Bojidar Stankov

Melatonin binding sites in the central nervous system

3. Central nervous system binding sites (receptors) for melatonin 3.1. The median eminence/pars tuberalis (MJWT) area 3.2. The suprachiasmatic nuclei (SCN) 3.3. Preoptic area (POA) of the anterior hypothalamus 3.4. Area postrema (AP) 3.5. CorticaI structures 3.6. Other areas

The melatonin receptor in the human brain: cloning experiments and distribution studies

The adult human cerebellum expresses melatonin receptors with high density in the external zone of the molecular layer. Cloning of the receptor cDNA isolated by RT-PCR from human cerebellar specimens and sequencing analysis of the full-length coding region revealed that the receptor protein is encoded by a transcript identical to that recently cloned from the human hypothalamus (Mel1a). In situ hybridization using an antisense cRNA-probe demonstrated that the melatonin receptor mRNA is localized in the cerebellar granule cells. Mapping of the messenger by RT-PCR with Mel1a specific primers in different areas of the human brain disclosed a quite widespread distribution of the transcript, although expressed at very low levels. Semi-quantitative comparison between the different brain regions allowed to establish the following relative mRNA abundance: cerebellum > or = occipital cortex > or = parietal cortex > temporal cortex > thalamus > frontal cortex > or = hippocampus. No mRNA was detected in white blood cells.

Melatonin and delayed sleep phase syndrome: Ambulatory polygraphic evaluation

THE present study objectively evaluated the efficacy of oral 5 mg day-1 melatonin in advancing the sleep-wake rhythm in patients with delayed sleep phase syndrome (DSPS). Six patients underwent ambulatory sleep monitoring for 72 h before, and 48 h after 1 month of melatonin treatment. In each patient melatonin was administered on the basis of his own estimated dim light melatonin onset (DLMO) delay. Mean advances in sleep onset time of 115 min, and in final awakening hour of 106 min were found after treatment, with no significant changes in sleep architecture parameters. Our study objectively confirms previous data obtained by a sleep-wake subjective diary on the efficacy of melatonin DSPS.

Characterization and Mapping of Melatonin Receptors in the Brain of Three Mammalian Species: Rabbit, Horse and Sheep

Melatonin receptors were characterized in the brains of three mammals (rabbit, horse and sheep) by an in vitro binding technique, using 2-[125I]iodomelatonin as labelled ligand. Although binding sites for melatonin have been described recently in several vertebrate species (including the sheep), the rabbit and the horse have not been the subject of investigation so far. Apart from characterization, the present report describes receptor distribution in a number of brain regions, thus allowing for direct interspecies comparison under the same methodological conditions. 2-[125I]iodomelatonin labelled high-affinity binding sites in crude membrane preparations from these species. A series of kinetic and saturation experiments revealed that the binding was rapid, stable, saturable, reversible, of high affinity (Kd in the low picomolar range) and low capacity (Bmax between 1 and 20 fmol/mg protein). The competition studies showed that the relative order of potency of a variety of indoles for inhibition of 2-[125I]iodomelatonin binding was as follows: 2-iodomelatonin greater than 6-chloromelatonin greater than melatonin much much greater than 5-methoxytryptophol greater than 5-methoxytryptamine, and that it was similar in the different brain regions. Prazosin, which has been reported as an extremely potent melatonin analog in the hamster brain, possessed no potency in all preparations from different regions in the three species under investigation. The regional distribution of the receptor showed insignificant species differences. Highest density was always recorded in the median eminence/pars tuberalis (ME/PT) area. Other regions (SCN, POA and certain cortical areas), showed lower, but significant, receptor content. Saturation and competition studies revealed that these binding sites were also of high affinity, low capacity and high specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Ligand efficacy and potency at recombinant human MT2 melatonin receptors: evidence for agonist activity of some mt1‐antagonists

NIH3T3 fibroblast cells transfected with the full‐length coding region of the MT2 human melatonin receptor stably expressed the receptor that is coupled to a pertussis toxin‐sensitive G protein and exhibits high affinity for melatonin (KI=261 pM). The order of apparent affinity for selected compounds was: 4‐phenyl‐2‐propionamidotetralin (4P‐PDOT)>2‐phenylmelatonin>2‐iodomelatonin>2‐bromomelatonin>6‐chloromelatoninmelatonin>luzindole>N‐acetyl‐tryptamineN‐[(2‐phenyl‐1H‐indol‐3‐yl)ethyl]cyclobutanecarboxamide (compound 6)>N‐acetylserotonin. 4P‐PDOT exhibited a very high selectivity (∼22,000 times) for the MT2 receptor with respect to the mt1 receptor subtype, as tested in comparative experiments with membrane preparations from NIH3T3 cells stably transfected with the human mt1 receptor. MT2 melatonin receptors mediated incorporation of [35S]‐GTPγS into isolated membranes via receptor catalyzed exchange of [35S]‐GTPγS for GDP. The relative intrinsic activity and potency of the compounds were subsequently studied by using [35S]‐GTPγS incorporation. The order of potency was equal to the order of apparent affinity. Melatonin and full agonists increased [35S]‐GTPγS binding by 250% over basal (taken as 100%). Luzindole did not increase basal [35S]‐GTPγS binding but competitively inhibited melatonin‐stimulated [35S]‐GTPγS binding, thus exhibiting antagonist action. The other two mt1 antagonists used here, 4P‐PDOT and N‐[(2‐phenyl‐1H‐indol‐3‐yl)ethyl]cyclobutanecarboxamide, behaved as partial agonists at the MT2 subtype, with relative intrinsic activities of 0.37 and 0.39, respectively. These findings show, for the first time, important differences in the intrinsic activity of analogues between the human mt1 and MT2 melatonin receptor subtypes.

Reduction of regional cerebral blood flow by melatonin in young rats

Melatonin (10 ng) was subcutaneously administered to 14-day-old Sprague-Dawley rats. Regional blood flow (rCBF) was measured in 22 anatomically defined structures 20 min later using iodo[14C]antipyrine and quantitative autoradiography. rCBF was markedly reduced in the cerebral areas supplied by circle of Willis and the basilar arteries. Melatonin also significantly decreased blood flow to choroid plexuses. These findings suggest that circulating melatonin may contribute to regulation of cerebral blood flow and brain fluid balance.

Distribution and characterization of melatonin receptors in the brain of the Japanese quail, Coturnix japonica

2-[125I]iodomelatonin was used to study the distribution and properties of the melatonin receptor in the Japanese quail brain. High receptor density was detected in the major targets of direct retinal input (optic tectum, nucleus of the optic basal rout, ventrolateral geniculate nucleus), as well as areas representing terminals in the visual pathways (nucleus rotundus, ectostriatum, thalamo-hyperstriatal pathway). Binding was also found in the piriform cortex, the hypophyseal pars tuberalis, the oculomotorius nucleus and the associated Edinger-Westphal nucleus, and in the nuclei of the third, fourth and sixth cranial nerves. A comparison of the receptor pharmacological profile to that of the mammalian brain demonstrated pharmacological identity of the two binding sites. In the saturation experiments, GPT gamma S decreased the binding affinity, numerical Kd values increasing from approximately 35 pM to approximately 150 pM.

Synthesis, pharmacological characterization and QSAR studies on 2-substituted indole melatonin receptor ligands

A number of 6-methoxy-1-(2-propionylaminoethyl)indoles, carrying properly selected substituents at the C-2 indole position, were prepared and tested as melatonin receptor ligands. Affinities and intrinsic activities for the human cloned mt1 and MT2 receptors were examined and compared with those of some 2-substituted melatonin derivatives recently described by us. A quantitative structure activity relationship (QSAR) study of the sixteen 2-substituted indole compounds, 5a-k, 1, 8-11, using partial least squares (PLS) and multiple regression analysis (MRA) revealed the existence of an optimal range of lipophilicity for the C2 indole substituent. There are also indications that planar, electron-withdrawing substituents contribute to the affinity by establishing additional interactions with the binding pocket. No mt1/MT2 subtype selectivity was observed, with the relevant exception of the 2-phenethyl derivative 5e, which exhibited the highest selectivity for the h-MT2 receptor among all the compounds tested (MT2/mt1 ratio of ca. 50). Conformational analysis and superposition of 5e to other reported selective MT2 ligands revealed structural and conformational similarities that might account for the MT2/mt1 selectivity of 5e.

Localization and characterization of melatonin binding sites in the brain of the rabbit (Oryctolagus cuniculus) by autoradiography and in vitro ligand-receptor binding

The distribution and the properties of the melatonin binding sites were characterized in the brain of the rabbit by combined use of autoradiography and in vitro ligand-receptor binding. Autoradiography revealed widespread specific binding in the brain. The pars tuberalis of the pituitary gland, suprachiasmatic nuclei, ventromedial hypothalamic nuclei, tapetum, hippocampus, indusium griseum, cingulate gyrus, cortex and the choroid plexus were intensely labelled. Diffuse specific binding was recorded in the olfactory bulb and the anterior hypothalamus. Series of in vitro ligand-receptor binding experiments, using the anterior hypothalamus, confirmed that the binding was of high affinity and specificity. Coincubation with a non-hydrolyzable GTP analogue provoked a shift in the binding affinity, the numerical values of the Kd increasing from 20-30 pM to 280-300 pM. Apparently the melatonin receptor in the rabbit brain is linked to its second messenger via a G protein, similarly to what has been described for the brain of other vertebrates.

The adult human cerebellum is a target of the neuroendocrine system involved in the circadian timing

In an investigation aimed at comprehensive mapping of the adult human brain with respect to receptor sites for the pineal hormone melatonin, we consistently observed specific binding in the cerebellum. Autoradiography and in vitro binding analysis with 125I-labeled melatonin were used to examine the location and the properties of these binding sites. In all cerebellar lobes, highest-density specific binding was localized to the external zone of the molecular layer. The binding was rapid, saturable, displaceable, specific and of high affinity. Physiological concentrations of NaCl decreased the affinity, while presence of calcium ions promoted it. The non-hydrolyzable GTP analog, GTP gamma S, inhibited binding in a dose-dependent manner and provoked a shift towards low affinity. The results strongly suggest that these binding sites may be functional melatonin receptors, and indicate that the adult human cerebellum is a target of melatonin, the pineal hormone involved in the control of the circadian timing.