Nitric oxide (NO) generated by neuronal NO synthase (nNOS) initiates penile erection, but has not been thought to participate in the sustained erection required for normal sexual performance. We now show that cAMP-dependent phosphorylation of nNOS mediates erectile physiology, including sustained erection. nNOS is phosphorylated by cAMP-dependent protein kinase (PKA) at serine(S)1412. Electrical stimulation of the penile innervation increases S1412 phosphorylation that is blocked by PKA inhibitors but not by PI3-kinase/Akt inhibitors. Stimulation of cAMP formation by forskolin also activates nNOS phosphorylation. Sustained penile erection elicited by either intracavernous forskolin injection, or augmented by forskolin during cavernous nerve electrical stimulation, is prevented by the NOS inhibitor L-NAME or in nNOS-deleted mice. Thus, nNOS mediates both initiation and maintenance of penile erection, implying unique approaches for treating erectile dysfunction.gasotransmitter | smooth muscle relaxation | endothelial NOS | cyclic GMP | phosphoantibody N itric oxide (NO) is well established as a mediator of penile erection (1, 2). Neuronal NO synthase (nNOS) is highly localized to the penile innervation (3, 4). Electrical stimulation of the cavernous nerve (CN) to the penis elicits penile erection, which is abolished with NOS inhibitors and markedly reduced in nNOS-α-deleted mice (nNOSα −/− ) (5-7). Neuronal depolarizationinduced production of NO reflects calcium entry activating calmodulin associated with nNOS to stimulate NO formation (8,9). This activation is short-lived and thus is believed to account only for the initiation of penile erection (10, 11). Sessa and colleagues (12) established that increased blood flow and associated shear stress, acting via PI3-kinase, augments the activity of the serine protein kinase Akt, which phosphorylates vascular endothelial NOS (eNOS) at serine(S)1179, causing prolonged NO formation at resting intracellular calcium levels (13). The increased penile blood flow through cavernosal vessels initiated by nNOS activation similarly stimulates penile Akt to phosphorylate eNOS, thereby promoting sustained maximal penile erection (14).nNOS possesses a phosphorylation consensus sequence at S1412 that closely resembles the sequence surrounding eNOS-S1179, and is thought to be a target for Akt in some systems (15-21). We wondered whether nNOS phosphorylation at S1412 might regulate penile erection. Using a highly selective antibody for phospho-S1412-nNOS (P-nNOS) we report electrically stimulated nNOS phosphorylation via cAMP-dependent protein kinase (PKA) and not through Akt. P-nNOS activity contributes to sustained erection in concert with phospho-eNOS stimulation, and the neuronal and endothelial NOS activities are independently regulated by separate signaling cascades. We propose a model integrating the posttranslational phospho-stimulation of normal erectile physiology. ResultsElectrical Stimulation of Penile Innervation Augments Phosphorylation of nNOS. We developed a C-terminal ...