Chromaffin Cell physiology: Novel molecular approaches

描述嗜铬粒蛋白A（CHGA）是一种48 kDa的酸性前蛋白，产生生物活性肽，包括一种新的片段（牛CHGA 344 -364;人CHGA 352 -372），我们已经发现并称之为“catestatin”，作为尼古丁诱导的儿茶酚胺（CA）分泌和激动剂脱敏的有效（IC 50约200 -400 nM）和特异性抑制剂。我们最近产生了CHGA敲除（Chga-/-）小鼠，其显示出更高的收缩压（SBP）和舒张压（DBF），并且没有下降。我们还鉴定了3种天然存在的人catestatin变体（Gly 364 Ser、Pro370 Leu、Arg 374 Gln）。我们发现Gly 364 Ser变异导致人类自主活动的深刻改变。本研究拟通过以下4个具体目的探讨Chga-/-小鼠高血压发生的机制、尼古丁的调节作用、catestatin变体在超灌注大鼠肾上腺中的作用机制以及catestatin变体的产生：目的I.探讨Chga-/-小鼠高收缩压和高舒张压的发生机制及烟碱对血压表型的调节作用。通过将含有人CHGA基因（CHGA +）的细菌人工染色体（BAG）导入Chga-/-小鼠的种系中，“拯救”Chga-/-小鼠中升高的BP表型。Aim II.在苯乙醇胺N-甲基转移酶（Chga-Pnmt-/-）产生细胞中产生条件性Chga-/-小鼠，以确定仅限于肾上腺素能细胞的Chga消融是否足以引起高血压及其相关的烟碱反应改变。Aim III.测定人catestatin变体（Gly 364 Ser、Pro370 Leu、Arg 374 Gln）对灌流大鼠肾上腺烟碱诱发的CA分泌和脱敏的影响。目标四。人CHGA的蛋白水解加工以产生人catestatin变体。这些研究，利用独特的基因敲除，转基因，和人类的catestatin变体，可能建立新的catestatin机制的发展，高血压和它的调制通过烟碱胆碱能受体。

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.