Protein kinase C- and reactive oxygen species-dependent stimulation of intracellular cAMP in human eosinophils. The role of extracellular signal-regulated protein kinases.
OBJECTIVE
The objective of this study was to investigate the role of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the signalling pathway of the novel protein kinase C (PKC)- and reactive oxygen species (ROS)-dependent stimulation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) production in human eosinophils.


METHODS
Immunomagnetically purified human eosinophils were stimulated in vitro with a PKC activator, phorbol myristate acetate (PMA), and the cAMP response in the presence of a phosphodiesterase inhibitor, rolipram, was determined. The role of ERK1/2 phosphorylation was investigated using specific inhibitors and Western blot analysis.


RESULTS
The PMA-stimulated eosinophils responded with a profound increase in intracellular levels of cAMP that was dependent on both PKC and ROS, as confirmed by the use of specific inhibitors: Ro 31-8220 for PKC and diphenyleneiodonium (DPI) for the ROS-generating enzyme NADPH oxidase. Pre-treatment of cells with the ERK1/2 inhibitor PD 98059, but not the p38-MAPK inhibitor SB203580, nor the PI3 kinase inhibitor, wortmannin, abolished the response. PMA treatment induced the phosphorylation of ERK1/2 with a time course that is consistent with a role in the cAMP response. The ERK1/2 phosphorylation was abolished by the ERK1/2 inhibitor PD 98059 and the PKC inhibitor Ro 31-8220, but not the NADPH oxidase inhibitor DPI.


CONCLUSIONS
These results reveal the involvement of ERK1/2 in the signalling mechanism of PMA-stimulated, PKC- and ROS-dependent stimulation of cAMP production in human eosinophils, and show that ERK1/2 phosphorylation is upstream of ROS production in the signalling pathway.
Department of Pharmacology and Toxicology, Faculty of Medicine, Kuwait University, Kuwait. ezeamuzie@hsc.edu.kw
Charles I
CI
Ezeamuzie
Najla
N
Taslim
D000242:Cyclic AMP / Q000096:biosynthesis*; D004804:Eosinophils / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020889:Rolipram / Q000494:pharmacology; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017382:Reactive Oxygen Species; D000242:Cyclic AMP; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020889:Rolipram; D013755:Tetradecanoylphorbol Acetate