THE ROLE OF PROTEIN-KINASE-C IN ALPHA-ADRENERGIC REGULATION OF NACL(K) COTRANSPORT IN HUMAN AIRWAY EPITHELIAL-CELLS

alpha(1)-Adrenergic (alpha(1)-AR) agents stimulate NaCl(K) cotransport and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P-2]-specific p hospholipase C in human trachea and nasal polyp epithelial cells. One second messenger generated by PtdIns(4,5)P-2 degradation is inositol t risphosphate. We now show that diglycerides (DG) are also generated du ring alpha(1)-AR stimulation. In cells prelabeled with [H-3]arachidoni c acid, alpha(1)-AR agents produced a biphasic DG generation in normal and cystic fibrosis (CF) cells that is blocked by pertussis toxin. Th e early DG peak closely paralleled PtdIns(4,5)P-2 degradation, stimula tion of cotransport by phorbol 12-myristate 13-acetate (PMA), and inhi bition of cotransport by the protein kinase C (PKC) inhibitor staurosp orine. This suggests that cotransporter activation requires PKC-protei n phosphorylation. This possibility was tested using the protein phosp hatase inhibitor okadaic acid. Okadaic acid elevated bumetanide-sensit ive Cl efflux. Staurosporine also blocked >63% of okadaic-acid-stimula ted Cl transport. The late DG peak did not support hormone-stimulated cotransport. The results demonstrate that DGs are a pivotal link betwe en alpha(1)-AR stimulation and NaCl(K) cotransport activation with a r ole for PKC and protein phosphorylation. (alpha(1)-AR intracellular si gnaling mechanisms apparently operate normally in CF cells.