Gene structures,biochemical characterization and distribution of rat melatonin receptors

G-protein coupled receptors for the pineal hormone melatonin have been partially cloned from rats. However, insufficient information about their cDNA sequences has hindered studies of their distribution and physiological responses to melatonin using rats as an animal model. We have cloned cDNAs of two rat membrane melatonin receptor subtypes, melatonin receptor 1a (MT1) and melatonin receptor 1b (MT2), using a rapid amplification of cDNA end (RACE) method. The rat MT1 and MT2 cDNAs encode proteins of 353 and 364 amino acids, respectively, and show 78–93% identities with the human and mouse counterparts. Stable expression of either rat MT1 or MT2 in NIH3T3 cells resulted in high affinity 2-[¹²⁵I]-iodomelatonin (¹²⁵I-Mel) binding (K _d = 73.2 ± 9.0 and 73.7 ± 2.9 pM, respectively), and exhibited a similar rank order of inhibition of specific ¹²⁵I-Mel binding by five ligands (2-iodomelatonin > melatonin > 6-hydroxymelatonin > luzindole > N-acetyl-5-hydroxytryptamine). RT-PCR analysis showed that MT1 is highly expressed in the hypothalamus, lung, kidney, adrenal gland, stomach, and ovary, while MT2 is highly expressed in the hippocampus, kidney, and ovary. We also performed multi-cell RT-PCR to examine the expression of mRNAs encoding MT1 and MT2 in adult GnRH neurons. MT1 was weakly expressed in male GnRH neurons, and was less expressed in the female neurons. MT2 expression was undetectable in GnRH neurons from either sex. This study delineates the gene structures, fundamental properties, and distribution of both rat melatonin receptor subtypes, and may offer opportunities to assess the physiological significance of melatonin in rats. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

褪黑素与褪黑素受体在成人甲状腺肿瘤中的表达及其意义

目的:研究甲状腺肿瘤患者褪黑素(MLT)与褪黑素受体(MR)表达的改变及意义。方法:取甲状腺肿瘤(腺瘤及腺癌)组织及腺瘤旁1cm正常组织匀浆及自身外周血,用ELISA方法检测MLT浓度改变,以免疫组化SP法检测MR蛋白改变,RT-PCR半定量分析MR亚型mRNA表达的改变。结果:ELISA方法显示,甲状腺肿瘤组织匀浆上清MLT浓度较自身外周血明显增高;甲状腺腺癌组高于腺瘤组和瘤旁组织组;甲状腺腺瘤组高于瘤旁组织组,但无统计学差异;各组外周血MLT浓度无统计学差异。免疫组化方法显示,成人甲状腺腺癌细胞膜、细胞质和细胞核中MT2亚型蛋白均呈棕褐色强阳性染色( ),较腺瘤及瘤旁正常甲状腺组织染色强度明显增加;mt1受体蛋白亚型表达无差异。RT-PCR结果显示,人甲状腺腺瘤、腺癌及瘤旁正常甲状腺组织均有mt1、MT2受体表达,甲状腺腺瘤组mt1、MT2受体亚型表达量与正常甲状腺组织无差异,甲状腺腺癌组MT2受体亚型表达量较正常甲状腺组织明显增加,mt1受体亚型表达量无差异。结论:成人甲状腺肿瘤组织中MLT浓聚,存在mt1、MT2受体亚型,其中腺癌的细胞膜、细胞质、细胞核内MT2呈高表达。提示MLT与甲状腺肿瘤有一定关联,可能主要通过MT2受体发挥抗甲状腺腺癌作用。     

褪黑素对自身免疫性肝炎的作用研究

目的研究褪黑素（MT）对自身免疫性肝炎大鼠模型外周血CD4^＋细胞、肝细胞特异性脂蛋白（LSP）抗体水平及辅助性淋巴细胞（Th）细胞因子水平的影响，并探讨三者之间的相关性。方法制作自身免疫性肝炎大鼠模型，随机分为模型组、MT注射组及猪促肝细胞生长素（pHGF）注射组，60d后检测各组动物的外周血淋巴细胞亚群浓度、LSP抗体水平及Th细胞因子的浓度，并分析三者之间的关系。结果MT注射组动物CD4^＋细胞百分比、LSP抗体水平、白细胞介素4（IL-4）及白细胞介索6（IL-6）均显著低于模型对照组动物，而白细胞介素2（IL-2）及γ干扰素（IF-γ）均低于模型对照组。结论外周血CD；百分比及IL-4、IL-6、IL-2、INF-γ和LSP抗体浓度之间存在一定的相关性，其变化均与肝组织病变有关。     

褪黑素对自身免疫性肝炎大鼠外周血Th细胞因子水平的影响

目的研究褪黑索（MT）对自身免疫性肝炎大鼠外周血Th细胞因子水平的影响。方法制作大鼠自身免疫性肝炎模型，并随机分为模型对照组、MT注射组和猪促肝细胞生长索（pHGF）注射组。60d后，检测各组动物外周血Th细胞因子的水平。结果（1）血浆Th1细胞因子浓度随低于肝组织病变较轻者，Th2细胞因子则相反；（2）MT注射组动物血浆IL-2为（53．7±12．8）ng／L，显著高于pHGF组[（42．9±9．7）ng／L）]、健康对照组[（48．9±11．2）ng／L]和模型对照组[（34．8±7．6）ng／L]（P〈0．01或P〈0．05）；（3）MT注射组血浆IFN-γ为[（351．2±84．6）ng／L]，显著高于pHGF组[（286．8±51．8）ng／L]、健康对照组[（322．4±67．5）ng／L]和模型对照组[（236．7±33．7）ng／L]（P〈0．01或P〈0．05）；（4）MT注射组血浆IL-4为[（48．0±8．1）ng／L]，显著低于pHGF组[（66．9±14．3）ng／L]、健康对照组[（52．7±12．5）ng／L]和模型对照组[（81．9±16．2）ng／L]（P〈0．01或P〈0．05）；（5）MT注射组血浆IL-6为（252．9 ±45．8）ng／L，显著低于pHGF组[（297．2±48．7）ng／L]和模型对照组[（405．8±45．8）ng／L]（P〈0．01或P〈0．05）。结论MT能刺激自身免疫性肝炎大鼠外周血Th1细胞因子，并抑制Th2细胞因子。     

褪黑素的神经保护作用

人体褪黑素是一种主要在松果体合成的激素，具有广泛的生理活性，主要包括调节生物节律，改善睡眠，以及抗氧化、免疫调节、抑制肿瘤等作用。介绍褪黑素的合成、代谢通路及其抗氧化、抗细胞凋亡等神经保护作用。阐述褪黑素的神经保护作用的临床意义。     

Is melatonin helpful in stopping the long-term use of hypnotics? A discontinuation trial

Objective To find out if administration of melatonin facilitates discontinuation of benzodiazepine (BD) therapy in patients with insomnia. Method A placebo controlled trial in nine general practices in the Netherlands. Long-term users of benzodiazepines were asked by their GP to participate in a discontinuation program in combination with melatonin or placebo. The intervention and follow-up period lasted one year. During this period participants received four questionnaires about their use of sleeping medication and several health instruments. The urine of all participants was tested for the presence of benzodiazepines, as proof of the discontinuation. Main outcome measure The discontinuation of benzodiazepine use measured by questionnaires and urine samples at three assessment points. Results A total of 503 long-term users were selected by the GPs, of whom 38 patients (16M/22F) participated. After one year 40% had stopped their benzodiazepine use, both in the intervention group on melatonin and in the placebo control group. Comparing stoppers and non-stoppers did not reveal significant differences in benzodiazepine use, or awareness of problematic use. Conclusion Our findings do not conclusively indicate that melatonin is helpful for the discontinuation of the use of benzodiazepines, but the average dose of benzodiazepines in the group was low. Further investigation is necessary, with special attention to the possible influence of the daily dose on the facilitation effect of melatonin. Frans H. J. A. Vissers passed away in September 2006     

Effects of melatonin on orofacial movements in rats

RATIONALE: While reserpine-induced oral movements (OM), an animal model of tardive dyskinesia, are more persistent in old than in adult rats, old animals present spontaneous OM, which are phenomenologically similar to those presented by reserpine-treated adult rats. We postulate that these OM may be the result of oxidative stress induced by both age and reserpine treatment.OBJECTIVES: We intended to determine the preventative effects of exogenous melatonin (one of the most important endogenous antioxidants) as well as suppression of endogenous melatonin via continuous exposure to light on reserpine- or age-induced OM in rats.METHODS: Adult (4 months of age) male Wistar rats were repeatedly treated with saline or melatonin (5 mg/kg, IP) and saline or reserpine and kept under a 12-h light/dark cycle for quantification of reserpine-induced OM as well as oxidative stress (via quantification of lipid peroxidation). To verify the effects of endogenous melatonin suppression on reserpine-induced OM, adult rats were repeatedly treated with saline or reserpine and continuously exposed to light. To verify the effects of exogenous melatonin on age-induced OM older (20 months of age) rats were long-term treated with saline or melatonin and kept under a 12-h light/dark cycle.RESULTS: Melatonin attenuated both reserpine- and age-induced OM. Reserpine enhanced striatal lipid peroxidation, that was prevented by melatonin co-administration. Continuous exposure to light increased spontaneous as well as reserpine-induced OM, indicating that endogenous melatonin may be involved in this movement disorder.CONCLUSIONS: We suggested that melatonin attenuates both reserpine- and age-induced OM in rats.     

Modulatory effects of melatonin on behavior, hemolymph metabolites, and neurotransmitter release in crayfish

Melatonin affects a variety of circadian processes such as behavior and neurotransmitter release in vertebrates. Crayfish melatonin production occurs in the eyestalks, and the cycle of production may change seasonally. To date, however, melatonin's roles and mechanisms of action in crustacean physiology are unclear. We injected melatonin or saline into crayfish in scotophase and monitored activity and hemolymph glucose/lactate over 24 h in early spring. Crayfish were significantly more active in photophase versus the expected scotophase, and had concurrent glucose/lactate peaks. Melatonin reversed the activity pattern, causing a scotophase activity peak, but not the glucose/lactate patterns. This study was repeated in late summer, during which control activity and glucose/lactate levels were elevated in scotophase. Melatonin decreased the amplitude of scotophase activity and glucose/lactate, eliminating activity and glucose cycles. We also injected melatonin or saline at various times of day in early summer and monitored locomotor activity for 1 h. Controls had high activity at 1200 (mid-photophase) and 2100 h (early scotophase), and melatonin increased activity at 1200 h but decreased it at 2100 h. Melatonin also increased activity at 1500 h but not 1800 h (late photophase). Next, we examined the influence of melatonin on crayfish neurophysiology. Melatonin (10 microM) enhanced synaptic transmission at the neuromuscular junction (NMJ). The presynaptic action resulted in more vesicles being released during evoked stimulation. Our study indicates that melatonin may have a phylogenetically conserved role in the transduction of circadian information in invertebrates as in vertebrates. Behavioral and physiological effects may be mediated by modulation of central pathways, enhanced at the peripheral level via neuromodulation of the NMJ.     

Neuroprotective action of melatonin on neonatal rat motoneurons after sciatic nerve transection

The neuronal isoform of nitric oxide synthase (nNOS), a NADPH-dependent diaphorase, is considered to play a role in motoneuron death induced by sciatic nerve transection in neonatal rats. Neuronal loss in these circumstances has been correlated with nitric oxide (NO) production and NADPH-diaphorase positivity in motoneurons after axotomy. In the present study we looked for a possible protective effect of melatonin, an antioxidant agent and inhibitor of nNOS, on spinal motoneurons after axonal injury. Neonatal Wistar rats (P2) were submitted to sciatic nerve transection and allowed to survive to P7. Melatonin at doses of 1, 5, 10, 50 and 100 mg/kg was given subcutaneously before and at intervals after the surgery. Controls operated in the same way received dilution vehicle or no treatment. The animals were killed by perfusion of fixative and the spinal cord was examined in serial paraffin sections. The motoneurons of the sciatic pool were counted in the axotomized and contralateral sides. Immunohistochemistry for nNOS and glial fibrillary acidic protein was used to evaluate nNOS expression in the axotomized cells and the astrocytic response. We found that melatonin at doses of 1-50 mg/kg decreased neuronal death. Astrocytic hypertrophy in melatonin treated animals was less intense. There were no differences in nNOS expression between treated and control rats, and surviving motoneurons of the sciatic pool did not express the enzyme, suggesting that nNOS may not be involved in neuronal death or survival in these experimental conditions. Possible mechanisms of melatonin neuroprotection, which was equally effective at doses of 1-50 mg/kg, are discussed. Doses of 50 and 100 mg/kg caused failure to thrive, seizures or death. The fact that neuroprotective doses were far smaller than toxic ones should encourage testing of melatonin in neurologic diseases.     

Interleukin-10 reverses acute detrimental effects of endotoxin-induced inflammation on perinatal cerebral hypoxia-ischemia

Dithiocarbamates, a class of compounds widely used in medicine and agriculture, have been reported to impair sleep structure. These effects have been attributed to the decrease in norepinephrine levels induced by these drugs. However, it has also been recently demonstrated that most of the mechanisms by which dithiocarbamates damage cell function involve changes in oxidative environment. To verify the potential relevance of the latter mechanism in the sleep impairment, we examined the sleep response of adult rats to an acute administration of diethyldithiocarbamate (DDTC). At the dose of 0.6 g/kg, DDTC induced fragmentation and a decrease in slow wave sleep (SWS), and a dramatic loss of paradoxical sleep (PS). These changes occurred soon after the treatment (day 0), persisted the following day (day 1), partially recovered on day 3, and regained near basal values on day 6. No sleep anomalies were observed with a lower dose of DDTC (0.06 mg/kg). On the other hand, when the higher dose of DDTC was given in association with either one of two antioxidants, alpha-tocopherol or melatonin, the amounts of SWS and PS significantly improved even on day 1, suggesting that the DDTC effects on sleep involved an impairment of the brain oxidative balance. Likewise, administration of the lower dose of DDTC 5 days before the higher dose induced a much earlier recovery of normal sleep, presumably due to the development of a tolerance to DDTC. On the whole, the data suggest that the brain oxidative environment may play a role in the mechanisms subserving sleep regulation.