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...

Impaired endothelial nitric oxide synthase (eNOS) function is associated with erectile dysfunction in diabetes mellitus, but the exact molecular basis for the eNOS defect in the diabetic penis remains unclear. We investigated whether hyperglycemia increases O-GlcNAc modification of eNOS in the penis, preventing phosphorylation at the primary positive regulatory site on the enzyme and hampering mechanisms of the erectile response. Type I diabetes mellitus was induced in male rats by alloxan (140 mg͞kg, i.p.). After 5 wk, the diabetic rat penis exhibited increased OGlcNAc modification of eNOS and decreased eNOS phosphorylation at Ser-1177 at baseline compared with the control rat penis; eNOS phosphorylation at Thr-495, Ser-615, and Ser-633 was not affected. In addition, eNOS phosphorylation at Ser-1177 was impaired in the diabetic rat penis in response to penile blood flow (shear stress) elicited by electrical stimulation of the cavernous nerve (ES) and to recombinant human VEGF165. Phosphorylation of Akt, a mediator of shear stress-induced eNOS phosphorylation at Ser-1177, was decreased in the diabetic penis at baseline, but it was restored by ES. Erectile response to shear stress elicited by ES and to VEGF was decreased in diabetic compared with control rats. This work demonstrates that eNOS inactivation occurs in the diabetic penis by a glycosylation mechanism specifically at Ser-1177, by which the enzyme is rendered incapable of activation by fluid shear stress stimuli and VEGF signaling. In vivo penile erection paradigm supports the physiologic relevance of O-GlcNAc modification in vascular disorders associated with diabetes.penis ͉ penile erection ͉ Akt ͉ VEGF T he role of endothelial nitric oxide synthase (eNOS) in penile erection is becoming increasingly recognized. Although initiation of erection is mediated by rapid, short-term calciumdependent release of NO from neuronal NOS-containing nerve terminals in the penis, full erection and maintenance of erection is achieved by the blood flow-induced phosphatidylinositol 3-kinase͞Akt͞eNOS (Ser-1177) phosphorylation cascade, resulting in reduced calcium dependence and sustained release of endothelial NO and continued cavernous smooth muscle relaxation (1). In addition, VEGF induces eNOS phosphorylation at Ser-1177 in the penis and promotes penile erection, but this action appears to be independent of Akt activation (2). In contrast to Ser-1177, Thr-495 acts on eNOS as a negative regulatory site, i.e., phosphorylation of this site is associated with a decrease in eNOS activity by increasing calcium͞calmodulin dependence of the enzyme (3-5). The physiological significance of phosphorylation of other sites on eNOS is less known and somewhat controversial. The phosphorylation of Ser-615 and Ser-633 causes activation of eNOS function (6, 7), whereas the phosphorylation of Ser-114 reduces eNOS function (8) (Fig. 1). Moreover, the phosphorylation of a site on eNOS may be affected by the state of phosphorylation of other residues on eNOS (9).Impairment of penile vascul...

These results document that PC3, but not DU145 cells retain the coregulators needed for AR tumor suppressor ability.

Purpose: Prostatic dihydrotestosterone (DHT) concentration is regulated by precursors from systemic circulation and prostatic enzymes of androgen metabolism, particularly 5a-reductases (i.e., SRD5A1 and SRD5A2). Therefore, the levels of expression SRD5A1 and SRD5A2 and the antiprostatic cancer growth response to finasteride, a selective SRD5A2 inhibitor, versus the dual SRD5A1and SRD5A2 inhibitor, dutasteride, were compared. Experimental Design: Real-time PCR and enzymatic assays were used to determine the levels of SRD5A1and SRD5A2 in normal versus malignant rat and human prostatic tissues. Rats bearing the Dunning R-3327H rat prostate cancer and nude mice bearing LNCaP or PC-3 human prostate cancer xenografts were used as model systems. Tissue levels of testosterone and DHT were determined using liquid chromatography-mass spectrometry. Results: Prostate cancer cells express undetectable to low levels of SRD5A2 but elevated levels of SRD5A1activity compared with nonmalignant prostatic tissue. Daily oral treatment of rats with the SRD5A2 selective inhibitor, finasteride, reduces prostate weight and DHTcontent but did not inhibit R-3327H rat prostate cancer growth or DHTcontent in intact (i.e., noncastrated) male rats. In contrast, daily oral treatment with even a low 1mg/kg/d dose of the dual SRD5A1and SRD5A2 inhibitor, dutasteride, reduces both normal prostate and H tumor DHTcontent and weight in intact rats while elevating tissue testosterone. Daily oral treatment with finasteride significantly (P < 0.05) inhibits growth of LNCaP human prostate cancer xenografts in intact male nude mice, but this inhibition is not as great as that by equimolar oral dosing with dutasteride. This anticancer efficacy is not equivalent, however, to that produced by castration. Only combination of dutasteride and castration produces a greater tumor inhibition (P < 0.05) than castration monotherapy against androgen-responsive LNCaP cancers. In contrast, no response was induced by dutasteride in nude mice bearing androgen-independent PC-3 human prostatic cancer xenografts. Conclusions: These results document that testosterone is not as potent as DHT but does stimulate prostate cancer growth, thus combining castration with dutasteride enhances therapeutic efficacy.