and specific aim): The focu of this application is the regulation of active sodium transport through amiloride-sensitive sodium channels in normoxic and hyperoxia-exposed rat lung cells. It was previously established that cyclic AMP/protein kinase A signal transduction system can stimulate active sodium and water clearance from the airspaces of the lungs via amiloride-sensitive sodium channels. The investigator proposes that alterations in the regulation of lung amiloride-sensitive sodium channels by endogenous ligands, mediated by intracellular messengers, contribute to the transport differences between normal and injured lungs.
Three specific aims have been proposed: 1) to determine whether cyclic nucleotides or cyclic nucleotide-dependent kinases ar directly involved in the regulation of amiloride-sensitive sodium channels; 2) to determine whether hyperoxia-induced lung injury changes Na+ transport acros the alveolar epithelium by changing functional expression of high vs. low affinity amiloride-sensitive sodium channels; and 3) to identify specific ligands whose signal transduction pathways are involved in regulating sodium transport through amiloride-sensitive sodium channels following hyperoxic exposure. Experiments will be conducted in cultured rat alveolar epithelial cells grown on plastic or filters. Short-circuit current and 22Na+-uptake assays will be done. Signal transduction assays include kinase, phosphatase, and cyclic nucleotide assays. Western blots will be used to localize guanylate cyclase.
