Chromaffin Cell physiology: Novel molecular approaches

描述(申请人提供)：嗜铬粒蛋白A(CHGA)是一种48 kDa的酸性蛋白，可产生生物活性多肽，其中包括我们发现的一段新片段(牛CHGA344-364；人CHGA352-372)，称为儿茶素，它对尼古丁诱导的儿茶酚胺(CA)分泌和激动剂脱敏起到有效的(IC50约200-400 NM)和特异性抑制作用。我们最近产生了CHGA基因敲除(CHGA-/-)小鼠，它们表现出更高的收缩压(SBP)和舒张压(DBF)，并且没有下降。我们还鉴定了3种自然存在的儿茶素变异体(Gly364Ser，Pro370Leu，Arg374Gln)。我们发现Gly364Ser变异会导致人类自主神经活动的深刻变化。本研究从4个方面探讨CHGA-/-小鼠高血压的发生机制、尼古丁的调节作用、儿茶素突变体在大鼠肾上腺的作用机制以及儿茶素突变体的产生：目的1.探讨CHGA-/-小鼠SBP和DBP升高的发生机制及烟碱对BP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，“挽救”CHGA-/-小鼠升高的BP表型。目的II.在苯乙醇胺N-甲基转移酶(Chga-Pnmt-/-)产生细胞中建立条件性Chga-/-小鼠，以确定去除仅限于肾上腺素能细胞的Chga是否足以引起高血压及其相关的尼古丁反应改变。目的III.研究人儿茶素突变体(Gly364Ser、Pro370Leu、Arg374Gln)对尼古丁诱导的大鼠肾上腺CA分泌的影响及脱敏作用。目的IV.人CHGA蛋白水解物对人儿茶素突变体的产生。这些研究利用独特的儿茶素基因敲除、转基因和人类变种，可能建立儿茶素在高血压发病中的新机制及其通过烟碱-胆碱能受体的调节。

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.