Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection

Abstract

In the penis, nitric oxide (NO) can be formed by both neuronal NO synthase and endothelial NOS (eNOS). eNOS is activated by viscous drag͞shear stress in blood vessels to produce NO continuously, a process mediated by the phosphatidylinositol 3-kinase (PI3-kinase)͞Akt pathway. Here we show that PI3-kinase͞Akt physiologically mediates erection. Both electrical stimulation of the cavernous nerve and direct intracavernosal injection of the vasorelaxant drug papaverine cause rapid increases in phosphorylated (activated) Akt and eNOS. Phosphorylation is diminished by wortmannin and LY294002, inhibitors of PI3-kinase, the upstream activator of Akt. The two drugs also reduce erection. Penile erection elicited by papaverine is reduced profoundly in mice with targeted deletion of eNOS. Our findings support a model in which rapid, brief activation of neuronal NOS initiates the erectile process, whereas PI3-kinase͞Akt-dependent phosphorylation and activation of eNOS leads to sustained NO production and maximal erection. N itric oxide (NO) serves many biological functions. As a neurotransmitter, it is produced by neuronal NOS (nNOS). Vascular tone is regulated by NO formed from endothelial NOS (eNOS). Inducible NOS accounts for diverse functions, especially responses to inflammatory stimuli (1-3). A substantial body of evidence implicates NO in normal erectile function: the nerves that regulate penile erection contain nNOS (4-7), NO donors and NOS inhibitors elicit and prevent erection, respectively (8-12), and mice lacking protein kinase G I (PKGI, a major target of NO͞cGMP signaling in the penis) demonstrate a pronounced reduction in reproductive capacity (13).Neurally derived NO is well established as a mediator of smooth muscle cell relaxation in the penis, engorgement of the cavernous sinusoids, and subsequent erection (14,15). eNOS is abundant in the endothelial lining of the penile vessels and trabecular meshwork and is also a potential source of [16][17][18] nNOS and eNOS are activated by calcium entry into the cell, binding to calmodulin associated with the enzymes (22). Whereas physiologic penile erection lasts several minutes, the calcium-dependent activation of nNOS or eNOS is quite transient. Recently, several groups showed that the phosphatidylinositol 3-kinase (PI3-kinase) pathway that activates the serine͞ threonine protein kinase Akt (also known as PKB) causes direct phosphorylation of eNOS, reducing the enzyme's calcium requirement and causing increased production of [23][24][25]. This pathway is responsible for both shear stress and growth-factor enhancement of blood flow that can last for hours (26-30). We have examined whether Akt regulation of eNOS occurs during penile erection and whether that regulation is important in producing and maintaining normal penile erection.We now show that electrical stimulation as well as druginduced relaxation of penile erectile tissue increases phosphorylation and activation of Akt as well as phosphorylation of eNOS. This response is reduced by PI3-kinase inhibitors. Mor...

Characterization of nitrergic function in monkey penile erection in vivo and in vitro

“…19 We speculated that the differential pattern of the responses in vitro and in vivo could be explained by the NO released from the sinusoidal endothelium as a result of stretching, which is due to the intracavernous pressure increase induced by neurogenic NO. This sequence of events has been postulated by Hurt et al 11 However, this is not the case in monkeys, as both the intracavernous pressure response to nerve stimulation and the relaxant response of isolated cavernous strips to electrical field stimulation rapidly decayed after nerve stimulation was terminated. In monkey preparations in vitro and in vivo, the cavernous smooth muscle relaxation 14 and intracavernous pressure increase (data not shown) persisted during nerve stimulation but faded after the stimulation was removed.…”

“…10 Recent studies suggest that NO released from the sinusoidal endothelium is also involved in the maintenance of penile erection. 11 Although cholinergic innervation has been shown in the penile corpus cavernosum, the functional role has not been elucidated. 12,13 Furthermore, reports in the literature have revealed interspecies variation in the responses to efferent nerve stimulation of the isolated corpus cavernosum.…”

Nitric oxide–cyclic GMP pathway with some emphasis on cavernosal contractility

“…10,11 Endothelial-derived NO plays a major role in the sustained erectile response and is a major source of NO in the penis. 8 Some suggest that NO is highly labile, therefore, it cannot be stored as a preformed neurotransmitter. 12 Alternatively, another neurotransmitter such as vasoactive intestinal polypeptide (VIP) may interact with either endothelial or smooth muscle cells in the corpus cavernosum to trigger the local formation of NO.…”

“…Neurally derived NO has been established as a mediator of smooth muscle relaxation in the penis, and it is thought that constitutive forms of nitric oxide synthase (NOS) work to mediate the erection. 8 Released NO activates sGC, which catalyzes the conversion of GTP to the intracellular second messenger cGMP in smooth muscle cells. An increase in cGMP modulates cellular events, such as relaxation of smooth muscle cells.…”

Tadalafil for the treatment of erectile dysfunction

“…NO is synthesized in nonadrenergic, noncholinergic fashion by neuronal nitric oxide synthase (nNOS) in the cavernous nerves to initiate erection and by endothelial nitric oxide synthase (eNOS) in the endothelium to maintain erection during sexual stimulation (Hurt et al 2002). NO passively crosses the cell membrane and activates soluble guanylyl cyclase (sGC) upon entering smooth muscle cytoplasm, which in turn increases the production of cyclic guanosine monophosphate (cGMP) by converting it from guanosine triphosphate (GTP) (Lincoln and Cornwell 1991).…”