TYPE-I CAMP-DEPENDENT PROTEIN-KINASE DELAYS APOPTOSIS IN HUMAN NEUTROPHILS AT A SITE UPSTREAM OF CASPASE-3

Current data suggest that apoptosis controls neutrophil numbers in tis sues. We analyzed roles for and the sites of action for the cAMP-depen dent protein kinases (cAPKs) in apoptosis induced in human neutrophils by in vitro storage, cycloheximide (CIM) exposure, and anti-Fas expos ure. Treatment with 8-chlorophenylthio-cAMP (8-CPT-cAMP) prolonged the time required for 50% of the cells to exhibit apoptotic morphology (t (50)) from 16.3 to 41.8 h (in vitro culture), from 2.4 to 7.8 h (CHX), and from 4.8 to 6.5 h (anti-Pas). CHX +/- 8-CPT-cAMP did not signific antly alter resting intracellular calcium levels and H-89, a selective inhibitor of cAPK, had no effect on apoptosis in the absence of the a nalogue. In contrast, site-selective cAMP analogues that specifically activated the type I cAPK, but not type II cAPK, synergistically atten uated apoptosis. Exposure to 8-CPT-cAMP delayed, in parallel, the acti vity of caspase-3 (CPP-32 beta), whereas mitogen-activated protein kin ase kinase (MAPKK) inhibitor, PD98059, had no effect on CHX-induced ap optosis +/- 8-CPT-cAMP. Together these results indicate that type I cA PK activation is necessary and sufficient to mediate cAMP-induced dela y in human neutrophil apoptosis induced by several mechanisms and sugg est that one of the major sites of cAPK action is upstream of caspase- 3 (CPP-32 beta) activation.