Molecular Dissection of Melatonin Synthesis In Vivo

描述：(申请人提供)褪黑激素是一种夜间荷尔蒙 有节律性地合成和释放松果体 四种酶：色氨酸羟化酶(TPFI)、芳香族氨基酸 脱羧酶(AAADC)、5-羟色胺N-乙酰转移酶(NAT)和 羟基吲哚O甲基转移酶(HIOMT)。尽管目前的证据表明 在大鼠中，cAMP信号调节转录和 褪黑素形成的转录后控制，对此知之甚少 松果体内cAMP的活体靶标。 我们推测cAMP主要通过PKA调节褪黑素的合成。 激活，使参与生物合成的关键蛋白磷酸化。 褪黑素；这些蛋白质的磷酸化导致结合 这些生物合成产物的转录增加和降解减少 酵素。我们计划研究cAMP信号通路在 用完整分子研究完整动物的松果体昼夜节律 和生理学方法。目标1将确立 CAMP依赖的蛋白激酶(PKA)和CREB在转录激活中的作用 体内NAT和褪黑素的形成。我们将确定 体内PKA催化的药物抑制物和激活剂 微透析及其对NAT转录激活和转录的影响 白天和晚上的镇压。我们还将提供重组人 表达A-CREB或固有活性PKA的腺病毒载体 完整的松果体，以检查它们对NAT mRNA和褪黑素产生的影响。 目的2评价FKA在体内NAT蛋白稳定性中的作用。我们会 利用磷酸化NAT特异性抗体研究磷酸化NAT的功能意义 PKA介导的NAT磷酸化；我们将从突变株中鉴定NAT 我们发现大鼠的NAT蛋白水平较低是由于点突变 在PKA磷酸化位点；最后，我们将使用体内病毒载体 缓释微透析法和体内在线微透析法研究其稳定性和功能 NAT突变体。目标3将描述cAMP在新陈代谢中的作用 我们新发现的松果体中5-羟色胺的三相昼夜节律释放 再次利用cAMP的药理和分子操作进行活体研究 信号转导结合体内5-羟色胺的生理测定 节奏。这些实验将进一步加深我们对体内信号的理解 控制转录和转录后转录的转导机制 褪黑素的激活和5-羟色胺的形成，这可能在 睡眠、精神和神经紊乱的发病机制。

DESCRIPTION: (provided by applicant) Melatonin is a nocturnal hormone rhythmically synthesized and released from the pineal gland due to the actions of four enzymes: tryptophan hydroxylase (TPFI), aromatic amino acid decarboxylase (AAADC), serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Although current evidence suggests that in rats cAMP signaling mediates both transcriptional and post-transcriptional control of melatonin formation, little is known about the in vivo targets of cAMP in the pineal. We hypothesize that cAMP regulates melatonin synthesis principally by PKA activation, which phosphorylates key proteins involved in the biosynthesis of melatonin; phosphorylation of these proteins results in a combination of increased transcription and decreased degradation of these biosynthetic enzymes. We plan to investigate the role of the cAMP signaling pathway in pineal circadian rhythms in the intact animal by using an integrated molecular and physiological approach. Aim 1 will establish the importance of the cAMP-dependent protein kinase (PKA) and CREB in transcriptional activation of NAT and melatonin formation in vivo. We will determine the effects of pharmacological inhibitors and activators of PKA catalysis in vivo by microdialysis and examine their influence on NAT transcriptional activation and repression during the day and night. We will also deliver recombinant adenoviral vectors expressing A-CREB or constitutively active PKA into the intact pineals to examine their effects on NAT mRNA and melatonin production. Aim 2 will evaluate the role of FKA in NAT protein stability in vivo. We will utilize a phospho-NAT specific antibody to study the functional significance of PKA-mediated NAT phosphorylation; we will characterize NAT from a mutant strain of rat we discovered that has lower NAT protein level due to a point mutation in a PKA phosphorylation site; lastly, we will use in vivo viral vector delivery and in vivo on-line microdialysis to study the stability and function of NAT mutants. Aim 3 will characterize the role of cAMP in the generation of our newly discovered tri-phasic circadian serotonin release in the pineal in vivo by again utilizing pharmacologic and molecular manipulations of cAMP signaling combined with in vivo physiological measurements of serotonin rhythms. These experiments will further our understanding of the in vivo signal transduction mechanisms that control transcriptional and post-transcriptional activation of melatonin and serotonin formation, which may play a role in the pathogenesis of sleep, psychiatric, and neurological disorders.