The relative contribution of monoamine oxidase and cytochrome p450 isozymes to the metabolic deamination of the trace amine tryptamine.
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Tryptamine is a trace amine in mammalian central nervous system that interacts with the trace amine TA(2) receptor and is now thought to function as a neurotransmitter or neuromodulator. It had been reported that deamination of tryptamine to tryptophol was mediated by CYP2D6, a cytochrome P450 that is expressed in human brain, suggesting that tryptamine may be an endogenous substrate for this polymorphic enzyme. We were unable to confirm this report and have reinvestigated tryptamine metabolism in human liver microsomes (HLM) and in microsomes expressing recombinant human cytochrome P450 and monoamine oxidase (MAO) isozymes. Tryptamine was oxidized to indole-3-acetaldehyde by HLM and recombinant human MAO-A in the absence of NADPH, and indole-3-acetaldehyde was further reduced to tryptophol by aldehyde reductase in HLM in the presence of NADPH. Steady-state kinetic parameters were estimated for each reaction step by HLM and MAO-A. The CYP2D6 substrates bufuralol and debrisoquine showed strong inhibition of both tryptophol production from tryptamine in HLM and the formation of indole-3-acetaldehyde from tryptamine catalyzed by recombinant MAO-A. Anti-CYP2D6 monoclonal antibody did not inhibit these reactions. Pargyline, a nonselective MAO inhibitor, did not show cross inhibition to debrisoquine 4-hydroxylation and dextromethorphan O-demethylation by HLM and recombinant CYP2D6 enzyme. This is the first unequivocal report of the selective conversion of tryptamine to tryptophol by MAO-A. CYP2D6 does not contribute to this reaction. (+info)
Melatonin modulates the light-induced sympathoexcitation and vagal suppression with participation of the suprachiasmatic nucleus in mice.
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In mammals, the autonomic nervous system mediates the central circadian clock oscillation from the suprachiasmatic nucleus (SCN) to the peripheral organs, and controls cardiovascular, respiratory and gastrointestinal functions. The present study was conducted in mice to address whether light signals conveyed to the SCN can control peripheral autonomic functions, and further examined the impact of centrally administered melatonin on peripheral autonomic functions via activation of melatonin receptor signalling. In vivo electrophysiological techniques were performed in anaesthetised, open-chest and artificially ventilated mice whilst monitoring the arterial blood pressure and heart rate. Light induced an increase of the renal sympathetic nerve activity, arterial blood pressure and heart rate immediately after lights on. Conversely, light rapidly suppressed the gastric vagal parasympathetic nerve activity, which was affected neither by hepatic vagotomy nor by total subdiaphragmatic vagotomy. These autonomic responses were mediated by the SCN since bilateral SCN lesion totally abolished the light-evoked neuronal and cardiovascular responses. Melatonin administered intracerebroventricularly (I.C.V.) attenuated the sympathetic and vagal nerve activities in a dose-dependent manner with a threshold of 0.1 ng and these effects were blocked by I.C.V. pre-treatment of the competitive melatonin receptor antagonist luzindole. These results suggest that light induces sympathoexcitation and vagal suppression through the SCN and that melatonin modulates the light-induced autonomic responses via activation of the central melatonin receptor signalling. (+info)
Short-term exposure to melatonin differentially affects the functional sensitivity and trafficking of the hMT1 and hMT2 melatonin receptors.
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The hormone melatonin mediates a variety of physiological functions in mammals through activation of pharmacologically distinct MT(1) and MT(2) G protein-coupled melatonin receptors. We therefore sought to investigate how the receptors were regulated in response to short melatonin exposure. Using 2-[(125)I]iodomelatonin binding, cAMP functional assays, and confocal microscopy, we demonstrated robust differences in specific 2-[(125)I]iodomelatonin binding, receptor desensitization, and cellular trafficking of hMT(1) and hMT(2) melatonin receptors expressed in Chinese hamster ovary (CHO) cells after short (10-min) exposure to melatonin. Exposure to melatonin decreased specific 2-[(125)I]iodomelatonin binding to CHO-MT(2) cells (70.3 +/- 7.6%, n = 3) compared with vehicle controls. The robust decreases in specific binding to the hMT(2) melatonin receptors correlated both with the observed functional desensitization of melatonin to inhibit forskolin-stimulated cAMP formation in CHO-MT(2) cells pretreated with 10 nM melatonin (EC(50) of 159.8 +/- 17.8 nM, n = 3, p < 0.05) versus vehicle (EC(50) of 6.0 +/- 1.2 nM, n = 3), and with the arrestin-dependent internalization of the receptor. In contrast, short exposure of CHO-MT(1) cells to melatonin induced a small decrease in specific 2-[(125)I]iodomelatonin binding (34.2 +/- 13.0%, n = 5) without either desensitization or receptor internalization. We conclude that differential regulation of the hMT(1) and hMT(2) melatonin receptors by the hormone melatonin could underlie temporally regulated signal transduction events mediated by the hormone in vivo. (+info)
Donitriptan selectively decreases jugular venous oxygen saturation in the anesthetized pig: further insights into its mechanism of action relevant to headache relief.
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The effects of donitriptan on systemic arterial-jugular venous oxygen saturation difference were evaluated in pentobarbitone-anesthetized pigs. Oxygen and carbon dioxide partial pressures in systemic arterial and jugular venous blood as well as hemoglobin oxygen saturation were determined by conventional blood gas analysis. Vehicle (40% polyethyleneglycol in saline, n = 9) or donitriptan (0.01, 0.04, 0.16, 0.63, 2.5, 10, and 40 microg/kg, n = 7) were cumulatively infused over 15 min/dose. The involvement of 5-hydroxytryptamine(1B) (5-HT(1B)) receptors was assessed in the presence of the 5-HT(1B/1D) receptor antagonist, GR 127935. Donitriptan decreased markedly and dose dependently jugular venous oxygen saturation [ED(50) 0.5 (0.3-1.1) microg/kg], in parallel with increases in carotid vascular resistance [ED(50) 0.9 (0.7-1.1) microg/kg]. Since arterial oxygen saturation and partial pressure remained unchanged, donitriptan significantly increased arteriovenous oxygen saturation difference from 0.63 microg/kg (maximal variation: 57 +/- 18%, P < 0.05 compared with vehicle). Unexpectedly, donitriptan from 2.5 microg/kg induced marked and significant increases in carbon dioxide partial pressure (pVCO(2)) in venous blood (maximal increase 18.8 +/- 5.7%; P < 0.05 compared with vehicle). Pretreatment with GR 127935 (0.63 mg/kg, n = 5) abolished the fall in venous oxygen saturation and the increase in carotid vascular resistance and reduced the increases in pVCO(2) induced by donitriptan. The results demonstrate that donitriptan, via 5-HT(1B) receptor activation, decreases the oxygen saturation of venous blood draining the head, concomitantly with cranial vasoconstriction. Since donitriptan also increased pVCO(2), an effect upon cerebral oxygen consumption and metabolism is suggested in addition to cranial vasoconstriction, which may be relevant to its headache-relieving effects. (+info)
Diurnal regulation of arylalkylamine N-acetyltransferase activity in chicken retinal cells in vitro: analysis of culture conditions.
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PURPOSE: Arylalkylamine N-acetyltransferase (AANAT) is a key regulatory enzyme in the synthesis of melatonin, which displays daily fluctuations in chicken retinal photoreceptors in vivo. The purpose of the present study was to determine if cultures of embryonic neural retina cells express diurnal rhythms of AANAT activity. METHODS: Cell cultures were prepared from chick embryonic day 6 neural retina and incubated for 4 to 8 days in vitro (DIV). Cells were incubated under a daily light-dark (LD) cycle and were harvested day and night. Culture conditions were modified to test the effects of cell density, serum concentration, incubation temperature, S-(4-nitrobenzyl)-6-thioinosine (NBTI), and taurine on AANAT activity. AANAT activity was assayed in cell homogenates by measuring the catalytic formation of N-acetyltryptamine from tryptamine and acetyl coenzyme A. RESULTS: Cells cultured in medium containing 10% fetal bovine serum (FBS) failed to show any diurnal fluctuation in AANAT activity on DIV 5 and 6. However, if the culture medium was replaced on DIV 4 with one containing 1% FBS, and 5 microM NBTI or 5 mM taurine, the cells expressed significant diurnal rhythms of enzyme activity. NBTI was more potent and effective than taurine. Culture conditions were optimized with respect to cell density, serum concentration, incubation temperature, and NBTI concentration. Under optimized conditions, overall cell survival and the density of photoreceptor cells were increased relative to that with the other culture conditions tested. CONCLUSIONS: The results indicate that diurnal rhythms of AANAT activity are expressed in embryonic retinal cells incubated under particular culture conditions. The results show that the mechanisms regulating melatonin synthesis in chicken retinal cells are established during early embryonic life. This culture preparation will be useful in elucidating the photic control mechanisms involved in regulation of melatonin biosynthesis in photoreceptor cells. (+info)
Ocular hypotensive effects of melatonin receptor agonists in the rabbit: further evidence for an MT3 receptor.
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(1) Melatonin is involved in the control of intraocular pressure during the night and day photoperiod. We have investigated the receptor that regulates intraocular pressure in New Zealand white rabbits by means of agonists and antagonists of melatonin receptors. (2) Melatonin and its analogues: 2-Phe-melatonin, 6-Cl-melatonin, 2-I-melatonin, 5- methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) and N-acetyltryptamine all produced a reduction in intraocular pressure. Dose-response analysis for these compounds gave pD(2) values of 9.3+/-0.24 for melatonin; 9.0+/-0.09 for 6-Cl-melatonin; 9.0+/-0.84 for 2-I-melatonin; 8.9+/-0.07 for 5-MCA-NAT; 8.7+/-0.18 for 2-Phe-melatonin and 9.4+/-0.30 for N-acetyltryptamine (all n=8). (3) At a dose of 0.5 nmol (in 10 micro l) melatonin and the selective melatonin MT(3) agonist 5-MCA-NAT, induced greater reductions of intraocular pressure (22.8+/-2.3% and 32.5+/-1.4%, respectively) than the other compounds. (4) The melatonin-receptor antagonists, prazosin, DH-97 and 4-P-PDOT, reversed the effect of 5-MCA-NAT in a dose-dependent manner, with pA(2) values of 13.5+/-0.17 for prazosin, 10.6+/-0.16 for DH-97 and 9.4+/-0.20 for 4-P-PDOT (n=8). (5) Cholinoceptor antagonists (hexamethonium and atropine) and alpha(2)- and beta(2)-adrenoceptor antagonists (yohimbine and ICI 118,551) partially reversed the effects produced by melatonin and 5-MCA-NAT, suggesting the possible involvement of cholinergic and noradrenergic systems in the hypotensive actions mediated by melatonin agonists. The alpha(1)-adrenoceptor antagonist, corynanthine, had no significant effect. (6) The strong hypotensive effect of the MT(3) agonist, 5-MCA-NAT, suggests that this compound may be a useful agent for treating those pathologies where intraocular pressure is abnormally elevated. (+info)
Identification of the human liver enzymes involved in the metabolism of the antimigraine agent almotriptan.
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Almotriptan is a novel highly selective 5-hydroxytryptamine(1B/1D) agonist developed for the acute oral treatment of migraine. The in vitro metabolism of almotriptan has been investigated using human liver subcellular fractions and cDNA-expressed human enzymes, to study the metabolic pathways and identify the enzymes responsible for the formation of the major metabolites. Specific enzymes were identified by correlation analysis, chemical inhibition studies, and incubation with various cDNA expressed human enzymes. Human liver microsomes and S9 fraction metabolize almotriptan by 2-hydroxylation of the pyrrolidine group to form a carbinolamine metabolite intermediate, a reaction catalyzed by CYP3A4 and CYP2D6. This metabolite is further oxidized by aldehyde dehydrogenase to the open ring gamma-aminobutyric acid metabolite. Almotriptan is also metabolized at the dimethylaminoethyl group by N-demethylation, a reaction that is carried out by five different cytochrome P450s, flavin monooxygenase-3 mediated N-oxidation, and MAO-A catalyzed oxidative deamination to form the indole acetic acid and the indole ethyl alcohol derivatives of almotriptan. The use of human liver mitochondria confirmed the contribution of MAO-A to the metabolism of almotriptan. Both, the gamma-aminobutyric acid and the indole acetic acid metabolites have been found to be the major in vivo metabolites of almotriptan in humans. In addition, different clinical trials conducted to study the effects of CYP3A4, CYP2D6, and MAO-A on the pharmacokinetics of almotriptan confirmed the involvement of these enzymes in the metabolic clearance of this drug and that no dose changes are required in the presence of inhibitors of these enzymes. (+info)
Schedules of controlled substances: temporary placement of alpha-methyltryptamine and 5-methoxy-N,N-diisopropyltryptamine into Schedule I. Final rule.
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The Deputy Administrator of the Drug Enforcement Administration (DEA) is issuing this final rule to temporarily place alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) into Schedule I of the Controlled Substances Act (CSA) pursuant to the temporary scheduling provisions of the CSA. This final action is based on a finding by the DEA Deputy Administrator that the placement of AMT and 5-MeO-DIPT into Schedule I of the CSA is necessary to avoid an imminent hazard to the public safety. As a result of this rule, the criminal sanctions and regulatory controls of Schedule I substances under the CSA will be applicable to the manufacture, distribution, and possession of AMT and 5-MeO-DIPT. (+info)