Mel1b Melatonin Receptor Research Articles

Mel1b and Mel1c melatonin receptors mediate green light-induced secretion of growth hormone in chick adenohypophysis cells via the AC/PKA and ERK1/2 signalling pathways.

A previous study showed that melatonin (MEL) membrane receptors 1b (Mel1b) and Mel1c promoted the secretion of growth hormone (GH) in chick adenohypophysis cells under monochromatic green light. However, the intracellular signalling pathways of these two receptors are unclear. Therefore, cultured adenohypophysis cells derived from chickens exposed to monochromatic green light were treated with MEL, Mel1b- and Mel1c-specific blockers, protein kinase A (PKA) inhibitors and adenylate cyclase (AC), or AC activator in vitro to explore the signal transduction mechanism that promote the secretion of GH. The results showed that Mel1b and Mel1c participate in MEL-mediated green light-induced secretion of GH in chick adenohypophysis cells. However, MEL increased cyclic adenosine monophosphate (cAMP) levels, and p-PKA protein levels were blocked by a Mel1b-specific antagonist but not a Mel1c-specific antagonist, which indicated that Mel1b affected the secretion of GH via the AC/cAMP/PKA signalling pathway. Moreover, Mel1b and Mel1c both activated ERK1/2 to regulate the secretion of GH. In addition, intracellular and extracellular Ca2+ channels were also involved in secretion of GH in chick adenohypophysis cells. These results demonstrate that the MEL mediated green light-induced secretion of GH in chick adenohypophysis via the Mel1b/AC/PKA/ERK1/2, Mel1c/ERK1/2, and intracellular and extracellular Ca2+ channel signalling pathways.

Eye and heart morphogenesis are dependent on melatonin signaling in chick embryos.

Calmodulin is vital for chick embryos morphogenesis in the incubation time 48-66 h when the rudimentary C-shaped heart attains an S-shaped pattern and the optic vesicles develop into optic cups. Melatonin is in the extraembryonic yolk sac of the avian egg; melatonin binds calmodulin. The aim of this study was to investigate the function of melatonin in the formation of the chick embryo optic cups and S-shaped heart, by pharmacological methods and immunoassays. Mel1a melatonin receptor immunofluorescence was distributed in the optic cups and rudimentary hearts. We separated embryonated chicken eggs at 48 h of incubation into basal, control and drug-treated groups, with treatment applied in the egg air sac. At 66h of incubation, embryos were excised from the eggs and analyzed. Embryos from the basal, control (distilled water), melatonin and 6-chloromelatonin (melatonin receptor agonist) groups had regular optic cups and an S-shaped heart, while those from the calmidazolium (calmodulin inhibitor) group did not. Embryos from the luzindole (melatonin receptor antagonist) and prazosin (Mel1c melatonin receptor antagonist) groups did not have regular optic cups. Embryos from the 4-P-PDOT (Mel1b melatonin receptor antagonist) group did not have an S-shaped heart. Previous application of the melatonin, 6-chloromelatonin or forskolin (adenylate cyclase enhancer) prevented the abnormal appearance of chick embryos from the calmidazolium, luzindole, prazosin and 4-P-PDOT groups. However, 6-chloromelatonin and forskolin only partially prevented the development of defective eye cups in embryos from the calmidazolium group. The results suggested that melatonin modulates chick embryo morphogenesis via calmodulin and membrane receptors.

The melatonin analog 5-MCA-NAT increases endogenous dopamine levels by binding NRH:quinone reductase enzyme in the developing chick retina

NRH:quinone reductase (QR2) is present in the retinas of embryonic and post-hatched (PH) chicks. 5-Methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) is a QR2 ligand that increases cAMP levels in developing retinas, but it does not affect cAMP levels in CHO-QR2 cells. The dopamine quinone reductase activity of QR2 retrieves dopamine, which increases cAMP levels in developing retinas. The objective of the present study was to investigate whether 5-MCA-NAT increases endogenous dopamine levels in retinas from chick embryos and post-hatched chicks. Endogenous dopamine was measured by enzyme-linked immunosorbent assay (ELISA). 5-MCA-NAT increased retinal endogenous dopamine levels at all developmental stages studied and in PH chicks (−logEC50=11.62±0.34M). This effect was inhibited by non-selective antagonists of receptors and melatonin binding sites N-acetyl-2-benzyltryptamine (luzindole, 5μM), but it was not inhibited by the Mel1b melatonin receptor antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT, 10nM). The QR2 cosubstrate, N-methyl-dihydronicotinamide (NMH) (−logEC50=6.74±0.26M), increased endogenous dopamine levels in controls and in retinas stimulated with 5-MCA-NAT (3nM). The QR2 inhibitor benzo[e]pyrene inhibited endogenous dopamine levels in both control (−logIC50=7.4±0.28M) and NMH-stimulated (at 100nM and 1μM benzo[e]pyrene concentrations) retinas. Theoretical studies using Molegro Virtual Docking software corroborated these experimental results. We conclude that 5-MCA-NAT increases the level of endogenous dopamine via QR2. We suggest that this enzyme triggers double reduction of the dopamine quinone, recovering dopamine in retinal development.

Melatonin receptors are anatomically organized to modulate transmission specifically to cone pathways in the retina of Xenopus laevis

Melatonin receptors have been identified in several retinal cell types, including photoreceptors, horizontal cells, amacrine cells, and ganglion cells. Recent reports suggest that melatonin potentiates signaling from rods to inner retinal neurons. However, the organization of the melatonin receptors mediating this action in the outer plexiform layer (OPL) is not clear. To assess melatonin receptor localization in the OPL, double-label confocal immunohistochemistry for Mel1a or Mel1b melatonin receptors was performed in combination with markers for cone photoreceptors (calbindin, XAP-1) and ON bipolar cells (guanine nucleotide binding protein alpha, Goα) on the retina of Xenopus laevis. Both Mel1a and Mel1b receptors were specifically associated with processes contacting the pedicles of cones, but localized to processes from different sets of second-order neurons. Mel1a receptors localized to the large axonal processes of horizontal cells, while Mel1b receptors localized to the dendrites of OFF bipolar cells. Both receptors also localized to third-order amacrine and ganglion cells and their processes in the inner plexiform layer. This study indicates that Mel1a and Mel1b melatonin receptors are expressed specifically in the Xenopus OPL to modulate transmission from cones to horizontal cells and OFF bipolar cells, respectively; they are second-order neurons that predominantly contact ribbon synapses and display OFF responses to light. When combined with results from recent physiological studies, the current results suggest a conserved function for melatonin in enhancing transmission from rods to second-order neurons across species, although the precise mechanisms by which melatonin enhances this transmission are likely to vary in a species-dependent manner.

Melatonin receptors are anatomically organized to modulate transmission specifically to cone pathways in the retina of Xenopus laevis

AbstractConfocal image of the relative distribution of the Mel1b melatonin receptor (green) and the ON bipolar cell marker guanine nucleotide binding protein alpha, Go α (red) in the retina of Xenopus laevis. Mel1b receptor immunoreactivity is absent from the cell bodies of ON bipolar cells, identifying the Mel1b receptorimmunoreactive bipolar cells as OFF bipolar cells. The Journal of Comparative Neurology, Volume 520, Number 6, pages 1115–1127.

Purification and Identification of G Protein-coupled Receptor Protein Complexes under Native Conditions

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.

Targeted deletions of Mel1a and Mel1b melatonin receptors affect pCREB levels in lactotroph and pars intermedia cells of mice

The actions of the pineal hormone melatonin depend on two types of membrane-bound, G-protein-coupled receptors: the MT1 (Mel1a) and MT2 (Mel1b) melatonin receptors. An important target of melatonin is the hypophysial pars tuberalis that controls the activity of lactotroph cells in the pars distalis (PD). To identify the melatonin receptor type responsible for regulation of the lactotroph cells in pars distalis we studied the levels of Ser133-phosphorylated pCREB in immunocytochemically identified lactotroph cells of wild-type mice (MelAABB) and of mice bearing targeted deletions of the Mel1a receptor (MelaaBB), the Mel1b receptor (MelAAbb) or of both receptor types (Melaabb) at five different time points of a light/dark cycle. Moreover, we analyzed whether pCREB levels in pars intermedia cells also depend on intact melatonin signal transduction cascades. In wild type and MelAAbb mice the percentage of lactotroph cells with nuclear pCREB immunoreactions varied significantly over a 24h period, whereas in MelaaBB and Melaabb mice no significant differences were found between the five time points analyzed. pCREB levels in the pars intermedia did not show rhythmic variation in wild type or Melaabb animals but wild type mice had higher pCREB levels than Melaabb. Our results indicate that Mel1a and Mel1b melatonin receptors are involved in the control of the activity state of lactotroph and pars intermedia cells of mice.

Localization of Mel 1b melatonin receptor-like immunoreactivity in ocular tissues of Xenopus laevis

The circadian signaling molecule, melatonin, is produced by pinealocytes and retinal photoreceptors. In the retina, melatonin is thought to diffuse into the inner retina to act as a paracrine signal of darkness by binding to specific receptors in retinal neurons. The retinal cell locations of the Mel 1a and Mel 1c melatonin receptor types have been reported, but the localization of the Mel 1b receptor, which is the most highly expressed melatonin receptor type in the retina, is unknown. To determine the cellular distribution of Mel 1b melatonin receptor protein in the Xenopus laevis retina and other ocular tissues, polyclonal antibodies were raised against a peptide fragment of the X. laevis Mel 1b receptor. Western blot analysis of several ocular tissues revealed the presence of one or more immunoreactive bands in the sclera, cornea, lens, retinal pigment epithelium (RPE)/choroid, and neural retina. In the neural retina, the major immunoreactive bands displayed electrophoretic mobilities corresponding to approximately 35, 42, 45, and 80 Kd. Sections of X. laevis eyes were analyzed by immunocytochemistry and confocal microscopy, in combination with antibodies against the Mel 1a melatonin receptor, a rod photoreceptor-specific protein, opsin, and two amacrine cell-specific markers, tyrosine hydroxylase (TOH; dopaminergic cells) and glutamic acid decarboxylase (GAD; GABA-ergic cells). Mel 1b immunoreactivity was localized to the apical membranes of RPE cells, and punctate Mel 1b immunoreactivity was observed in both rod and cone photoreceptor inner segments. Presumptive horizontal cells that ramify in the outer plexiform layer (OPL) were immunoreactive for Mel 1b, and were exclusive of the Mel 1a immunoreactivity present in the OPL. Neither TOH nor GAD co-localized with the Mel 1b immunoreactivity that was present in the inner plexiform layer (IPL), suggesting that Mel 1b is not expressed in dopaminergic or GABA-ergic amacrine cells. Mel 1b immunoreactivity was observed in ganglion cells of the retina, a population of cells covering the outer surface of the outer fibrous layer of the sclera, and in lens fibers located in the outer regions of the lens. These results suggest that melatonin may influence retinal function by binding to receptors on RPE and photoreceptor cells, and by acting on neurons of the inner retina that do not use dopamine or GABA as a neurotransmitter. Furthermore, melatonin may bind to receptors on cells located in the sclera and lens, perhaps to modify the growth or function of these ocular tissues.

Clock gene expression in the suprachiasmatic nucleus and the pars tuberalis of mel1b melatonin receptor knockout mice

Clock gene expression in the suprachiasmatic nucleus and the pars tuberalis of mel1b melatonin receptor knockout mice

Targeted disruption of the mouse Mel(1b) melatonin receptor.

Two high-affinity, G protein-coupled melatonin receptor subtypes have been identified in mammals. Targeted disruption of the Mel(1a) melatonin receptor prevents some, but not all, responses to the hormone, suggesting functional redundancy among receptor subtypes (Liu et al., Neuron 19:91-102, 1997). In the present work, the mouse Mel(1b) melatonin receptor cDNA was isolated and characterized, and the gene has been disrupted. The cDNA encodes a receptor with high affinity for melatonin and a pharmacological profile consistent with its assignment as encoding a melatonin receptor. Mice with targeted disruption of the Mel(1b) receptor have no obvious circadian phenotype. Melatonin suppressed multiunit electrical activity in the suprachiasmatic nucleus (SCN) in Mel(1b) receptor-deficient mice as effectively as in wild-type controls. The neuropeptide, pituitary adenylyl cyclase activating peptide, increases the level of phosphorylated cyclic AMP response element binding protein (CREB) in SCN slices, and melatonin reduces this effect. The Mel(1a) receptor subtype mediates this inhibitory response at moderate ligand concentrations (1 nM). A residual response apparent in Mel(1a) receptor-deficient C3H mice at higher melatonin concentrations (100 nM) is absent in Mel(1a)-Mel(1b) double-mutant mice, indicating that the Mel(1b) receptor mediates this effect of melatonin. These data indicate that there is a limited functional redundancy between the receptor subtypes in the SCN. Mice with targeted disruption of melatonin receptor subtypes will allow molecular dissection of other melatonin receptor-mediated responses.

Receptor-mediated modulation of avian caecal muscle contraction by melatonin: role of tyrosine protein kinase.

Abstract: Melatonin receptors in the quail caecum were studied by 2[125I]iodomelatonin binding assay and the involvement of tyrosine protein kinase in the melatonin-induced contraction was explored. The binding of 2[125I]iodomelatonin in the quail caecum membrane preparations was saturable, reversible and of high affinity with an equilibrium dissociation constant (Kd) of 24.6 +/- 1.1 pm (n = 7) and a maximum number of binding sites (Bmax) of 1.95 +/- 0.09 fmol (mg/protein) (n = 7). The relative order of potency of indoles in competing for 2[125I]iodomelatonin binding was: 2-iodomelatonin > melatonin > 2-phenylmelatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin, indicating that ML(1) receptors are involved. The binding was inhibited by Mel1b melatonin receptor antagonists, luzindole and 4-phenyl-2-propionamidotetralin (4-P-PDOT) as well as by non-hydrolyzable analogs of GTP like GTPgammaS and Gpp(NH)p but not by adenosine nucleotides. The latter suggests that the action of melatonin on the caecum is G-protein linked. Cumulative addition of melatonin (1-300 nM) potentiated both the amplitude and frequency of spontaneous contractions in the quail caecum. The potentiation of rhythmic contractions was blocked by both luzindole and 4-P-PDOT. Antagonists of tyrosine kinase, genistein(2 microM) and erbstatin(4 microM) suppressed the modulation of spontaneous contractions by melatonin, but not inhibitors of protein kinase C (PKC) or protein kinase A (PKA). Melatonin-induced increment in spontaneous contraction was blocked by nifedipine (0.4 nM). Thus, we suggest that melatonin potentiates spontaneous contraction in the quail caecum via interacting with G-protein-coupled Mel(1b) receptor which may activate L-type Ca2+ channels by mobilizing tyrosine kinases.

First cloning and functional characterization of a melatonin receptor in fish brain: a novel one?

Melatonin, a neuroendocrine transducer of photoperiod, influences a number of physiological functions and behaviors through specific seven transmembrane domains receptors. We report here the first full-length cloning and functional characterization of a melatonin receptor (P2.6) in a fish, the pike (Teleost). P2.6 encodes a protein that is approximately 80% identical to melatonin receptors previously isolated partially in non-mammals and classified as members of the Mel(1b) subtype; but, it shares only 61% identity with the full-length human Mel(1b) melatonin receptor (hMT2). Expression of P2.6 results in ligand binding characteristics similar to that described for endogenous melatonin receptors. Selective antagonists of the hMT2 (4-phenyl-2-propionamidotetraline and luzindole) were poor competitors of 2-[125I]iodomelatonin binding to the recombinant receptor. In Chinese hamster ovary cells expressing both the cystic fibrosis transmembrane conductance regulator chloride channel and P2.6 receptor, melatonin counteracted the forskolin induced activation of the channel. The results are best explained by a selective inhibition of the adenylyl cyclase. By reverse transcription-polymerase chain reaction, P2.6 mRNA appeared expressed in the optic tectum and, to lesser extent, in the retina and pituitary. In conclusion, these results, together with those of a phylogenetic analysis, suggest that P2.6 might belong to a distinct subtype group within the vertebrate melatonin receptor family.

Functional expression of MT2 (Mel1b) melatonin receptors in human PAZ6 adipocytes.

Several reports have demonstrated that the pineal hormone, melatonin, plays an important role in body mass regulation in mammals. To date, however, the target tissues and relevant biochemical mechanisms involved remain uncharacterized. As adipose tissue is the principal site of energy storage in the body, we investigated whether melatonin could also act on this tissue. Semiquantitative RT-PCR analysis revealed the expression of MT1 and MT2 melatonin receptor mRNAs in the human brown adipose cell line, PAZ6, as well as in human brown and white adipose tissue. Binding analysis with 2-[(125)I]iodomelatonin ((125)I-Mel) revealed the presence of a single, high affinity binding site in PAZ6 adipocytes with a binding capacity of 7.46 +/- 1.58 fmol/mg protein and a K(d) of 457 +/- 5 pM. Both melatonin and the MT2 receptor-selective antagonist, 4-phenyl-2-propionamidotetraline, competed with 2-[(125)I]iodomelatonin binding, with respective K(i) values of 3 x 10(-11) and 1.5 x 10(-11) M. Functional expression of melatonin receptors in PAZ6 adipocytes was indicated by the melatonin-induced, dose-dependent inhibition of forskolin-stimulated cAMP levels and basal cGMP levels with IC(50) values of 2 x 10(-9) and 3 x 10(-10) M, respectively. Modulation of the cGMP pathway by melatonin further supports functional expression of MT2 receptors, as this pathway was shown to be specific for that subtype in humans. In addition, long-term melatonin treatment of PAZ6 adipocytes was found to decrease the expression of the glucose transporter Glut4 and glucose uptake, an important parameter of adipocyte metabolism. These results suggest that melatonin may act directly at MT2 receptors on human brown adipocytes to regulate adipocyte physiology.

Functional Expression of MT2 (Mel1b) Melatonin Receptors in Human PAZ6 Adipocytes

Functional Expression of MT2 (Mel1b) Melatonin Receptors in Human PAZ6 Adipocytes

Melatonin receptor RNA is expressed in photoreceptors and displays a diurnal rhythm in Xenopus retina

Melatonin is an output signal of an endogenous circadian clock of retinal photoreceptors, with highest levels occurring at night. Melatonin synthesized in the retina appears to act as a paracrine signal by binding to specific receptors in the eye. We have previously demonstrated that RNA encoding the Mel 1b and Mel 1c melatonin receptor subtypes is expressed in the Xenopus laevis retina. The goal of this study was to determine the distribution of the Mel 1b and Mel 1c receptor subtype RNA expression in the retina, and to determine if the level of expression of these receptors exhibits a diurnal rhythm. Sections of frog neural retina were analyzed by in situ hybridization with 35S-labeled Xenopus Mel 1c and Mel 1b riboprobes. Hybridization was present in cells of the inner nuclear layer and the ganglion cell layer. Moreover, there was hybridization in the photoreceptors, which has not been previously reported. To test the hypothesis that retinal melatonin receptor mRNA undergoes a diurnal rhythm of expression, total RNA was isolated from frog neural retinas obtained at 3-h intervals during a 24-h period. The total RNA was used in real-time PCR assays to quantify the differences in Mel 1b and Mel 1c receptor mRNA expression at various circadian times. Both the Mel 1b and Mel 1c receptor RNA demonstrated a diurnal rhythm of expression, with peak levels occurring late in the light period, and lowest levels late in the dark period. These results support the hypothesis that RNA encoding melatonin receptors undergo a diurnal rhythm of expression. To further investigate the possible expression of the Mel 1a receptor subtype in Xenopus retina, we generated Mel 1a PCR products in genomic DNA, and in reverse-transcribed neural retina and retinal pigment epithelium (RPE) RNA. The identity of the PCR product was confirmed by sequencing. Therefore, all three known Xenopus melatonin receptor subtypes appear to be expressed in the neural retina and RPE.

Melatonin limits transcriptional impact of phosphoCREB in the mouse SCN via the Mel1a receptor.

In the mouse, activity phase-shifts of the endogenous clock in the suprachiasmatic nucleus (SCN) are associated with phosphorylation of the transcription factor Ca2+/cAMP responsive element binding protein (CREB). CREB phosphorylation is induced by the retino-hypothalamic transmitter pituitary adenylate cyclase-activating polypeptide (PACAP). As detected by immunohistochemistry in SCN slices from wild-type mice, melatonin completely blocked PACAP-stimulated CREB phosphorylation at low concentrations (1 nM). In Mel1a melatonin receptor-deficient mice, the PACAP-induced CREB phosphorylation was inhibited only at melatonin concentrations of 100 nM. This inhibition was, however, blunted by blocking the Mel1b melatonin receptor. Thus, melatonin modulates PACAP-mediated retinal stimuli for clock entrainment primarily via the Mel1a melatonin receptor through molecular interaction within the cAMP-signalling pathway.

Melatonin receptors in human uveal melanocytes and melanoma cells.

Previous work has demonstrated that melatonin inhibits growth of cultured human uveal melanoma cells. The goal of this study was to determine the expression of mRNA encoding the melatonin receptor subtypes and the effect of specific melatonin receptor agonists on cell growth of uveal melanoma cells and melanocytes. RNA expression of the human melatonin Mel1a and Mel1b receptor subtypes was determined by reverse transcription-polymerase chain reaction (RT-PCR) amplification of RNA isolated from two melanoma cell lines and from one cell line of normal melanocytes. PCR-amplified cDNA encoding the Mel1b melatonin receptor subtype, but not the Mel1a subtype, was detected in reverse-transcribed RNA obtained from both normal uveal melanocytes and melanoma cell lines. Uveal melanoma cells and melanocytes were cultured for 24 hr, then melatonin or one of its membrane receptor agonists, 6-chloromelatonin (Mel1a-1b) or S-20098 (Mel1b) or its putative nuclear agonist, CGP-52608 (Mel2), was added to the medium. After 5 days, the cells were detached, counted, and compared to untreated controls. Melatonin and its membrane receptor agonists (Mel1a-1b and Mel1b), but not its putative nuclear receptor agonist (Mel2), inhibited the growth of uveal melanoma cells, but not normal melanocytes, at very low concentrations. In uveal melanoma cells, the expression of RNA encoding the Mel1b receptor suggests that the growth inhibiting effect of melatonin on uveal melanoma cells is related to activation of the melatonin Mel1b membrane receptor. Furthermore, the expression of RNA encoding melatonin receptors in normal uveal melanocytes suggests that melatonin may play a role in the function of these cells.

Differential signaling of human Mel1a and Mel1b melatonin receptors through the cyclic guanosine 3′-5′-monophosphate pathway

Cyclic guanosine 3′-5′-monophosphate (cGMP) has recently been shown to constitute a second messenger for Xenopus laevis melatonin Mel1c receptors. To verify whether cGMP levels are also modulated by mammalian melatonin receptors, we cloned the genes encoding the human Mel1a and Mel1b receptor subtypes and expressed them in human embryonic kidney cells. Pharmacological profiles and inhibition of forskolin-stimulated adenosine 3′-5′-cyclic monophosphate levels by melatonin confirmed functional expression of high-affinity melatonin receptors. Mel1b receptor-transfected cells modulated cGMP levels in a dose-dependent manner via the soluble guanylyl cyclase pathway. In contrast, Mel1a receptors had no effect on cGMP levels. These results demonstrate that mammalian melatonin receptors modulate cGMP levels and reveal for the first time differences in signaling between melatonin receptor subtypes, which may explain the necessity to express different receptor subtypes.

Vasoconstrictor effects of various melatonin analogs on the rat tail artery in the presence of phenylephrine.

We performed a pharmacologic analysis of the increase in perfusion pressure induced by melatonin and related analogues in the perfused rat tail artery precontracted by 1 microM phenylephrine. Melatonin, 2-iodomelatonin, 6-chloromelatonin, and S20098 (N-[2-(7-methoxy-1-naphthyl)ethyl]acetamide) produced a concentration-dependent enhancement of the vasoconstrictor response evoked by 1 microM phenylephrine with a rank order of potency compatible with the pharmacologic profile defined for high-affinity melatonin receptors. Melatonin had no effect on electrically induced [3H]noradrenaline release, but the neurogenic vasoconstriction was increased at melatonin concentrations of 100 and 300 nM. Increasing concentrations of the naphthalenic-based antagonist S20928 (N-[2-(1-naphthyl)ethyl]cyclobutanecarboxamide) caused a parallel rightward shift in the melatonin concentration-response curve without depressing the maximal response. The pA2 value of S20928 was 7.01 +/- 0.08. Luzindole, 1 microM, an antagonist of Mel1b melatonin receptors, was without effect on melatonin-induced responses. By using reverse transcription-polymerase chain reaction (RT-PCR), we found that messenger RNA (mRNA) encoding for Mel1a is transcribed in the rat tail artery. In conclusion, the results show that melatonin produced an enhancement of the contractile response elicited by phenylephrine in the perfused rat tail artery. This vasoconstrictor response appears to be mediated through activation of Mel1a receptors located on smooth-muscle cells. No evidence for an action of melatonin on either the endothelium or sympathetic nerve endings was found.

Melatonin receptor subtype expression in human cerebellum.

We report the distinct cellular distribution in the human cerebellar cortex of the mammalian mt1 (Mel1a) and MT2 (Mel1b) (1) melatonin receptor subtypes. Specific binding of the non-selective radioligand 2-[125I]iodomelatonin to the outer molecular layer was significantly higher than to the granule cell layer. Melatonin receptor subtype expression was assessed by in situ hybridization using selective and specific digoxigenin-labeled antisense oligonucleotide probes. This is the first demonstration of MT2 melatonin receptor mRNA expression in human cerebellar Bergmann glia and astrocytes. On the other hand, the mt1 melatonin receptor mRNA was expressed in both basket-stellate cells and granule cells. We conclude that mt1 and MT2 melatonin receptors are heterogeneously expressed in human cerebellar cortex.