Hypotensive activity of the pineal indoleamine hormones melatonin, 5-methoxytryptophol and 5-methoxytryptamine

Injection of the pineal indoles melatonin, 5-methoxytryptophol and 5-methoxytryptamine via the external jugular vein elicited a dose-dependent depression in mean arterial pressure. Melatonin and 5-methoxytryptophol were approximately equipotent and a dose of 150 micromol/kg brought about a reduction of about 40 mmHg in mean arterial pressure. Methoxytryptamine exerted a much more potent hypotensive action. An abrupt decrement in mean arterial pressure by 30 mmHg occurred when the dose was only 2 nmol/kg. Subsequent increases in the dose further lowered the mean arterial pressure, but more gently. The other pineal indoles tested including 5-methoxyindoleacetic acid and 5-hydroxyindoleacetic acid, as well as 6-methoxy-2-benzoxazolinone, did not affect the mean arterial pressure when tested up to 80 micromol/kg. Methylene blue, a guanylate cyclase inhibitor, was not able to antagonize the hypotensive activity of melatonin, suggesting that the mechanism of action of melatonin does not involve guanylate cyclase. Lidocaine, which blocks sodium channels in perivascular nerves, antagonized the hypotensive action of melatonin.     

Expression of human organic anion transporters in the choroid plexus and their interactions with neurotransmitter metabolites

The purpose of the present study was to elucidate the expression of human organic anion transporter 1 (hOAT1) and hOAT3 in the choroid plexus of the human brain and their interactions with neurotransmitter metabolites using stable cell lines. Immunohistochemical analysis revealed that hOAT1 and hOAT3 are expressed in the cytoplasmic membrane and cytoplasm of human choroid plexus. Neurotransmitter metabolites, namely, 5-methoxyindole-3-acetic acid (5-MI-3-AA), homovanillic acid (HVA), vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HI-3-AA), N-acetyl-5-hydroxytryptamine (NA-5-HTT), melatonin, 5-methoxytryptamine (5-MTT), 3,4-dihidroxymandelic acid (DHMA), 5-hydroxytryptophol, and 5-methoxytryptophol (5-MTP), but not methanephrine (MN), normethanephrine (NMN), and 3-methyltyramine (3-MT), at 2 mM, inhibited para-aminohippuric acid uptake mediated by hOAT1. On the other hand, melatonin, 5-MI-3-AA, NA-5-HTT, 5-MTT, 5-MTP, HVA, 5-HI-3-AA, VMA, DOPAC, 5-hydroxytryptophol, and MN, but not 3-MT, DHMA, and NMN, at 2 mM, inhibited estrone sulfate uptake mediated by hOAT3. Differences in the IC(50) values between hOAT1 and hOAT3 were observed for DHMA, DOPAC, HVA, 5-HI-3-AA, melatonin, 5-MI-3-AA, 5-MTP, 5-MTT, and VMA. HOAT1 and hOAT3 mediated the transport of VMA but not HVA and melatonin. These results suggest that hOAT1 and hOAT3 are involved in the efflux of various neurotransmitter metabolites from the cerebrospinal fluid to the blood across the choroid plexus.     

Neuropharmacology of pineal secretions

A connection between pineal function and several psychiatric diseases has been shown recently. The diurnal and seasonal rhythmicity of melatonin production is associated with affective disorders and several types of endogenous depression. The cortisolmelatonin ratio was significantly higher in depressed individuals than in healthy controls. Alterations in melatonin secretion may also occur in non-affective psychiatric disorders, such as chronic schizophrenia. Antidepressants and other psychotropic drugs modify melatonin synthesis. In rodents, monoamine oxidase (MAO) inhibitors (e.g. pheniprazine, harmine or nialamide) increase pineal concentrations of the melatonin precursors, serotonin (5HT) and N-acetyl serotonin (NAS), by enhancing N-acetyl transferase activity. These drugs also increase melatonin, 5HT and NAS in the cerebrospinal fluid. Chronically administered tricyclic antidepressants reduce pineal and serum melatonin content in rodents. In humans, both the MAO-A selective inhibitor clorgyline and the non-selective inhibitor tranylcypromine increase serum melatonin levels. In contrast, serum melatonin remains unaltered by the MAO-B selective inhibitor L-deprenyl. The actions of other drugs on melatonin production, including lithium, propranolol, amphetamine and several monoamine precursors, are in accordance with their psychotropic effects and with their effect on monoamine functions.     

Potentiation by deprenyl of the autoreceptor-mediated inhibition of [3H]-5-hydroxytryptamine release by 5-methoxytryptamine

Summary The 5-hydroxytryptamine (5HT) receptor agonist, 5-methoxytryptamine, inhibited in a concentration-dependent manner the electrically-evoked release of ³H-5HT from superfused rat hypothalamic slices, with an IC₅₀ of 560 nmol/l, without affecting the spontaneous outflow of radioactivity. In the presence of the selective monoamine oxidase B (MAO B) inhibitor, (–)-deprenyl (1 mol/l), the concentration-effect curve for 5-methoxytryptamine was shifted significantly to the left, and the IC₅₀ was decreased to 25 nmol/l. Under the same experimental conditions, the potency of the 5HT receptor agonist lysergic acid diethylamide (LSD) at inhibiting the electrically-evoked release of ³H-5HT was the same in the presence as well as in the absence of (–)-deprenyl. The IC₅₀ values for LSD were 34 nmol/l in the absence of deprenyl, and 31 nmol/l in the presence of the MAO B inhibitor. It is concluded that deprenyl potentiates the inhibition by 5-methoxytryptamine of ³H-5HT release, by preventing its inactivation through MAO B. Since 5-methoxytryptamine may be present in the pineal gland of some species, the potent effects of this 5-HT receptor agonist on seretoninergic neutrotransmission may be of physiological relevance.     

Melatonin and synthetic melatonergic agonists: actions and metabolism in the central nervous system

The CNS is both source and target of melatonin. This methoxyindole formed in the pineal gland is also produced in other CNS regions and additionally enters the brain by uptake from the circulation as well as via the pineal recess. The mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN), not only controls the pineal, but also receives a feedback information on darkness. Two G protein-coupled melatonin receptors, MT1 and MT2, are responsible for the transduction of many melatonergic actions. High receptor densities are especially found in the SCN, but their presence at lower expression levels in other areas is functionally important. Various metabolites and analogs are formed in the CNS, such as N-acetylserotonin, 5-methoxytryptamine, 5-methoxytryptophol, 5-methoxylated kynuramines, and even 6-sulfatoxymelatonin. The chronobiological effects of melatonin go beyond the resetting of a single circadian oscillator. They contribute to phase relationships between oscillatory subsets and are required for robust rhythm amplitudes. CNS effects of melatonin comprise sleep initiation, antiexcitatory, antiepileptic, antinociceptive, anxiolytic, proneurotrophic, antiinflammatory, antioxidant and other neuroprotective actions. The role as a sleep-promoting compound, which is limited by its short half-life in the circulation, has led to the development of controlled-release formulations and of various synthetic agonists, such as ramelteon, agomelatine, tasimelteon, TIK-301, UCM765 and UCM924. Their differences concerning receptor affinities, preferences for receptor subtypes, and pharmacokinetics are discussed, as well as additional antidepressive actions of agomelatine and TIK-301 based on properties as antagonists of the serotonergic 5-HT2C receptor. Indirect antidepressive effects by melatonergic drugs are largely explained by circadian readjustments.     

Ontogenetic effects of MAO-A inhibition on rat pineal n-acetylserotonin and melatonin during the first month of neonatal life

Inhibitors of monoamine oxidase A (MAO-A) but not MAO-B stimulate the activity of pineal serotonin N-acetyltransferase (AANAT) in the adult rat pineal leading to increased formation of N-acetyl serotonin (NAS) and melatonin (MEL). The pineal gland of the neonatal rat has AANAT activity, but the second enzyme in melatonin biosynthesis, HIOMT (hydroxyindole-O-methyltransferase) converting NAS to MEL, is absent during the first week of neonatal life. In this study we examined the effects of acute clorgyline treatment in vitro and in vivo, on pineal indoles over the first month of neonatal life. The results show that clorgyline stimulates NAS production by pineal both in vitro and in vivo from day five on with a marked increase between day 14 and day 21. In contrast, MEL is not increased until day 21, with a sharp rise thereafter. Copyright 2000 John Wiley & Sons, Ltd.     

Carbaryl-induced changes in indoleamine synthesis in the pineal gland and its effects on nighttime serum melatonin concentrations.

The effects of different doses of chronically administered carbaryl on rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during darkness (2300 h and 0100 h) when pineal melatonin synthesis is high were studied. Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Carbaryl was administered at total doses (over 6 days) of either 50, 125 or 250 mg/kg by gastric gavage. Control rats received vehicle (corn oil) only. During the study, the rats were exposed to light/dark cycles of 14:10 with lights off at 2100 h. Pineal NAT and HIOMT activities and pineal melatonin were increased at 0100 h following carbaryl administration at all three doses. Conversely, serum melatonin was increased at 2300 h after the 250 mg/kg dose of carbaryl while all three doses of the pesticide reduced serum melatonin levels at 0100 h. Pineal 5-HTP, 5-HT and 5-HIAA levels were usually increased at 2300 h but unaffected at 0100 h. The results indicate that carbaryl has significant effects on pineal melatonin synthesis and secretion.     

Effect of venlafaxine on pineal melatonin and noradrenaline in the male rat

Studies in vitro indicate that the antidepressant drug, venlafaxine (VEN), inhibits the reuptake of both serotonin (5-hydroxytryptamine, 5-HT) and noradrenaline (NA) but has little activity on other neurotransmitter receptors. There are, however, few studies on the effects of VEN on monoamine neurotransmission in vivo. In the present study we examined the effect of VEN treatment on the melatonin content of the rat pineal gland because the synthesis of melatonin is regulated by the release of NA onto pinealocyte beta-adrenoceptors. Acute treatment with higher doses (15 mg/kg) of VEN significantly increased pineal melatonin and NA but this effect was attenuated by subchronic treatment. These data are consistent with in vitro data suggesting that VEN increases NA neurotransmission at higher doses and that repeated treatment can desensitize pinealocyte beta-adrenoceptors.     

Melatonin metabolism in the central nervous system

The metabolism of melatonin in the central nervous system is of interest for several reasons. Melatonin enters the brain either via the pineal recess or by uptake from the blood. It has been assumed to be also formed in some brain areas. Neuroprotection by melatonin has been demonstrated in numerous model systems, and various attempts have been undertaken to counteract neurodegeneration by melatonin treatment. Several concurrent pathways lead to different products. Cytochrome P(450) subforms have been demonstrated in the brain. They either demethylate melatonin to N-acetylserotonin, or produce 6-hydroxymelatonin, which is mostly sulfated already in the CNS. Melatonin is deacetylated, at least in pineal gland and retina, to 5-methoxytryptamine. N(1)-acetyl-N(2)-formyl-5-methoxykynuramine is formed by pyrrole-ring cleavage, by myeloperoxidase, indoleamine 2,3-dioxygenase and various non-enzymatic oxidants. Its product, N(1)-acetyl-5-methoxykynuramine, is of interest as a scavenger of reactive oxygen and nitrogen species, mitochondrial modulator, downregulator of cyclooxygenase-2, inhibitor of cyclooxygenase, neuronal and inducible NO synthases. Contrary to other nitrosated aromates, the nitrosated kynuramine metabolite, 3-acetamidomethyl-6-methoxycinnolinone, does not re-donate NO. Various other products are formed from melatonin and its metabolites by interaction with reactive oxygen and nitrogen species. The relative contribution of the various pathways to melatonin catabolism seems to be influenced by microglia activation, oxidative stress and brain levels of melatonin, which may be strongly changed in experiments on neuroprotection. Many of the melatonin metabolites, which may appear in elevated concentrations after melatonin administration, possess biological or pharmacological properties, including N-acetylserotonin, 5-methoxytryptamine and some of its derivatives, and especially the 5-methoxylated kynuramines.     

N2-Acetylphenelzine : effects on rat brain GABA,alanine and biogenic amines

Summary The neurochemical properties of N ²-acetyl-phenelzine were compared with those of phenelzine in a rat model. N ²-Acetylphenelzine is a relatively potent inhibitor of monoamine oxidase-A and -B and causes increases in whole-brain levels of noradrenaline and 5-hydroxytryptamine, and decreases in homovanillic acid, 5-hydroxyindole-3-acetic acid, and 3,4-dihydroxyphenylacetic acid after acute i.p. administration of the drug. Phenelzine is a more potent monoamine oxidase inhibitor than is N ²-acetylphenelzine. The most marked difference in the profile was that N ²-acetylphenelzine had no effect on whole brain levels of the amino acid neurotransmitters alanine and -aminobutyric acid, whereas phenelzine caused dramatic increases. Acetylation of phenelzine at the N² position presumably interferes with the inhibition of the transaminase enzymes for -aminobutyric acid and alanine.     

Effects of pinealectomy and exogenous melatonin on serum leptin levels in male rat.

The effects of pinealectomy and exogenous melatonin (N-acetyl-5-methoxytryptamine) on serum leptin levels were investigated in rats. Exogenous administration of melatonin to intact rats resulted in significant decreases in serum leptin levels (P < 0.05) compared to those of the intact control group. Serum leptin levels were significantly elevated in the pinealectomised rats in comparison to the sham-pinealectomised animals (P < 0.001) and were significantly suppressed by exogenous administration of melatonin compared to those of non-treated pinealectomised rats (P < 0.001). Hormone concentrations in the melatonin-treated pinealectomised group were found to be similar to those seen in the sham-pinealectomised group. These results suggest that pineal gland has an effect on leptin release.     

Differential effect of benserazide (Ro4-4602) on the concentration of indoleamines in rat pineal and hypothalamus.

1 Low doses (50 and 80 mg/kg) of benserazide (Ro4-4602), an aromatic amino acid decarboxylase inhibitor, markedly reduced 5-hydroxytryptamine and melatonin in the rat pineal gland without affecting hypothalamic 5-hydroxytryptamine. 2 This differential effect shows that inhibition of the pineal gland decarboxylase activity is possible, and confirms that the rat pineal gland is accessible to peripherally acting agents.     

Marked enhancement by clorgyline of nocturnal and daytime melatonin release in rhesus monkeys

The type A monoamine oxidase (MAO)-inhibiting antidepressant clorgyline (1 mg/kg/24 days) administered to rhesus monkeys increased night-time cerebrospinal fluid (CSF) melatonin concentrations 3-fold and day-time maltonin values 5-fold. Other circadian parameters of melatonin release, including the peak time and duration of nocturnal melatonin elevation measured during continuous CSF collection periods of 90 min duration over 24-h cycles, were unaffected by clorgyline. While pinealocytes are thought to contain only MAO-B, treatment with the selective MAO-B inhibitor deprenyl (2 mg/kg/24 days) did not alter day or night-time melatonin concentrations. These results are consistent with MAO-A and non-selective MAO inhibitors acting via blockade of degradation of the preferential substrates of MAO-A, serotonin and/or norepinephrine, in adrenergic neurons entering the pineal gland. Further study is needed to evaluate the relative contributions of an increased availability of the melatonin precursor, serotonin, or a sustained net increase in alpha₁-or beta adrenoceptor-mediated input on pinealocytes to these marked changes in melatonin production.     

Amantadine tremor,a 5-hydroxytryptamine-mediated response?

Amantadine-induced tremor has been investigated using mice. Experiments with, mebanazine, reserpine, diethyl-dithiocarbamate, and p-chlorophenylalanie suggest that the tremorgenic action of amantadine is influenced by a balance between three putative central nervous system (CNS) transmitters: noradrenaline, dopamine and 5-hydroxytryptamine (5-HT). Drugs which reduce the concentration of the catecholamines in brain increase amantadine-induced tremor. p-Chlorophenylalanine, which specifically depletes brain 5-HT, antagonises amantadine-induced tremor. An ED₅₀ (tremor) dose of amantadine decreases the concentration of 5-hydroxyindoleacetic acid (5-HIAA) in rat brain, particularly when this is elevated due to pretreatment with 5-hydroxytryptophan. Neither inhibition of monoamine oxidase nor reduction of 5-HT-reuptake appear to be responsible for this decrease. Experiments on rat fundus suggest that amantadine increased the sensitivity of receptors to 5-HT. A similar mechanism of action in the CNS could explain both the tremor and the decrease in brain 5-HIAA. The possible relevance of these findings is discussed with respect to the known anti-Parkinson action of amantadine.     

Hypotensive Activity of the Pineal Indoleamine Hormones Melatonin, 5‐Methoxytryptophol and 5‐Methoxytryptamine

Abstract: Injection of the pineal indoles melatonin, 5‐methoxytryptophol and 5‐methoxytryptamine via the external jugular vein elicited a dose‐dependent depression in mean arterial pressure. Melatonin and 5‐methoxytryptophol were approximately equipotent and a dose of 150 μmol/kg brought about a reduction of about 40 mmHg in mean arterial pressure. Methoxytryptamine exerted a much more potent hypotensive action. An abrupt decrement in mean arterial pressure by 30 mmHg ocurred when the dose was only 2 nmol/kg. Subsequent increases in the dose further lowered the mean arterial pressure, but more gently. The other pineal indoles tested including 5‐methoxyindoleacetic acid and 5‐hydroxyindoleacetic acid, as well as 6‐methoxy‐2‐benzoxazolinone, did not affect the mean arterial pressure when tested up to 80 μmol/kg. Methylene blue, a guanylate cyclase inhibitor, was not able to antagonize the hypotensive activity of melatonin, suggesting that the mechanism of action of melatonin does not involve guanylate cyclase. Lidocaine, which blocks sodium channels in perivascular nerves, antagonized the hypotensive action of melatonin.     

Characterisation of imidazoline I2 binding sites in pig brain

The imidazoline I2 binding sites in the central nervous system have previously been described in several different species including rat, mouse, rabbit and frog. The present study has investigated the imidazoline I2 binding site, and its relationship to the monoamine oxidase isoforms, in pig whole brain and compared the results obtained with data from other species. Results from saturation binding studies revealed that the imidazoline I2-selective ligand, [3H]2BFI (2-(2-benzofuranyl)-2-imidazoline) labelled a single saturable population of sites with a KD=6.6 nM and Bmax=771.7 fmol/mg protein. The pharmacological characterisation of the sites was similar to that previously reported with a rank order of potency for the imidazoline I2 ligands of 2BFI>BU224>Idazoxan>BU226. Displacement by the imidazoline I1 ligands was low affinity and the monoamine oxidase inhibitors displaced with micromolar affinity. The majority of compounds displaced the binding in a monophasic manner, however, displacement by the putative endogenous ligand, harmane was biphasic. The relative populations of the two monoamine oxidase isoforms revealed a 10 fold greater expression of monoamine oxidase B relative to monoamine oxidase A. These data confirm the presence of imidazoline I2 binding sites in pig brain and show that their pharmacology is characteristic of that seen in other species. The proportion of monoamine oxidase A and B expressed in the pig brain is similar to that seen in the human brain therefore, given the association between imidazoline I2 binding sites and monoamine oxidase, the pig may provide a more useful model for human imidazoline I2 binding sites than other species such as the rat.     

The influence of L-tryptophan and monoamine oxidase inhibitors on catecholamine metabolism in rat brain.

1. L-Tryptophan (100 mg/kg) was administered to rats with or without pretreatment with a monoamine oxidase inhibitor and the concentration of 5-hydroxyindoleacetic acid, homovanillic acid, dihydroxyphenylacetic acid, 3-methoxy 4-hydroxyphenyl glycol, normetanephrine, noradrenaline and dopamine measured in whole brain one hour later. 2. L-Tryptophan increased the concentration of 5-hydroxyindoleacetic acid, homovanillic acid, dihydroxyphenylacetic acid, 3-methoxy 4-hydroxyphenyl glycol and normetanephrine. The concentration of noradrenaline did not change whilst that of dopamine increased significantly. 3. In animals pretreated chronically with a monoamine oxidase inhibitor, tryptophan increased the concentration of dihydroxyphenylacetic acid and homovanillic acid compared to monoamine oxidase alone. 4. The results suggest either a release of dopamine and noradrenaline by 5-hydroxytryptamine, with a compensatory increase in their synthesis, or an increase in the firing of dopaminergic and noradrenergic neurones after L-tryptophan.     

Differential effects of indolepyruvic acid and 5-hydroxytryptophan on indole metabolism in the pineal gland of the rat during the light-dark cycle.

The effect of two serotonin precursors, 5-hydroxytryptophan (5-OH-TRP) and indolepyruvic acid (IPA), a tryptophan ketoanalogue, on rat pineal indole metabolism during the light-dark cycle was investigated. 5-OH-TRP drastically increased the production of 5-hydroxyindoleacetic acid at a dose of only 10 mg/kg, whereas 50-100 mg/kg was needed to reach higher serotonin levels. It had no effect on the pathway leading to the production of N-acetylserotonin and melatonin. IPA, on the other hand, led to a marked dose-related increase in tryptophan, 5-OH-TRP, serotonin and 5-OH-indoleacetic acid, and was also active on N-acetylserotonin and melatonin synthesis in both phases. The different behaviour of these two substances with regard to melatonin synthesis was also confirmed by their effects on N-acetyltransferase, since IPA increased, whereas 5-OH-TRP decreased its activity. These data suggest that an increase in serotonin does not necessarily lead to an increase in melatonin, and that IPA may in fact induce this effect by altering the activity on N-acetyltransferase, which is regarded as a key enzyme in pineal hormone synthesis.     

Effect of psychotropic drugs on the contents of melatonin, serotonin and N-acetylserotonin in rat pineal gland

In the dark phase, the effects of the psychotropic drugs on the contents of melatonin, serotonin (5-HT) and N-acetylserotonin (NAS) in rat pineal gland were examined. The pineal gland was removed at a certain period of time after subcutaneous injection of the drugs. 5-HT, NAS and melatonin contents in the pineal gland were determined by high performance liquid chromatography with fluorometric detection. A dose-dependent decrease was observed for melatonin content in the administration of diazepam (DZP), hydroxyzine (HYZ), chlorpromazine (CPZ) or haloperidol (HPD). When imipramine (IPM) or amitriptyline (APL) was given to rats, pineal 5-HT content was significantly decreased. On the other hand, the administration of IPM or APL caused increases in pineal NAS and melatonin. Furthermore, the administration of phenytoin (PYT) revealed no changes in the content of pineal indoleamines. These results suggest that the psychotropic drugs widely used in clinical applications could cause significant changes in pineal indoleamine content.     

The effects of acute melatonin and ethanol treatment on antioxidant enzyme activities in rat testes.

The pineal hormone melatonin (N -acetyl, 5-methoxytryptamine) was recently accepted to act as an antioxidant under both in vivo and in vitro conditions. In this study, we examined the possible preventive effect of melatonin on ethanol-induced lipid peroxidation in rat testes. Thirty-seven male Wistar albino rats, 5.5--6 months old, were randomly divided into four groups (9--10 animals in each). The first group (control animals) received 4% ethanol at similar intervals to the experimental groups to equalize the stress effect. The second group received only melatonin i.p. 7 mg kg(-1)bw three times over 1.5 h intervals. The third group received only 30% alcohol 3 g kg(-1)bw twice daily. The fourth group were treated with melatonin and ethanol according to the above protocol, melatonin injections preceding ethanol treatments. The product of lipid peroxidation, malondialdehyde (MDA) and antioxidant enzymes, superoxide dismutase (Cu--Zn SOD), glutathione peroxidase (GPx) and catalase (CAT) were measured in the post-mitochondrial fraction of the testes. MDA levels were significantly increased due to acute ethanol intoxication. GPx activity was higher in the three experimental groups than the control levels. The activity of CAT was increased significantly in the melatonin plus ethanol-treated group but the other groups appeared not to be influenced by acute ethanol treatment. Cu--Zn SOD activity remained unaltered. These results suggest that antioxidants may be a protective agent for the testicular injury caused by ethanol consumption.