Shiu F. Pang

Melatonin and Its Receptors in the Gastrointestinal Tract

Melatonin and its synthesizing enzymes have been demonstrated in the gastrointestinal tract, suggesting the in situ biosynthesis of melatonin by the gut tissues. There appears to be a diurnal rhythm of melatonin in the gastrointestinal tissues of birds and rodents, with high levels in the dark period. Release of gut melatonin into the general circulation, however, was recorded following tryptophan treatment. Melatonin has direct gastrointestinal functions: it decreased serotonin-induced gut concentration, alleviated serotonin-induced reduction in gastric glandular mucosal blood flow, diminished epithelial sodium absorption, and inhibited the proliferation of jejunal epithelium. The use of a radiolabeled melatonin agonist, 2-[125I]iodomelatonin, has allowed the study of putative melatonin receptors in the gut of duck, mouse, chicken and human. The gastrointestinal 2-[125I]iodomelatonin binding to the duck, chicken and human was rapid, stable, saturable, reversible, specific and with a high affinity. The 2-[125I]iodomelatonin binding sites in the mouse gut were of a much lower affinity. In the duck gut, there was a significant variation in the densities of 2-[125I]iodomelatonin binding sites in different regions of the gut, with the following descending order of density: ileum, jejunum > duodenum, colon > cecum > esophagus. Autoradiographical studies have showed that the highest concentration of 2-[125I]iodomelatonin binding sites was in the villi of the small intestine and also in the mucosal layers of the cecum and colon. In contrast to the diurnal rhythms of 2-[125I]iodomelatonin binding sites reported in other tissues, 2-[125I]iodomelatonin binding in the duck gut showed no daily rhythm. The subcellular distribution of binding sites was in the following descending order: nuclear > microsomal > mitochondrial >> cytosolic fraction. These findings are consistent with a paracrine and/or hormonal action of melatonin in the gastrointestinal tract.

2[125I]iodomelatonin binding sites in spleens of guinea pigs

2-[125I]Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8 +/- 4.12 pmol/l and binding site density (Bmax) of 0.69 +/- 0.082 fmol/mg protein at mid-light (n = 10). There was no significant change in the Kd (41.8 +/- 3.16 pmol/l) or the Bmax (0.58 +/- 0.070 fmol/mg protein) at mid-dark (n = 10). Kinetic analysis showed a Kd of 23.13 +/- 4.81 pmol/l (mean +/- SE, n = 4), in agreement to that derived from the saturation studies. The 2-[125I]iodomelatonin binding sites have the following order of potency: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than N-acetylserotonin, 6-hydroxymelatonin greater than 5-methoxytryptamine, 5 methoxytryptophol greater than serotonin, 5-methoxyindole-3-acetic acid greater than 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan greater than tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction (65.5%), the rest are distributed in the microsomal fraction (17.4%), mitochondrial fraction (14.7%) and cytosolic fraction (0.3%). The demonstration of 2-[125I]iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system.

Melatonin-induced inhibition of proliferation and G1/S cell cycle transition delay of human choriocarcinoma JAr cells: Possible involvement of MT2 (MEL1B) receptor

Abstract: Melatonin, the pineal neurohormone, is an evolutionarily conserved photoperiodic signaling molecule with diverse functions that include the entrainment of human circadian rhythms. Although evidence supporting a direct inhibitory action of melatonin on human cancer cell proliferation exists in the literature, the molecular and cellular signaling mechanisms involved are largely undefined. In our study, significant inhibition of human choriocarcinoma JAr cell proliferation at physiological and pharmacological concentrations of melatonin was observed. 2‐Iodomelatonin, a high affinity melatonin receptor agonist, was more potent than melatonin in inhibiting JAr cell proliferation. In addition, the presence of putative melatonin receptors in choriocarcinoma was suggested by the demonstration of specific 2‐[125I]iodomelatonin binding to the tumor. Interestingly, the selective MT2 melatonin receptor ligand, 4‐phenyl‐2‐propionamidotetraline (4‐P‐PDOT), was found to exert not only concentration‐dependent anti‐proliferative actions on JAr cells, but also additive effects with melatonin in inhibiting JAr cell proliferation. Furthermore, MT2 melatonin receptor gene expression by JAr cells was demonstrated by reverse transcription‐polymerase chain reaction (RT‐PCR) and in situ hybridization (ISH). Taken together, our data suggest that the reported anti‐proliferative action of melatonin on human choriocarcinoma JAr cells may be mediated, in part, by MT2 melatonin receptor. Moreover, analysis of melatonin effect on cell cycle kinetics indicated that G1/S transition delay may underlie the observed inhibition of choriocarcinoma cell proliferation by melatonin.

Identification and characterization of melatonin binding sites in the gastrointestinal tract of ducks.

Utilizing 2-[125I]iodomelatonin as the radioligand, melatonin binding sites were identified and characterized in the jejunum of ducks. These sites were found to be reversible, saturable, specific and exhibited high affinity for melatonin. Scatchard analyses have established the equilibrium dissociation constant (Kd) for tissues collected during mid-photophase to be 40.9 +/- 7 pM and the maximum quantity of binding sites (Bmax) to be 2.0 +/- 0.4 fmol/mg protein while Kd of samples collected during mid-scotophase was found to be 54.1 +/- 10 pM with a corresponding Bmax of 1.5 +/- 0.3 fmol/mg protein. These Kd values are in good proximity to the kinetically derived equilibrium dissociation constant of 47.3 +/- 20 pM. No significant difference in Kd or Bmax was detected between the mid-light and mid-dark samples. Pharmacological profile of these binding sites, developed by their interactions with other indoles and compounds, indicated that these binding sites are highly specific for melatonin. Subcellularly, different densities of binding sites were localized to various fractions in the following order: nuclear greater than microsomal greater than mitochondrial greater than cytosolic. These binding sites in the jejunum might be the receptors accountable for promoting paracrine activities for the locally synthesized gastrointestinal melatonin and/or responsible for eliciting hormonal actions via interactions with melatonin of pineal origin.

Potential involvement of mt1 receptor and attenuated sex steroid-induced calcium influx in the direct anti-proliferative action of melatonin on androgen-responsive LNCaP human prostate cancer cells

Melatonin, a pineal secretory product, has been shown to exert a direct anti‐proliferative action on the androgen‐sensitive LNCaP prostate cancer cell line through hitherto undefined mechanisms. In this communication, expression of mt1 melatonin receptor protein in human prostate cancer tissues and LNCaP cells was demonstrated by immunohisto(cyto)chemistry and western blotting, hence supporting the use of LNCaP cell line as a model for the study of melatonin signaling in prostate cancer cell growth. Using H‐thymidine incorporation assay, LNCaP cell proliferation was inhibited by 2‐iodomelatonin, a high‐affinity melatonin receptor agonist. Furthermore, melatonin inhibited H‐thymidine incorporation into LNCaP cells and attenuated 5α‐dihydrotestosterone (DHT) or 17β‐estradiol (E2)‐induced stimulation of LNCaP cell proliferation at physiological and pharmacological concentrations. Similar concentration‐dependent inhibition of sex steroid‐induced stimulation of thymidine incorporation into LNCaP cells by 2‐iodomelatonin was also observed. Interestingly, attenuation of sex steroid‐stimulated calcium influx into LNCaP cells by pharmacological concentrations of melatonin was recorded, whereas 2‐iodomelatonin had no effect on cytosolic calcium changes induced by sex steroids. In addition, proliferative and cytosolic calcium changes were associated with inhibition of total prostate‐specific antigen (PSA) production by LNCaP cells at high physiological and pharmacological concentrations of melatonin. Our data suggest that activated mt1 receptor and attenuated sex steroid‐induced calcium influx are two important mechanisms mediating the direct anti‐proliferative action of melatonin on androgen‐responsive human prostate cancer cells.

Limited Blood‐Brain Barrier Transport of Polyamines

Abstract: Transport of polyamines across the blood‐brain barrier of adult rats was examined by measuring the amount of radioactivity that reached the forebrain 5 s after a “bolus’ intracarotid injection. The values were expressed by the brain uptake index (BUI), which is the percentage of material transported in relation to freely diffusible water in a single passage through the brain. Transport was restricted as indicated by the respective BUI values, presented as means ± SD (number of animals): putrescine, 5.3 ± 0.8 (11); spermidine, 6.1 ± 1.3 (7); and spermine, 5.8 ± 0.5 (4). A kinetic study of the transport of [14C]putrescine showed that transport due to passive diffusion accounted for the majority of the observed influx (66% at 1 mM putrescine). However, a small saturable component exists with a Km value of 4–5 mM and a Vmax of 30 nmol ± min−1± g−1. This Km value is considerably higher than the circulating levels of the polyamine in the normal mature animal, and thus is unlikely to be of physiological significance. Competition studies indicated that putrescine does not interact with carriers for adenosine, arginine, choline, or leucine.

2-[125I]Iodomelatonin Binding Sites in the Testis and Ovary: Putative Melatonin Receptors in the Gonads

Through the synthesis and secretion of the hormone, melatonin, the pineal has been assigned the role of synchronizing a reproductive response to appropriate environmental conditions. Theoretical melatonin target sites may occur at several levels of the hypothalamic-pituitary-gonadal hierarchy, including a direct action on the gonads. The availability of a biologically active radioligand, 2-[125I]iodomelatonin, has provided the opportunity to examine the possible direct melatonin action on the gonads. 2-[125I]Iodomelatonin binding sites were identified in the testes and ovaries of chickens, ducks and quail but were not measurable in mammalian gonads, with the exception of tree shrew testes. The avian gonadal 2-[125I]iodomelatonin binding sites were stable, saturable, reversible, specific and of high affinity. 2-[125I]Iodomelatonin appeared to label a single class of binding sites as evidenced by the linearity of Rosenthal analysis of the specific binding data, the Hill coefficients close to unity and the monophasic competition curves. The high affinity on the gonadal 2-[125I]iodomelatonin binding sites, characterized by apparent equilibrium dissociation constants in the low picomolar range, was in accordance with circulating levels of melatonin suggesting that they may be physiologically relevant. Autoradiography indicated that these 2-[125I]iodomelatonin binding sites were widely distributed throughout the testes but localized in ovarian follicles in the birds studied. Specific inhibition of testicular 2-[125I]iodomelatonin binding by a guanine nucleotide analog has provided evidence that the 2-[125I]iodomelatonin binding sites in chicken testes may be coupled to a guanine nucleotide binding protein-effector system, thus promoting the idea that testicular 2-[125I]iodomelatonin binding sites may mediate a cascade of intracellular events. Although no circadian rhythm in the density or affinity of 2-[125I]iodomelatonin binding in chicken ovaries was found, there was a decrease in 2-[125I]iodomelatonin binding affinity at middark in chicken testes with no change in the number of testicular 2-[125I]iodomelatonin binding sites. The present evidence is in line with the hypothesis of a direct melatonin action on the gonads and further investigations on the above problem will be rewarding.

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-[125I]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-[125I]iodomelatonin binding sites to be mt1 (MEL1A) and MT2 (MEL1B) 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 Gi 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 MT2 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-carbazone, 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 mt1 and MT2 melatonin receptors resulting in the inhibition of cAMP signaling and an increase in epithelial cell proliferation.

Regional concentrations of melatonin in the rat brain in the light and dark period

The levels of melatonin in five brain regions, whole brain, pineal and serum samples were studied in rats adapted under a photoperiod of 12 h light and 12 h dark. It was found that the melatonin levels for all the tissues obtained in the dark period were significantly higher than those obtained in the light period. Regional study of melatonin levels in the brain in the light and dark period demonstrated a high level in the hypothalamus, intermediate levels in the mid-brain, cerebellum and pons-medulla and low level in the telencephalon. Our findings indicate that melatonin in the brain is unevenly distributed and that there are diurnal rhythms of melatonin in all the five brain regions studied.

Pharmacological characterization, molecular subtyping, and autoradiographic localization of putative melatonin receptors in uterine endometrium of estrous rats.

The objective of this study was to determine the biochemical characteristics, subtypes, and localization of melatonin receptors in the rat uterus in estrous stage. Autoradiography with the melatonin ligand, 2-[125I]iodomelatonin, showed that melatonin receptors were localized in the rat uterine endometrium. Binding of 2-[125I]iodomelatonin in crude membrane preparations of rat uterine endometrium in estrous stage was stable, saturable, reversible and of high affinity. Rosenthal analysis yielded an equilibrium dissociation constant (Kd) of 28.9 +/- 3.59 pmol/l (n = 8) and a maximum number of binding sites (Bmax) of 1.6 +/- 0.15 fmol/mg protein (n = 8). The Kd value determined from kinetic analysis was 16.5 +/- 3.02 pmol/l (n = 3). Competition studies using various indoles and neurotransmitters demonstrated that 2-iodomelatonin, melatonin, 6-chloromelatonin, 6-hydroxymelatonin and N-acetylserotonin showed significant inhibition of the 2-[125I]iodomelatonin binding, while the other indole compounds tested had no significant inhibition. The expression of rat uterine endometrial melatonin receptor subtypes was studied by reverse transcription-polymerase chain reaction (RT-PCR) using mt1 and MT2 receptor gene-specific primers. mt1 receptor cDNA was amplified and confirmed by nucleotide sequencing. These findings indicate that mt1 receptors were present in the rat uterine endometrium, and suggest that melatonin plays an integral part in uterine physiology.