BIOLOGY OF ATRIAL NATRIURETIC FACTOR AND ITS RECEPTORS

研究的长期目标是了解功能 心房钠尿因子 (ANF) 及其两种主要受体。 动力学和功能的分子决定因素研究 ANF（GCA 受体）的生物受体本身将与 第293章 CHO细胞。 测试受体的解离是否非常快 配体复合物通过 ATP 与激酶的相互作用进行调节 与 GCA 受体的结构域一样，突变体将通过以下方式构建：i) 总细胞内激酶样和鸟苷酸环化酶的截短 域； ii) 激酶中氨基酸的缺失和取代 可能决定其与 ATP 相互作用的结构域。 细胞表达 转染的受体将用于确定受体的解离 配体复合物、cGMP 的生成以及物质的影响 延迟受体-配体解离（例如阿米洛利）。 隔离 重组细胞的膜将用于评估效果 添加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激活。 分子研究 ANF 清除 (C) 受体内吞作用的决定因素将 使用重组人野生型和突变型 C-ANF 受体进行 转染至CHO细胞中。 Tyr508、Phe538 和 Cys473 将是 通过寡核苷酸定向诱变取代丙氨酸进行测试 细胞质结构域中的芳香族氨基酸和 胞质外结构域中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 类型和突变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。 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 分离的大鼠肾灌注制剂和使用新型的全大鼠肾制剂 GCA受体拮抗剂，命名为HS-142-1。 该假设将被检验 ANF 在调节压力-容量稳态方面具有重要作用，通过 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 离体灌注大鼠肾。 HS-142-1 的急性和慢性影响 对肾功能和血压的影响将在血压正常时确定， 高血压（Goldblatt 和 SHR 大鼠）和扩容（盐水 输液、高盐饮食）清醒且麻醉的大鼠。 研究 预计将为 ANF 受体功能和 ANF 在健康和疾病中的作用。

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.