High-affinity Melatonin Binding Sites Research Articles

From the Pineal Gland to the Central Clock in the Brain: Beginning of Studies of the Mammalian Biological Rhythms in the Institute of Physiology of the Czech Academy of Sciences.

The Institute of Physiology of the Czech Academy of Sciences (CAS) has been involved in the field of chronobiology, i.e., in research on temporal regulation of physiological processes, since 1970. The review describes the first 35 years of the research mostly on the effect of light and daylength, i.e., photoperiod, on entrainment or resetting of the pineal rhythm in melatonin production and of intrinsic rhythms in the central biological clock. This clock controls pineal and other circadian rhythms and is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. During the early chronobiological research, many original findings have been reported, e.g. on mechanisms of resetting of the pineal rhythm in melatonin production by short light pulses or by long exposures of animals to light at night, on modulation of the nocturnal melatonin production by the photoperiod or on the presence of high affinity melatonin binding sites in the SCN. The first evidence was given that the photoperiod modulates functional properties of the SCN and hence the SCN not only controls the daily programme of the organism but it may serve also as a calendar measuring the time of a year. During all the years, the chronobiological community has started to talk about "the Czech school of chronobiology". At present, the today´s Laboratory of Biological Rhythms of the Institute of Physiology CAS continues in the chronobiological research and the studies have been extended to the entire circadian timekeeping system in mammals with focus on its ontogenesis, entrainment mechanisms and circadian regulation of physiological functions. Key words: Pineal, Melatonin, AA-NAT rhythm, Light entrainment, Photoperiod, SCN clock.

Evidence of a role for melatonin in fetal sheep physiology: direct actions of melatonin on fetal cerebral artery, brown adipose tissue and adrenal gland

Although the fetal pineal gland does not secrete melatonin, the fetus is exposed to melatonin of maternal origin. In the non-human primate fetus, melatonin acts as a trophic hormone for the adrenal gland, stimulating growth while restraining cortisol production. This latter physiological activity led us to hypothesize that melatonin may influence some fetal functions critical for neonatal adaptation to extrauterine life. To test this hypothesis we explored (i) the presence of G-protein-coupled melatonin binding sites and (ii) the direct modulatory effects of melatonin on noradrenaline (norepinephrine)-induced middle cerebral artery (MCA) contraction, brown adipose tissue (BAT) lypolysis and ACTH-induced adrenal cortisol production in fetal sheep. We found that melatonin directly inhibits the response to noradrenaline in the MCA and BAT, and also inhibits the response to ACTH in the adrenal gland. Melatonin inhibition was reversed by the melatonin antagonist luzindole only in the fetal adrenal. MCA, BAT and adrenal tissue displayed specific high-affinity melatonin binding sites coupled to G-protein (K(d) values: MCA 64 +/- 1 pm, BAT 98.44 +/- 2.12 pm and adrenal 4.123 +/- 3.22 pm). Melatonin binding was displaced by luzindole only in the adrenal gland, supporting the idea that action in the MCA and BAT is mediated by different melatonin receptors. These direct inhibitory responses to melatonin support a role for melatonin in fetal physiology, which we propose prevents major contraction of cerebral vessels, restrains cortisol release and restricts BAT lypolysis during fetal life.

Calreticulin-melatonin. An unexpected relationship.

Increasing evidence suggests that melatonin can exert some effect at nuclear level. Previous experiments using binding techniques clearly showed the existence of specific melatonin binding sites in cell nucleus of rat liver. To further identify these sites, nuclear extracts from rat hepatocytes were treated with different percentages of ammonium sulfate and purified by affinity chromatography. Subsequent ligand blot analysis shows the presence of two polypeptides of approximately 60 and approximately 74 kDa that bind specifically to melatonin. N-Terminal sequence analysis showed that the 60 kDa protein shares a high homology with rat calreticulin, whereas the 74 kDa protein shows no homology with any known protein. The binding of melatonin to calreticulin was further characterized incubating 2-[125I]melatonin with recombinant calreticulin. Binding kinetics show a Kd = 1.08 +/- 0.2 nm and Bmax = 290 +/- 34 fmol.mg protein-1, compatible with other binding sites of melatonin in the cell. The presence of calreticulin was further identified by Western blot analysis, and the lack of endoplasmic reticulum contamination in our material was assessed by Western blot and immunostaining with anti-calnexin Ig. The results suggest that calreticulin may represent a new class of high-affinity melatonin binding sites involved in some functions of the indoleamine including genomic regulation.

Down-regulation of mt 1 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 (K D=83 pM; B max=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt 1 melatonin receptor antibody, we have also detected mt 1 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 × 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 B max=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a B max=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt 1 melatonin receptors in rat ovary were down-regulated (∼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 =5.5 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.

Melatonin receptor genes

Melatonin is produced rhythmically by the pineal gland and the retina with increased synthesis during darkness. Pineal melatonin serves as the ‘chemical expression of darkness’ conveying information on the ambient light-dark cycle into rhythmic bodily functions. On-going debate on modes and sites of action ranges from views of melatonin affecting each and every cell (‘cure-all’) to those of melatonin having restricted actions through specific high-affinity receptors. The present review deals with the latter view. The use of 2-[125I]-iodomelatonin has allowed the exact localization and characterization of high-affinity melatonin receptors that signal through the Gi/o class of G proteins. Molecular cloning of melatonin receptor genes has confirmed that most, if not all, high-affinity melatonin-binding sites represent the G-protein-coupled melatonin receptors. Based on sequence dissimilarities, melatonin receptors are classified into three subtypes, Mella, Mellb and Mel1c. A distribution wider than originally thought of melatonin receptors in the human brain and peripheral sites has brought these receptors into focus of several drug companies, promising exciting times for research on melatonin and new therapeutic possibilities.

Putative melatonin receptors in benign human prostate tissue.

Melatonin, secreted by the pineal gland at night, inhibits pubertal development of rats and presumably men. In addition, it may directly suppress prostate growth in the adult rat. To investigate the possibility for a causal relationship between the age-related decline in melatonin production and increase in prevalence of benign prostate hypertrophy (BPH) in man, the presence of melatonin binding sites in human BPH tissue was examined. In vitro autoradiography indicated specific 125I-labeled melatonin (125I-melatonin) binding in the prostate, localized to the glandular epithelium. Separation and subcellular fractionation indicated that these sites were associated with the microsomal fraction of the epithelial cells. Kinetic and equilibrium 125I-melatonin binding experiments revealed that the binding was time dependent and reversible, with an apparent half saturation at 140 pmol/L. Competition experiments indicated high and low affinity melatonin binding sites; binding was inhibited by melatonin (IC50 1 nmol/L and 1 micromol/L, respectively) and partially by the putative melatonin antagonist, N-(2,4 dinitrophenyl)-5-methoxytryptamine (ML-23; IC50 0.1 nmol/L). Serotonin and 6-hydroxymelatonin were less potent, whereas up to 0.1 mmol/Lol/L of 5-methoxytryptamine, 6-methoxymelatonin, and tryptamine caused only a partial reduction in specific binding. The guanine nucleotide analogs, guanosine 5'-O-[3-thiotriphosphate] and guanosine 5'-O-[2-thio-diphosphate, inhibited specific 125I-melatonin binding, whereas 5'-guanylyl imidodiphosphate was less potent. The results indicate putative melatonin receptors in the human prostate epithelium.

No high affinity melatonin binding sites are detected in murine melanoma cells and in normal human melanocytes cultured in vitro

Saturation studies of 2-(125I)-iodomelatonin binding to membranes from normal human melanocytes, mouse melanoma cells B16F10 and amelanotic S91 revealed no specific binding. Using 2-(125I)-iodomelatonin in the concentration range of 6 to 566 pM, no high affinity melatonin binding sites were detectable in any of the cell types. Even when the concentration of radioligand was increased up to 2000 pM, specific binding was either low or absent and not reproducible. These results suggest that in the culture conditions used in this study, no high affinity melatonin binding sites are detected in pigmented cells.

Daily and circadian regulation of 2-[125I]iodomelatonin binding in the chick brain.

The pineal gland and its hormone melatonin are important in the control of circadian rhythms in birds. Recent research has indicated the presence of high affinity melatonin-binding sites in a wide array of avian cerebral structures. In this study melatonin-binding sites were localized and characterized in the brains of 2-week-old chicks using in vitro autoradiography and image analysis of 2-[125I]iodomelatonin (IMEL) binding. The IMEL binding predominated in the major components of the chick's visual and auditory systems. These brain binding sites showed a daily variation in IMEL binding, with a higher density of binding during the daytime. A significant peak in IMEL binding was observed in the late afternoon, Zeitgeber time 10. This rhythm of IMEL binding continued in constant darkness, with the peak at circadian time 10. The amplitude of the peak was increased in constant darkness in all structures, with the exception of the ectostriatum and neostriatum. Scatchard analysis of the binding revealed an average increase of 45 +/- 5% in the number of binding sites in the daytime vs. nighttime samples, with little change in the binding affinities, indicating that the rhythm in binding is caused by an increase in the total number of binding sites available in the daytime tissue, rather than an increase in the affinity of the binding sites. The data suggest that a rhythm of melatonin sensitivity in a diverse set of cerebral structures may regulate a temporal control of their function.

Daily and circadian regulation of 2-[125I]iodomelatonin binding in the chick brain

The pineal gland and its hormone melatonin are important in the control of circadian rhythms in birds. Recent research has indicated the presence of high affinity melatonin-binding sites in a wide array of avian cerebral structures. In this study melatonin-binding sites were localized and characterized in the brains of 2-week-old chicks using in vitro autoradiography and image analysis of 2-[125I]iodomelatonin (IMEL) binding. The IMEL binding predominated in the major components of the chick's visual and auditory systems. These brain binding sites showed a daily variation in IMEL binding, with a higher density of binding during the daytime. A significant peak in IMEL binding was observed in the late afternoon, Zeitgeber time 10. This rhythm of IMEL binding continued in constant darkness, with the peak at circadian time 10. The amplitude of the peak was increased in constant darkness in all structures, with the exception of the ectostriatum and neostriatum. Scatchard analysis of the binding revealed an average increase of 45 +/- 5% in the number of binding sites in the daytime vs. nighttime samples, with little change in the binding affinities, indicating that the rhythm in binding is caused by an increase in the total number of binding sites available in the daytime tissue, rather than an increase in the affinity of the binding sites. The data suggest that a rhythm of melatonin sensitivity in a diverse set of cerebral structures may regulate a temporal control of their function.

Localization and quantification of high-affinity melatonin binding sites in Rana pipiens retina.

Melatonin binding was localized to the inner plexiform layer (IPL) of the frog retina by in vitro autoradiography, using 2-125I-melatonin as the radioligand. Radioreceptor binding assays of frog retinal homogenate demonstrated saturable melatonin binding. Scatchard analysis revealed a single population of binding sites with an apparent dissociation constant (Kd) of 125 pM, with a Bmax of 0.138 fmoles/mg of protein. These results suggest that high-affinity melatonin binding sites are present in the IPL of the frog retina, which may reflect the presence of melatonin receptors in this synaptic layer.

Distribution of central melatonin binding sites in the goldfish ( Carassius auratus)

In fish, as well as in birds and mammals, the pineal hormone melatonin acts as a neuroendocrine transducer of photoperiod and as such has a profound influence over the timing of seasonal behavior, especially reproduction. With the aim of better understanding how melatonin may exert its influence in the fish, we have studied the distribution and characteristics of the melatonin binding sites in the brain and pituitary of the goldfish ( Carassius auratus) using the ligand [2- 125I]iodomelatonin and a combination of in vitro autoradiography and membrane homogenate receptor assays. Specific binding of the ligand was defined as that displaced by 1 μM melatonin. High-affinity melatonin binding sites were found to have a widespread distribution within the brain of the goldfish and measurements of the relative optical densities on X-ray films using image analysis showed that the highest concentrations were present in the visual system (optic tract, pretectal area, optic tectum), the molecular layer of the cerebellum, and the hypothalamus. No specific binding could be detected in either the pituitary gland or the olfactory bulbs. Displacement of binding by increasing concentrations of melatonin measured by comparative optical density readings on serial sections revealed an ED 50 for melatonin of 10 −9M. No displacement of binding by increasing concentrations of serotonin up to a concentration of 10 −6 M could be detected. Binding was shown to be time dependent with maximal binding occurring after 1 h at room temperature (22°C). Addition of 1 μM melatonin after 2 h resulted in a time-dependent dissociation of ligand binding. Saturation studies carried out on crude membrane homogenates of whole brains gave rise to a straight line Scatchard plot with a K D of 69 p M and a B max of 14 fmol/mg protein. The widespread distribution of high-affinity melatonin binding sites in the brain of the goldfish suggests that melatonin may influence circadian and seasonal behavior in fish via numerous sites involving sensory and neuroendocrine systems but does not have a direct action on the pituitary.

Short days induce changes in specific melatonin binding in hamster median eminence and anterior pituitary

Autoradiography of 125I-melatonin binding to hamster brain sections revealed a competitive 125I-melatonin binding in median eminence only. Saturation studies on crude membrane fraction revealed high-affinity melatonin binding sites in median eminence ( K a = 59 pM) and anterior pituitary ( K d=97 pM). In the hamsters maintained on LD 14:10, the concentration of the binding sites was 10.4 and 6.0 fmol/mg protein in median eminence and anterior pituitary respectively; long-term exposure to LD 8:16 decreased the concentration to less than a half.

Hypothalamic melatonin receptor sites revealed by autoradiography

125I-Melatonin was used to localize and characterize the melatonin receptor sites in the rat hypothalamus. Autoradiography revealed that displaceable 125I-melatonin binding occurred in suprachiasmatic nuclei and median eminence only. Further studies performed on crude membrane fractions from median eminences revealed high affinity ( K d = 21 pM) melatonin binding sites ( B max = 8.5 fmol/mg protein). The order of potency of various indole amines to inhibit 125I-melatonin binding was melatonin ⪢ N-acetyl-5-hydroxytryptamine > 5-methoxytryptamine > 5-hydroxytryptamine.