title: Superoxide generation from nNOS splice variants and its potential involvement in redox signal regulation.
abstract: We previously demonstrated different spacial expression profiles of the neuronal nitric oxide (NO) synthase (nNOS) splice variants nNOS-µ and nNOS-α in the brain; however, their exact functions are not fully understood. Here, we used electron paramagnetic resonance to compare the electron-uncoupling reactions of recombinant nNOS-µ and nNOS-α that generate reactive oxygen species (ROS), in this case superoxide. nNOS-µ generated 44% of the amount of superoxide that nNOS-α generated. We also evaluated the ROS production in HEK293 cells stably expressing nNOS-α and nNOS-µ by investigating these electron-uncoupling reactions as induced by calcium ionophore A23187. A23187 treatment induced greater ROS production in HEK293 cells expressing nNOS-α than those expressing nNOS-µ. Also, immunocytochemical analysis revealed that A23187-treated cells expressing nNOS-α produced more 8-nitroguanosine 3',5'-cyclic monophosphate, a second messenger in NO/ROS redox signaling, than did the cells expressing nNOS-µ. Molecular evolutionary analysis revealed that the ratio of nonsynonymous sites to synonymous sites for the nNOS-µ-specific region was higher than that for the complete gene, indicating that this region has fewer functional constraints than does the complete gene. These observations shed light on the physiological relevance of the nNOS-µ variant and may improve understanding of nNOS-dependent NO/ROS redox signaling and its pathophysiological consequences in neuronal systems.
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan ihara@b.s.osakafu-u.ac.jp.
authors.forename: Hideshi
authors.initials: H
authors.lastname: Ihara
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kitamura
authors.affiliation: Departments of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Kasamatsu
authors.affiliation: Departments of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Ida
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kakihana
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Tsutsuki
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Sawa
authors.affiliation: Department of Pharmacology, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Watanabe
authors.affiliation: Departments of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Akaike
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000001:Calcimycin / Q000494:pharmacology; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000096:biosynthesis; D004583:Electrons; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013481:Superoxides / Q000378:metabolism*; D014162:Transfection
chemical_list: C525106:8-nitroguanosine 3',5'-cyclic monophosphate; D007527:Isoenzymes; D011994:Recombinant Proteins; D013481:Superoxides; D000001:Calcimycin; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D006152:Cyclic GMP
keywords: 8-nitro-cGMP; ROS; electron uncoupling; nNOS; redox signaling; splice variants