The putative melatonin receptor antagonist GR128107 is a partial agonist on Xenopus laevis melanophores

1. GR128107 (3-(1-acetyl-3-methyl-piperidine)-5-methoxyindole) has previously been reported to be a competitive melatonin receptor antagonist in blocking melatonin inhibition of [3H]-dopamine release from rabbit retina, a response mediated by the MT2 receptor subtype. 2. GR128107, like melatonin, induced a rapid (maximum response in 60-90 min) pigment aggregation in a clonal line of Xenopus laevis melanophores. GR128107 behaved as a partial agonist (pEC50 8.58+/-0.03, n=3) with an Emax of 0.83 (relative to melatonin, pEC50 10.09+/-0.03, n=3). 3. The concentration-response curve for pigment granule aggregation to both melatonin and GR128107 was displaced in a parallel, rightward manner by melatonin receptor antagonists with very similar potencies; estimated pKB RJ252 (against melatonin 4.60/against GR128107 4.54) < GR135533 (6.40/6.14) < Luzindole (6.45/6.49) < S20929 (6.58/6.65) < 4-P-PDOT (6.73/6.85). 4. Both melatonin- and GR128107-induced pigment granule aggregation was prevented by pretreatment of melanophores with pertussis toxin (10-1000 ng ml(-1)). 5. Prolonged pre-treatment of melanophores with melatonin desensitized the pigment aggregation response to GR128107. In desensitized cells, the maximal aggregation produced by GR128107 was only 0.27+/-0.01 (n=4) and the pEC50 was reduced (vehicle 8.57+/-0.12; melatonin pre-treated 7.84+/-0.09, n=4). The maximal response to melatonin in desensitized melanophores was unchanged but the pEC50 was reduced (vehicle 10.49+/-0.03; melatonin pre-treated 9.83+/-0.04, n=4). 6. These results demonstrate that GR128107 induces pigment granule aggregation in Xenopus melanophores by activating a cell membrane melatonin receptor coupled via a pertussis toxin-sensitive G-protein. 7. The partial agonist activity of GR128107 in melanophores may be apparent because of the very high density of melatonin receptors in these cells (Bmax 1223 fmol mg protein(-1)) compared to the low density of sites in rabbit retina (Bmax 3.1 fmol mg protein(-1)). This suggestion is supported by the finding that GR128107, like melatonin, acted as a full agonist and inhibited forskolin-stimulation of cyclic AMP accumulation in NIH-3T3 cells expressing a high density of human mt1 or MT2 receptors.     

Aggregation of pigment granules in single cultured Xenopus laevis melanophores by melatonin analogues.

1. Isolated melanophores were differentiated from aggregates of neural crest obtained from neurula stage Xenopus laevis embryos after 2 days in culture. 2. Condensation of pigment granules in these cells by melatonin (5-methoxy N-acetyltryptamine, aMT) and various novel analogues was monitored with an image analysis system to quantitate the area occupied by pigment in individual cells. 3. Melanophores exposed to vehicle (a maximum of 0.1% MeOH) showed little (less than 5%) change in pigment area. aMT produced a dramatic condensation of pigment granules (EC50 = the concentration producing a half maximal condensation, 9 pM). The response was rapid, reached a maximum (approximately 80% decrease in pigmented area) by 10 min, and was reversible after removal of aMT from the culture medium. 4. Aggregation to aMT was blocked by treating melanophores with pertussis toxin (1 microgram ml-1, 7 h) indicating a role for a guanosine 5' triphosphate (GTP)-binding protein in transducing the aMT receptor signal. 5. Structure-activity studies indicated that analogues of aMT lacking a side-chain N-acyl substituent (5-methoxytryptamine, MT) or a group at the 5-position of the indole ring (N-acetyltryptamine, aT) were unable to induce pigment aggregation (EC50 greater than 10 microM). 6. Lengthening the side-chain N-acyl group (N-propionyl, N-butanoyl) was tolerated to some degree but eventually (N-valeroyl and larger) activity diminished. Of the 5-position analogues tested 5-methoxy (aMT) was by far the most potent. 7. Halogen substitution in the 6-position of the indole ring led to some loss of activity as did a 6-OH substitution. The 6-OCH3 compound was inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

2-Phenylmelatonin: a partial agonist at enteric melatonin receptors

The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guinea-pig proximal colon. 2-Phenylmelatonin (0.01 nM-1 microM) caused a concentration-dependent contractile response. The potency value (-log EC50) was 9.3 +/- 1.0. The maximum effect was 25 +/- 4%, of that elicited by the maximally effective concentration (0.3 microM) of 5-HT and 43 +/- 3%, of that by the maximally effective concentration (10 microM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 microM), a selective antagonist of melatonin MT3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 microM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.     

Analogues of diverse structure are unable to differentiate native melatonin receptors in the chicken retina, sheep pars tuberalis and Xenopus melanophores.

1. The pineal hormone melatonin exerts its biological effects through specific, high affinity G-protein coupled receptors. Recently, three melatonin receptor subtypes (Mel1a, Mel1b and Mel1c) have been cloned. Neither the cloned subtypes, nor the native receptors have yet been compared in a detailed pharmacological analysis. 2. The present study examined the structure-activity relationships of a series of 21 melatonin analogues, by comparing their potency on the pigment aggregation response in Xenopus laevis melanophores with their affinity in radioligand binding competition studies in chicken retina and sheep pars tuberalis (PT), two tissues in which melatonin is known to mediate a biological response. 3. All but four of the analogues were full melatonin receptor agonists producing a concentration-related redistribution of pigment granules in cultured Xenopus melanophores. The remaining analogues produced little pigment aggregation at 10 microM. 4. Saturation studies with 2-[125I]-iodomelatonin identified a single binding site in the chicken retina and sheep PT membranes, with a KD of 36.6 +/- 2.8 and 37.3 +/- 4.3 pM, and a maximal number of binding sites (Bmax) of 16.6 +/- 0.5, and 40.1 +/- 1.7 fmol mg-1 protein, respectively. 5. Comparison of the potency/affinity of the analogues for the binding sites gave a highly significant correlation in each case, retina/melanophore, r = 0.97 (P < 0.001, n = 17), PT/melanophore, r = 0.97 (P < 0.001, n = 17) and PT/retina, r = 0.98 (P < 0.001, n = 21). 6. Despite their large range in affinity and structural diversity these melatonin agonists were unable to distinguish between melatonin receptors in the chicken retina, sheep pars tuberalis and Xenopus melanophores.     

An endogenous 5-HT(7) receptor mediates pigment granule dispersion in Xenopus laevis melanophores

Melatonin (5-methoxy N-acetyltryptamine) and serotonin (5-HT) exert rapid, but opposite effects on pigment granule distribution in Xenopus laevis melanophores. Low concentrations of melatonin (10(-11) - 10(-9) M) cause a dramatic perinuclear aggregation of the melanin-containing granules, while 5-HT (10(-8) - 10(-5) M) disperses pigment granules throughout the cell. The present study found that pharmacological doses of melatonin (> or =10(-6) M) induced a time- and concentration-dependent pigment granule dispersion, which was mediated by an endogenous melanophore 5-HT receptor. 5-HT produced a concentration-dependent elevation of melanophore cyclic AMP, and 5-HT-induced dispersion was blocked by H89 (10(-4) M), an inhibitor of protein kinase A (PKA), but not by a PKC inhibitor (Ro 31-8220, 10(-5) M), indicating a vital role for cyclic AMP in 5-HT-induced dispersion. 5-HT-mediated dispersion was not blocked by antagonists selective for G(s)-coupled 5-HT(4) (GR113808) or 5-HT(6) (Ro 04-6790, Ro 63-0563, olanzepine) receptors, nor by 5-HT(1 - 3) (pindolol, ketanserine, metoclopramide, MDL72222, tropisetron) receptor antagonists, but was inhibited by a selective 5-HT(7) receptor antagonist, DR4004, and other antagonists with a high affinity for 5-HT(7) receptors. The rank order of antagonist potency was: risperidone (mean pK(B) 7.82)>methiothepin (7.43)>DR4004 (6.92)>mesulergine (6.83)>methysergide (6.60)>[+/-]-sulpiride (5.81)>spiperone (5.52). The agonist potency order [mean pEC(50), 5-CT (8.68)>5-HT (7.13)>5-MT (6.94)>8-OH-DPAT (4.79)>sumatriptan (<4)] was also consistent with an action on 5-HT(7) receptors. RT - PCR confirmed that melanophores express 5-HT(7) receptor mRNA. The pigment dispersing effect of high melatonin concentrations in melanophores is most likely mediated by activation of 5-HT(7) receptors. Conceivably some of the effects attributed to pharmacological doses of melatonin in mammals may be mediated by activation of 5-HT(7) receptors.     

The action of melatonin on single amphibian pigment cells in tissue culture.

1. Neural crest material from neurula stage Xenopus embryos was tissue cultured as small aggregates of cells or a single cell suspension. Isolated pigment cells differentiated after 2 days in culture. 2. Melatonin (10(-15) to 10(-13) M) induced pigment granule condensation; it was 10,000 times more effective than any other compound tested. 3. Tests with appropriate agents showed the pigment cells to have beta-adrenoceptors and 5-hydroxytryptamine receptors; these sites could be blocked without affecting the response to melatonin. Phentolamine blocked the effect of melatonin. 4. Removal of Na+ or Ca2+ from the bathing medium inhibited melatonin-induced pigment granule condensation; 10 mM K+ induced pigment granule condensation. D600, Mn2+ and La3+, which inhibit Ca2+ entry, blocked the effect of melatonin. 5. Cyclic GMP induced pigment condensation and cyclic AMP pigment dispersion (10(-2) to 10(-4) M). 6. It is suggested that the action of melatonin is accompanied by depolarization and the entry of calcium ions. 7. Pigment cells in tissue culture could provide a useful bioassay method for melatonin.     

Comparison of the structure-activity relationships of melatonin receptor agonists and antagonists: lengthening the N-acyl side-chain has differing effects on potency on Xenopus melanophores

The potency and affinity of two series of melatonin receptor ligands were examined using the pigment aggregation response in a clonal line of Xenopus laevis melanophores and radioligand binding assays on native receptors in chicken brain, recombinant human mt₁ and MT₂ and Xenopus laevis mel_1c receptor subtypes. One series was based on melatonin and had a methoxy group at the 5-position of the indole ring, while the other was based on luzindole and lacked this substituent but did have a 2-benzyl moiety; the N-acyl group of each series of analogues was varied from one to five carbon atoms. All analogues in the melatonin series were full agonists in melanophores (pEC₅₀ 7.76–10.24), while all compounds in the luzindole series were competitive melatonin antagonists (pA ₂ 5.47–6.60). With the agonist series, increasing the N-acyl side-chain from one to three carbon atoms was well tolerated in both the functional and binding assays, but further lengthening of the side-chain progressively and dramatically reduced potency and affinity. In contrast, for the antagonist series neither potency nor binding affinity changed substantially with the length of the N-acyl chain, except at the recombinant MT₂ subtype where two of the analogues had a lower affinity. In binding assays, three of the five antagonists were MT₂-selective; the most selective analogue (N-pentanoyl 2-benzyltryptamine, MT₂ pK _i 8.03) having 89- and 229-fold higher affinity than at mt₁ or mel_1c receptor subtypes. The different structure-activity relationships of these receptor agonists and antagonists is discussed with regard to the possible binding sites of agonists and antagonists within the receptor protein. Received: 26 June 1998 / Accepted: 6 September 1998     

Possible mechanisms of action in melatonin reversal of morphine tolerance and dependence in mice

In our earlier study, we reported the ability of melatonin to reverse the development of morphine tolerance and dependence in mice. In the present study, we attempted to analyse the possible involvement of putative melatonin receptors, central and peripheral benzodiazepine receptors and the nitric oxide (NO) system in the mechanism of melatonin reversal of morphine tolerance and dependence in mice. Co-administration of L-N(G)-nitro arginine methyl ester (L-NAME) or melatonin with morphine during the induction phase (days 1-9) delayed the development of tolerance to the anti-nociceptive action of morphine and also reversed naloxone precipitated withdrawal jumpings. L-arginine administration during the induction phase enhanced the development of tolerance to the anti-nociceptive effect of morphine but had no effect on the naloxone-precipitated withdrawal response. During the expression phase (day 10), acute administration of melatonin or L-NAME reversed, whereas L-arginine facilitated, naloxone-precipitated withdrawal jumping in morphine-tolerant mice, but none of these drugs affected the nociceptive threshold in morphine-tolerant mice. Further, co-administration of melatonin or L-NAME with L-arginine during the induction phase antagonized later the effects on the development of morphine tolerance. Also, prior administration of melatonin or L-NAME reversed the L-arginine potentiation of naloxone-precipitated withdrawal jumping in morphine tolerant mice. Among the antagonists for putative melatonin receptors studied, neither luzindole (melatonin MT1 and MT2 receptor antagonist) nor prazosin (melatonin MT3 receptor antagonist) antagonized the melatonin reversal of morphine tolerance and dependence. 1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK11195), a peripheral but not central benzodiazepine receptor antagonist, flumazenil, partially antagonized the melatonin reversal of naloxone-precipitated withdrawal jumping in morphine-dependent mice, but had no effect on the reversal of morphine tolerance induced by melatonin. Overall, the present observations suggest that the melatonin-induced reversal of morphine tolerance and dependence may involve its ability to suppress nitric oxide synthase (NOS) activity. Further, the melatonin-induced reversal of morphine tolerance and dependence is not mediated through its actions via putative melatonin receptors. The agonistic activity of melatonin towards peripheral benzodiazepine receptors may partially contribute to the suppression of morphine dependence but not to the reversal of tolerance to the analgesic activity of morphine.     

Radioligand binding affinity and biological activity of the enantiomers of a chiral melatonin analogue

Melatonin, a hormone secreted by the pineal gland, can act on the central circadian oscillator in the suprachiasmatic nucleus of the hypothalamus. It has been proposed that melatonin or its analogues may be useful in restoring disturbed circadian rhythms in jet-lag, shift-work and some blind subjects, and as sleep-promoting agents. In the present study, the (−)- and (+)-enantiomers of N-acetyl-4-aminomethyl-6-methoxy-9-methyl-1,2,3,4-tetrahydrocarbazole (AMMTC) were separated and tested. The affinity of the enantiomers at the specific 2-[¹²⁵I]iodomelatonin binding site in chick brain membranes was compared in competition assays, and their biological activity in a specific melatonin receptor bioassay, aggregation of pigment granules in Xenopus laevis melanophores. The (−)-enantiomer of AMMTC was 130-fold and 230-fold more potent than the (+)-enantiomer in competition radioligand binding assays and melanophores, respectively. Both enantiomers are melatonin receptor agonists; (−)-AMMTC is slightly more potent than melatonin itself. As the tetrahydrocarbazole nucleus holds the C-3 amido side-chain of AMMTC in a restricted conformation, the analogues will be useful in modelling the melatonin receptor binding site.     

Effects of melatonin on rat pial arteriolar diameter in vivo.

1. Based on our finding that melatonin decreased the lower limit of cerebral blood flow autoregulation in rat, we previously suggested that melatonin constricts cerebral arterioles. The goal of this study was to demonstrate this vasoconstrictor action and investigate the mechanisms involved. 2. The effects of cumulative doses of melatonin (10-10 to 10-6 M) were examined in cerebral arterioles (30 - 50 microM) of male Wistar rats using an open skull preparation. Cerebral arterioles were exposed to two doses of melatonin (3x10-9 and 3x10-8 M) in the absence and presence of the mt1 and/or MT2 receptor antagonist, luzindole (2x10-6 M) and the Ca2+-activated K+ (BKCa) channel blocker, tetraethylammonium (TEA+, 10(-4) M). The effect of L-nitro arginine methyl ester (L-NAME, 10-8 M) was examined on arterioles after TEA+ superfusion. Cerebral arterioles were also exposed to the BKCa activator, NS1619 (10(-5) M), and to sodium nitroprusside (SNP, 10-8 M) in the absence and presence of melatonin (3x10-8 M). 3. Melatonin induced a dose-dependent constriction with an EC50 of 3.0+/-0.1 nM and a maximal constriction of -15+/(-1%). Luzindole abolished melatonin-induced vasoconstriction. TEA+ induced significant vasoconstriction (-10+/(-2%). No additional vasoconstriction was observed when melatonin was added to the aCSF in presence of TEA+, whereas L-NAME still induced vasoconstriction (-10+/(-1%). NS1619 induced vasodilatation (+11+/(-1%) which was 50% less in presence of melatonin. Vasodilatation induced by SNP (+12+/(-2%) was not diminished by melatonin. 4. Melatonin directly constricts small diameter cerebral arterioles in rats. This vasoconstrictor effect is mediated by inhibition of BKCa channels following activation of mt1 and/or MT2 receptors.     

Mapping the melatonin receptor. 7. Subtype selective ligands based on beta-substituted N-acyl-5-methoxytryptamines and beta-substituted N-acyl-5-methoxy-1-methyltryptamines

A series of beta-substituted and beta,beta-disubstituted N-acyl 5-methoxy-1-methyltryptamines and 5-methoxytryptamines have been prepared as melatonin analogues to investigate the nature of the binding site of the melatonin receptor. The affinity of analogues was determined in a radioligand binding assay using cloned human MT(1) and MT(2) receptor subtypes expressed in NIH 3T3 cells. Agonist and antagonist potency of all analogues was measured using the pigment aggregation response of a clonal line of Xenopus laevis melanophores. beta-Methylmelatonin (17a) and beta,beta-dimethylmelatonin (17b), though showing a slight decrease in binding at human receptors, show an increase in potency on Xenopus. N-Butanoyl 5-methoxy-1-methyl-beta,beta-trimethylenetryptamine (12c) is an antagonist at human MT(1) receptors but an agonist at MT(2), while N-butanoyl 5-methoxy-1-methyl-beta,beta-tetramethylenetryptamine (13c) is an antagonist at MT(1) but had no action at MT(2) and is one of the first examples of an MT(1) selective antagonist.     

Melatonin modulates the GABAergic response in cultured rat hippocampal neurons

In the present study, we investigated the effect of melatonin on the GABA-induced current (I(GABA) and GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in cultured rat hippocampal neurons using the whole-cell patch-clamp technique. We found that melatonin rapidly and reversibly enhanced I(GABA) in a dose-dependent manner, with an EC50 of 949 μM. Melatonin markedly enhanced the peak amplitude of a subsaturating I(GABA) but not that of a saturating I(GABA). Interestingly, melatonin was effective only when GABA and melatonin were applied together. Furthermore, the effect of melatonin on I(GABA) was voltage-independent and did not change the ion selectivity of the GABA(A) receptor. The melatonin enhancement on I(GABA) can not be blocked by luzindole, a melatonin receptor antagonist, indicating that melatonin-induced I(GABA) enhancement was not via activation of its own membrane receptors. However, this enhancement may be mediated via high-affinity benzodiazepine sites as it was inhibited by the classical benzodiazepine antagonist flumazenil, suggesting an allosteric modulation of melatonin by binding to the sites of GABA(A) receptors. In addition, melatonin increased both amplitude and frequency of GABAergic mIPSCs, indicating that melatonin enhances GABAergic inhibitory transmission. Hence, our observation that melatonin has an enhancing effect on the GABAergic system may implicate a potential pathway for the neuroprotective effects of melatonin.     

Pharmacological characterization of human recombinant melatonin mt(1) and MT(2) receptors

We have pharmacologically characterized recombinant human mt(1) and MT(2) receptors, stably expressed in Chinese hamster ovary cells (CHO-mt(1) and CHO-MT(2)), by measurement of [(3)H]-melatonin binding and forskolin-stimulated cyclic AMP (cAMP) production. [3H]-melatonin bound to mt(1) and MT(2) receptors with pK(D) values of 9.89 and 9.56 and B(max) values of 1.20 and 0.82 pmol mg(-1) protein, respectively. Whilst most melatonin receptor agonists had similar affinities for mt(1) and MT(2) receptors, a number of putative antagonists had substantially higher affinities for MT(2) receptors, including luzindole (11 fold), GR128107 (23 fold) and 4-P-PDOT (61 fold). In both CHO-mt(1) and CHO-MT(2) cells, melatonin inhibited forskolin-stimulated accumulation of cyclic AMP in a concentration-dependent manner (pIC(50) 9.53 and 9.74, respectively) causing 83 and 64% inhibition of cyclic AMP production at 100 nM, respectively. The potencies of a range of melatonin receptor agonists were determined. At MT(2) receptors, melatonin, 2-iodomelatonin and 6-chloromelatonin were essentially equipotent, whilst at the mt(1) receptor these agonists gave the rank order of potency of 2-iodomelatonin>melatonin>6-chloromelatonin. In both CHO-mt(1) and CHO-MT(2) cells, melatonin-induced inhibition of forskolin-stimulated cyclic AMP production was antagonized in a concentration-dependent manner by the melatonin receptor antagonist luzindole, with pA(2) values of 5.75 and 7.64, respectively. Melatonin-mediated responses were abolished by pre-treatment of cells with pertussis toxin, consistent with activation of G(i)/G(o) G-proteins. This is the first report of the use of [(3)H]-melatonin for the characterization of recombinant mt(1) and MT(2) receptors. Our results demonstrate that these receptor subtypes have distinct pharmacological profiles.     

The action of reserpine on teleost melanophores

Reserpine caused darkening of both pencil fish and angelfish, this effect lasted for at least 10 and 25 days respectively.The aggregation of pigment granules within the melanophores evoked by stimulation of the nerves supplying the melanophores was inhibited by reserpine in both species. Reserpine caused a lowering of the catecholamine content of pencil fish skin. The time course both these effects paralleled the time course of darkening. The response of the melanophores of the angelfish to adrenaline, noradrenaline, dopamine, and melatonin was not affected by treatment with reserpine for 3 days. However, reserpine treatment for 14 days induced a marked sensitivity to noradrenaline. Reserpine partially abolished the response to tyramine in both pencil fish and angelfish. Small doses of noradrenaline and adrenaline given to reserpine-treated angelfish partially restored the response of the melanophores to nerve stimulation. No action of ACTH, MSH, nor direct effect of reserpine could be demonstrated on melanophores of either species. It was concluded that reserpine acts on the melanophores of pencil fish and angelfish in an indirect way by depletion of catecholamines from the sympathetic nerves supplying the melanophores.     

Tight association of the human Mel(1a)-melatonin receptor and G(i): precoupling and constitutive activity.

If stably expressed in human embryonic kidney (HEK)293 cells, the human Mel(1a)-melatonin receptor activates G(i)-dependent, pertussis toxin-sensitive signaling pathways, i.e., inhibition of adenylyl cyclase and stimulation of phospholipase Cbeta; the latter on condition that G(q) is coactivated. The antagonist luzindole blocks the effects of melatonin and acts as an inverse agonist at the Mel(1a) receptor in both intact cells and isolated membranes. This suggests that the Mel(1a) receptor is endowed with constitutive activity, a finding confirmed on reconstitution of the Mel(1a) receptor with G(i). Because the receptor density is in the physiological range, constitutive activity is not an artifact arising from overexpression of the receptor. In addition, the following findings indicate that the Mel(1a) receptor forms a very tight complex with G(i) which can be observed both in the presence and absence of an agonist. 1) In intact cells and in membranes, high-affinity agonist binding is resistant to the destabilizing effect of guanine nucleotides. 2) The ability to bind an agonist with high affinity is preserved even after exposure of the cells to pertussis toxin, because a fraction of G(i) is inaccessible to the toxin in cells expressing Mel(1a) receptors (but not the A(1)-adenosine receptor, another G(i)-coupled receptor). 3) An antiserum directed against the Mel(1a) receptor coprecipitates G(i) even in the absence of an agonist. We therefore conclude that the Mel(1a) receptor is tightly precoupled and that its constitutive activity may play a role in pacing the biological clock, an action known to involve the melatonin receptors in the suprachiasmatic nucleus.     

The melatonin receptor antagonist luzindole induces the activation of cellular stress responses and decreases viability of rat pancreatic stellate cells

In this study, we have examined the effects of luzindole, a melatonin receptor-antagonist, on cultured pancreatic stellate cells. Intracellular free-Ca²⁺ concentration, production of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPK), endoplasmic reticulum stress and cell viability were analyzed. Stimulation of cells with the luzindole (1, 5, 10 and 50 μm ) evoked a slow and progressive increase in intracellular free Ca²⁺ ([Ca²⁺]_i) towards a plateau. The effect of the compound on Ca²⁺ mobilization depended on the concentration used. Incubation of cells with the sarcoendoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (1 μm ), in the absence of Ca²⁺ in the extracellular medium, induced a transient increase in [Ca²⁺]_i. In the presence of thapsigargin, the addition of luzindole to the cells failed to induce further mobilization of Ca²⁺. Luzindole induced a concentration-dependent increase in ROS generation, both in the cytosol and in the mitochondria. This effect was smaller in the absence of extracellular Ca²⁺. In the presence of luzindole the phosphorylation of p44/42 and p38 MAPKs was increased, whereas no changes in the phosphorylation of JNK could be noted. Moreover, the detection of the endoplasmic reticulum stress-sensor BiP was increased in the presence of luzindole. Finally, viability was decreased in cells treated with luzindole. Because cellular membrane receptors for melatonin have not been detected in pancreatic stellate cells, we conclude that luzindole could exert direct effects that are not mediated through its action on melatonin membrane receptors.     

Antidepressant-like activity of the melatonin receptor antagonist, luzindole (N-0774), in the mouse behavioral despair test

The anti-immobility effect of the selective melatonin receptor antagonist, luzindole, was investigated in the behavioral despair test using three different strains (C3H/HeN, C57BL/6J and albino ND/4) of mice. The time of immobility of the C3H/HeN during the 240 s swimming period measured at noon (12:00 to 14:00 h) was 47.8 +/- 3.0 s (n = 63) and at midnight (00:00 to 02:00 h) was 67.7 +/- 2.8 s (n = 68) (P less than 0.001, when compared with the noon value), when the levels of endogenous melatonin are presumably low and high, respectively. Melatonin (30 mg/kg) given i.p. did not modify the time of immobility at either time of measurement. Luzindole (30 mg/kg i.p.) reduced the time of immobility in a dose-dependent manner, the effect being more pronounced at midnight (60% reduction) than at noon (39% reduction). The effect of luzindole was time-dependent, showing a maximal effect at 60 min. The anti-immobility effect of luzindole (10 mg/kg i.p.) was prevented by the administration of melatonin (30 mg/kg i.p.). Luzindole (30 mg/kg i.p.) did not modify the time of immobility either at noon or midnight in the albino ND/4 mouse, or in the C57BL/6J mouse, which does not produce melatonin. Our results suggest that endogenous melatonin plays a role during swimming in the C3H/HeN mouse behavioral despair test. We conclude that luzindole may exert antidepressant-like activity in the C3H/HeN mouse by antagonizing the action of endogenous hormone.     

Serotonin and antidepressant drugs antagonize melatonin-induced behavioural changes after injection into the nucleus accumbens of rats

Small doses of melatonin (0.1-100 ng), injected into the nucleus accumbens of rats, decreased locomotor activity and rearing, and increased grooming and sniffing behaviour when the animals were tested in small test-cages. Larger doses of melatonin appeared to be less effective. The action of melatonin is apparently not mediated by dopaminergic systems, because the behavioural changes were not antagonized by local pretreatment with haloperidol or sulpiride. Injection of serotonin antagonists (methysergide and cyproheptadine) into the nucleus accumbens resulted in similar behavioural changes as was found after treatment with melatonin. Treatment with serotonin and various antidepressant drugs (zimelidine, mianserin, nortriptyline, clomipramine, desipramine) injected into the nucleus accumbens, completely inhibited the melatonin-induced behavioural responses. The antidepressants did not significantly interfere with the decrease of locomotor activity and rearing induced by injection of small doses of the dopamine agonist, apomorphine, into the nucleus accumbens. These results suggest that there is an interrelationship between melatonin and serotonin systems in the nucleus accumbens and showed that various antidepressant drugs, similar to serotonin, antagonized the behavioural effects of melatonin after injection into the nucleus accumbens.     

Mechanisms of melatonin-induced vasoconstriction in the rat tail artery: a paradigm of weak vasoconstriction

1. Vasoconstrictor effects of melatonin were examined in isolated rat tail arteries mounted either in an isometric myograph or as cannulated pressurized segments. Melatonin failed by itself to mediate observable responses but preactivation of the arteries with vasopressin (AVP) reliably uncovered vasoconstriction responses to melatonin with maxima about 50% of maximum contraction. Further experiments were conducted with AVP preactivation to 5-10% of the maximum contraction. 2. Responses to melatonin consisted of steady contractions with superimposed oscillations which were large and irregular in isometric but small in isobaric preparations. Nifedipine (0.3 microM) reduced the responses and abolished the oscillations. Charybdotoxin (30 nM) increased the magnitude of the oscillations with no change in the maximum response. 3. Forskolin (0.6 microM) pretreatment increased the responses to melatonin compared to control and sodium nitroprusside (1 microM) treated tissues. The AVP concentration required for preactivation was 10 fold higher than control in both the forskolin and nitroprusside treated groups. 4. In isometrically-mounted arteries treated with nifedipine, melatonin receptor agonists had the potency order 2-iodomelatonin > melatonin > S20098 > GR196429, and the MT2-selective antagonist luzindole antagonized the effects of melatonin with a low pK(B) of 6.1+/-0.1. 5. It is concluded that melatonin elicits contraction of the rat tail artery via an mt1 or mt1-like receptor that couples via inhibition of adenylate cyclase and opening of L-type calcium channels. Calcium channels and charybdotoxin-sensitive K channels may be recruited into the responses via myogenic activation rather than being coupled directly to the melatonin receptors. 6. It is proposed that the requirement of preactivation for overt vasoconstrictor responses to melatonin results from the low effector reserve of the melatonin receptors together with the tail artery having threshold inertia. Potentiative interactions between melatonin and other vasoconstrictor stimuli probably also result from the threshold inertia. A simple model is presented and a general framework for consideration of interactions between weak vasoconstrictor agonists and other vasoconstrictor stimuli is discussed.