nNOS phosphorylatability Presages NANC Inhibition: Investigation with a Novel Phosphorylation-deficient nNOS Mutant Mouse

Phosphorylation of the C-terminal Akt/PKA consensus site activates endothelial NOS (eNOS) and decreases its calcium/calmodulin requirement, but no definite physiological role for phosphorylation of the homologous site in neuronal NOS (nNOS) has been described. Therefore, we created a knock-in mouse in which nNOS Serine-1412 (S1412) is mutated to non-phosphorylatable alanine (nNOSS1412A). We tested whether nNOSS1412A ileal rings have altered nitrergic inhibitory non-adrenergic, non-cholinergic (NANC) signaling in a physiological organ bath. Under NANC conditions, electrical field stimulation (EFS; 20V, 2msec, 1Hz, 30sec train) relaxed WT to a greater degree than nNOSS1412A ileum (73 ± 20% inhibition vs 31 ± 17%; N=3). Similarly, pharmacological treatment with forskolin (FSK) also relaxed WT more effectively than nNOSS1412A ileum (IC50 95% CI 35-44nM; N=42 vs 78-107nM; N=29). Application of selective nNOS (NANT) or pan-NOS antagonists (L-NAME) reduced FSK dependent relaxation identically in WT and nNOSS1412A ilea (IC50 ~150nM for both genotypes), confirming that a portion of FSK relaxation was due to nNOS activity. Akt and PKA are putative nNOS S1412 kinases. While there was no difference between WT and nNOSS1412A relaxation with FSK using the Akt inhibitor MK-2206, PKA inhibitors (H-89, Myr-PKI, and Rp-cAMPs) only decreased FSK relaxation in WT ileum (~3-fold increase in IC50) but not in nNOSS1412A ileum, suggesting a critical link between PKA and nNOS. Together, our data identify a novel NANC neuroinhibitory mechanism in which PKA phosphorylation of nNOS S1412 leads to calcium independent neuronal NO production and smooth muscle relaxation. This previously unrecognized NO physiology might be important in other cell types and organ systems as well.