Phosphorylation is considered a main mechanism modulating nNOS (neuronal nitric oxide synthase) function to reduce NO production. In the present study, the effects of nNOS phosphorylation on redox signalling, including that of NO, ROS (reactive oxygen species), and 8-nitro-cGMP (8-nitroguanosine 3′,5′-cyclic monophosphate), a downstream messenger of redox signalling, were investigated. In vitro experiments revealed that a phosphorylation-mimic mutant of nNOS (Ser847 replaced with aspartic acid, 847D) increased uncoupling to produce a superoxide. In addition, nicotine, which triggers an influx of Ca2+, induced more ROS and 8-nitro-cGMP production in 847D-expressing PC12 cells than WT (wild-type)-expressing cells. Additionally, nicotine-induced phosphorylation of nNOS at Ser847 and increased ROS and 8-nitro-cGMP production in rat CGNs (cerebellar granule neurons). In CGNs, the NOS (nitric oxide synthase) inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and superoxide dismutase completely inhibited ROS and 8-nitro-cGMP production, whereas the CaMK (Ca2+/calmodulin-dependent protein kinase) inhibitor KN93 mildly reduced this effect. Nicotine induced HO-1 (haem oxygenase 1) expression in CGNs and showed cytoprotective effects against apoptosis. Moreover, 8-nitro-cGMP treatment showed identical effects that were attenuated by KN93 pre-treatment. The present paper provides the first substantial corroboration for the biological effects of nNOS phosphorylation at Ser847 on redox signalling, including ROS and intracellular 8-nitro-cGMP generation in neurons, which possibly play roles in neuroprotection.
Redox signal regulation via nNOS phosphorylation at Ser847 in PC12 cells and rat cerebellar granule neurons
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Shingo Kasamatsu, Yasuo Watanabe, Tomohiro Sawa, Takaaki Akaike, Hideshi Ihara; Redox signal regulation via nNOS phosphorylation at Ser847 in PC12 cells and rat cerebellar granule neurons. Biochem J 15 April 2014; 459 (2): 251–263. doi: https://doi.org/10.1042/BJ20131262
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