Julie K. Crone

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

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.

Ablation of renal tumors in a rabbit model with interstitial saline-augmented radiofrequency energy: preliminary report of a new technology

OBJECTIVES To evaluate the efficacy of interstitial saline radiofrequency energy for reproducibly ablating nonmalignant (control) and malignant (the VX-2 tumor) renal tissue in a rabbit model, and to determine the ability of conventional gray-scale and power sonography to image the tumor and ablative process in real time before, during, and after treatment. METHODS The VX-2 tumor was implanted beneath the renal capsule in 18 rabbit kidneys. Twelve days after implantation, 50 W of 500-kHz radiofrequency energy was delivered into the surgically externalized renal tumor and contralateral control kidney for 30 or 45-second treatment intervals using an interstitial saline-augmented radiofrequency probe (the virtual electrode). Localization of the tumor and response to treatment were imaged with gray-scale and power Doppler ultrasonography. The effect of radiofrequency and extent of the destructive process on benign and malignant renal tissue were evaluated histologically. RESULTS Mean tumor size was 1.3 x 0.7 cm. Both 30 and 45-second treatment intervals provided marked tissue/tumor ablation. Gross anatomic and histologic analysis showed time-dependent ablated lesions averaging 1.4+/-0.3 x 1.0+/-0.3 cm (30-second treatment) and 1.8+/-0.4 x 1.5+/-0.3 cm (45-second treatment), with clear demarcation of the surrounding parenchyma. Conventional gray-scale sonography allowed visualization of the ablative process, and power Doppler ultrasound demonstrated changes in the vascular pattern of the tumor both before and after ablation. No immediate treatment-related complications were observed. CONCLUSIONS These preliminary studies in a rabbit model demonstrate the feasibility of using the interstitial saline-augmented electrode to ablate small renal tumors and the ability to simultaneously visualize the ablative process using real-time ultrasonography. This technology may have the potential to treat small renal tumors in a minimally invasive manner in the clinical setting.

Combined loss of neuronal and endothelial nitric oxide synthase causes premature mortality and age-related hypertrophic cardiac remodeling in mice.

Deficiency of either neuronal nitric oxide synthase (NOS1) or endothelial nitric oxide synthase (NOS3) leads to cardiac hypertrophy in mice. Loss of both produces concentric left ventricular (LV) remodeling, in which increased wall thickness is accompanied by reduced cavity size. In humans, this phenotype develops in elderly hypertensive patients and independently predicts mortality. Accordingly, we tested the hypothesis that NOS1/3(-/-) mice have reduced longevity compared to either NOS1(-/-) or NOS3(-/-). Survival data on colonies of NOS1(-/-) (n = 295), NOS3(-/-) (n = 525), and NOS1/3(-/-) (n = 331) mice were collected for 2 years. NOS1(-/-) mice had increased mortality compared to NOS3(-/-) (relative risk, RR 2.5, P < 0.001), whereas NOS1/3(-/-) fared significantly worse (RR 7.3, P < 0.001 vs. NOS3(-/-)). Importantly, gender did not affect survival in NOS1(-/-) or NOS3(-/-), but male NOS1/3(-/-) mice had 2-fold increased mortality compared to females. NOS1/3(-/-) mice developed progressive myocyte hypertrophy and interstitial fibrosis with age. NOS1/3(-/-) mice underwent in vivo hemodynamic analysis with a combined pressure-volume catheter to assess age-related cardiovascular changes. Compared with control, NOS1/3(-/-) demonstrated hypertension and hypercontractility at all ages, and developed passive diastolic dysfunction with increasing age. Thus, combined deficiency of NOS1 and NOS3 causes increased mortality, myocyte hypertrophy, and an age-associated increase in ventricular stiffness. These findings suggest that cardiac NO signals may play an essential role in successful cardiac aging.

Phosphorylated Endothelial Nitric Oxide Synthase Mediates Vascular Endothelial Growth Factor-Induced Penile Erection

Abstract The objective of the present study was to evaluate whether vascular endothelial growth factor (VEGF)-induced penile erection is mediated by activation of endothelial nitric oxide synthase (eNOS) through its phosphorylation. We assessed the role of constitutively activated eNOS in VEGF-induced penile erection using wild-type (WT) and eNOS-knockout (eNOS−/−) mice with and without vasculogenic erectile dysfunction. Adult WT and eNOS−/− mice were subjected to sham operation or bilateral castration to induce vasculogenic erectile dysfunction. At the time of surgery, animals were injected intracavernosally with a replication-deficient adenovirus expressing human VEGF145 (109 particle units) or with empty virus (Ad.Null). After 7 days, erectile function was assessed in response to cavernous nerve electrical stimulation. Total and phosphorylated protein kinase B (Akt) as well as total and phosphorylated eNOS were quantitatively assessed in mice penes using Western immunoblot and immunohistochemistry. In intact WT mice, VEGF145 significantly increased erectile responses, and in WT mice after castration, it completely recovered penile erection. However, VEGF145 failed to increase erectile responses in intact eNOS−/− mice and only partially recovered erectile function in castrated eNOS−/− mice. In addition, VEGF145 significantly increased phosphorylation of eNOS at Serine 1177 by approximately 2-fold in penes of both intact and castrated WT mice. The data provide a molecular explanation for VEGF stimulatory effect on penile erection, which involves phosphorylated eNOS (Serine 1177) mediation.

The effect of androgen on nitric oxide synthase in the male reproductive tract of the rat**Supported by United States Public Health Service grants HD30137 and DK19300–18 and training grant H32–6647, Bethesda, Maryland.††Presented at the Conjoint Meeting of The American Fertility Society and the Canadian Fertility and Andrology Society, Montreal, Quebec, Canada, October 11 to 14, 1993.

OBJECTIVE To determine if nitric oxide synthase activity within the male reproductive tract is regulated by androgen. DESIGN Nitric oxide synthase activity was measured in the reproductive organs of three groups of mature rats: unoperated controls, 1-week castrates, and 1-week castrates given T capsules at the time of surgery. The presence of nitric oxide synthase activity was confirmed by using the nitric oxide synthase-specific inhibitor N-nitro-L-arginine methyl ester (L-NAME). RESULTS After castration, nitric oxide synthase activity was significantly reduced by 88%, 73%, and 54% in the caput, corpus, and cauda epididymidis, respectively. In the penis, nitric oxide synthase activity decreased 45% and nitric oxide synthase protein decreased 57% after castration. In the seminal vesicle and lateral prostate, nitric oxide synthase activity increased significantly after castration from nondetectable levels in controls. Nitric oxide synthase activity in the coagulating gland and ventral and dorsal prostate did not change after castration. The changes in nitric oxide synthase activity in all organs after castration were prevented by T replacement. Additionally, the activity measured in every organ in all three treatment groups was > 90% inhibited by L-NAME. CONCLUSION These data demonstrate that androgen differentially affects nitric oxide synthase activity in the male reproductive tract. To the best of our knowledge this is the first time that nitric oxide synthase activity has been shown to be influenced by androgen in any tissue.