Exocytosis in Rat Parotid Acini After in vitro Treatment with Forskolin is Accompanied by Cellular Redistribution of Regulatory Subunits of Cyclic AMP-dependent Protein Kinase

Abstract

Incubation of rat parotid acinar tissue with 1 μmol/L forskolin resulted in progressive exocytosis which was virtually complete after a 30-minute incubation period. Cyclic AMP binding to protein kinase (cA-PK) regulatory (R) subunits, determined by photo-affinity labeling with [³²P]-8-azido cyclic AMP, was found to increase in a time-dependent manner in the 10,000-g supernatant fraction of a broken cell preparation. An apparent redistribution of protein kinase R subunits took place in the 600-g supernatant after in vitro treatment of cells with forskolin. In control cells, R_I and R_II subunits and a 35-to-40-kdal fragment were present in approximately equal amounts throughout the incubation. Azido labeling of R_II appeared either to increase or to remain unchanged, while that of R_I decreased in the 600-g pellet. Only type I isozyme R subunits were found in the 600-g pellet in either the absence or presence of forskolin. These findings indicate that a temporal relationship exists between stimulated protein exocytosis and cyclic AMP-dependent protein kinase activation. Forskolin stimulation of adenylate cyclase in salivary gland cells therefore provides a defined system for the study of cyclic AMP-mediated protein secretion.