| Scope and method of study. We began this project from a target gene GABRP, identified from DNA microarray. We firstly examined the expression patterns of GABRP in various culture conditions by using real-time PCR, western blot and immunostaining. Then we isolated lung tissue or type II cells from fetal, neonate, and adult rats, as well as hyperoxia-exposed rats. By using real-time PCR, we demonstrated the differential expression of all the ionotropic GABA receptor subunits during fetal lung development and after hyperoxia exposure. We further performed the functional study on fresh or cultured type II cells and rats, by using real-time PCR, western blot, immunostaining, biotinylation, immunproecipitation, isotope, RNA intereference, and lung fluid clearance assays.; Findings and conclusions. (1) The GABRP expression was regulated by culture conditions and closely associated with the type II cell-phenotype. (2) Among 19 known GABA receptor subunits, 17 were expressed in rat lungs and type II cells throughout the fetal and adult stages. Those subunits were differentially regulated during lung development and most of them were decreased by hyperoxia. (3) We found that adult rat type I cells and type II cells both expressed multiple ionotropic GABA receptor subunits. GABA and GAD were synthesized in type II cells. We identified one GABA A receptor complex, a1 alpha3beta2gamma2pi in the type II cells. This receptor was localized on the apical but not basolateral plasma membranes. GABA increased Cl- efflux dose-dependently, which was not due to CFTR, NKCC and CaCC. When type II cells were cultured on the air-liquid interface, GABA inhibited the basal and isoproterenol-stimulated Cl - absorptions. Knock-down of the pi-subunit eliminated the GABA-dependent Cl- efflux. In anesthetized rats, GABA inhibited both basal and isoproterenol-stimulated alveolar fluid clearance. Therefore, we have identified a novel fluid transport pathway in adult rat alveolar epithelial cells, contributing to the ionotropic GABA receptors. |
