In this proposal we aim to investigate the mechanism of regulation of lung surfactant secretion by cytosolic pH. Using isolated perfused lung preparation and lung epithelial type II cells we have shown that the secretion of lung surfactant (phosphatidylcholine) is stimulated by alkalosis. The alkalosis-stimulated secretion was inhibited by acidification with diffusible weak acids or by inhibitors of protein kinase C mediated phosphorylation. We hypothesize that a rise in intracellular pH increases the degradation (and turnover) of phosphatidylinositol-4, 5- bisphosphate to increase the diacylglycerol and inositol triphosphate (IP3) levels, which activates protein kinase C and elevates cytosolic free calcium respectively. This is followed by increased phosphorylation of specific proteins in various subcellular compartments of type Ii cells. Using isolated cells, we will investigate cytosolic free calcium changes with alkalosis and compare it to that with B-adrenergic and purinergic agonists. Alkalosis in lung cells will be produced by lowering CO2 in the gas mixture, by removal of a pulse of high CO2 (acidosis), and with NH4Cl. We will determine the degree and duration of change in intracellular pH and quantitate changes in phosphatidylcholine (PC), phosphatidylinositol (PI), diacylglycerols and IP3 in isolated type II cells, and isolated plasma membranes on stimulation of secretion with alkalosis, ATP, calcium ionophores, and terbutaline. The substrate for the diacylglycerol release will be determined with a pulse of cells with [3H-methy]choline, [14C]glycerol, [14C]myoinositol, or 32P and chase of the label in PI, PI- mono- and bisphosphate, PC, diacylglycerol and IP3 in the presence of various stimuli. Protein phosphorylation will be investigated in intact cells, prelabeled with 32P and exposed to secretagogues, and the intracellular sites determined by isolation of plasma membranes, microsomal cytosolic, and lamellar body fractions. These will be confirmed with phosphorylation of proteins of these fractions from control cells with AT32P and using membrane fractions from control and secretagogue exposed cells. Detailed investigations of protein kinase as to cofactor requirement and inhibition with various inhibitors will enable us to identify the type of protein kinase involved in the pH-dependent stimulation of surfactant in type II cells. These studies will also help establish a temporal relationship between the elevations in intracellular pH, diacylglycerols (and Ca2+), and protein phosphorylation in the stimulation of lung surfactant secretion.
