BIOLOGY OF ATRIAL NATRIURETIC FACTOR AND ITS RECEPTORS

心房利尿钠因子及其受体的生物学

• 批准号: 2227348
• 负责人: THOMAS M MAACK
• 金额: $ 35.17万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2227348
• 关键词: CHO cells; adenine nucleoside; amiloride; atrial natriuretic peptide; blood pressure; blood volume; crosslink; cyclic GMP; endocytosis; guanylate cyclase; homeostasis; hypertension; isolation perfusion; kidney function; molecular biology; phenylalanine; receptor; site directed mutagenesis; spontaneous hypertensive rat; transfection; tyrosine

The long-term objective of the research is to understand the functions of atrial natriuretic factor (ANF), and of its two main receptors. Studies on the molecular determinants of the dynamics and function of the biological receptor proper of ANF (GCA receptor) will be performed with recombinant wild-type and mutated receptors transfected into 293 and/or CHO cells. To test whether the very rapid dissociation of receptor- ligand complexes is modulated by the interaction of ATP with the kinase- like domain of GCA receptors, mutants will be constructed with: i) truncations of the total intracellular, kinase-like and guanylate cyclase domains; and ii) deletion and substitutions of amino acids in the kinase domain that may determine its interactions with ATP. Cells expressing transfected receptors will be used to determine dissociation of receptor- ligand complexes, generation of cGMP, and influence of substances that retard receptor-ligand dissociation (e.g., amiloride). Isolated membranes from the recombinant cells will be used to assess the effects of addition of adenine nucleotides and amiloride on kinetics of ligand binding and activation of guanylate cyclase. The study on molecular determinants of endocytosis of the clearance (C) receptors of ANF will be performed with recombinant human wild-type and mutated C-ANF receptors transfected into CHO cells. Tyr508, Phe538, and Cys473 will be substituted for alanine by oligonucleotide-directed mutagenesis to test the hypothesis that aromatic amino acids in the cytoplasmic domain and the unpaired cysteine residue in the extracytoplasmic domain are main determinants of C-ANF receptor endocytosis. CHO cells expressing wild- type and mutated receptors will be used to determine receptor-ligand internalization, lysosomal hydrolysis, and receptor internalization and recycling. Chemical cross-linking will be performed to investigate for the presence of surface dimer and monomer forms of wild-type and mutated receptors. The physiological role of ANF will be investigated in the isolated perfused rat kidney preparation and in whole rats using a novel GCA receptor antagonist, named HS-142-1. The postulate will be tested that ANF has a major role in regulating pressure-volume homeostasis, via activation of GCA receptors. The effects of HS-142-1 on dose-response curves of ANF effect on the kidney, and on specific binding of ANF to whole kidney, kidney cortex and medulla will be investigated in the isolated perfused rat kidney. The acute and chronic effects of HS-142-1 on renal function and blood pressure will be determined in normotensive, hypertensive (Goldblatt and SHR rats) and volume expanded (saline infusion, high salt diet) conscious and anesthetized rats. The studies are expected to provide novel insights on ANF receptor function and the role of ANF in health and disease.

这项研究的长期目标是了解其功能 心钠素(ANF)及其两种主要受体。 细胞动力学和功能的分子决定因素研究 ANF的生物受体本身(GCA受体)将与 重组野生型和突变型受体转染293和/或 CHO细胞。为了测试受体的快速解离是否- 配体复合体受三磷酸腺苷与激酶相互作用的调节。 与GCA受体的结构域一样，突变体将被构建为：i) 细胞内总环化酶、蛋白激酶样环化酶和鸟苷环化酶的截短 结构域；以及ii)该激酶中氨基酸的缺失和取代 可决定其与ATP相互作用的区域。细胞表达 转基因受体将用于确定受体的解离- 配体络合物，cGMP的产生，以及 延缓受体-配体解离(例如，阿米洛利)。孤立 重组细胞的膜将被用来评估效果 腺嘌呤核苷酸和阿米洛利对配基动力学的影响 鸟苷环化酶的结合和激活。分子生物学的研究进展 心钠素清除(C)受体内吞作用的决定因素 重组人野生型和突变型C-ANF受体 将其导入CHO细胞。Tyr508、Phe538和Cys473将是 用寡核苷酸定向突变替代丙氨酸进行检测 细胞质结构域中的芳香族氨基酸和 胞质外区未配对的半胱氨酸残基是主要的 C-ANF受体内吞作用的决定因素。表达野生型的CHO细胞 将使用类型和突变的受体来确定受体配体 内化、溶酶体水解和受体内化 回收利用。将进行化学交联以调查 野生型和突变型表面二聚体和单体的存在 感受器。ANF的生理作用将在 分离灌流大鼠肾脏制剂和在整个大鼠中使用一种新的 GCA受体拮抗剂，命名为HS-142-1。这一假设将受到检验 ANF在调节压力-容量动态平衡方面起着重要作用，通过 激活GCA受体。HS-142-1对剂量效应的影响 ANF对肾脏的作用曲线及其与ANF特异性结合的研究 全肾、肾皮质和髓质将在 分离的灌流大鼠肾脏。HS-142-1的急性和慢性效应 对肾功能和血压的影响将在血压正常的情况下进行， 高血压(Goldblatt和SHR大鼠)和容量扩大(生理盐水 输液，高盐饮食)清醒和麻醉大鼠。这些研究 有望为ANF受体的功能和ANF受体的 ANF在健康和疾病中的作用。