Akt phosphorylation of neuronal nitric oxide synthase regulates gastrointestinal motility in mouse ileum

Nitric oxide (NO) is a major inhibitory neurotransmitter that mediates nonadrenergic noncholinergic (NANC) signaling. Neuronal NO synthase (nNOS) is activated by Ca²⁺/calmodulin to produce NO, which causes smooth muscle relaxation to regulate physiologic tone. nNOS serine1412 (S1412) phosphorylation may reduce the activating Ca²⁺ requirement and sustain NO production. We developed and characterized a nonphosphorylatable nNOS^S1412A knock-in mouse and evaluated its enteric neurotransmission and gastrointestinal (GI) motility to understand the physiologic significance of nNOS S1412 phosphorylation. Electrical field stimulation (EFS) of wild-type (WT) mouse ileum induced nNOS S1412 phosphorylation that was blocked by tetrodotoxin and by inhibitors of the protein kinase Akt but not by PKA inhibitors. Low-frequency depolarization increased nNOS S1412 phosphorylation and relaxed WT ileum but only partially relaxed nNOS^S1412A ileum. At higher frequencies, nNOS S1412 had no effect. nNOS^S1412A ileum expressed less phosphodiesterase-5 and was more sensitive to relaxation by exogenous NO. Under non-NANC conditions, peristalsis and segmentation were faster in the nNOS^S1412A ileum. Together these findings show that neuronal depolarization stimulates enteric nNOS phosphorylation by Akt to promote normal GI motility. Thus, phosphorylation of nNOS S1412 is a significant regulatory mechanism for nitrergic neurotransmission in the gut.