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...

Nitric oxide synthase (NOS), which catalyzes the production of nitric oxide (NO), was characterized within the reproductive tract of adult male Sprague-Dawley rats by means of biochemical and immunohistochemical techniques. Tissues examined included the testis, epididymis (caput, corpus, and cauda regions), vas deferens, ejaculatory duct, seminal vesicle, and coagulating gland. NOS activity was measured by use of an assay based on the stoichiometric conversion of [3H]-L-arginine to [3H]-L-citrulline and NO, catalyzed by NOS. Low levels of NOS activity were detected in the testis and seminal vesicle (< 0.5 fmol [3H]-L-citrulline formed/min/mg protein in each tissue). The highest levels of NOS activity were present in the cauda segment of the epididymis and in the vas deferens, each having a sevenfold greater amount of NOS activity than the testis (p < 0.05). Intermediate levels of NOS activity were detected in the coagulating gland (0.863 +/- 0.248 fmol [3H]-L-citrulline formed/min/mg protein), caput epididymidis (0.457 +/- 0.180 fmol [3H]-L-citrulline formed/min/mg protein), and corpus epididymidis (0.631 +/- 0.215 fmol [3H]-L-citrulline formed/min/mg protein). NADPH diaphorase histochemistry and NOS immunohistochemistry localized NOS to neuronal fibers coursing throughout the smooth musculature and subepithelial regions of the epididymis, vas deferens, and ejaculatory duct. Endothelial cells and nerve plexuses within the adventitia of blood vessels supplying reproductive tissues were also positive for NOS. Additional localizations of NOS were within epithelial cells of the epididymis and coagulating gland.(ABSTRACT TRUNCATED AT 250 WORDS)

With the current understanding that nitric oxide (NO) mediates penile erection, the endothelial isoform of NO synthase (eNOS) has been implicated in this function. We undertook this study applying transgenic mice with targeted deletion of the eNOS gene (eNOSϪ/Ϫ mice) as an experimental approach to evaluate the importance of eNOS in cholinergically stimulated erectile function in vivo. Combined pharmacostimulation with intracavernosal carbachol (3 ng) administration and submaximal cavernous nerve (CN) electrical stimulation (16 Hz, 5 millisecond, 1 V) simultaneous with intracavernosal pressure (ICP) monitoring, and both biochemical assay of NO synthase activity and Western blot analysis of eNOS protein content in penile tissue, were performed on eNOSϪ/Ϫ mice and wild-type controls. Combined intracavernosal carbachol administration and submaximal CN electrical stimulation raised the recorded ICP, elicited by CN electrical stimulation alone in wild-type mice (from 35.7 Ϯ 2.7 to 48.1 Ϯ 5.5 mm Hg, P Ͻ .05) but not in eNOSϪ/ Ϫ mice (from 54.9 Ϯ 6.3 to 51.0 Ϯ 9.5 mm Hg, not significant [NS]). Pretreatment with the nonselective nitric oxide synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 100 mg intracavernosally) blocked electrically stimulated ICP responses in eNOSϪ/Ϫ mice to baseline levels (37.8 Ϯ 4.4 vs 12.7 Ϯ 4.0 mm Hg, P Ͻ .05). In penes of eNOSϪ/Ϫ mice, approximately 60% NO synthase activity of wildtype penis levels was retained (NS), and eNOS protein was absent. We concluded that eNOSϪ/Ϫ mice preserve erectile function on the basis of a noncholinergic but NO-dependent mechanism and that eNOS physiologically mediates penile erection under cholinergic stimulation.