The acquisition of catecholamine sensitivity by the fetal lung for two crucial adaptive responses, the release of surface active material and the resorption of lung water, has been shown to occur very late in gestation. Because neonatal pulmonary immaturity kills and disables more newborns than any other disorder, it is important to understand how the developing lung becomes sensitive to catecholamines. This study proposes to examine the development of the catecholamine sensitive adenylate cyclase (AC) in fetal rabbit lung. The characterization of the important regulatory components of this system, the beta-adrenergic receptor and the guanyl nucleotide sensitive regulatory protein (G-protein) will be performed in membrane particulates so that the integrated function of the fetal system can be evaluated and in reconstituted systems so that functional characteristics of individual components may be resolved. Radiolabelling and protein separation techniques will be used so that structural correlates to functional alterations with development can be sought for the two important regulatory components of AC, the beta-adrenergic receptor and the G-protein. Information regarding structure and function of intact fetal AC and resolved reconstituted AC components will be determined during gestation and compared with adult rabbit lung AC and AC components. Irreversible photo-activated beta-adrenergic ligands will be used to examine apparent molecular size during gestation, to prepare peptide maps of the fetal beta receptor, and to determine glycogen content by pre-incubation with glycolytic enzymes. G-protein structure will be studied by cholera toxin-32PNAD labeling and subsequent electrophoresis. Resolved G-protein activity will be studied by solubilization of fetal lung membranes and reconstitution of G-protein activity into S49 cyc-membranes, demonstrated to lack functional G-protein. Resolved beta receptor activity will be determined by partial affinity purification and reconstitution into membranes which contain G-protein and catalytic cyclase but not beta-adrenergic receptors. Attempts to probe the mechanisms of catecholamine insensitivity of fetal adenylate cyclase will involve experiments designed to modify G-protein function and others designed to """"""""repair"""""""" the fetal cyclase by incubation with various adult and fetal subcellular fractions.
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