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 (O2-)-generating NADPH oxidase and effects of cAMP on the pathway using electropermeabilized human neutrophils. The permeabilized cells produced O2- by the addition of protein kinase C (PKC) activator, phorbol myristate acetate (PMA), and a non-hydrolyzable GTP analogue, GTP gamma S in the presence of ATP and Mg2+. The O2- production by PMA not by GTP gamma S was inhibited by inhibitors of PKC. The production by PMA and GTP gamma S was inhibited by a GDP analogue, GDP beta S, in the same dose-dependent manner and the production by PMA was not enhanced by the addition of GTP gamma S and vice versa. These findings suggest the presence of a GTP-binding protein which follows PKC in the activation pathway. The O2- production by PMA and GTP gamma S 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 activating pathway at the site between the GTP-binding protein located downstream of PKC and the NADPH oxidase by activating cAMP-dependent protein kinase. The activation of the oxidase by sodium dodecyl sulfate (SDS) seemed to be different from the above pathway. It needed higher concentrations of GDP beta S for inhibition, did not absolutely need ATP and was inhibited by neither cAMP nor protein kinase C inhibitors. Moreover, the O2- production by the combination of GTP gamma S 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 the signaling pathway involving PKC for the activation of the oxidase is distinct from the pathway induced by SDS: the former is blocked by cAMP at the site between the GTP-binding protein located downstream of PKC and the oxidase and the latter is cAMP-insensitive.
Mitsuyama
T
T
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Takeshige
K
K
Minakami
S
S
D001665:Binding Sites / Q000187:drug effects; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D016897:Respiratory Burst / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D013481:Superoxides; D012967:Sodium Dodecyl Sulfate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate