title: Modulation of microglial superoxide production by alpha-tocopherol in vitro: attenuation of p67(phox) translocation by a protein phosphatase-dependent pathway.
abstract: As in other phagocytic cells, the NADPH-oxidase system in microglia is thought to be primarily responsible for the production of superoxide anion radicals (O2(-.), a potentially cytotoxic reactive oxygen species. The assembly of a functional NADPH-oxidase complex at the plasma membrane depends on the phosphorylation and subsequent translocation of several cytosolic subunits. Immunocytochemical and subcellular fractionation experiments performed during the present study revealed that the NADPH-oxidase subunit p67(phox) translocates from the cytosol to the plasma membrane upon stimulation. Pre-incubation of microglia in alpha-tocopherol (alphaTocH) containing medium decreased O2(-.) production in a time- and concentration-dependent manner, findings attributed to attenuated p67(phox) translocation to the plasma membrane. Moreover, alphaTocH-supplementation of the culture medium resulted in decreased microglial protein kinase C (PKC) activities, an effect that could be partially or completely reversed by the addition of protein phosphatase inhibitors (okadaic acid and calyculin A). The addition of the PKC-inhibitor staurosporine inhibited the microglial respiratory burst in a manner comparable to alphaTocH. The addition of okadaic acid or calyculin A completely restored O2(-.) production in alphaTocH-supplemented cells. The present findings suggest that alphaTocH inactivates PKC via a PP1 or PP2A-mediated pathway and, as a consequence, blocks the phosphorylation-dependent translocation of p67(phox) to the plasma membrane. As a result, O2(-.) production by the microglial NADPH-oxidase system is substantially inhibited.
authors.affiliation: Institute of Medical Biochemistry and Molecular Biology, Karl Franzens University Graz, Graz, Austria.
authors.forename: T
authors.initials: T
authors.lastname: Egger
authors.forename: A
authors.initials: A
authors.lastname: Hammer
authors.forename: A
authors.initials: A
authors.lastname: Wintersperger
authors.forename: D
authors.initials: D
authors.lastname: Goti
authors.forename: E
authors.initials: E
authors.lastname: Malle
authors.forename: W
authors.initials: W
authors.lastname: Sattler
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008387:Marine Toxins; D051379:Mice; D017628:Microglia / Q000187:drug effects* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology*; D010750:Phosphoproteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D016897:Respiratory Burst / Q000187:drug effects* / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D013552:Swine; D024502:alpha-Tocopherol / Q000494:pharmacology*
chemical_list: D000975:Antioxidants; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D009419:Nerve Tissue Proteins; D010080:Oxazoles; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D019319:Okadaic Acid; C059041:calyculin A; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D024502:alpha-Tocopherol; D019311:Staurosporine