title: Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages.
abstract: This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca(2+)-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-kappaB (NF-kappaB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca(2+)-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca(2+)-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC-alpha and -beta activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-kappaB-specific DNA-protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-kappaB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-kappaB activation. These data suggest that persistent inhibition of ER Ca(2+)-ATPase by TG would influence calcium release from ER Ca2+ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-kappaB activation that induces iNOS expression and NO production.
authors.affiliation: Department of Medical Technology, Institute of Biotechnology, Yuanpei University of Science and Technology, 306 Yuanpei St., Hsinchu 30015, Taiwan. yjchen@mail.yust.edu.tw
authors.forename: Yu-Jen
authors.initials: YJ
authors.lastname: Chen
authors.forename: Kai-Wen
authors.initials: KW
authors.lastname: Hsu
authors.forename: Jen-Ning
authors.initials: JN
authors.lastname: Tsai
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Hung
authors.forename: Tsun-Cheng
authors.initials: TC
authors.lastname: Kuo
authors.forename: Yun-Liang
authors.initials: YL
authors.lastname: Chen
mesh_terms: D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D006873:Hydroquinones / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology*
chemical_list: D006873:Hydroquinones; D008070:Lipopolysaccharides; D016328:NF-kappa B; C019359:2,5-di-tert-butylhydroquinone; D009569:Nitric Oxide; D000001:Calcimycin; D015759:Ionomycin; D019284:Thapsigargin; D052247:Nitric Oxide Synthase Type II; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha