REGULATION OF PNMT ACTIVITY BY ALTERNATIVE MRNA SPLICING

描述：本研究的长期目标是确定分子 控制组织特异性表达的机制 苯乙醇胺甲基转移酶（PNMT）。PNMT与健康相关 重要性，因为它是将去甲肾上腺素转化为 神经递质肾上腺素我们最近发现，在体内， 脑干，以及在体外培养的PC 12细胞，两种 PNMT mRNA是通过一种称为“选择性剪接”的机制产生的。 内含子保留我们假设这种剪接机制， 在哺乳动物中遇到的，形成了组织特异性调节的基础， PNMT在各种肾上腺素能组织中的表达。在脑干， 剪接的无内含子信息的表达在表达后被下调， 而内含子限制的mRNA种类是组成性的， 表达。激素控制这种物种的反应是由我们的研究表明， 初步数据，如PC 12细胞体外暴露于合成的 糖皮质激素（GC），地塞米松，诱导剪接变异体，而在 对照细胞，或在暴露于神经生长因子（NGF）时，仅 观察到内含子保留的变体。剪接变体的出现 伴随着PNMT活性的诱导。在肾上腺，主要器官 对于PNMT活性和肾上腺素合成，只有香料变体是 很明显。我们建议研究PNMT的生理重要性 脑干、肾上腺和上级颈神经节mRNA剪接 RT-PCR。所有这些组织都以发育调节的方式表达PNMT。 方式我们将通过以下方法生理性调节胚胎PNMT活性： 给怀孕的动物以及它们的后代注射 地塞米松或糖皮质激素受体（GCR）的特异性抑制剂。 mRNA剪接变体的相对水平将与 功能的测量，即，通过放射酶测定的酶活性， 蛋白质印迹法检测PNMT蛋白表达，免疫印迹法检测儿茶酚胺表达 高效液相色谱法在体外，GC和GCR在该剪接反应中的作用将被研究。 采用PC 12细胞、纯化的嗜铬细胞和宫颈癌细胞系， 神经节，在有或没有地塞米松的情况下培养， 没有GCR。PNMT mRNA剪接变体的相对水平将被 与PNMT功能相关。了解分子基础， PNMT功能的差异调节。了解分子 在大脑中PNMT活性的差异调节的基础， 肾上腺可能揭示治疗干预的可能靶点， 肾上腺素分泌紊乱的病态

DESCRIPTION: The long-term goal of this study is to identify molecular mechanisms controlling the tissue-specific expression of phenylethanolamine methyl transferase (PNMT). PNMT has health-related importance, as it is the enzyme that converts noradrenaline to the neurotransmitter adrenaline. We recently discovered that both in vivo in the brainstem, as well as in vitro in cultured PC12 cells, two species of PNMT mRNA are produced by an alternative splicing mechanism known as intron retention. We hypothesize that this splicing mechanism, rarely encountered in mammals, forms the basis for tissue-specific regulation of PNMT expression in various adrenergic tissues. In brainstem, the expression of a spliced intron-less message is down-regulated post- natally, while an intron-retrained mRNA species is constitutively expressed. Hormonal control of this species reaction is indicated by our preliminary data, as in vitro exposure of PC12 cells to the synthetic glucocorticoid (GC), dexamethasone, induce the spliced variant while in control cells, or upon exposure to nerve growth factor (NGF), only the intron-retained variant is observed. The appearance of the spliced variant is accompanied by induction of PNMT activity. In adrenal, the main organ for PNMT activity and epinephrine synthesis, only the spiced variant is evident. We propose to investigate the physiological importance of PNMT mRNA splicing in brainstem, adrenal and superior cervical ganglia using the RT-PCR. All these tissues express PNMT in a developmentally-regulated manner. We will physiologically modulate embryonic PNMT activity by injecting both pregnant animals, as well as their offspring, with dexamethasone or a specific inhibitor of glucocorticoid receptors (GCRs). The relative levels of mRNA spliced variants will be correlated with measurements of function, viz., enzyme activity by radio-enzyme assay, expression of PNMT protein by Western blotting, and catecholamines by HPLC. In vitro, the role of GCs and GCRs in this splicing reaction will be investigated using PC12 cells, purified chromaffin cells and cervical ganglia, cultured with or without dexamethasone and in the presence or absence of GCRs. Relative levels of PNMT mRNA spliced variants will be correlated with PNMT function. Understanding the molecular basis for the differential regulation of PNMT function. Understanding the molecular basis for the differential regulation of PNMT activity in the brain and adrenal may reveal possible targets for therapeutic intervention in diseased states with perturbed adrenaline production.