Protein Kinase C- and Reactive Oxygen Species-Dependent Stimulation of Intracellular cAMP in Human Eosinophils

Abstract

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. Materials and 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. Conclusion: 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.

C3b‐induced eosinophil degranulation involves PI3‐kinases and is inhibited by protein kinase C activity

“…Ezeamuzie et al. found that PD98059 inhibited the PMA‐induced production of cAMP, and suggested a signalling pathway with ERK1/ERK2 located downstream of PKC, and where the end product was cAMP (41). This hypothesis could thus explain the increased degranulation caused by staurosporine, GÖ6976 and PD98059; however, it remains to be proven that this is the pathway elicited by C3b and serum‐opsonized Sephadex particles.…”

Anti-tussive and bronchodilator mechanisms of action for the enaminone E121

“…This method has been described recently (Ezeamuzie and Taslim, 2008). In brief, fresh blood was obtained from consenting individuals who were mildly atopic, but had no allergic disease nor taken any medication in the last 72 h. The protocol for the study was reviewed and approved by the Ethics Committee of the Faculty of Medicine, Kuwait University, Kuwait.…”

“…This was done using a method recently reported (Ezeamuzie and Taslim, 2008). In brief, eosinophils, re-suspended at a concentration of 10 7 cells/ml in BSA-free reaction buffer, were dispensed in 50 μl aliquots into each well of a 96-well plate.…”

Influence of plasma-activated compounds on melanogenesis and tyrosinase activity

“…The cAMP stimulation results in the up-regulation of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related proteins (Tyrp1 and Tyrp 2) 5 6 7 . The stimulation of cAMP can be affected by several factors such as adrenocorticotropic hormone (ACTH), prostaglandin E2 (PGE2), and alpha-melanocyte stimulating hormone (α-MSH) 3 4 8 9 . The increase in melanogenesis without affecting the skin is very important, because it can protect the skin from diseases such as cancer 10 and vitiligo 11 .…”