NNOS modulates NO–ROS signaling in neurons via phosphorylation at Ser847

Abstract

Background nNOS produces NO and reactive oxygen species (ROS). nNOS activity is regulated thorough phosphorylation at Ser847. However, the physiological relevance of this phosphorylation remains unclear. In the present study, we aimed to determine the implications of this phosphorylation on NO–ROS signaling in neurons, including 8-nitro-cGMP formation, a downstream molecule of NO–ROS signaling. Method We prepared a phosphorylation-mimic mutant of nNOS (Ser847 to Asp; 847D) and examined the enzyme activities in vitro, including NO and ROS production, NADPH oxidation, and uncoupling efficiency. Furthermore, we generated PC12 cells expressing the nNOS mutant and measured the generation of NO, ROS, and 8-nitro-cGMP in these cells. Next, we determined nicotine-induced ROS and 8-nitro-cGMP generation in cerebellar granule neurons (CGNs). Results In vitro analysis showed that compared to the wild-type (WT) enzyme, the 847D mutant had decreased NO production and increased ROS production and NADPH oxidation. The uncoupling efficiency of the WT and 847D mutant was 49% and 62%, respectively. In vivo analysis showed that 847D-expressing PC12 cells exhibited lower NO production and higher ROS production than WT-expressing PC12 cells. We observed strong immunoreactivity only in nNOS-expressing cells on immunostaining with an 8-nitro-cGMP-specific antibody; the fluorescent intensity in 847D-expressing cells was greater than that in WT-expressing cells. We detected nicotine-induced ROS and 8-nitro-cGMP production in CGNs. These production reduced on pretreatment of the CGNs with a calmodulin kinase II inhibitor. These results suggest that nNOS regulates NO-ROS signaling, including 8-nitro-cGMP production, in neurons via phosphorylation at Ser847.