rottlerin and zinc-chloride

Peroxynitrite has been suggested to be the potent oxidant causing toxicity to neurons and oligodendrocytes (OLs). Our previous studies have illustrated that intracellular zinc liberation contributes to peroxynitrite toxicity to mature OLs. In this study, we further investigated the signaling pathways involved in this event and identified protein kinase C (PKC) as an important early signaling molecule. We found that a non-selective PKC inhibitor bisindolylmaleimide-1 blocked OL toxicity induced by a peroxynitrite generator SIN-1 and exogenous zinc. The protective effects were due to its inhibition on ERK1/2 phosphorylation and ROS generation. The same phenomenon was also observed in OLs following prolonged treatment with phorbol 12 myristate 13 acetate (PMA), which downregulates the conventional and the novel PKC isoforms (cPKCs and nPKCs). To determine the role of specific PKC isoforms, we found that a specific nPKC inhibitor rottlerin significantly reduced SIN-1- or zinc-induced toxicity, whereas Go6976, a cPKC inhibitor, reduced OL toxicity triggered by zinc, but not by SIN-1 at high concentrations. Rottlerin was more potent than Go6976 to attenuate ERK1/2 phosphorylation and ROS generation induced by SIN-1 or zinc. Surprisingly, zinc only induced phosphorylation of PKCθ, but not PKCδ. Knockdown of PKCθ using lentiviral shRNA attenuated SIN-1- or zinc-induced toxicity. These results suggest that PKCθ might be the major PKC isoform involved in peroxynitrite and zinc toxicity to mature OLs, and provide a rationale for development of specific inhibitors of PKCθ in the treatment of multiple sclerosis and other neurodegenerative diseases, in which peroxynitrite formation plays a pathogenic role.

Topics: Acetophenones; Animals; Benzopyrans; Carbazoles; Cells, Cultured; Chlorides; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Indoles; Isoenzymes; Maleimides; Molsidomine; Oligodendroglia; Peroxynitrous Acid; Protein Kinase C; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Tetradecanoylphorbol Acetate; Zinc Compounds

Eosinophils play a primary role in the pathophysiology of asthma. In the lung, the activation state of the infiltrating eosinophils determines the extent of tissue damage. Interleukin-5 (IL-5) and leukotriene B4 (LTB4) are important signaling molecules involved in eosinophil recruitment and activation. However, the physiological processes that regulate these activation events are largely unknown. In this study we have examined the mechanisms of human eosinophil NADPH oxidase regulation by IL-5, LTB4, and phorbol ester (PMA). These stimuli activate a Zn2+-sensitive plasma membrane proton channel, and treatment of eosinophils with Zn2+ blocks superoxide production. We have demonstrated that eosinophil intracellular pH is not altered by IL-5 activation of NADPH oxidase. Additionally, PKCdelta inhibitors block PMA, IL-5 and LTB4 mediated superoxide formation. Interestingly, the PKCdelta-selective inhibitor, rottlerin, does not block proton channel activation by PMA indicating that the oxidase and the proton conductance are regulated at distinct phosphorylation sites. IL-5 and LTB4, but not PMA, stimulated superoxide production is also blocked by inhibitors of PI 3-kinase indicating that activation of this enzyme is an upstream event common to both receptor signaling pathways. Our results indicate that the G-protein-coupled LTB4 receptor and the IL-5 cytokine receptor converge on a common signaling pathway involving PI 3-kinase and PKCdelta to regulate NADPH oxidase activity in human eosinophils.

Topics: Acetophenones; Benzopyrans; Cells, Cultured; Chlorides; Dose-Response Relationship, Drug; Electric Conductivity; Enzyme Inhibitors; Eosinophils; Humans; Hydrogen-Ion Concentration; Interleukin-5; Isoenzymes; Kinetics; Leukotriene B4; NADPH Oxidases; Patch-Clamp Techniques; Protein Kinase C; Protein Kinase C-delta; Proton Pumps; Superoxides; Tetradecanoylphorbol Acetate; Zinc Compounds