The overall goal of this project is to determine how pulmonary surfactant secretion is regulated. It is known that secretion of the major surfactant phospholipid, phosphatidylcholine (PC), can be influenced by a variety of physiological and pharmacological agents. Using primary cultures of type II pneumocytes from adult, fetal and newborn rats it is proposed to elucidate the signal-transduction pathways by which selected secretagogues stimulate PC secretion. The major emphasis will be on P1 (adenosine and its analogs) and P2 (ATP) purinoceptor agonists but interactions with other secretogogues will also be examined. Steps in the signal-transduction process that will be investigated in adult cells include effector enzyme activation, involvement of guanine nucleotide binding proteins (G proteins) and protein phosphorylation. It will be determined if phosphodiesteratic cleavage of PC by the actions of phospholipase C or phospholipase D is an event in the signal-transduction process leading to PC secretion in response to ATP, tetradecanoylphorbol-13-acetate (TPA) and ionomycin. Generation of diacylglycerol in this manner might result in sustained activation of protein kinase C and account for the prolonged stimulation of PC secretion noted in response to these agonists. The involvement of G proteins in coupling purinergic and beta-adrenergic receptors with activation of intracellular effector enzymes will be investigated in plasma membrane fractions as well as in permeabilized type II cells. The pattern of protein phosphorylation in response to secretagogues alone and in combination will be examined to determine if synergistic interactions on PC secretion occur at this level. Secretion of the major surfactant- associated protein SP-A will be measured to determine if it is regulated similarly to that of PC. To investigate the mechanism by which PC secretion is inhibited by SP-A and other lectins, their effects on effector enzyme activation and protein phosphorylation pattern will be examined. Surfactant secretion in type II cells isolated from fetal and postnatal rats will be studied to investigated the mechanism of the developmental change in response to PC secretagogues and to determine if similar changes occur with respect to SP-A secretion. The proposed research will generate information critical to the understanding and prevention of human disease conditions in which pulmonary surfactant deficiency is known to occur. These include respiratory distress syndrome (RDS) of the newborn as well as adult RDS.
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