Melatonin Receptors as Therapeutic Targets in Primates

我们研究的总体目标是了解褪黑素调节神经内分泌、昼夜节律、视觉和心血管功能的机制，并将这些知识应用于新的治疗策略的设计。本研究的目的是确定褪黑激素受体[MT1（以前称为Mel1a）， MT2（以前称为Mel1b）]在昼夜活动的非人灵长类动物的昼夜节律和边缘系统中的分布。此外，我们将验证褪黑激素促进睡眠独立于一天中的时间的假设，并分别通过激活MT1和MT2褪黑激素受体诱导活动、睡眠和温度的昼夜节律的相移。这里描述的实验将在非人类灵长类动物——猕猴身上进行，猕猴是研究睡眠和昼夜节律的良好模型，对褪黑激素促进睡眠的作用很敏感。褪黑素对睡眠参数、运动活动昼夜节律阶段和核心体温的影响将通过活动描记术和无线电遥测术进行评估。我们将首先通过放射性配体2-[125I]碘化褪黑素（MT1/MT2）和3H-4P-PDOT （MT2）的定量受体放射自显影技术，确定MT1和MT2褪黑素受体在视交叉上核和边缘系统区域的分布、亲和力、密度和mRNA表达。mRNA的表达将通过使用地高辛和33P-UTP标记低聚探针进行原位杂交来测定。然后，我们将评估褪黑素促进睡眠和运动活动和核心体温的相移昼夜节律（CT2, CT6, CT10）的昼夜节律敏感性周期。使用特异性但非选择性的褪黑激素受体拮抗剂luzindole阻断褪黑激素受体，使用-肾上腺素受体拮抗剂心得安抑制松果体褪黑激素的产生，我们将评估内源性夜间褪黑激素是否通过褪黑激素受体激活促进睡眠。最后，使用非选择性（MT1/MT2）褪黑激素受体拮抗剂luzindole和选择性MT2拮抗剂4P-PDOT，我们将评估外源性褪黑激素是否通过激活MT1受体介导CT 6和CT 10的睡眠促进作用，以及通过激活MT2褪黑激素受体介导CT 10的阶段推进作用。这些研究的结果将首次证明，激活不同的褪黑激素受体可以调节昼夜活动的物种的睡眠和昼夜节律的相移，这些物种表现出与人类相似的生理和行为反应。这些知识将刺激设计治疗失眠、睡眠和情绪昼夜节律障碍的新治疗策略，并发现受体选择性和治疗有效的褪黑激素受体类似物。

The overall goal of our research is to understand the mechanism(s) by which melatonin regulates neuroendocrine, circadian, visual, and cardiovascular functions and to apply this knowledge to the design of new therapeutic strategies. The goal of this study is to determine the distribution of melatonin receptors [MT1 (formerly Mel1a), MT2 (formerly Mel1b)] in the circadian and limbic system of a diurnal non-human primate. Furthermore, we will test the hypothesis that melatonin promotes sleep independently of time of day and induces phase shifts of circadian rhythms of activity, sleep and temperature through activation of the MT1 and MT2 melatonin receptors, respectively. The experiments described here will be conducted in the diurnal non-human Primate, Macaca mulata which is a well established model for studies of sleep and circadian rhythms and is sensitive to the sleep promoting effects of melatonin. The effects of melatonin on sleep parameters, and circadian phase of motor activity and core body temperature will be assessed by actigraphy and radiotelemetry. We will first determine the distribution, affinity, density and mRNA expression of MT1 and MT2 melatonin receptors in the suprachiasmatic nucleus and areas of the limbic system by quantitative receptor autoradiography with the radioligands 2-[125I] iodomelatonin (MT1/MT2) and 3H-4P-PDOT (MT2). The mRNA expression will be determined by in situ hybridization using digoxigenin and 33P-UTP label oligoprobes. We will then assess the periods of circadian sensitivity for melatonin to promote sleep and to phase shift circadian rhythms (CT2, CT6, CT10) of motor activity and core body temperature. Using, the specific but non-selective melatonin receptor antagonist luzindole to block melatonin receptors, and the beta- adrenoceptor antagonist propranolol to inhibit pineal melatonin production, we will assess whether endogenous nocturnal melatonin promotes sleep by melatonin receptor activation. Finally, using the non selective (MT1/MT2) melatonin receptor antagonist luzindole and the selective MT2 antagonist, 4P-PDOT we will assess whether exogenous melatonin mediates the sleep promoting effect at CT 6 and CT 10 by activation of the MT1 receptor and the phase advance at CT 10 by activation of the MT2 melatonin receptor. Results from these studies will provide, for the first time, proof of the principle that activation of distinct melatonin receptors can regulate sleep and phase shift of circadian rhythms in a diurnal species showing physiological and behavioral responses similar to man. Such knowledge would stimulate the design of novel therapeutic strategies for the treatment of insomnia, circadian disorders of sleep and mood and the discovery of receptor-selective and therapeutically effective melatonin receptor analogues.