Chromaffin Cell physiology: Novel molecular approaches

描述（由申请人提供）：嗜铬粒蛋白A （CHGA）是一种48 kDa的酸性蛋白，产生生物活性肽，包括一个新的片段（牛CHGA344-364；人CHGA352-372），我们已经发现并称为“catestatin”，它是一种有效的（IC50约200-400 nM）和尼古丁诱导的儿茶酚胺（CA）分泌的特异性抑制剂和激动剂脱敏。我们最近培育了CHGA敲除（CHGA -/-）小鼠，这些小鼠表现出更高的收缩压（SBP）和舒张压（DBF），并且不下沉。我们还鉴定了3种天然存在的人类睾丸素变异（Gly364Ser, Pro370Leu, Arg374Gln）。我们发现Gly364Ser变异引起人类自主神经活动的深刻改变。本研究拟从四个方面探讨Chga-/-小鼠高血压的发生机制、尼古丁对其的调节作用、catestatin变异在灌注大鼠肾上腺中的作用机制以及catestatin变异的产生。目的1 .探讨Chga-/-小鼠高收缩压和舒张压的发生机制以及尼古丁对BP表型的调节作用。通过将含有人类Chga基因（Chga +）的细菌人工染色体（BAG）引入Chga-/-小鼠种系，“挽救”Chga-/-小鼠BP表型升高。目的二世。生成条件Chga-/-产生苯乙醇胺n -甲基转移酶（Chga- pnmt -/-）细胞的小鼠，以确定Chga仅限于肾上腺素能细胞的消融是否足以引起高血压及其相关的尼古丁反应改变。第三目标。测定人catestatin变体（Gly364Ser, Pro370Leu, Arg374Gln）对尼古丁诱导的大鼠肾上腺CA分泌和脱敏的影响。目的四：人CHGA蛋白水解过程对人睾丸素变异的产生。这些研究利用了独特的catestatin基因敲除、转基因和人类变体，很可能在高血压的发展及其通过尼古丁-胆碱能受体的调节中建立新的catestatin机制。

DESCRIPTION (provided by applicant): Chromogranin A (CHGA) is a 48 kDa acidic proprotein giving rise to biologically active peptides including a novel fragment (bovine CHGA344-364; human CHGA352-372) which we have discovered and called "catestatin," that acts as a potent (IC50 about200-400 nM) and specific inhibitor of nicotine-induced catecholamine (CA) secretion and agonist desensitization. We have recently generated CHGA knockout (Chga-/-) mice that displayed higher systolic (SBP) and diastolic blood pressure (DBF), and non-dipping. We also identified 3 naturally occurring human catestatin variants (Gly364Ser, Pro370Leu, Arg374Gln). We found that Gly364Ser variant causes profound alteration in human autonomic activity. This proposal explores the mechanism of development of hypertension in Chga-/- mice, modulation by nicotine, mechanism of action of catestatin variants in su perfused rat adrenal gland and generation of catestatin variants through 4 specific aims: Aim I. Explore the mechanism of development of higher SBP and DBP in Chga-/- mice and nicotinic modulation of BP phenotype. "Rescue" the elevated BP phenotype in Chga-/- mice by introduction of a bacterial artificial chromosome (BAG) containing the human CHGA gene (CHGA +) into the germline of Chga-/- mice. Aim II. Generation of mice with conditional Chga-/- in phenylethanolamine N-methyl transferase (Chga-Pnmt-/-) producing cells, to determine whether ablation of Chga restricted to adrenergic cells is sufficient to cause hypertension and its associated nicotinic response alterations. Aim III. Determine the effects of human catestatin variants (Gly364Ser, Pro370Leu, Arg374Gln) on nicotine-evoked CA secretion from superfused rat adrenal gland and desensitization. Aim IV. Proteolytic processing of human CHGA to generation of human catestatin variants. These studies, utilizing unique knockout, transgenic, and human variants of catestatin, are likely to establish novel catestatin mechanisms in the development of hypertension and its modulation through nicotinic-cholinergic receptors.