Protein kinase C activation inhibits eosinophil degranulation through stimulation of intracellular cAMP production

The mechanism of inhibition of eosinophil degranulation by protein kinase C (PKC) was investigated in complement C5a (C5a)‐stimulated degranulation of highly purified human eosinophils using the specific PKC activator – phorbol 12‐myristate 13‐acetate (PMA). C5a‐induced release of eosinophil peroxidase and eosinophil cationic protein was potently inhibited in a concentration‐dependent manner by PMA (IC 50 : 3 and 5 n M , respectively). The inhibition by PMA, but not histamine, was significantly reversed by the specific, but isoform nonselective, PKC inhibitor Ro 31‐8220 (1 μ M ). In the presence of phosphodiesterase inhibitor rolipram (5 μ M ), PMA stimulated a pronounced concentration‐dependent increase in intracellular cAMP, with a potency 400 times that of histamine (EC 50 : 55 n M vs 22.5 μ M ). The inactive PMA analogue, 4 α ‐PMA, had no such effect. The cAMP production by PMA, but not histamine, was significantly reversed by Ro 31‐8220 (1 μ M ) and the selective inhibitor of the novel PKC δ , rottlerin (1–3 μ M ), but not the selective inhibitor of the classical PKC isoforms, Gö 6976 (0.01–0.1 μ M ). Western blot analysis revealed the presence of six PKC isoforms ( α , β I, β II, δ , ι and ζ ) in isolated eosinophils. Chelation of internal or external calcium had no effect on PMA‐induced cAMP response, but abolished that induced by histamine. There was a good correlation between increase in intracellular cAMP and inhibition of degranulation. These results show, for the first time, that in human eosinophils, PMA, via activation of PKC δ isoform, can stimulate cAMP production, and that this may be the basis for its potent anti‐degranulatory effect.British Journal of Pharmacology (2004) 143 , 725–732. doi: 10.1038/sj.bjp.0706028