CYCLIC-AMP INHIBITS THE RESPIRATORY BURST OF ELECTROPERMEABILIZED HUMAN NEUTROPHILS AT A DOWNSTREAM SITE OF PROTEIN-KINASE-C

We studied a signaling pathway for the activation of the superoxide (O 2-)-generating NADPH oxidase and effects of cAMP on the pathway using electropermeabilized human neutrophils. The permeabilized cells produc ed O2- by the addition of protein kinase C (PKC) activator, phorbol my ristate acetate (PMA), and a non-hydrolyzable GTP analogue, GTPgammaS in the presence of ATP and Mg2+. The O2- production by PMA not by GTPg ammaS was inhibited by inhibitors of PKC. The production by PMA and GT PgammaS was inhibited by a GDP analogue, GDPbetaS, in the same dose-de pendent manner and the production by PMA was not enhanced by the addit ion of GTPgammaS and vice versa. These findings suggest the presence o f a GTP-binding protein which follows PKC in the activation pathway. T he O2- production by PMA and GTPgammaS was dose-dependently inhibited by cAMP and the inhibition was completely restored by an inhibitor of cAMP-dependent protein kinase, H-89, indicating that cAMP blocks the a ctivating pathway at the site between the GTP-binding protein located downstream of PKC and the NADPH oxidase by activating cAMP-dependent p rotein kinase. The activation of the oxidase by sodium dodecyl sulfate (SDS) seemed to be different from the above pathway. It needed higher concentrations of GDPbetaS for inhibition, did not absolutely need AT P and was inhibited by neither cAMP nor protein kinase C inhibitors. M oreover, the O2- production by the combination of GTPgammaS and SDS or of PMA and SDS was essentially the same as the sum of the production by each stimulant alone. We may conclude from the observations that th e signaling pathway involving PKC for the activation of the oxidase is distinct from the pathway induced by SDS: the former is blocked by cA MP at the site between the GTP-binding protein located downstream of P KC and the oxidase and the latter is cAMP-insensitive.