Abstract
Recently, we showed that phorbol 12‐myristate 13‐acetate (PMA) can cause a direct, PKC‐dependent, stimulation of intracellular adenosine 3′, 5′‐cyclic monophosphate (cAMP) production in human eosinophils. Since PMA also stimulates oxidative burst and release of reactive oxygen species (ROS) in these cells, we have investigated whether ROS is involved in the cAMP response. Provided eosinophils were incubated for less than 20 min at 37°C before stimulation, PMA induced a concentration‐dependent stimulation of cAMP that surpassed that of histamine. Pre‐treatment of the cells with diphenyleneiodonium (DPI), a NADPH oxidase inhibitor, caused a concentration‐dependent inhibition of cAMP production induced by PMA, but not that induced by histamine. This treatment also strongly inhibited the release of superoxide anions (O2−). The cAMP response was also inhibited by pre‐treatment with the specific peroxide scavenger, ebselen, but not superoxide dismutase, or L‐NAME, thus, suggesting the possible involvement of hydrogen peroxide (H2O2) rather than O2− or NO. PMA treatment induced the phosphorylation of extracellular signal‐regulated protein kinase 1/2 (ERK1/2) with a time‐course comparable with that of cAMP production. Both the PMA‐induced ERK1/2 phosphorylation and cAMP production were abolished by pre‐treatment with both the specific ERK inhibitor PD 98059 and the PKC inhibitor Ro 31–8220, but not the NADPH oxidase inhibitor, DPI. These results reveal a novel PKC‐dependent, ROS‐mediated stimulation of cAMP production in human eosinophils that appears to depend on a signaling pathway in which ROS production is downstream of ERK phosphorylation.
This work was supported by Grant # MR 02/01 from Research Administration, Kuwait University, Kuwait.