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: 2906157
• 负责人: THOMAS M MAACK
• 金额: $ 27.03万
• 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 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2906157
• 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.

研究的长期目标是了解 心钠素（ANF）及其两种主要受体。 分子决定因素的研究 ANF的生物受体（GCA受体）将与 转染到293和/或 CHO细胞。 为了测试受体的快速解离- 配体复合物通过ATP与激酶的相互作用来调节， 类似于GCA受体的结构域，突变体将用以下构建：i） 总细胞内激酶样和鸟苷酸环化酶的截短 ii）激酶中氨基酸的缺失和取代 这可能决定了它与ATP的相互作用。 细胞表达 转染的受体将用于测定受体的解离， 配体复合物，cGMP的产生，以及 延迟受体-配体解离（例如，阿米洛利）。 分离 将使用重组细胞的膜来评估 腺嘌呤核苷酸和阿米洛利对配体动力学的影响 鸟苷酸环化酶的结合和活化。 分子生物学研究 ANF的清除（C）受体的内吞作用的决定因素将 用重组人野生型和突变的C-ANF受体进行 转染到CHO细胞中。 Tyr 508、Phe 538和Cys 473将被 用丙氨酸取代丙氨酸， 假设芳香族氨基酸在细胞质结构域和 胞质外结构域中的未配对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- 型和突变的受体将用于确定受体-配体 内化、溶酶体水解和受体内化， 回收利用。 将进行化学交联以研究 野生型和突变型表面二聚体和单体形式的存在 受体。 ANF的生理作用将在本研究中进行研究。 分离的灌流大鼠肾脏制备物和在整个大鼠中使用新的 GCA受体拮抗剂，命名为HS-142-1。 假设将被检验 ANF在调节压力-容量稳态中具有重要作用， GCA受体的激活。 HS-142-1对剂量反应的影响 ANF对肾脏的作用曲线，以及ANF与 整个肾脏、肾皮质和髓质将在 离体灌流大鼠肾。 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， 高血压（Goldblatt和SHR大鼠）和体积扩张（盐水 输注、高盐饮食）清醒和麻醉的大鼠。 研究 有望提供新的见解ANF受体的功能和 ANF在健康和疾病中的作用。