Journal of Pineal Research

Melatonin has potent antioxidant, analgesic, and antinociceptive properties. However, the effects of melatonin against oxidative stress-induced cytotoxicity and inflammatory mediators in human chondrocytes remain poorly understood. This study examined the effects and underlying mechanism of melatonin in hydrogen peroxide (H(2) O(2) )-stimulated human chondrocytes and rabbit osteoarthritis (OA) model. Melatonin markedly inhibited hydrogen peroxide (H(2) O(2) )-stimulated cytotoxicity, iNOS, and COX-2 protein and mRNA expression, as well as the downstream products, NO and PGE(2) . Incubation of cells with melatonin decreased H(2) O(2) -induced Sirtuin 1 (SIRT1) mRNA and protein expression. SIRT1 inhibition by sirtinol or Sirt1 siRNA reversed the effects of melatonin on H(2) O(2) -mediated induction of pro-inflammatory cytokines (NO, PGE(2) , TNF-α, IL-1β, and IL-8) and the expression of iNOS, COX-2, and cartilage destruction molecules. Melatonin blocked H(2) O(2) -induced phosphorylation of PI3K/Akt, p38, ERK, JNK, and MAPK, as well as activation of NF-κB, which was reversed by sirtinol and SIRT1 siRNA. In rabbit with OA, intra-articular injection of melatonin significantly reduced cartilage degradation, which was reversed by sirtinol. Taken together, this study shows that melatonin exerts cytoprotective and anti-inflammatory effects in an oxidative stress-stimulated chondrocyte model and rabbit OA model, and that the SIRT1 pathway is strongly involved in this effect.

Anterior-posterior and lateral skull roentgenograms of 1,044 children aged 0-18 yr were examined for pineal gland calcification. Eighty children with pineal calcification were identified. Cranial computed tomograms (CCT) existing for half of the 80 cases provided confirmation. In contrast to existing reports on pineal calcification in the first decade of life, we found a significant percentage of "physiological" calcification even between 0 and 6 yr of age (range 2.9-4.2%). Contrary to current opinion we were not able to detect any signs of pineal gland tumors in these cases. We were able to confirm other reports which note a steep rise of the incidence of pineal calcification during the second decade of life.

We tested the hypothesis that melatonin acts as a powerful hydroxyl radical (*OH) scavenger in vivo in the brain, and interferes with oxidative stress caused by the parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We investigated the effect of melatonin on in vitro *OH production employing a Fenton-like reaction in test tubes, and ex vivo *OH generation in isolated mitochondria induced by 1-methyl-4-phenyl pyridinium (MPP+), as well as on in vivo *OH formation in the mouse striatum following systemic administration of MPTP. We also measured reduced glutathione (GSH) levels, and superoxide dismutase (SOD) activity in the nucleus caudatus putamen (NCP) and substantia nigra (SN), 7 days following MPTP and/or melatonin administration. Melatonin caused a significant and dose-dependent inhibition of the production of *OH in the in vitro, ex vivo and in vivo experimental conditions. Melatonin caused no changes in monoamine oxidase-B activity, in vitro in mitochondrial P2 fractions or in vivo following systemic administration. MPTP treatment in mice caused a significant depletion of GSH, and increased the specific activity of SOD both in SN and NCP on the seventh day. MPTP-induced GSH depletion was dose-dependently blocked in SN and NCP by melatonin. Higher doses of melatonin exhibited a synergistic effect on MPTP-induced increase in the SOD activity in the SN. These results suggest that while GSH inhibition is a direct consequence of *OH generation following neurotoxin administration, the increase in SOD activity is a compensatory mechanism for removing superoxide radicals generated as the result of MPTP. Our results not only point to the potency of melatonin in blocking the primary insults caused by MPTP, but also provide evidence for triggering secondary neuroprotective mechanisms, suggesting its use as a therapeutic agent in neurodegenerative disorders, such as Parkinson's disease.

The effects of melatonin in mammalian cells are exerted via specific receptors or are related to its free radical scavenging activity. It has previously been reported that melatonin inhibits insulin secretion in the pancreatic islets of the rat and in rat insulinoma INS1 cells via Gi-protein-coupled MT1 receptors and the cyclic adenosine 3',5'-monophosphate pathway. However, the inositol-1,4,5-trisphosphate (IP3) pathway is involved in the insulin secretory response as well, and the melatonin signal may play a part in its regulation. This paper addresses the involvement of the second messengers IP3 and intracellular Ca2+ ([Ca2+]i) in the signalling cascade of melatonin in the rat insulinoma INS1 cell, a model for the pancreatic beta-cell. For this purpose melatonin at concentrations ranging from 1 to 100 nmol/L, carbachol and the nonselective melatonin receptor antagonist luzindole were used to stimulate INS1 cell batches, followed by an IP3-mass assay and Ca2+ imaging. Molecular biological studies relating to the mRNA of IP3 receptor (IP3R) subtypes and their relative abundance in INS1 cells showed expression of IP3R-1, IP3R-2 and IP3R-3 mRNA. In conclusion, we found that in rat insulinoma INS1 cells there is a dose-dependent stimulation of IP3 release by melatonin, which is accompanied by a likewise transient increase in [Ca2+]i concentrations. The melatonin effect observed mimics carbachol action. It can be abolished by 30 micromol/L luzindole and is sustained in Ca2+-free medium, suggesting a mechanism that includes the depletion of Ca2+ from intracellular stores.

A recent prospective study indicated that melatonin supplements may reduce the progression of idiopathic scoliosis, the most common deformity of the spine. This form of scoliosis occurs during rapid skeletal growth. To date, however, there is no direct evidence regarding an antiproliferative effect of melatonin at the level of osteoblasts. Herein, we investigated whether melatonin inhibits cell proliferation in a normal human fetal osteoblastic cell line hFOB 1.19. MTT staining showed that at 1 mm concentrations, melatonin significantly inhibited osteoblast proliferation in time-dependent manner. Flow cytometry demonstrated that melatonin significantly increased the fraction of cells in G(0) /G(1) phase of the cell cycle, while simultaneously reducing the proportion in the G(2) /M phase rather than the S phase. Western blot and real-time PCR analyses further confirmed that melatonin's inhibitory effect was possibly because of downregulation of cyclin D1 and CDK4, related to the G(1) phase, and of cyclin B1 and CDK1, related to the G(2) /M phase. There was no downregulation of cyclin E, CDK2, and cyclin A, which are related to G(1) /S transition and S phase. In addition, the trypan blue dye exclusion assay showed that cell viability was not changed by melatonin relative to control cells. These findings provide evidence that melatonin may significantly delay osteoblast proliferation in a time-dependent manner and this inhibition involves the downregulation of cyclin D1 and CDK4, related to the G(1) phase, and of cyclin B1 and CDK1, related to the G(2) /M phase.

Melatonin regulates mitogen-activated protein kinase (MAPK) and Akt signaling pathways. The MAPK family mainly includes extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Our previous study documented that melatonin delays osteoblast proliferation; however, the mechanism of action of melatonin remains unclear. Here, we demonstrate that melatonin significantly inhibited phosphorylation of ERK but not p38, JNK, or Akt in a human osteoblastic cell line 1.19 (hFOB), as measured by western blot. The expression of ERK, p38, JNK, and Akt was not altered. PD98059 (a selective inhibitor of MEK that disrupts downstream activation of ERK) and melatonin alone, and especially in combination, significantly induced an antiproliferative effect, G(1) and G(2) /M phase arrest of the cell cycle, and downregulation of the expression at both the protein and mRNA levels of cyclin D1 and CDK4, related to the G(1) phase, and of cyclin B1 and CDK1, related to the G(2) /M phase, as measured by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method, flow cytometry after propidium iodide staining, and both western blot and real-time PCR, respectively. Moreover, the combination of PD98059 and melatonin synergistically and markedly augmented the action of either agent alone. Coimmunoprecipitation further confirmed that there was an interaction between phosphorylation of ERK and cyclin D1, CDK4, cyclin B1, or CDK1, which was weaken in the presence of melatonin or PD98059. These results suggest that the prevention of ERK activation is involved in melatonin-induced G(1) and G(2) /M phase arrest, and this inhibitory effect is potentially via the ERK, but not p38, JNK, or Akt, pathway.

In the mouse suprachiasmatic nucleus (SCN), melatonin activates MT1 and MT2 G-protein coupled receptors, which are involved primarily in inhibition of neuronal firing and phase shift of circadian rhythms. This study investigated the ability of melatonin to phase shift circadian rhythms in wild type (WT) and MT1 melatonin receptor knockout (KO) C57BL/6 mice. In WT mice, melatonin (90 microg/mouse, s.c.) administered at circadian time 10 (CT10; CT12 onset of activity) significantly phase advanced the onset of the circadian activity rhythm (0.60 +/- 0.09 hr, n = 41) when compared with vehicle treated controls (-0.02 +/- 0.07 hr, n = 28) (P < 0.001). In contrast, C57 MT1KO mice treated with melatonin did not phase shift circadian activity rhythms (-0.10 +/- 0.12 hr, n = 42) when compared with vehicle treated mice (-0.12 +/- 0.07 hr, n = 43). Similarly, in the C57 MT1KO mouse melatonin did not accelerate re-entrainment to a new dark onset after an abrupt advance of the dark cycle. In contrast, melatonin (3 and 10 pm) significantly phase advanced circadian rhythm of neuronal firing in SCN brain slices independent of genotype with an identical maximal shift at 10 pm (C57 WT: 3.61 +/- 0.38 hr, n = 3; C57 MT(1)KO: 3.45 +/- 0.11 hr, n = 4). Taken together, these results suggest that melatonin-mediated phase advances of circadian rhythms of neuronal firing in the SCN in vitro may involve activation of the MT2 receptor while in vivo activation of the MT1 and possibly the MT2 receptor may be necessary for the expression of melatonin-mediated phase shifts of overt circadian activity rhythms.

The role of melatonin in humans still remains unclear. Uncertainties persist about its effects on neurophysiology regarding its levels in human cerebrospinal fluid (CSF), as the bulk of knowledge on this subject mainly derives from studies conducted on animals. In this study, CSF was micro-sampled with a simple, new method from each cerebral ventricle of patients undergoing neuroendoscopy for hydrocephalus. Our purpose was to measure CSF melatonin levels and determine possible differences in its concentration among various significant areas in the cerebral ventricles (e.g. pineal recess, pituitary recess, lateral ventricle, fourth ventricle) and lumbar cistern. From 2002 to 2004, 10 hydrocephalic patients were operated on using a neuroendoscopic technique. The CSF specimens were investigated for melatonin concentrations (free plus protein-bound) after deproteinization; the measurement technique was high-performance liquid chromatography. The preliminary data obtained with this endoscopic micro-sampling technique (applied to humans for the first time) suggest that melatonin is more concentrated within the ventricles and its highest concentration is found in the third ventricle (IIIv), although the difference detected between the CSF of the IIIv and that of the pineal recess was not significant.

Melatonin enters cells and causes cytoskeletal rearrangements in unicellular organisms, plants and vertebrates. This pineal secretory product causes microtubule enlargement and neurite outgrowth by a calmodulin antagonism in N1E-115 cells. Recently, direct in vitro activation of protein kinase C by melatonin was described. Vimentin intermediate filaments are attached to microtubules and their organization depends on both microtubule distribution and phosphorylation of specific proteins. Protein kinase C is a serine threonine kinase which phosphorylates vimentin and through this mechanism causes intermediate filament disassembly. In this work the effects of melatonin on protein kinase C activation, content, and subcellular distribution were studied in N1E-115 cells. Also, melatonin effects on vimentin phosphorylation and subcellular distribution were explored. The results show that melatonin both activates and increases protein kinase C content in the membrane cytoskeletal fraction. Melatonin protein kinase C activation was followed by an increase in both vimentin phosphorylation and by vimentin subcellular redistribution. Moreover, staurosporine, a serine threonine kinase inhibitor, prevented increased vimentin phosphorylation elicited by melatonin. Similar effects to those caused by melatonin were obtained with the protein kinase C activator phorbol 12-myristate 13-acetate. Data support the idea that melatonin modulates vimentin organization through protein kinase C activation.

Melatonin increases neurite formation in N1E-115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) activation. Microfilament rearrangement is also a necessary process in growth cone formation during neurite outgrowth. In this work, we studied the effect of melatonin on microfilament rearrangements present at early stages of neurite formation and the possible participation of PKC and the Rho-associated kinase (ROCK), which is a downstream kinase in the PKC signaling pathway. The results showed that 1 nm melatonin increased both the number of cells with filopodia and with long neurites. Similar results were obtained with the PKC activator phorbol 12-myristate 13-acetate (PMA). Both melatonin and PMA increased the quantity of filamentous actin. In contrast, the PKC inhibitor bisindolylmaleimide abolished microfilament organization elicited by either melatonin or PMA, while the Rho inhibitor C3, or the ROCK inhibitor Y27632, abolished the bipolar neurite morphology of N1E-115 cells. Instead, these inhibitors prompted neurite ramification. ROCK activity measured in whole cell extracts and in N1E-115 cells was increased in the presence of melatonin and PMA. The results indicate that melatonin increases the number of cells with immature neurites and suggest that these neurites can be susceptible to differentiation by incoming extracellular signals. Data also indicate that PKC and ROCK are involved at initial stages of neurite formation in the mechanism by which melatonin recruits cells for later differentiation.

Despite the fact that many physiological and pharmacological actions of melatonin (MEL) have been described, its mechanism of action at the subcellular level remains unclear. It has been suggested that MEL has effects on cellular processes that involve microfilaments and microtubules. In the present study MEL effects on the cytoskel-eton were evaluated in MDCK and N1E-115 cells in which the microfilaments have been shown to participate in cell morphology and dome formation (MDCK) and the microtubules in neurite outgrowths. After one day of culture with 10-11 -10-7 M MEL MDCK cells showed an increase in the number of elongated cells. After four days with the hormone, an increase in the incidence of MDCK cells contacting neighboring cells through long cytoplasmic elongations was observed. Actin antibody stain showed the appearance of thicker fluorescent fibres beneath the cell membrane and over the nucleus in the MEL treated cells. An increase in dome formation in confluent cells was also observed. In N1E-115 cells MEL (10-3-10-5 M) induced an increase in cell with neurite processes. Neurite outgrowth is clearly seen at 24 h after plating. MEL-treated cells grow in clusters with neurites forming intricate networks. Antitubulin antibody stain showed long fluorescent neurites in the N1E-115 MEL-treated cells. A decrease in N1E-115 neurite formation was observed with either serotonin or 6-hydroxymelatonin (6OH-MEL). However, the number of MDCK cells with cytoplasmic elongations was decreased only after 6OH-MEL. We conclude that MEL action at the cellular level involves a modification of the cytoskeletal organization.

The purpose of this study was to examine the influence of exogenously administered melatonin on cataract formation and lipid peroxidation in newborn rats treated with buthionine sulfoximine (BSO), a drug which inhibits the rate-limiting enzyme in glutathione (GSH) synthesis, gamma-glutamylcysteine synthase, thereby depleting animals of their stores of the important intracellular antioxidant, GSH. BSO (3 mmol/kg BW) was given for three consecutive days beginning on postnatal day 2; melatonin (4 mg/kg) was injected daily beginning on postnatal day 2 and continuing until the animals were killed (either day 9 or day 17 after birth). None of the control animals (rats treated with neither BSO nor with melatonin) developed lenticular opacification during the observation period. In the BSO-treated rats, 16 of 18 animals (89%) had observable cataracts when they were examined. In rats that received both BSO and melatonin, the incidence of cataracts was highly significantly decreased, i.e., only 3 of 18 rats (7%) had observable cataracts. In addition to cataracts, the level of lipid peroxidation products (malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA)) was examined in the lens, brain, liver, lung, and kidney of control and experimental animals. In BSO-treated rats, the lens, kidney, and lung exhibited increased levels of MDA plus 4-HDA relative to those measured in the control rats; these increases were reversed in the BSO-treated rats who were injected with melatonin daily. While BSO administration did not increase basal levels of MDA plus 4-HDA in either the brain or liver, melatonin reduced levels of lipid peroxidation products below those measured in the control rats (at 17 days after birth). The changes induced by melatonin are consistent with the free-radical scavenging and antioxidative properties of this indole.

Recio J, Mediavilla MD, Cardinali DP, Sánchez-Barceló EJ. Pharmacological profile and diurnal rhythmicity of 2-[125I]-iodomelatonin binding sites in murine mammary tissue. J. Pineal Res. 1994: 16: 10–17. Recent studies demonstrated that melatonin treatment decreased the growth of mammary glands in pubertal and pregnant mice. In vitro, melatonin inhibited murine mammary gland growth at μM concentrations and increased it at pM concentrations. Melatonin-induced changes of cyclic nucleotide synthesis was also demonstrated in mammary gland slices in vitro. The objective of the present study was to assess the possible existence of specific binding sites for melatonin in murine mammary gland by using 2-[125I]-iodomelatonin as a probe. The specific binding of 2-[125I]-iodomelatonin to murine mammary gland membranes was rapid, saturable, and reversible, showed an affinity in the low nM range, and displayed time, temperature, and pH dependence. Scatchard analysis indicated the existence of a single class of binding sites that exhibited a diurnal rhythmicity in affinity (Kd) and receptor density (Bmax). A maximum in Bmax (267 ± 42 fmol/mg protein) was found at the light period, while affinity was maximal during darkness (Kd= 1.33 ± 0.22 nM). In competition studies dopamine and dopamine-related agents, as well as 6-hydroxymelatonin and serotonin, but not melatonin, effectively displaced 2-[125I]-iodomelatonin from mammary binding sites. The results demonstrated a specific binding of 2-[125I]-iodomelatonin to murine mammary glands, with affinity in the low nM range, and a pharmacological profile that differed from that reported for 2-[125I]-iodomelatonin acceptor sites in other tissues.

The purpose of this study was to identify sites of action of melatonin in the human fetal brain by in vitro autoradiography and in situ hybridization. Specific, guanosine triphosphate (GTP) sensitive, binding of 2-[(125)I]iodomelatonin was localized to the leptomeninges, cerebellum, thalamus, hypothalamus, and brainstem. In the hypothalalmus, specific binding was present in the suprachiasmatic nuclei (SCN) as well as the arcuate, ventromedial and mammillary nuclei. In the brainstem specific binding was present in the cranial nerve nuclei including the oculomotor nuclei, the trochlear nuclei, the motor and sensory trigeminal nuclei, the facial nuclei, and the cochlear nuclei. The localization of MT1 receptor subtype gene expression as determined by in situ hybridization matched the localization of 2-[(125)I]iodomelatonin binding. No MT2 receptor subtype gene expression was detected using this technique. Thus, melatonin may act on the human fetus via the MT1 receptor subtype at a number of discrete brain sites. A major site of action of melatonin in both fetal and adult mammals is the pars tuberalis of the pituitary gland. However, no 2-[(125)I]iodomelatonin binding or melatonin receptor gene expression was detected in the pituitary gland in the present study, indicating that the pituitary, particularly the pars tuberalis, is not a site of action of melatonin in the human fetus.

In-vitro autoradiography was utilized to compare the distribution of 2[125I]iodomelatonin binding sites or putative melatonin receptors in the gastrointestinal tracts of humans, guinea pigs, mice, rats, hamsters, rabbits, ducks, chickens, pigeons, and quail. In humans, binding was detected in the mucosa of the colon, caecum, appendix, and on their blood vessels but not in the ileum. In the other mammals, significant binding was only demonstrated in the mucosa of the rabbit rectum, mouse colon, mouse rectum, and guinea pig ileum. The distribution of 2[125I]iodomelatonin binding in the avian gut varied with species. In the esophagus, binding was present in the lamina propria and blood vessels of all four birds. However, only the lamina propria of the chicken and quail proventriculus and ventriculus showed positive binding. For the duodenum and ileum, binding was very strong in the duck lamina propria, weak in the chicken lamina propria, and absent in the quail. In contrast, the pigeon muscle layer was weakly positive. The most striking species difference was found in the caecum where the duck lamina propria showed very strong binding, while the chicken lamina propria was only weakly positive. Conversely, the caecal muscle layer was strongly positive in chicken and quail but negative in duck and pigeon. In the rectum, a similar but less intense pattern of distribution was observed. The tremendous diversity in the distribution of 2[125I]iodomelatonin binding sites in the gastrointestinal tract is in accord with the hypothesis that melatonin may serve different functions in the gut of different species.

2[125I]Iodomelatonin ([125I]Mel) binding sites were characterized on membrane preparations of young chick hearts. [125I]Mel binding was rapid, saturable, stable, reversible, specific and of picomolar affinity and femtomolar density. Guanosine 5'-O-(3-thiotriphosphate) significantly lowered the binding affinity by one- to twofold, supporting G-protein linkage of melatonin receptors. Binding was detected as early as embryonic day-9 (E9), and increased steadily peaking at E13 before it slowly declined to about 15% of the peak level a week posthatch. Specific [125I]Mel binding was significantly increased by in ovo administration of inotropic agents dopamine and isoproterenol. Melatonin or 2-iodo-N-butanoyl-tryptamine inhibited isoproterenol-stimulated cAMP accumulation in primary heart cell cultures and the effect was attenuated after pretreatment with pertussis toxin (PTX). Localization of melatonin receptors using autoradiography showed intense labeling in the coronary arteries in all age groups whereas those in the myoblasts decreased as the heart matured. While the myoblasts and undifferentiated developing coronary arteries expressed melatonin MT1 receptor subtype in E11 hearts as detected by immunostaining with anti-MT1 receptor serum, immunoreactivities were observed mostly on the endothelium/subendothelium and smooth muscle cells of the well developed coronary vessels in posthatch hearts. Collectively, our data suggest the presence of PTX-sensitive, G protein-coupled melatonin receptors, whose expression is up-regulated by dopamine and isoproterenol, in the chick heart. Activation of these receptors, which include MT1 subtype, may modulate beta-adrenergic receptor-mediated cAMP signaling in the control of chick heart and coronary artery physiology.

Melatonin binding sites were characterized in rat spleen crude membranes. The specific binding of 2-[125I]iodomelatonin by spleen crude membranes fulfills all the criteria for binding to a receptor site. Thus, binding was dependent on time and temperature, stable, specific, and increased under constant light exposure and after pinealectomy. In competition studies, the specific binding of 2-[125I]iodomelatonin to spleen crude membranes was inhibited by increasing concentrations of native melatonin. Scatchard analysis showed that the data were compatible with the existence of two classes of binding sites: a high affinity site with a Kd of 0.53 nM and a binding capacity of 2.52 pM, and a low-affinity site with a Kd of 374 nM and binding capacity of 820 pM. Moreover, binding of 2-[125I]iodomelatonin exhibited day-night variations with the highest binding observed late during the light period, and the lowest binding was observed late at night. However, binding of 2-[125I]iodomelatonin to membranes remained high when animals were kept under light exposure at night. Results support the hypothesis of a regulatory role of melatonin on the immune system in which melatonin downregulates its own binding site.

Using quantitative autoradiography, 2-125I-melatonin binding was investigated throughout the light/dark cycle in the pars tuberalis (PT) of the pituitary of adult Syrian hamsters kept for 8 weeks either in long or short photoperiod (LP or SP, respectively). Melatonin receptor density in the PT displayed photoperiod dependent daily variations (maximal values in LP). Indeed, in LP, melatonin receptor density underwent strong daily variations with maximal values during the first half of the light period and minimal values at the end of the night. These variations depended on changes in the maximal binding (Bmax) without differences in the dissociation constant (Kd). In contrast, PT melatonin receptor density was constant and at a very low level throughout the light:dark cycle in SP exposed animals. Daily PT melatonin receptor density variations of LP exposed animals were abolished by pinealectomy or continuous light exposure. These results show clearly that both at the daily and at the seasonal level the regulation of PT melatonin receptors is strongly dependent on circulating melatonin concentrations in the Syrian hamster, but that other regulatory factors, yet unclarified, might also play a role.

2-[125]Iodomelatonin binding sites in membrane preparations of pigeon spleen have been characterized. The binding was stable, saturable, reversible, and of high affinity. Rosenthal and Hill analyses showed that the radioligand-receptor interaction involved a single class of binding sites. Analysis of the binding results of spleens collected during mid-light revealed an equilibrium dissociation constant (Kd) of 36.6 +/- 4.8 pmol/l (mean +/- sem, n = 10) and a maximum density (Bmax) of 2.3 +/- 0.2 fmol/mg protein. There was no significant difference in the Kd (46.9 +/- 5.0 pmol/l) or the Bmax values (2.4 +/- 0.3 fmol/mg protein) for spleens collected during mid-dark (n = 9), although the mid-dark serum and pineal melatonin levels were significantly higher (P < 0.05) than the corresponding mid-light values. Kinetic analysis showed a Kd of 8.6 +/- 2.0 pmol/l (n +/- 4), in agreement with that derived from the saturation studies. Except for inhibition by 2-iodomelatonin, melatonin, 6-chloromelatonin, 6-hydroxymelatonin and N-acetylserotonin, the other indoles or neurotransmitters tested have little inhibition on the binding. In addition, guanosine 5'-O-(3-thiophosphate) (GTP gamma S), a nonhydrolysable analog of GTP, was found to inhibit the binding in a dose-dependent manner. Saturation studies revealed that this is due to a decrease in both the affinity and density of the binding sites. These data suggest that a single type of melatonin receptor is found in the pigeon spleen and that the site is coupled to a guinine nucleotide binding protein (G-protein). Our findings support a direct pineal melatonin action on the immune system.

The [125I]-iodomelatonin binding sites in chicken brain membrane preparations were studied. The binding of [125I]-iodomelatonin to the membrane preparations of chicken brain was rapid, stable, saturable, and reversible. The order of pharmacological affinities of [125I]-iodomelatonin binding sites in the chicken brain membrane preparations was: melatonin greater than 6-chloromelatonin greater than N-acetylserotonin greater than 5-hydroxytryptamine greater than tryptamine greater than 5-methoxytryptophol, much greater than 1-acetylindole-3-carboxaldehyde, 5-hydroxyindole-3-acetic acid, L-tryptophan, 5-hydroxytryptophan, 3-acetylindole. Compounds known to act on the receptor of norepinephrine or acetylcholine were inactive as compared to melatonin. Among the various brain regions studied, melatonin binding had maximal level in the hypothalamus, intermediate levels in the mid-brain, ponsmedulla, and telencephalon, and minimum level in the cerebellum. Subcellular fraction studies indicated that 40% of the binding was located in the mitochondrial fraction, 27% in the nuclear, 26% in the microsomal, and 6% in the cytosol fraction. Scatchard analysis of the membrane preparations revealed a dissociation constant (Kd) of 199.6 +/- 17 pM and a total number of binding sites (Bmax) of 16.6 +/- 0.75 fmol/mg protein at midlight. Thus, our results showed the presence of specific melatonin binding sites in the chicken brain membrane preparations. Saturation studies demonstrated that [125I]-iodomelatonin binding capacity in chicken brain membrane preparations were 40% greater at midlight (16.6 +/- 0.75 fmol/mg protein) than at middark (10.6 +/- 0.56 fmol/mg protein), with no significant variation in their binding affinities.

The binding sites for 2-[125I]iodomelatonin in chicken spleens were characterized. The binding was rapid, stable, saturable, reversible, and of high affinity. Both melatonin and 6-chloromelatonin strongly inhibited the binding. The dissociation constant (Kd) obtained from the Scatchard analysis was 31.4 +/- 5.19 pmol/l (3-weeks old, n = 4), which was in good agreement with the Kd (50.6 pmol/l) calculated from the kinetic study. The maximum number of binding sites (Bmax) was 1.09 +/- 0.11 fmol/mg protein (3-weeks old, n = 4). Twelve 11-week-old chicks were killed in two groups at mid-light or mid-dark. Saturation studies indicated no significant difference (P greater than 0.05) in the Kd between mid-light (42.1 +/- 3.9 pmol/l) and mid-dark (31.6 +/- 4.9 pmol/l). The maximum number of binding sites (Bmax) at mid-light and mid-dark were 1.52 +/- 0.16 and 1.35 +/- 0.08 fmol/mg protein, respectively, with no significant variation (P greater than 0.05) recorded. However, when the whole spleen was taken into consideration, the Bmax per spleen protein of the mid-light samples (253 +/- 36 fmol/spleen protein) was significantly greater than that (129 +/- 16 fmol/spleen protein) of the mid-dark samples (P less than 0.05). This indicated that in our study a diurnal rhythm of the total number of 2-[125I]iodomelatonin binding sites might exist in the chicken spleen.

The present study analyzes the effect of temperature-dependent modifications on the binding of the analog 2-[125I]-melatonin to melatonin receptors in isolated neural retina membranes from the greenfrog Rana perezi. Association and dissociation rate constants (K+1, K-1) were exponentially increased by the assay temperature. At 15 degrees C, association and dissociation required several hours; meanwhile, at 35 degrees C, rate constants were 100- and 34-fold faster, respectively. However, the Kd constant calculated as K-1/K+1 was unmodified by the assay temperature. When frogs were acclimated at either 5 or 22 degrees C for 1 month, K+1, and K-1 constants determined at 15 and 25 degrees C were identical in both cold- and warm-acclimated groups. Thus, the binding kinetics of melatonin receptors in frog retinas did not shown any thermal compensation. Results from saturation curves and pharmacological profiles of melatonin binding sites support a lack of effect of assay temperature on the affinity of melatonin receptors in the frog retina. The inhibition of [125I]Mel binding by GTPgammaS showed clearly that the coupling of melatonin receptors to G proteins is temperature-dependent. Higher concentrations of the GTP analog were needed to inhibit specific binding when temperature decreased. The temperature effect on binding kinetics and on the G protein coupling to melatonin receptors suggests that the melatonin signal could be transduced distinctly depending on the temperature. Thus, temperature plays a major role, not only on melatonin synthesis, but also in the transduction of melatonin signal in ectotherms.

Using 2[125I]iodomelatonin as the radioligand, we characterized 2[125I]iodomelatonin binding sites in guinea pig platelet membrane preparations. Saturation radioreceptor studies indicated that these 2[125I]iodomelatonin binding sites were of picomolar affinity and femtomolar density. The dissociation constant (Kd) and maximum number of receptor sites (Bmax) were 42.5 +/- 1.79 pM and 11.8 +/- 0.8 fmol/mg protein (n = 6), respectively. 2[125I]Iodomelatonin competition studies with indoles or drugs indicate the following rank order of potency: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-methoxytryptophol, whereas serotonin and its analogs had less than 20% inhibition at 0.1 mM. Guanosine 5'-O-(3-thiotriphosphate) significantly increased the Kd by twofold suggesting that these binding sites are coupled to the guanine nucleotide binding proteins. Immunoblotting studies using anti-MT(1) IgG demonstrated one peptide blockable band with an apparent molecular mass of 37 kDa. Melatonin had no effect on prostacyclin or forskolin-stimulated intracellular 3',5'-cyclic adenosine monophosphate accumulation. A diurnal variation in binding density, which was abolished after the animals were adapted to constant light conditions, was observed. Age related studies demonstrated that Bmax increased as the animal matured. Physiological melatonin concentrations potentiated whereas those at pharmacological levels inhibited adenosine diphosphate- or arachidonic acid-stimulated platelet aggregation. Our study demonstrated G-protein coupled, saturable, reversible and highly specific picomolar affinity 2[125I]iodomelatonin binding sites in guinea pig platelets. Pharmocological and physiological data indicate that they may be different from the nanomolar [3H]melatonin binding sites in human platelets previously reported.

Pang CS, Tang PL, Song Y, Pang SF, Ng KW, Guardiola‐Lemaitre B, Delagrange P, Brown G.M. Differential inhibitory effects of melatonin analogs and three naphthalenic ligands on 2‐[ l25 I]iodomelatonin binding to chicken tissues. J. Peneal Res. 1997; 23:148–155. © Munksgaard, Copenhagen Abstract We have compared the 50% inhibition values of 2‐[ l25 I]iodomelatonin ([ ¹²⁵ I]Mel) competition curves by melatonin and 3 naphthalenic ligands, N‐[2‐(7‐methoxy‐l‐naphthyl) ethyl] cyclobutane carboxamide (S20642), N‐propyl N‐[2‐(7‐methoxy‐l‐naphtyl) ethyl] urea (S20753), and N‐[2‐(7‐methoxy‐l‐naphthyl) ethyl] crotonamide (S20750), using membrane preparations of four tissues (lung, spleen, brain, and kidney) of the chicken simultaneously. In retired breeders, we have demonstrated that the affinities of S20642 were similar in the lung and spleen. However they were 2‐fold lower in the brain and 80‐fold lower in the kidney. Similar differential binding affinities to the melatonin receptors were observed in the four tissues of young male chicks. This suggests that age and sex have little influence on the differential inhibitory properties of melatonin and S20642 in the tissues studied. The addition of guanosine 5'‐0‐thiotriphosphate (GTPyS), which encouraged the uncoupling of melatonin receptor to the G protein complex, lowered the binding affinity of melatonin and S20642 in the tissues studied but their differentia] affinities in the four tissues were however maintained. The affinities of 5‐methoxy‐N‐cyclopropanoyltryptamine (CPMT) in the kidney were also 5–10‐fold lower than those in the lung, spleen, and brain of young male chicks. The distinctive differential affinities of melatonin, S20642, and CPMT for [ l25 I]Mel binding sites in the chicken lung, spleen, brain, and kidney indicated that the binding sites in these tissues are heterogeneous. Our study implicated that the naphthalenic ligand S20642 may be a useful melatonin analog to distinguish melatonin receptor subtypes in tissues and a possible drug candidate worthwhile for further investigations.

Siberian hamsters undergo reproductive quiescence during exposure to a short day photoperiod, but this response appears to diminish with age. This study investigated whether age-related losses in photoperiodic responsiveness may be related to decreases in specific 2-[125I]-iodomelatonin binding sites in the suprachiasmatic nuclei or pars tuberalis. Adult male Siberian hamsters (group 1: 3-6 months of age and group 2: 9-12 months of age) were exposed to short photoperiod (10 hr of light/day) for 10 weeks. Profound testicular regression was evident in the majority of the hamsters in group 1, but in only a few of the hamsters in group 2. There were no significant differences between the groups in the density or affinity of the specific 2-[125I]-iodomelatonin binding sites in either the suprachiasmatic nuclei or the pars tuberalis. These findings suggest that the failure of older hamsters to respond to short photoperiod is not caused by a loss of specific 2-[125I]-iodomelatonin binding sites in the suprachiasmatic nuclei or pars tuberalis.