzylphenethylamines based on the 2,5-dimethoxy-4-iodophenethylamine structure ('25I') and twelve substituted N-benzyl-5-methoxytryptamines ('5MT') have been prepared and extensively characterized. Techniques used for characterization were gas chromatography/ion trap mass spectrometry in electron and chemical ionization mode, liquid chromatography/diode array detection (DAD), infrared spectroscopy, electrospray high mass accuracy quadrupole time-of-flight tandem mass spectrometry, and triple quadrupole tandem mass spectrometry.The characterization of 18 'NBOMe' compounds provided a comprehensive collection of chromatographic and spectral data. Four groups of three positional isomers, i.e. 25I-NB2OMe, 25I-NB3OMe, 25I-NB4OMe, 25I-NB2B, 25I-NB3B, 25I-NB4B and their 5-methoxytryptamine counterparts, were included and assessed for ability to obtain differentiation. Six meta-substituted N-benzyl derivatives of 5-methoxytryptamine (CF3, F, CH3, Cl, I, SCH3) were also studied.The implementation of mass spectral techniques was helpful for the differentiation between isomers, for example, when considering the difference in a number of ion ratios. This was considered beneficial in cases where chromatographic separation was only partially achieved under liquid chromatography (LC) conditions. The use of LC/DAD analysis was also found to be valuable for this particular purpose, which confirmed the integrative value of complementary techniques used in areas related to forensic toxicology.

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.

5-Methoxytryptamine and L-tryptophan methyl ester were acylated with malonic acid, dimethyl malonate, or succinic anhydride to produce the corresponding N, N'-dicarbonyltryptamine derivatives. The analgesic activity was evaluated by the tail flick test. All of the compounds exhibited desirable analgesic potency. This result is consistent with that of N-(N-acetyl-L-tryptophanyl)-5-methoxytryptamine and confirmed that introducing substituted tryptamine into the amide chain of melatonin does enhance analgesic potency.

The inhibitory effects of endogenous and synthetic compounds on the nitration and oxidation of l-tyrosine by peroxynitrite were examined. Nitration and oxidation activities of l-tyrosine by peroxynitrite were estimated by monitoring the formation of 3-nitrotyrosine and dityrosine with a high-performance liquid chromatography-ultraviolet (HPLC-UV)-fluorescence detector system. Glutathione and synthetic compounds ((2S,3R,4S)-N-ethylmercapto-3,4-dihydroxy-2-hydroxymethylpyrrolidine, l-N-dithiocarboxyproline) inhibited both the nitration and the oxidation reactions of l-tyrosine effectively. On the other hand, 5-methoxytryptamine and lipoic acid inhibited only the nitration reaction of l-tyrosine, and instead increased the oxidation reaction. It was assumed that 5-methoxytryptamine and lipoic acid reacted only with the nitrating species of peroxynitrite. This is the first report of a selective inhibitor for the nitrating reaction of peroxynitrite.

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.

A fatal overdose involving case by 5-methoxy- N , N -diisopropyltryptamine (5-MeO-DIPT) is reported. 5-MeO-DIPT and its two metabolites, 5-hydroxy- N , N -diisopropyltryptamine (5-OH-DIPT) and 5-methoxy- N -isopropyltryptamine (5-MeO-NIPT), were identified by LC–MS. The level of 5-MeO-DIPT, 5-OH-DIPT and 5-MeO-NIPT in blood and urine was 0.412, 0.327 and 0.020μg/ml, and 1.67, 27.0 and 0.32μg/ml, respectively. These blood and urine levels were higher than published data for such poisoning.

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.

Two mutations of the rat serotonin 5-HT2A receptor were made, expressed and examined for their ability to bind and be stimulated by certain tryptamines as well as their ability to bind antagonists. Mutation of Ser207 to an Ala (S207A) resulted in no substantial changes in binding of either 5-HT2A antagonists or agonists. In contrast, mutation of Ser239 to an Ala (S239A) resulted in significant changes in the 5-HT2A receptor with some but not all agonists and antagonists examined. Specifically, 5-HT had decreased affinity for the S239A mutated 5-HT2A receptor, showing over a 10-fold decrease in receptor-binding displacement, while still being capable of stimulating IP3 formation. However, the agonists tryptamine, 5-methoxytryptamine (5-MeOT), and N-1-isopropyl-5-methoxytryptamine; and the antagonists ketanserin, LY 86057, and LY 53857 were significantly less affected by a S239A mutation. These results suggest that while 5-HT might have a direct interaction with the Ser239 of the 5-HT2A receptor, tryptamine and 5-MeOT interact with this receptor in a different manner.

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).