nNOS phosphorylation mediates gastrointestinal motility

Purpose One third of the world’s population suffers from gastrointestinal (GI) dysmotility, a common feature of which is disrupted nitric oxide (NO) signaling and spastic contractility. In the healthy gut, neuronal NO synthase (nNOS) induces cGMP-dependent GI smooth muscle relaxation, which facilitates digestion. However, regulation of nNOS activity is poorly understood. PKA or Akt phosphorylation of nNOS S1412 enhances hypothalamic and penile NO synthesis. We hypothesized that nNOS S1412 phosphorylation promotes GI motility. Methods We quantified the contractile force of ileal segments from C57Bl/6 wildtype and nNOS S1412A mutant mice in an organ bath under non-adrenergic, non-cholinergic (NANC) conditions. Relaxation stimuli—neuronal depolarization (EFS; 20V, 2msec pulsewidth) or adenylate cyclase activation (FSK)—were applied to ileal segments in the presence of kinase, nNOS, or cGMP pathway inhibitors. We measured nNOS S1412 phosphorylation by western blot. We determined non-NANC ileal contraction velocity with the gastrointestinal motility monitor. Results EFS (1Hz) and FSK (300nM) increased nNOS S1412 phosphorylation by 310% (p S1412A ileal segments were less sensitive to relaxation induced by FSK (2.4-fold higher IC 50 , p S1412A FSK relaxation was only sensitive to Akt inhibition. Under non-NANC conditions, nNOS S1412A ileal segments contracted 1.38 times faster (p Conclusions nNOS S1412 phosphorylation promotes GI motility by decreasing spastic contractility in murine ileal segments. Neuronal depolarization elicits Akt-dependent phosphorylation, while adenylate cyclase activation elicits PKA-dependent phosphorylation of nNOS. Common GI motility disorders like irritable bowel syndrome may benefit from therapies that upregulate nNOS S1412 phosphorylation.