大鼠阴茎海绵体NOSⅠ、NOSⅢ mRNA和蛋白表达研究

目的 :探讨正常大鼠阴茎海绵体中一氧化氮合酶 (NOS) Ⅰ和 Ⅲ mRNA和蛋白的表达。　方法 :取雄性SD大鼠阴茎组织 ,应用高度特异性抗体经免疫沉淀、Western印迹分析技术分别检测NOSⅠ 、NOS Ⅲ 蛋白的表达 ;RT PCR方法检测NOSⅠ和NOS Ⅲ mRNA的表达。　结果 :正常大鼠阴茎海绵体组织中有NOSⅠ 和NOS ⅢmRNA和蛋白的表达 ,并且NOSⅢmRNA和蛋白的表达明显多于NOSⅠmRNA和蛋白的表达。　结论 :不同的NOS亚型可能共同参与阴茎勃起的调控。     

Structural and functional investigations of the murine cavernosal nerve: a model system for serial spatio-temporal study of autonomic neuropathy

OBJECTIVE: To illustrate the ultrastructural fibre composition of the rat cavernosal nerve at serial levels, from its origin in the main pelvic ganglion to its termination in the corpus cavernosum of the distal penile shaft, and to develop a technique that permits repeated electrophysiological recording from the fibres that form the cavernosal nerve distinct from the axons of the dorsal nerve of the penis (DNP). MATERIALS AND METHODS: For the light microscope and ultrastructural studies, Sprague-Dawley rats were anaesthetized and the pelvic organs and lower limbs were perfused with glutaraldehyde through the distal aorta. Tissue samples were embedded in epoxy resin and prepared for light and electron microscopy. Frozen tissue was used for the immunohistochemical studies and sections were stained with rabbit anti-nitric oxide synthetase 1 (NOS1). For the electrophysiology, anaesthetized rats were used in sterile conditions. Nerve conduction velocity for the cavernosal nerve was assessed from a point 2 mm below the main (major) pelvic ganglion after stimulating the nerve at the crus penis; multi-unit averaging techniques were used to enhance the recording of slow-conduction activity. Recordings from the DNP were obtained over the proximal shaft after stimulation at the base of the penis. RESULTS: Step-serial sections of the cavernosal nerve revealed numerous ganglion cells in the initial segments and gradually fewer myelinated fibres at distal levels. At the point of crural entry, the nerve contained almost exclusively unmyelinated axons. As it descended the penile shaft, the nerve separated into small fascicles containing only one to four axons at the level of the distal shaft. In the corpus cavernosum, vesicle-filled presynaptic axon preterminals were close to smooth muscle fibres, but did not seem to be in direct contact. Immunohistochemical evaluation of NOS1 activity showed intense staining of the fibres of the DNP and most of the neurones in the main pelvic ganglion. There was also scattered NOS1 activity in the nerve bundles of the corpus cavernosum. Electrophysiology identified activity in C fibres on the cavernosal nerve and in Aalpha-Adelta fibres in the DNP. CONCLUSION: These results show that it is possible to perform integrated cavernosal pressure monitoring and ultrastructural and electrophysiological studies in this model. These yielded accurate data about the erectile status of the penis, and the state of unmyelinated and myelinated fibres in the DNP and cavernosal nerves of the same animal. This study provides a useful template for future studies of experimental diabetic autonomic neuropathy.     

Nitric oxide-cyclic GMP pathway with some emphasis on cavernosal contractility

Nitric oxide (NO) is formed from the conversion of L-arginine by nitric oxide synthase (NOS), which exists in three isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). nNOS is expressed in penile neurons innervating the corpus cavernosum, and eNOS protein expression has been identified primarily in both cavernosal smooth muscle and endothelium. NO is released from nerve endings and endothelial cells and stimulates the activity of soluble guanylate cyclase (sGC), leading to an increase in cyclic guanosine-3',5'-monophosphate (cGMP) and, finally, to calcium depletion from the cytosolic space and cavernous smooth muscle relaxation. The effects of cGMP are mediated by cGMP dependent protein kinases, cGMP-gated ion channels, and cGMP-regulated phosphodiesterases (PDE). Thus, cGMP effect depends on the expression of a cell-specific cGMP-receptor protein in a given cell type. Numerous systemic vasculature diseases that cause erectile dysfunction (ED) are highly associated with endothelial dysfunction, which has been shown to contribute to decreased erectile function in men and a number of animal models of penile erection. Based on the increasing knowledge of intracellular signal propagation in cavernous smooth muscle tone regulation, selective PDE inhibitors have recently been introduced in the treatment of ED. Phosphodiesterase 5 (PDE5) inactivates cGMP, which terminates NO-cGMP-mediated smooth muscle relaxation. Inhibition of PDE5 is expected to enhance penile erection by preventing cGMP degradation. Development of pharmacologic agents with this effect has closely paralleled the emerging science.     

Soluble guanylate cyclase and cGMP-dependent protein kinase I expression in the human corpus cavernosum

Nitric oxide (NO) as a mediator in smooth muscle cells causes rapid and robust increases in cGMP levels. The cGMP-dependent protein kinase I has emerged as an important signal transduction mediator for smooth muscle relaxation. The purpose of this study was to examine the existence and distribution of two key enzymes of the NO/cGMP pathway, the cGMP-dependent kinase I (cGK I) and the soluble guanylate cyclase (sGC) in human cavernosal tissue. The expression of the enzymes were examined in corpus cavernosum specimens of 23 patients. Eleven potent patients suffered from penile deviations and were treated via Nesbit's surgical method. Nine long-term impotent patients underwent implantation of flexible hydraulic prothesis. Three potent patients underwent trans-sexual operations. Expression of the sGC and cGK I were examined immunohistochemically using specific antibodies. In all specimens of cavernosal tissue a distinct immunoreactivity was observed in different parts and structures. We found a high expression of sGC and cGK I in smooth muscle cells of vessels and in the fibromuscular stroma. The endothelium of the cavernosal sinus, of the cavernosal arteries, and the cavernosal nerve fibers showed an immunoreactivity against sGC. The distribution analysis of cGK I revealed a predominately vesicular localization in smooth muscle cells. The examination of the endothelium showed no clear immunoreactivity against cGK I. There was no distinct difference in immunoreactivity and cellular distribution between potent and impotent patients.     

Nitric Oxide and the Cyclic GMP System in the Penis

Since the discovery that nitric oxide is the mediator of penile erection, considerable evidence has been accumulated in experimental animals and men regarding the pharmacology and molecular biology of the nitric oxide/cGMP pathway in the penis. This review examines the expression and tissue localization of nitric oxide synthase isoforms (NOS) in this organ and the functional significance of their variants. The disregulation of the balance between nitric oxide synthesis and the compliance of the corpora cavernosa smooth muscle in relation to erectile dysfunction in animal models is discussed. The possible up-regulation of NOS levels and the interaction of NOS variants with protein modulators of their activity is analyzed in the context of novel concepts of gene therapy of erectile dysfunction.     

Nitric oxide may mediate nipple erection

The nipple is a specialized structure that can become erect by cold, sexual arousal, breast-feeding, or other tactile stimulations, which can induce the milk ejection reflex and sexual arousal because of intense sensory innervation. The studies that have been conducted thus far to identify the mechanism of nipple erection (NE) are not sufficient. It has been stated that NE occurs via activation of the sympathetic nervous system and smooth muscle contraction. The purposes of this study were to investigate the existence of nitric oxide synthase (NOS) in the nipple-areola complex (NAC) to explain the NE mechanism. Considering that smooth muscle relaxation might be effective in NE, endothelial and neuronal NOS expression and localization were investigated via immunohistochemical methods on sagittal sections from 17 human NACs. The results of this study indicate that eNOS is expressed in the vascular endothelium, ductal epithelium, and smooth muscles, whereas nNOS is expressed in the neural fibers, smooth muscles, ductal epithelium, and vascular endothelium in the NAC. Sinusoidal spaces with endothelial layers similar to those found in penile cavernosal tissue are not found in the NAC. Various mediators are known to affect the function of the NAC smooth muscles; however, this study demonstrates that enzymes (eNOS and nNOS) that synthesize nitric oxide are expressed in the NAC.     

Histopathological changes and nitric oxide synthase activity in corpus cavernosum from rats with neurogenic erectile dysfunction

OBJECTIVE: To investigate changes in histology and nitric oxide synthase (NOS) activity in cavernosal tissues from rats with neurogenic erectile dysfunction induced experimentally. MATERIALS AND METHODS: Twenty-four adult male Sprague-Dawley rats were divided equally into three groups and underwent a sham operation (control, group 1), unilateral (group 2) or bilateral (group 3) cavernosal nerve resection. Three months later they were killed and the cavernosal tissues analysed histologically by light and transmission electron microscopy, with NOS activity detected using an NADPH-diaphorase staining technique. RESULTS: On light and electron microscopy, while penile nerves and cavernosal smooth muscle cells had a normal morphological appearance in the eight control rats, there were degenerative changes of the myelinated penile nerves and axonal fibrosis in groups 2 and 3. However, these changes were not significant. Using NADPH-diaphorase staining, NOS activity was detected in all three groups in endothelial cells and cavernosal structures. However, the staining was more intense in endothelial cells and cavernosal muscles of rats in group 2 than in the other groups. CONCLUSION: NOS activity was increased in the cavernosal tissue after cavernosal denervation, but the pharmacological action of nitric oxide may be impaired.     

Advanced glycation end products in human penis: elevation in diabetic tissue, site of deposition, and possible effect through inos or enos

Objectives

We hypothesized that advanced glycation end product (AGE) formation contributes to erectile dysfunction (ED) by quenching nitric oxide. Our first goal was to identify the specific AGE pentosidine in the diabetic human penis. Because AGE-mediated effects may involve inducible nitric oxide synthase (iNOS), we performed immunohistochemical and Western blot analysis of diabetic and nondiabetic human penile tissue for iNOS. Finally, because AGEs may act intracellularly to affect proteins, we set out to identify endothelial NOS (eNOS) in the human penis as an initial step in examining a possible intracellular interaction between eNOS and AGEs.

Methods

We performed high-performance liquid chromatographic analysis of diabetic human penile corpus cavernosum and serum for pentosidine and performed immunohistochemical, electron microscopic (EM), and Western blot analysis of the diabetic and nondiabetic penile corpus cavernosum and tunica for pyrraline, iNOS, and eNOS (and neural NOS [nNOS]for comparative purposes) via standard methods.

Results

We found a significant elevation of pentosidine in the penile tissue but not the serum of diabetic patients (average age 55.6 ± 2.3 years) compared with that of nondiabetic patients (average age 61.8 ± 3.6 years). Pentosidine was 117.06 ± 9.19 pmol/mg collagen in the diabetic tunica versus 77.58 ± 5.5 pmol/mg collagen in the nondiabetic tunica (P < 0.01) and 74.58 ± 8.49 pmol/mg collagen in the diabetic corpus cavernosum versus 46.59 ± 2.53 pmol/mg collagen in the nondiabetic corpus cavernosum (P < 0.01), suggesting a tissue-specific effect of the AGEs. We localized the site of deposition of the specific AGE pyrraline to the human penile tunica and the penile corpus cavernosum collagen. Immunohistochemical and EM analysis localized eNOS and iNOS to the cavernosal endothelium and smooth muscle. Western blot analysis in 6 patients revealed the following: iNOS, but no eNOS, in penile tissue from 1 insulin-dependent diabetic man; eNOS only in 1 man after radical prostatectomy; both eNOS and iNOS in 2 men with Peyronie's disease, as well as in 2 other men with impotence and hypertension. Finally, the specific iNOS inhibitor PNU-19451A significantly augmented relaxation of precontracted human cavernosal tissue, from 64.7% ± 5.58 to 80.03% ± 4.55 at 10 μM acetylcholine and 65.06% ± 2.84 to 86.16% ± 3.96 at 0.1 mM acetylcholine (n = 4, P < 0.002 and P < 0.02, respectively).

Conclusions

AGEs are elevated in diabetic human penile tissue, but not in serum, and are localized to the collagen of the penile tunica and corpus cavernosum. We identified eNOS and iNOS in the human penile cavernosal smooth muscle and endothelium. The augmentation of cavernosal relaxation with a specific iNOS inhibitor, combined with the identification of iNOS protein, but not eNOS, in a patient with severe diabetes and ED, allows for speculation of a pathophysiologic mechanism for AGE-mediated ED via upregulation of iNOS and downregulation of eNOS. These data provide further insight into the mechanisms of advanced glycation end product-mediated ED and provide a foundation for further study. UROLOGY 50: 1016-1026, 1997.     

Expression of inducible nitric oxide synthase in smooth muscle cells from rat penile corpora cavernosa

Nitric oxide (NO), the main mediator of penile erection, is assumed to be synthesized in the penis by the neuronal constitutive nitric oxide synthase (nNOS). However, nNOS has not been identified in the penile smooth muscle, the target of NO action. The other NOS isozymes, the inducible NOS (iNOS) and the endothelial NOS (eNOS) have not been reported in any penile tissue. The smooth muscle vascular and trabecular tissue from rat corpora cavernosa is represented in vitro by cell cultures designated RPSMC. To determine whether iNOS can be expressed in penile smooth muscle, RPSMC were treated with different lymphokines and/or bacterial lipopolysaccharide (LPS). The selected inducer, LPS/interferon, elicited at 48 hours up to a 50-fold increase in nitrites in the medium; the nitroarginine methyl ester (L-NAME), aminoguanidine, actinomycin D, cycloheximide, transforming growth factor-beta1 (TGF-beta1), and dexamethasone, but was resistant to nifedipine and platelet-derived growth factor AB (PDGF-AB). iNOS induction increased with cell passage. The [3H]L-arginine/citrulline measurement of NO synthesis with intact cells confirmed these results. Incubations of soluble and particulate fractions showed that the cytosol contained most of the activity (Km = 43 microM), which was partially inhibited by ethyleneglycal-bis-tetraacetic acid (EGTA). The 4.4-kb iNOS mRNA peaked at a late period (24-30 hours) and remained high for up to 72 hours. iNOS mRNA induction was strongly inhibited by actinomycin D and dexamethasone, partially inhibited by TGF-beta1, inhibited slightly by PDGF-AB, and unaffected by nifedipine. These results show that iNOS can be expressed in RPSMC in a cell passage-dependent fashion that has so far not been reported for other cell lines, and that the induction reaches much higher levels than in rat or human vascular smooth muscle cells. The expression pattern is also distinctive for the penile cells in time course of induction, Ca2+ dependence, response to certain agents, and mRNA stability.     

Immunocytochemical distribution of nitric oxide synthase in the human corpus cavernosum: an electron microscopical study using the tyramide signal amplification technique

Nitric oxide has proven to be an important mediator in the relaxation of human cavernosal smooth muscle. Nevertheless, there are many inconsistencies in the literature regarding the cellular and subcellular distribution of endothelial nitric oxide synthase in the human penis. The purpose of this study was to reexamine the localization of eNOS and nNOS in the cellular anatomy of the human cavernous body by means of electron microscopical immunocytochemistry in combination with the tyramide signal amplification technique (TSA). Using specific antibodies against eNOS and nNOS, the NAPDH-diaphorase reaction and advanced protocols for fixation and staining procederes, the occurrence of NOS isoenzymes eNOS and nNOS were examined in cavernosal specimens of ten male patients who were subjected to surgery for penile deviation. eNOS immunoreactivity and NADPH-d staining was seen to be significantly present in the endothelial cells covering the cavernous spaces and in the endothelium of helicine arteries. In endothelial cells, the NADPH-d reaction product BSPT-formazan was abundantly detectable attached to membranes of the endoplasmatic reticulum and the mitochondria whereas posititve eNOS immunostaining was seen in the endothelial cells throughout their cytoplasm without any particular relation to organelles. No considerable eNOS immunoreactivity was detectable in the trabecular smooth muscle cells. nNOS staining was found in nerve fibers innervating the cavernous body and cavernosal arteries. Our results counteract the hypothesis of the cavernous smooth muscle as a local source of NO and underline the importance of an intact endothelial function for penile erection and the contribution of eNOS to this process. Received: 14 September 2000 / Accepted: 9 February 2001     

Novel nitric oxide signaling mechanisms regulate the erectile response

Nitric oxide (NO) is a physiologic signal essential to penile erection, and disorders that reduce NO synthesis or release in the erectile tissue are commonly associated with erectile dysfunction. NO synthase (NOS) catalyzes production of NO from L-arginine. While both constitutively expressed neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms mediate penile erection, nNOS is widely perceived to predominate in this role. Demonstration that blood-flow-dependent generation of NO involves phosphorylative activation of penile eNOS challenges conventional understanding of NO-dependent erectile mechanisms. Regulation of erectile function may not be mediated exclusively by neurally derived NO: Blood-flow-induced fluid shear stress in the penile vasculature stimulates phosphatidyl-inositol 3-kinase to phosphorylate protein kinase B, which in turn phosphorylates eNOS to generate NO. Thus, nNOS may initiate cavernosal tissue relaxation, while activated eNOS may facilitate attainment and maintenance of full erection.     

Effects of castration and testosterone replacement on veno-occlusion during penile erection in the rat

AIM: To determine if androgens directly regulate veno-occlusion or if androgens act indirectly to maintain the penile structures which control outflow. METHODS: Using CASTRATE and TESTO rats, measurement was made of mean arterial pressure (MAP), intracavernosal pressure (CCP), and intracavernosal flow (CCF) during erection resulting from stimulation of the autonomic innervation of the penis. CCP and CCF were also measured during saline infusion into the cavernosal sinuses before and after treatment with sodium nitroprusside (SNP, a nitric oxide donor drug) to fully relax cavernosal smooth muscle. Penile tissue was also collected to measure the content of alpha actin and proline and hydroxyproline to determine if brief withdrawal of androgenic support led to changes in the number of smooth muscle cells or the collagen content of the tissue. RESULTS: Infusion of saline into the cavernosal sinuses demonstrated that veno-occlusion was defective in CASTRATE rats while veno-occlusion was fully functional in TESTO animals. Furthermore, veno-occlusion could be induced in CASTRATE rats if they were first treated with SNP. This observation suggests that failure of veno-occlusion in the CASTRATE rats is due to a deficiency in the production of NO resulting in a reduction in the degree of relaxation of the penile smooth muscle. The measurements of smooth muscle a actin and proline and hydroxyproline content of collagen showed that both were unaffected by castration and that the basic structure of the penis did not degenerate after one week without androgenic support. CONCLUSION: These results can be interpreted to mean that androgens control the veno-occlusive mechanism indirectly via a NO dependent mechanism and not by maintaining the structures of the penis which are essential to veno-occlusion.     

Neuronal and endothelial nitric oxide synthase isoforms: quantification of protein and mRNA in the normal rat penis

Nitric oxide synthase (NOS) is an important enzyme for erection. We evaluated the content of neuronal (nNOS) and endothelial (eNOS) isoforms and their mRNA in the penis and major pelvic ganglion (MPG) of adult male rats by Western and Northern blot analysis. The cerebellum was evaluated as a control. nNOS protein and its mRNA were detected in abundance in the MPG, cerebellum, pelvic urethra and within the crura of the penis. In contrast, the penile urethra, neurovascular bundle and the shaft of penis contained smaller amounts of this protein. eNOS protein was most abundant in the penile and pelvic parts of the urethra, whereas a moderate level was found in the penile shaft, crura, neurovascular bundle, MPG and cerebellum. Similarly eNOS mRNA was abundant in the penile and pelvic parts of the urethra, MPG and cerebellum. Penile shaft, crura and neurovascular bundle showed moderate amounts of eNOS mRNA. In conclusion, nNOS and its mRNA are most abundant in the MPG and crura of penis whereas eNOS is most abundant in the urethra and to a lesser extent present in the penis. Importantly eNOS protein and mRNA were demonstrated in the MPG, where eNOS function has to be studied.     

Gene transfer of endothelial nitric oxide synthase partially restores nitric oxide synthesis and erectile function in streptozotocin diabetic rats

PURPOSE: We determined whether adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis of streptozotocin induced diabetic rats could improve the impaired erectile response. MATERIALS AND METHODS: Two experimental groups of animals were transfected with adenoviruses, including streptozotocin (Sigma Chemical Company, St. Louis, Missouri) diabetic rats with AdCMVbetagal and streptozotocin diabetic rats with AdCMVeNOS. At 1 to 2 days after transfection these study animals underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age matched control rats. In control and transfected streptozotocin diabetic rats eNOS and neuronal NOS (nNOS) were examined by Western blot analysis. Constitutive and inducible NOS activities were evaluated in the presence and absence of calcium by L-arginine to L-citrulline conversion and nitrate plus nitrite levels were measured. In control and streptozotocin diabetic penes beta-galactosidase activity and localization were determined. RESULTS: After transfection with AdCMVbetagal beta-galactosidase was localized to the endothelium and smooth muscle cells of the streptozotocin diabetic rat penis. Streptozotocin diabetic rats had a significant decrease in erectile function, as determined by peak and total intracavernous pressure (area under the curve) after cavernous nerve stimulation compared with control rats. Streptozotocin diabetic rats transfected with AdCMVeNOS had peak intracavernous pressure and area under the curve similar to those in control animals. This change in erectile function was a result of eNOS over expression with an increase in eNOS protein expression and constitutive NOS activity as well as an increase in nitric oxide biosynthesis, as reflected by an increase in cavernous nitrate plus nitrite formation. There was no change in nNOS protein expression or calcium independent conversion of NOS (inducible NOS activity). CONCLUSIONS: Adenoviral gene transfer of eNOS significantly increased peak and total intracavernous pressure to cavernous nerve stimulation in streptozotocin diabetic rats to a value similar to the response observed in control rats. Our results suggest that eNOS contributes significantly to the physiology of penile erection. These data demonstrate that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the streptozotocin diabetic rat.     

RhoA/Rho激酶在阴茎勃起中的作用

阴茎海绵体血管的张力受血管收缩和舒张因子的调控。通常认为NO舒张阴茎小动脉和海绵体平滑肌在阴茎勃起中具有重要作用。最近研究发现,RhoA/Rho激酶参与收缩因子去甲肾上腺素(NE)和内皮素1(ET-1)介导的阴茎小动脉和海绵体平滑肌收缩过程,与NO介导的海绵体平滑肌舒张过程有相互作用,NO的勃起效应可能与阻断RhoA/Rho激酶介导的阴茎海绵体血管平滑肌收缩有关。RhoA/Rho激酶抑制剂在ED的治疗中有广阔的应用前景。     

Human and rabbit cavernosal smooth muscle cells express Rho-kinase

Rho-kinase is an enzyme involved in the Ca2+-sensitizing pathway in smooth muscle cells. Inhibition of this enzyme has been recently demonstrated to elicit penile erection by relaxing cavernosal smooth muscle. We aimed to investigate the presence and activity of Rho-kinase in human cavernosal smooth muscle. Primary culture of smooth muscle cells from human and rabbit penile corpus cavernosum was developed, and cells showed characteristic myocyte morphology and alpha-actin immunoreactivity. The presence of Rho-kinase was demonstrated by indirect immunofluorescence and Western blotting. A specific inhibitor of Rho-kinase, Y-27632, inhibited in a concentration-dependent manner the kinase activity of the protein immunoprecipitated with anti-Rho-kinase antibody. These results demonstrate for the first time expression and activity of Rho-kinase in human penile cavernosal smooth muscle cells and suggest that these cells can provide a cellular model for the study of enzymes involved in Ca2+-sensitizing pathways.     

Adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis improves age-related erectile dysfunction in the rat

Nitric oxide (NO) is the principal mediator of penile erection. NO is synthesized by a variety of nitric oxide synthases (NOS). It has been demonstrated that a decrease in NOS activity, as observed in aging, is associated with a diminished erectile response. The objective of this study was to determine if adenoviral-mediated gene transfer of eNOS could reverse age-related erectile dysfunction in the rat. Two groups of animals were transfected with adenoviruses: (1) aged rats (60 weeks) with AdRSVbetagal; and (2) aged rats (60 weeks) with AdRSVeNOS. Five days after transfection, these study animals underwent cavernosal nerve stimulation (CNS) to assess erectile function and their responses were compared with young (20 weeks) control rats. Cross-sections of the rat penises transfected with AdRSVeNOS were examined after trichrome staining. Adenoviral transduction efficiency of beta-galactosidase reporter gene was measured by a galacto-light chemiluminescent reporter gene assay in cavernosal tissues of rats administered AdRSVbetagal. The transgene expression of eNOS was examined by RT-PCR in rats transfected with AdRSVbetagal and AdRSVeNOS. eNOS and iNOS protein levels were measured by Western blot analysis, and cGMP levels were assessed in cavernosal tissue by enzyme immunoassay. Adenoviral expression of the beta-galactosidase reporter gene was observed in cavernosal tissue for up to 30 days, with peak expression registered at 5 days after intracavernosal administration of AdRSVbetagal. Cross-sections of the rat penises transfected with the AdRSVeNOS revealed no pathological (morphological or histological) changes. Five days after administration of AdRSVeNOS, eNOS protein, mRNA and cGMP levels in the corpora cavernosa were significantly increased (P<0. 05), while iNOS protein levels remained unchanged (P>0.05). In conclusion, enhanced expression of eNOS employing an adenoviral vector significantly increased the erectile response to cavernosal nerve stimulation in the aged rat, similar to the response observed in younger rats. These data suggest that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the aged rat.