Chimeric melatonin mt1 and melatonin-related receptors. Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt1 receptor

Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt(1) receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[(125)I]iodomelatonin binding. Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[(125)I]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt(1) receptor sequences identified specific 2-[(125)I]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt(1) receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.     

Biological basis and possible physiological implications of melatonin receptor-mediated signaling in the rat epididymis

The mammalian epididymis plays an important role in sperm maturation, an important process of male reproduction. Specific high-affinity 2-[(125)I]iodomelatonin binding sites, satisfying the pharmacokinetic properties of specific receptors, have been found in the rat corpus epididymis, suggesting a direct melatonin action on epididymal physiology. Subsequent molecular and cell biology studies have identified these 2-[(125)I]iodomelatonin binding sites to be mt(1) (MEL(1A)) and MT(2) (MEL(1B)) melatonin receptor subtypes. Changes in the binding characteristics of these receptors in the rat corpus epididymis in response to castration and steroid hormones like testosterone and hydrocortisone indicated that these membrane melatonin receptors are biologically functional receptors, whose activities are differentially regulated by testosterone and hydrocortisone. These melatonin receptors are coupled to pertussis toxin (PTX)-sensitive G(i) protein and probably participate in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions. Furthermore, rat corpus epididymal epithelial cell proliferation was stimulated by melatonin, whose action was dependent on the concentration and duration of exposure to the hormone. Interestingly, an MT(2) receptor ligand (4-phenyl-2-propionamidotetraline, 4-P-PDOT) induced a stimulatory effect on epididymal epithelial cell proliferation similar to that produced by melatonin. In contrast, a nuclear melatonin receptor agonist (1-[3-allyl-4-oxo-thiazolidine-2-ylidene]-4-methyl-thiosemi-car bazone , CGP52608) and 8-bromo-cAMP inhibited epididymal epithelial cell proliferation. Taken together, our data lead us to postulate that one of the possible physiological functions of melatonin on the rat epididymis is the stimulation of mt(1) and MT(2) melatonin receptors resulting in the inhibition of cAMP signaling and an increase in epithelial cell proliferation.     

N1E-115 mouse neuroblastoma cells express MT1 melatonin receptors and produce neurites in response to melatonin

Melatonin, a pineal hormone that induces sleep, has become a popular over-the-counter drug. The cellular effects of melatonin, however, are only beginning to be studied. We have recently shown that stimulation of the MT1 melatonin receptor induces rapid and dramatic cytoskeletal rearrangements in transformed non-neuronal cells (Witt-Enderby et al., Cell. Motil. Cytoskel. 46 (2000) 28). These cytoskeletal changes result in the formation of structures that closely resemble neurites. In this work, we show that the N1E-115 mouse neuroblastoma cell line rapidly responds to melatonin stimulation and forms neurites within 24 h. We also demonstrate that these cells readily bind 2-[125I]iodomelatonin at levels consistent with what is noted for native tissues (B(max)=3.43+/-1.56 fmol/mg protein; K(d)=240 pM). Western analysis shows that these cells possess and express melatonin receptors of the MT1 subtype. Treatment with pertussis toxin eliminates neurite formation whereas treatment with the MT2 subtype-specific activator, BMNEP, does not induce neurite formation. We have previously shown that increases in MEK 1/2 and ERK 1/2 phosphorylation are correlated with the shape changes in transformed CHO cells. Western analysis of the MEK/ERK signaling pathway in N1E-115 cells shows that this pathway is most likely maximally and constitutively stimulated. This may account for the spontaneous production of neurites noted for this cell line after long culture periods. The results of this work show that melatonin receptor stimulation in a neuronal cell type results in the formation of neurites and that the receptors responsible for melatonin-induced neurite formation in N1E-115 cells are most likely of the MT1 subtype.     

Pharmacology and function of melatonin receptors

The hormone melatonin is secreted primarily from the pineal gland, with highest levels occurring during the dark period of a circadian cycle. This hormone, through an action in the brain, appears to be involved in the regulation of various neural and endocrine processes that are cued by the daily change in photoperiod. This article reviews the pharmacological characteristics and function of melatonin receptors in the central nervous system, and the role of melatonin in mediating physiological functions in mammals. Melatonin and melatonin agonists, at picomolar concentrations, inhibit the release of dopamine from retina through activation of a site that is pharmacologically different from a serotonin receptor. These inhibitory effects are antagonized by the novel melatonin receptor antagonist luzindole (N-0774), which suggests that melatonin activates a presynaptic melatonin receptor. In chicken and rabbit retina, the pharmacological characteristics of the presynaptic melatonin receptor and the site labeled by 2-[125I]iodomelatonin are identical. It is proposed that 2-[125I]iodomelatonin binding sites (e.g., chicken brain) that possess the pharmacological characteristics of the retinal melatonin receptor site (order of affinities: 2-iodomelatonin greater than 6-chloromelatonin greater than or equal to melatonin greater than or equal to 6,7-di-chloro-2-methylmelatonin greater than 6-hydroxymelatonin greater than or equal to 6-methoxymelatonin greater than N-acetyltryptamine greater than or equal to luzindole greater than N-acetyl-5-hydroxytryptamine greater than 5-methoxytryptamine much greater than 5-hydroxytryptamine) be classified as ML-1 (melatonin 1). The 2-[125I]iodomelatonin binding site of hamster brain membranes possesses different binding and pharmacological characteristics from the retinal melatonin receptor site and should be classified as ML-2. In summary, the recent advances in the pharmacological characterization of melatonin receptors in the central nervous system will further stimulate the search for potent and selective melatonin receptor agonists and antagonists, and should aid in our understanding of the mechanism of action of melatonin in mammalian brain.     

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in syrian hamster hypothalamus

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [125I]iodomelatonin, was examined using an incubation temperature (30 degrees C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [125I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.     

Localization of 2-[125I]Iodomelatonin Binding Sites in Mammalian Retina

Specific melatonin binding sites were localized in the mammalian retina using the selective radioligand 2-[125I]iodomelatonin. Frozen sections obtained from both pigmented and albino rabbit eyes and albino mouse eyes were incubated with 2-[125I]iodomelatonin in the absence and presence of competing agents. In eyecups from albino rabbits, the highest density of specific 2-[125I]iodomelatonin binding sites was localized over the inner plexiform layer. Approximately 40-60% of the binding was specific, as determined with both the agonist 6-chloromelatonin and the antagonist luzindole. A high density of binding sites was observed over the choroid and retinal pigmented epithelium, but no statistical difference between total and nonspecific binding was detected. Results were similar with eyecups from pigmented rabbits. Albino mice showed a significant extent of 2-[125I]iodomelatonin binding in both the inner plexiform and the outer and inner segment layers. The specific binding of 2-[125I]iodomelatonin in retinas from albino rabbits maintained in the light for 24 h before decapitation was increased in the inner retina compared with the control. The distribution of 2-[125I]iodomelatonin binding sites in the various layers of the mammalian retina is consistent with the described functions for this hormone in retinal physiology.     

3-[(3-Cholamidopropyl)dimethylammonio]-1-Propane Sulfonate-Solubilized Binding Sites for 2-[125I]Iodomelatonin in Chick Brain Retain Sensitivity to Guanine Nucleotides

Binding of 2-[125I]iodomelatonin to 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized sites from chick forebrain was rapid. reversible, saturable, of high affinity, and of pharmacological selectivity. Scatchard analyses showed that 2-[125I]iodomelatonin binds to a single site with equilibrium dissociation constant (KD) values of 328 +/- 22 (n = 4) and 302 +/- 26 pM (n = 3) and a maximal number of binding sites (Bmax) of 36.2 +/- 2.0 and 49.5 +/- 6.6 fmol/mg of protein in solubilized and membrane fractions, respectively. The KD values obtained from the ratio of kinetic constants (k2/k1) in solubilized and membrane preparations were 228 and 216 pM, respectively. Inhibition studies indicated the following order of pharmacological affinities for both membrane and solubilized sites: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than prazosin greater than N-acetylserotonin much greater than serotonin greater than metergoline greater than ketanserin greater than propranolol greater than phentolamine greater than cyproheptadine. Guanyl nucleotides inhibited binding of 2-[125I]iodomelatonin to solubilized and membrane fractions, by converting binding sites from a high-affinity to a low-affinity state. These findings show that solubilized binding sites for melatonin exhibit the specific binding and pharmacological characteristics present in membrane-bound sites. Moreover, the retention of sensitivity to guanine nucleotides in fractions solubilized with CHAPS suggests that this solubilization procedure is suitable for further studies aimed at the isolation, purification, and molecular characterization of active melatonin binding sites.     

mt(1) Receptor-mediated antiproliferative effects of melatonin on the rat uterine antimesometrial stromal cells.

It has been shown that melatonin regulates uterine function. Our previous studies have demonstrated the presence of melatonin receptors in the rat uterine endometrium, indicating that melatonin may act directly on the uterus. In the present study, the histological localization of the rat uterine melatonin binding was revealed by autoradiography and the molecular subtyping was studied by in situ hybridization in the stromal cells. The signal transduction process and effects of melatonin on stromal cell proliferation was also investigated. Our autoradiograms showed that 2[(125)I]iodomelatonin binding sites were localized in the antimesometrial endometrial stroma. In situ hybridization with specific mt(1) receptor cDNA probe in the primary culture of antimesometrial stromal cells demonstrated the expression of mt(1) receptor mRNAs. Melatonin dose-dependently inhibited forskolin-stimulated cAMP accumulation, which was reversed by pertussis toxin. This indicates that the rat uterine melatonin receptors are negatively coupled to adenylate cyclase via pertussis toxin sensitive G(i) protein. Melatonin also inhibited the incorporation of [(3)H]thymidine in the rat uterine antimesometrial stromal cells, showing that melatonin has an anti-proliferative effect on the uterus. Our results suggest that melatonin may act directly on the mt(1) melatonin receptors in the rat uterine antimesometrial stromal cells to inhibit their proliferation. Its action may be mediated through a pertussis toxin-sensitive adenylate cyclase coupled G(i)-protein.     

A novel and sensitive radioreceptor assay for serum melatonin levels.

A simple and sensitive radioreceptor assay (RRA) has been developed to measure melatonin levels in serum. The assay is based on competition between 2-[125I]iodomelatonin ([125I]MEL) and melatonin for binding to high-affinity binding sites in chick forebrain. To measure the amount of melatonin present in a serum sample, it was extracted with dichloromethane and added to the assay medium. The percentage inhibition of radioligand binding in the presence of the extracted serum was determined and compared to the percent displacement by known amounts of melatonin in a standard curve. There was little or no cross-reactivity with other structurally related compounds. The sensitivity of the assay is approximately 1.5pg/0.15 mL and the intra- and inter-assay variations are approximately 8%. Since the RRA results are comparable to that of an established radioimmunoassay (RIA), it provides a sensitive and rapid alternative to the more time consuming RIA.     

Melatonin binding sites in senegal sole: day/night changes in density and location in different regions of the brain

We localized melatonin binding sites in different brain regions (optic tectum, telencephalon, cerebellum, hypothalamus, olfactory bulbs, and medulla oblongata) of Senegal sole, a species of aquaculture interest, and checked day/night changes in density (B(max)) at mid-light (ZT06) and mid-dark (ZT18). Plasma melatonin was measured using a radioimmunoassay, while binding assays were performed using 2-[(125)I]iodomelatonin as a radioligand. Plasma melatonin concentrations were significantly lower at mid-light (189.5+/-46 pg/ml) than mid-dark (455.5+/-163 pg/ml). Values of B(max) were statistically significantly higher in the optic tectum (5.6+/-0.6 and 12.3+/-1 fmol/mg prot, at mid-light and mid-dark, respectively) and in the cerebellum (7.7+/-1.1 and 10.6+/-1.3 fmol/mg prot, at mid-light and mid-dark, respectively). Significant day/night differences were only observed in these two tissues. These results show for the first time the distribution of melatonin binding sites within the brain of a flatfish species and their lack of down-regulation.     

5-Methoxytryptamine Inhibits Cyclic AMP Accumulation in Cultured Retinal Neurons Through Activation of a Pertussis Toxin-Sensitive Site Distinct from the 2-[125I]Iodomelatonin Binding Site

Abstract: 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 n M for melatonin and 7.2 n M 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 K _i value (0.8 n M ) similar to the EC₅₀ for inhibition of cyclic AMP formation. In contrast, 5-methoxytryptamine only inhibited 2-[¹²⁵I]iodomelatonin binding at very high concentrations ( K _i = 650 n M ). 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.     

Circadian rhythms of melatonin release from chicken pineal in vitro: modified melatonin radioimmunoassay

An improved and simplified radioimmunoassay for measuring pineal, serum, and in vitro cultured medium melatonin is described. Using 2-[125I]iodomelatonin as radiolabeled ligand and a polyclonal rabbit antimelatonin antiserum, melatonin concentrations were determined in all three types of samples by a 2-day direct equilibrium double-antibody assay method without prior extraction. Serial dilutions of pineal homogenates, serum, and cultured medium all gave parallel displacement curves. Cross-reactivity of the antisera with other indoles was negligible. Intraassay coefficients of variation (n = 3) were 5.09, 3.32, and 5.05% at 7.81, 62.5, and 500 pg/tube, respectively, and the interassay coefficients of variation (n = 20) were 12.18% at 62.5 pg/tube. A characteristic diurnal rhythm of melatonin was observed using this direct assay for measuring daytime and nighttime chicken pineal and serum samples. An in vitro incubation of chicken pineal glands with a lighting cycle of 12-hr light:12-hr dark showed that the diurnal rhythm of melatonin secretion into the cultured medium was maintained. The direct assay method described in this report for measuring chicken melatonin using 2-[125I]iodomelatonin as radiolabeled ligand coupled with the in vitro cultured chicken pineal gland clearly offers great potential for studying the chicken pineal circadian oscillator and its underlying mechanism.     

2-(125I) iodomelatonin binding sites in rat adrenals: pharmacological characteristics and subcellular distribution.

Specific binding sites for 2-[125I] iodomelatonin, a selective radiolabeled melatonin receptor ligand, were detected and characterized in rat adrenal membranes. Saturation studies demonstrated that 2-[125I]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 541 pM and a total binding capacity (Bmax) of 3.23 fmol/mg protein. Competition experiments revealed that the relative order of potency of compounds tested was as follows: 6-chloromelatonin greater than 2-iodomelatonin greater than melatonin greater than 5-methoxytryptamine greater than 5-methoxytryptophol. The highest density of binding sites was found in membranes from nuclear (0.76 fmol/mg protein) and mitochondrial (1.82 fmol/mg protein) subcellular fractions.     

Melatonin binding proteins identified in the rat brain by affinity labeling

N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM), a novel derivative of the biologically active melatonin analog, 2-iodomelatonin, was prepared and used to identify melatonin binding proteins in rat brain synaptosomes. Incubation of the synaptosomes with BIM resulted in a time and concentration dependent, irreversible inhibition of 2-[125I]iodomelatonin binding. In parallel, the radioactive form of BIM, N-bromoacetyl-2-[125I]iodo-5-methoxytryptamine ([125I]BIM) became incorporated into the synaptosomes. The incorporation of [125I]BIM was inhibited by BIM, 2-iodomelatonin and melatonin but not by 5-methoxytryptamine or N-acetyl serotonin. [125I]BIM became covalently attached to three polypeptides with apparent molecular weight values of 92, 55 and 45 kDa; the labeling of all three proteins was markedly inhibited by melatonin. These results indicate that the 92, 55 and 45 kDa polypeptides are melatonin binding proteins.     

Synthesis and biological evaluation of novel 4-benzylpiperazine ligands for sigma-1 receptor imaging

We report the synthesis and evaluation of 4-benzylpiperazine ligands (BP-CH(3), BP-F, BP-Br, BP-I, and BP-NO(2)) as potential σ(1) receptor ligands. The X-ray crystal structure of BP-Br, which crystallized with monoclinic space group P2(1)/c, has been determined. In vitro competition binding assays showed that all the five ligands exhibit low nanomolar affinity for σ(1) receptors (K(i)=0.43-0.91nM) and high subtype selectivity (σ(2) receptor: K(i)=40-61nM; K(i)σ(2)/K(i)σ(1)=52-94). [(125)I]BP-I (1-(1,3-benzodioxol-5-ylmethyl)-4-(4-iodobenzyl)piperazine) was prepared in 53±10% isolated radiochemical yield, with radiochemical purity of >99% by HPLC analysis after purification, via iododestannylation of the corresponding tributyltin precursor. The logD value of [(125)I]BP-I was found to be 2.98±0.17, which is within the range expected to give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of radiolabeled substances in organs known to contain σ(1) receptors, including the brain, lung, kidney, heart, and spleen. Administration of haloperidol 5min prior to injection of [(125)I]BP-I significantly reduced the concentration of radioactivity in the above-mentioned organs. The accumulation of radiolabeled substance in the thyroid was quite low suggesting that [(125)I]BP-I is relatively stable to in vivo deiodination. These findings suggest that the binding of [(125)I]BP-I to σ(1) receptors in vivo is specific.     

Down-regulation of mt1 melatonin receptors in rat ovary following estrogen exposure.

In this study, we have demonstrated that 2-[125I]-iodomelatonin binds specifically to rat ovarian granulosa cell (GC) membranes with high affinity (KD=83 pM; Bmax=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt1 melatonin receptor antibody, we have also detected mt1 melatonin receptors in rat ovary. Because melatonin has been reported to alter the steroidogenic responses of ovarian tissues to gonadotropins, a physiological role for intra-ovarian melatonin may exist. Thus, in order to investigate a possible intra-ovarian role for melatonin, we have used both an in vivo and in vitro model of follicular development. Treatment of immature (day 21) female rats with estradiol (E; 0.2 mg/d x 3 d; subcutaneous) was used to induce follicular growth. Membranes from both untreated (U) and E-treated animals' ovaries contained high-affinity 2-[125I]-iodomelatonin (I-MEL) binding sites (Kd=83 and 23 pM, respectively). Estradiol treatment in vivo caused a significant decrease (P<0.05) in binding of 2-[125I]-iodomelatonin to ovarian membranes with untreated animals' ovaries having a Bmax=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a Bmax=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt1 melatonin receptors in rat ovary were down-regulated (approximately 95%) using immunoblot analysis. Granulosa cells isolated from E-treated rats were further matured in vitro with testosterone (T) and the pituitary gonadotropin follicle-stimulating hormone (FSH). Granulosa cells were cultured with either T (10 ng/ml) or FSH (5.71 ng ovine FSH-20/ml) alone, or both FSH and T for 48 h. There was no statistically significant specific binding of 2-[125I]-iodomelatonin to GC membranes cultured with T or FSH alone. However, following a 48-h exposure to FSH and T in vitro specific 2-[125I]-iodomelatonin binding occurred with total 2-[125I]-iodomelatonin binding =3.15 [corrected] fmol/mg protein. Therefore, the existence of hormonally-regulated expression of high-affinity melatonin binding sites suggests that melatonin may have an important intra-ovarian physiological role.     

Synthesis and characterization of [¹²⁵I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine transporter 1

In this study, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which predominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide (5) showed excellent inhibitory activity of [(3)H]glycine uptake (IC(50)=2.4 nM). Saturation assay in rat cortical membranes revealed that [(125)I]5 had a single high affinity binding site with a K(d) of 1.54 nM and a B(max) of 3.40 pmol/mg protein. In vitro autoradiography demonstrated that [(125)I]5 showed consistent accumulation with GlyT1 expression. The in vitro binding was greatly inhibited by GlyT1 inhibitors but not by other site ligands, which suggested the high specific binding of [(125)I]5 with GlyT1. In the biodistribution and ex vivo autoradiography studies using mice, [(125)I]5 showed high blood-brain barrier permeability (1.68-2.17% dose/g at 15-60 min) and similar regional brain distribution pattern with in vitro results. In addition, pre-treatment of GlyT1 ligands resulted in significant decrease of [(125)I]5 binding in the GlyT1-rich regions. This preliminary study demonstrated that radio-iodinated 5 is a promising SPECT imaging probe for GlyT1.     

Synthesis and characterization of styrylchromone derivatives as beta-amyloid imaging agents

Several promising agents have been synthesized and evaluated for in vivo imaging probes of beta-amyloid plaques in Alzheimer's disease (AD) brain. Recently, we have developed flavone derivatives, which possess the basic structure of the 2-phenylchromone, as useful candidates for amyloid imaging agents. In an attempt to further develop novel tracers, we synthesized and evaluated a series of 2-styrylchromone derivatives, which replace the 2-phenyl substituent of flavone backbone with the 2-styryl. A series of radioiodinated styrylchromone derivatives were designed and synthesized. The binding affinities for amyloid plaques were assessed by in vitro binding assay using pre-formed synthetic Abeta(1-40) aggregates. The new series of styrylchromone derivatives showed high binding affinity to Abeta aggregates at the K(d) values of 32.0, 17.5 and 8.7nM for [(125)I]6, [(125)I]9, and [(125)I]12, respectively. In biodistribution studies using normal mice, [(125)I]6 and [(125)I]9 examined in normal mice displayed high brain uptakes with 4.9 and 2.8%ID/g at 2min post injection. The radioactivity washed out from the brain rapidly (1.6 and 1.0%ID/g at 60min post injection for [(125)I]6 and [(125)I]9, respectively). But [(125)I]12 did not show marked brain uptake, and the washout rate from the brain was relatively slow throughout the time course (1.1 and 1.4%ID/g at 2 and 30min post injection, respectively). Although additional modifications are necessary to improve the brain uptake and rapid clearance of non-specifically bound radiotracer, the styrylchromone backbone may be useful as a backbone structure to develop novel beta-amyloid imaging agents.     

Activation of MT(2) melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock

The aim of this study was to identify the melatonin receptor type(s) (MT(1) or MT(2)) mediating circadian clock resetting by melatonin in the mammalian suprachiasmatic nucleus (SCN). Quantitative receptor autoradiography with 2-[(125)I]iodomelatonin and in situ hybridization histochemistry, with either (33)P- or digoxigenin-labeled antisense MT(1) and MT(2) melatonin receptor mRNA oligonucleotide probes, revealed specific expression of both melatonin receptor types in the SCN of inbred Long-Evans rats. The melatonin receptor type mediating phase advances of the circadian rhythm of neuronal firing rate in the SCN slice was assessed using competitive melatonin receptor antagonists, the MT(1)/MT(2) nonselective luzindole and the MT(2)-selective 4-phenyl-2-propionamidotetraline (4P-PDOT). Luzindole and 4P-PDOT (1 nM-1 microM) did not affect circadian phase on their own; however, they blocked both the phase advances (approximately 4 h) in the neuronal firing rate induced by melatonin (3 pM) at temporally distinct times of day [i.e., subjective dusk, circadian time (CT) 10; and dawn, CT 23], as well as the associated increases in protein kinase C activity. We conclude that melatonin mediates phase advances of the SCN circadian clock at both dusk and dawn via activation of MT(2) melatonin receptor signaling.