Physiological responsiveness of isolated rabbit tracheal epithelial cells

Abstract

Surface tracheal epithelial cells (tracheocytes) from rabbit were isolated by treating intact tissue with chelators and proteolytic enzymes. The cells were viable as assessed by the following criteria: fluorescent viability staining, sequestration of lactate dehydrogenase, and maintenance of constant ATP levels. Radiolabeled Na+ was transported into cells with a rate constant of 0.06/min and an initial velocity of 1.6 nmol X 10(6) cells-1 X min-1 X beta-adrenergic agonists increased adenosine 3',5'-cyclic monophosphate (cAMP) levels in a time- and dose-dependent manner. The beta-adrenergic effects were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and blocked by propranolol. The tracheocytes retained the capacity to respond to beta-adrenergic agonists for at least 90 min after isolation. Two major cAMP binding proteins of apparent molecular weights of 50,000 and 54,000 were identified in tracheocytes with the photoaffinity label 8-N3-[32P]cAMP. Agents that increased cAMP levels in intact cells and unlabelled cAMP added to homogenates of cells that were not exposed to drugs decreased photoaffinity labeling. The two proteins correspond in electrophoretic mobility to the regulatory subunits of cAMP-dependent protein kinases I and II, respectively. The results demonstrate that the beta-adrenergic receptors and cAMP binding proteins identified in rabbit tracheal mucosa submucosa are present on tracheocytes, suggesting a role for these receptors in the regulation of tracheocyte physiological events.