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 drag/shear stress in blood vessels to produce NO continuously, a process mediated by the phosphatidylinositol 3-kinase (PI3kinase)/Akt pathway. Here we show that PI3-kinase/Akt 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-kinase/Akt-dependent phosphorylation and activation of eNOS leads to sustained NO production and maximal erection.

Figure 1

Electrical stimulation of the cavernous nerve enhances phosphorylation of Akt-S473 and eNOS-S1177 in the rat penis. (a) Representative Western immunoblots of phospho-Akt-S473 and total Akt (top two rows) or phospho-eNOS-S1177 and total eNOS (bottom two rows) after electrical stimulation of the cavernous nerve for 0 sec, 15 sec, or 5 min. Gel shift of phosphorylated form in the total Akt blot is consistent with phospho-Akt staining. HEK293 cells transfected with a plasmid expressing Akt or eNOS were starved of serum for 24 h [(−) serum] to abolish Akt activity or starved and then stimulated with 50 ng/ml insulin-like growth factor-1 for 5 min [(+) serum] as controls for each set of blots. (b) Changes in phospho-eNOS after electrical stimulation for 0 sec, 15 sec, 5 min, or 5 min followed by 10 min of rest. Bands were quantified in arbitrary units by densitometry. Each bar represents the mean ± SE of phospho-eNOS/eNOS expressed relative to sham control result. The number of measurements is indicated in each bar. *, P < 0.05 vs. control by t test.

Figure 2

Immunohistochemical localization of Akt and eNOS in serial sections of rat penis shows increased staining of phosphorylated forms in the endothelium after electrical stimulation. Rats were sham-treated or electrically stimulated for 5 min before excising the penis and staining for the indicated proteins. Low-power views (×40) show a cross-section of the penis with major structures marked in the last micrograph. (Insets) Segments of penile vascular endothelium stained for phospho-Akt or phospho-eNOS (×400) are shown. Factor VIII staining is specific for the endothelium. A, artery; V, dorsal vein. The arrows indicate dorsal nerves.

Figure 3

Wortmannin and LY294002 reduce ICP increases elicited by electrical stimulation of the cavernous nerve. (a) Representative ICP responses for individual rats stimulated 10 min after intracavernosal injection of wortmannin (260 ng), LY294002(900 ng), or DMSO alone (0.15%). The stimulus interval is indicated by a solid bar. (b) Mean maximal ICP, total ICP, and tumescence slope calculated for rats treated with the indicated doses of wortmannin (left side) or LY294002 (right side). The slope and area under the curve were calculated from ICP recordings normalized to mean arterial pressure (MAP). Each bar depicts the mean ± SE for n = 7 (wortmannin) or 8 (LY294002) animals. *, P < 0.05 vs. vehicle alone by ANOVA.

Figure 4

Increased ICP after intracavernous papaverine injection (PAP) is reduced by PI3-kinase inhibitors. (a) Representative ICP tracings for erections elicited in rats by 1.5 mg of papaverine 10 min after intracavernous injection of wortmannin (WORT, 260 ng), LY294002 (LY, 900 ng), or DMSO alone (0.15%). (b) Mean ± SE for maximal ICP of the indicated number of animals in each treatment group. *, P < 0.05 vs. DMSO alone by ANOVA.

Figure 5

eNOS^−/− mice show decreased erectile response to papaverine injection (PAP) compared with wild-type and nNOS^−/− animals. (a) Representative ICP tracings showing 0.4 mg of papaverine's effect on ICP in individual mice. Injection of papaverine is indicated, with elevation of ICP occurring ≈5 min later. (b) Mean ± SE for maximal ICP response in wild-type, eNOS^−/−, and nNOS^−/− mice after intracavernous injection of papaverine. The number of animals tested is indicated in each bar. *, P < 0.01 vs. wild-type response by ANOVA.

Figure 6

A model depicting initiation of erection by nNOS and its maintenance by eNOS. Initial stimuli from higher neural centers elicit a relatively short-lived burst of NO from nerve fibers throughout the penile tissue that relaxes smooth muscle, enhancing blood flow. PI3-kinase/Akt activation by the resulting shear stress phosphorylates eNOS (indicated with P) in endothelial cells and provides a sustained increase in NO leading to maximal relaxation that does not require continuous neuronal depolarization. CaM, calmodulin.