Perry Barrett

Melatonin receptors: Localization, molecular pharmacology and physiological significance

A pre-requisite to understanding the physiological mechanisms of action of melatonin is the identification of the target sites where the hormone acts. The radioligand 2-[125I]iodo-melatonin has been used extensively to localize binding sites in both the brain and peripheral tissues. In general these binding sites have been found to be high affinity, with Kd in the low picomolar range, and selective for structural analogues of melatonin. Also the affinity of these sites can generally be modulated by guanine nucleotides, consistent with the notion that they are putative G-protein coupled receptors. However, only a few studies have demonstrated that these putative receptors mediate biochemical and cellular responses. In the pars tuberalis (PT) and pars distalis (PD) of the pituitary, the amphibian melanophore and vertebrate retina, evidence indicates that melatonin acts to inhibit intracellular cyclic AMP through a G-protein coupled mechanism, demonstrating that this is a common signal transduction pathway for many melatonin receptors. However in the pars distalis the inhibition of calcium influx and membrane potential are also important mediators of melatonin effects. How many different forms or states of the melatonin receptor exist is unknown, but clearly the identification of the structure of the melatonin receptor(s) and its ability to interact with different G-proteins and signal transduction pathways are quintessential to our understanding of the physiological mechanisms of action of melatonin. In parallel the recent development of new melatonin analogues will greatly aid our understanding of the pharmacology of the melatonin receptor both in terms of the development of potent melatonin receptor antagonists and for the definition of receptor sub-types. The wide species and phylogenic diversity of melatonin binding sites in the brain has probably generated more questions than answers. Nevertheless the localization of melatonin receptors to the suprachiasmatic nucleus of the hypothalamus is at least consistent with circadian effects within the foetus and the adult. In contrast the PT of the pituitary presents an enigma in relation to the seasonal effects of melatonin. A model of how melatonin might mediate the timing of the circannual events through the PT is proposed.

Digging deep--structure-function relationships in the melatonin receptor family.

Abstract: The melatonin receptor family is a small group of receptors within the G protein‐coupled receptor (GPCR) superfamily. The group comprises of three subtypes which bind melatonin and one member, the melatonin related receptor (MRR), that shares >40% sequence identity with the other melatonin receptors but does not bind melatonin. Identification of two subtypes expressed in the mouse suprachiasmatic nucleus, one of which (MT1) inhibits neuronal firing and the other (MT2) mediating the phase advancing properties of melatonin has given renewed interest to the development of subtype specific compounds for each of the mammalian melatonin receptors. Towards this goal site‐directed and chimaeric receptor mutagenesis studies have been performed which have provided some insight into the structure–function relationships of the melatonin receptors. Furthermore, these studies may lead to the identification of the ligand for the orphan MRR.

Localization of the Melatonin-Related Receptor in the Rodent Brain and Peripheral Tissues

Previous studies have provided a limited examination of the expression of the orphan melatonin‐related receptor in the pituitary and hypothalamus of human and sheep and retinal tissue in the sheep. The present study reports evidence of conservation of expression in regions of the hypothalamus (dorsal medial hypothalamus, lateral hypothalamus, arcuate nucleus), the epithelial layer lining the third ventricle and the paraventricular thalamic nucleus of the mouse, rat and hamster. An extensive and detailed analysis of melatonin‐related receptor mRNA expression in the mouse central nervous system and peripheral tissues is presented. Mapping the distribution throughout the entire mouse brain has revealed new sites of expression in a number of brain nuclei, including preoptic areas, parabrachial nuclei and widespread distribution in the olfactory bulb. Reverse transcriptase‐polymerase chain reaction was performed with RNA isolated from peripheral tissues revealing expression of the melatonin‐related receptor mRNA in the mouse kidney, adrenal gland, intestine, stomach, heart, lung, skin, testis and ovary. These results suggest a conserved function in neuroendocrine regulation and a potential role in coordinating physiological responses in the central nervous system and peripheral tissues.

Photoperiod regulates growth, puberty and hypothalamic neuropeptide and receptor gene expression in female Siberian hamsters.

In seasonal mammals, both the growth and reproductive axes are regulated by photoperiod. Female Siberian hamsters were kept, for up to 12 weeks, in long-day (LD) or short-day (SD) photoperiod, from weaning at 3 weeks of age (Exp 1). LD hamsters had characteristically faster growth and higher asymptotic body weight, adiposity, and leptin gene expression in adipose tissue. Only LD females attained puberty. Gene expression in the hypothalamic arcuate nucleus for leptin receptor (OB-Rb), POMC, and melanocortin 3-receptor (MC3-R) was higher in LD but did not change from weaning levels in SD. In contrast, gene expression in the arcuate nucleus for cocaine and amphetamine-regulated transcript (CART) was higher in SD than LD, a difference that was apparent at 2 weeks post weaning. Transfer of SD females to LD at 15 weeks post weaning (Exp 2) increased body weight, leptin signal, and gene expression for POMC but failed to induce normal puberty onset or to increase gene expression for OB-Rb and MC3-R. Therefore, ph...

The Regulation of Seasonal Changes in Food Intake and Body Weight

Seasonal rhythms of body weight, reflecting altered food intake, energy storage and expenditure, are a common feature of mammals inhabiting temperate and arctic latitudes. They have evolved so that predictable annual changes in the external environment can be anticipated and animals can adjust their physiology and behaviour in preparation for the changing demands of the seasons. These long‐term changes in energy balance are not simply effected by the brain centres and peptidergic pathways known to underlie short‐term homeostatic regulation. Screens of altered gene expression in Siberian hamsters comparing the anabolic summer state in long photoperiods and the catabolic ‘winter’ state in short photoperiods have identified differential gene expression in the hypothalamus. The majority of gene expression changes are confined to two restricted areas: the dorsomedial posterior arcuate nucleus, and the ventral ependymal layer of the third ventricle. Functions encoded by these ‘seasonal’ genes include thyroid hormone metabolism, retinoic acid and histaminergic signalling, and VGF and secretogranin production. The changes in thyroid hormone availability that are brought about by differential activity of deiodinase enzymes are of particular importance because experimental manipulation of central thyroid levels can prevent seasonal cyclicity. Given the importance of thyroid hormone in the initial development of the brain, we hypothesise that thyroid hormone‐dependent plasticity of hypothalamic connections and neurogenesis underlie seasonal cycles of food intake and body weight.

Neuromedin U and Neuromedin U receptor-2 expression in the mouse and rat hypothalamus: effects of nutritional status

Neuromedin U (NMU) has been associated with the regulation of food‐intake and energy balance in rats. The objective of this study was to identify the sites of gene expression for NMU and the NMU receptor‐2 (NMU2R) in the mouse and rat hypothalamus and ascertain the effects of nutritional status on the expression of these genes. In situ hybridization studies revealed that NMU is expressed in several regions of the mouse hypothalamus associated with the regulation of energy balance. Analysis of NMU expression in the obese ob/ob mouse revealed that NMU mRNA levels were elevated in the dorsomedial hypothalamic (DMH) nucleus of obese ob/ob mice compared to lean litter‐mates. In addition, NMU mRNA levels were elevated in the DMH of mice fasted for 24 h relative to ad libitum fed controls. The pattern of expression of NMU and NMU2R were more widespread in the hypothalamus of mice than rats. These data provide the first detailed anatomical analysis of the NMU and NMU2R expression in the mouse and advance our knowledge of expression in the rat. The data from the obese rodent models supports the hypothesis that NMU is involved in the regulation of nutritional status.

Both Pertussis Toxin‐Sensitive and Insensitive G‐Proteins Link Melatonin Receptor to Inhibition of Adenylate Cyclase in the Ovine Pars Tuberalis

Bordetella pertussis toxin (islet activating protein, IAP) has been used to investigate the G‐proteins involved in mediating the action of the melatonin receptor. Melatonin inhibits iorskolin‐stimulated cyclic AMP production in ovine pars tuberalis (PT) cells. In cells treated with IAP for 16 h this response is attenuated in a dose‐dependent manner, but not abolished. IAP catalyses the incorporation of [32 P‐ADP]ribose into a 41 kd protein present in PT membranes, but this labelling can be reduced if PT cells are preincubated with IAP for 16 h. Treatment of crude membrane preparations with IAP (20 /ig/ml) suppresses the binding of 2‐[125 l]iodomelatonin by 20%, whereas 1 mM GTP alone reduces binding by 40%, and in combination with IAP its effect is additive (60% inhibition). Therefore, these results indicate that the melatonin receptor acts via two G‐proteins, one pertussis toxin‐sensitive and the other pertussis toxin‐insensitive.

Cloning and functional analysis of a polymorphic variant of the ovine Mel 1a melatonin receptor.

We have isolated a novel variant of the Mel 1a melatonin receptor from an ovine PT cDNA library. Relative to the reported sequence for the Mel 1a melatonin receptor there are 8 changes in the DNA sequence. Only 3 of these result in amino acid substitutions, one in extracellular loop 3 and two in the carboxy-terminal tail. We have designated the novel variant of the sheep Mel 1a receptor Mel 1a(beta), and correspondingly the previously reported variant Mel 1a(alpha). As minor changes in the primary amino acid sequence of G-protein-coupled receptors can influence their functional characteristics we have accordingly characterized this novel variant of the Mel 1a melatonin receptor. This melatonin receptor displays high affinity binding and inhibits the cAMP second messenger pathway in transfected L-cells demonstrating that this receptor is fully functional. PCR analysis shows Mel 1a(beta) is present in several breeds of sheep and suggests that the Mel 1a(beta) receptor was established early in the evolution of the sheep species.

REGIONAL DISTRIBUTION OF IODOMELATONIN BINDING SITES WITHIN THE SUPRACHIASMATIC NUCLEUS OF THE SYRIAN HAMSTER AND THE SIBERIAN HAMSTER

The pineal hormone melatonin is a potent regulator of seasonal and circadian rhythms in vertebrates. In order to characterize potential target tissues of melatonin, the distribution of iodomelatonin (IMEL)‐binding sites was examined within neurochemically and anatomically defined subdivisions of the suprachiasmatic nucleus (SCN), a structure necessary for seasonal and circadian rhythms in mammals. Studies were carried out in both the adult Syrian (Mesocricetus auratus) and Siberian (Phodopus sungorus) hamster. The retinoreceptive zone of the SCN was identified anatomically by immunocytochemical (ICC) visualization of cholera toxin B subunit tracer (ChTB‐ir) following its intra‐ocular injection. Photically‐responsive SCN cells were identified by immunostaining for the protein product of the immediate‐early gene c‐fos (Fos‐ir) following exposure of the animal to light. The non‐photoresponsive zone of the SCN was identified using in situ hybridization (ISH) for arginine vasopressin (AVP) mRNA, whilst sites of IMEL‐binding in the SCN were identified by in vitro film autoradiography using the specific ligand 2‐[125l]‐iodomelatonin. To compare directly the distribution of IMEL‐binding sites and one of the functional zones of the nucleus, alternate serial coronal sections through the SCN were processed for autoradiography for IMEL and one of the following: ICC for ChTB‐ir or Fos‐ir, or ISH for AVP mRNA. Overall, the regional distribution of the various markers within the SCN was comparable in the two species. The retinorecipient (ChTB‐ir) and photically‐responsive (Fos‐ir) zones of the SCN mapped together to the middle and caudal thirds of the nucleus, predominantly in its ventro‐lateral division. IMEL‐binding was present throughout the full rostro‐caudal extent of the SCN, but by far the most extensive area of IMEL‐binding was in the rostral half of the nucleus, leading to a clear dissociation along the rostro‐caudal axis of the principal zone of IMEL‐binding and the retinorecipient zone of the nucleus. In the Syrian hamster, in coronal sections of the caudal SCN which did contain significant amounts of both IMEL‐binding and Fos‐ir, IMEL‐binding was confined to the medial zone, distinct from the Fos‐ir region of the ventro‐lateral SCN. The segregation was less clear‐cut in the Siberian hamster where the area of IMEL‐binding was more extensive. The dissociation of IMEL‐binding and photically‐responsive cells in the Syrian hamster was confirmed in a series of sagittal sections which were processed alternately for Fos‐ir and IMEL‐binding. Whereas Fos‐ir was confined to the ventro‐lateral SCN, IMEL‐binding was concentrated in the medial zone of the nucleus. In both species, mRNA for AVP was found throughout the rostro‐caudal extent of the SCN, but the peak area was located in the rostral half, and so was segregated from the principal retinorecipient zone. The distribution of mRNA for AVP along the rostro‐caudal and medio‐lateral axes was in direct register with the IMEL‐binding in both species. These studies suggest that melatonin acts upon pathways within the SCN different to those addressed by light, and that it may influence directly the efferent activity of the nucleus, possibly via an effect on vasopressinergic cells.

The Ovine Melatonin-Related Receptor: Cloning and Preliminary Distribution and Binding Studies

A melatonin‐related receptor was cloned from an ovine genomic library. The sequenced gene has a similar structure to that of the melatonin receptor gene family and consists of two exons separated by an intron of approximately 3 kb. Exon 1 and exon 2 of the ovine melatonin‐related receptor encode a protein of 575 amino acids which is 73.8% homologous to the human melatonin‐related receptor and shows 40.9% homology with the ovine Mel1a melatonin receptor. COS‐7 cells transiently expressing ovine melatonin‐related receptors did not bind 2‐[125I]iodomelatonin or 3H‐melatonin. Reverse transcription‐polymerase chain reaction (RT‐PCR) and in situ hybridization studies revealed expression of the ovine melatonin‐related receptor in the hypothalamus, pituitary, retina and retinal pigment epithelium. Furthermore, expression of the ovine melatonin‐related receptor is shown to be coincident with Mel1a and 2‐[125I]iodomelatonin binding in the pituitary and serotonin N‐acetyl transferase (arylalkylamine N‐acetyl transferase, AANAT) expression in the retina. Expression patterns and similarity with the melatonin receptor gene family suggest a role for this novel G protein‐coupled receptor in control and regulation of endocrine function and retinal physiology.