BIOLOGY OF ATRIAL NATRIURETIC FACTOR AND ITS RECEPTORS

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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.