Melatonin (MLT) exerts its physiological effects principally through two high‐affinity membrane receptors MT1 and MT2. Understanding the exact mechanism of MLT action necessitates the use of highly selective agonists/antagonists to stimulate/inhibit a given MLT receptor. The respective distribution of MT1 and MT2 within the CNS and elsewhere is controversial, and here we used a “knock‐in” strategy replacing MT1 or MT2 coding sequences with a LacZ reporter. The data show striking differences in the distribution of MT1 and MT2 receptors in the mouse brain: whereas the MT1 subtype was expressed in very few structures (notably including the suprachiasmatic nucleus and pars tuberalis), MT2 subtype receptors were identified within numerous brain regions including the olfactory bulb, forebrain, hippocampus, amygdala and superior colliculus. Co‐expression of the two subtypes was observed in very few structures, and even within these areas they were rarely present in the same individual cell. In conclusion, the expression and distribution of MT2 receptors are much more widespread than previously thought, and there is virtually no correspondence between MT1 and MT2 cellular expression. The precise phenotyping of cells/neurons containing MT1 or MT2 receptor subtypes opens new perspectives for the characterization of links between MLT brain targets, MLT actions and specific MLT receptor subtypes.
Mechanisms underlying the daily and photoperiodic variations in mt1 melatonin receptors were investigated in the pars tuberalis (PT) and suprachiasmatic nuclei (SCN) of Siberian and Syrian hamsters. Whatever its daily profile, melatonin receptor density was strongly increased in both structures and species after constant light exposure or pinealectomy, and decreased after a single melatonin injection, indicating melatonin involvement in the daily regulation of the receptor protein. This was confirmed by a strong inverse correlation between melatonin binding capacity and plasma melatonin concentration. In contrast, regulation of mt1 mRNA appeared more complex. The circadian clock, the light/dark cycle and melatonin are all implicated in mt1 gene daily fluctuations, but the extent of their involvement depends upon the structure and the species studied. The photoperiodic decrease in melatonin receptor density observed in short photoperiod (PT of the two hamster species and Syrian hamster SCN) seems to be the consequence of a long-term mt1 gene repression induced by the lengthening of the melatonin peak. Altogether, these results show that during daily variations, mt1 melatonin receptor mRNA and protein are differentially regulated, while at the photoperiodic level, the mt1 protein status depends on mRNA transcription.
Melatonin regulates circadian and seasonal physiology via melatonin receptors expressed in the brain. However, little is known about the signal transduction mechanisms that mediate the action of melatonin in neuronal cells. To begin to address this issue, we expressed the human MT(1) receptor in the human neuroblastoma SH-SY5Y cell line. In this cell line, melatonin acutely stimulated cAMP synthesis through a calcium-calmodulin dependent pathway. This stimulatory effect was independent of an interaction with G(i) or G(s) G proteins and dependent upon internal calcium stores. Melatonin also potentiated forskolin-activated cAMP synthesis. Differentiation of the neuroblastoma cells with retinoic acid to the neuronal phenotype did not alter the ability of melatonin to acutely stimulate cAMP. These data may be relevant to the neuronal action of melatonin and highlight the importance of the cellular context of expression of melatonin and other G protein-coupled receptors.
The pars tuberalis (PT) of the pituitary is a major neuroendocrine target site for melatonin as it contains a large number of high-affinity melatonin receptors. We have previously shown that melatonin autoregulates the density of its own receptors in the PT. However, whether melatonin regulation includes mRNA expression in vivo is unclear. In the present study we have used quantitative in situ hybridization to (1) follow the daily profile of mt1 mRNA expression in the rat PT and (2) investigate whether mt1 mRNA expression could be regulated in vivo by melatonin. We found clear diurnal variations of mt1 mRNA expression that persist in constant darkness. We also showed, on pinealectomized animals, that the rhythmic pineal melatonin secretion is necessary for the expression of these daily variations. In a second step, we studied the effect of an acute suppression of endogenous melatonin synthesis on mt1 melatonin receptors by applying a 1-hour light pulse during the night. We found that light induced a dramatic increase in mt1 mRNA which was totally prevented by a melatonin injection showing that the acute effect of melatonin on the receptor mRNA is strongly inhibitory. A light pulse applied to animals with a chronic absence of melatonin was ineffective showing that light only affects melatonin receptors via the light-induced plasma melatonin suppression. Altogether our results show that melatonin regulates mt1 melatonin receptor mRNA expression. However, this regulation seems to be complex: acute changes in plasma melatonin concentration regulate negatively the gene transcription, even if the daily endogenous nocturnal melatonin peak seems a prerequisite for variations in its receptor expression.
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