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...

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Phospholipase C gamma 1 (PLC-gamma 1) hydrolyses phosphatidylinositol-4,5-bisphosphate to the second messengers inositol-1,4,5-trisphosphate and diacylglycerol. PLC-gamma 1 also has mitogenic activity upon growth-factor-dependent tyrosine phosphorylation; however, this activity is not dependent on the phospholipase activity of PLC-gamma 1, but requires an SH3 domain. Here, we demonstrate that PLC-gamma 1 acts as a guanine nucleotide exchange factor (GEF) for PIKE (phosphatidylinositol-3-OH kinase (PI(3)K) enhancer). PIKE is a nuclear GTPase that activates nuclear PI(3)K activity, and mediates the physiological activation by nerve growth factor (NGF) of nuclear PI(3)K activity. This enzymatic activity accounts for the mitogenic properties of PLC-gamma 1.