Noncholinergic penile erection in mice lacking the gene for endothelial nitric oxide synthase.

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 (eNOS-/- 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 eNOS-/- 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 +/- 2.7 to 48.1 +/- 5.5 mm Hg, P < .05) but not in eNOS-/ - mice (from 54.9 +/- 6.3 to 51.0 +/- 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 eNOS-/- mice to baseline levels (37.8 +/- 4.4 vs 12.7 +/- 4.0 mm Hg, P < .05). In penes of eNOS-/- mice, approximately 60% NO synthase activity of wild-type penis levels was retained (NS), and eNOS protein was absent. We concluded that eNOS-/- mice preserve erectile function on the basis of a noncholinergic but NO-dependent mechanism and that eNOS physiologically mediates penile erection under cholinergic stimulation.     

Rho激酶及血红素氧合酶在自发性高血压大鼠阴茎海绵体中的表达

目的:探讨NOS/NO、HO/CO、RhoA/Rho激酶等信号通路在自发性高血压大鼠(SHR)阴茎海绵体中的表达及相互关系。方法:健康成年雄性SPF级SHR与对照组WKY大鼠各7只,16周龄,体重250~300g。麻醉后颈动脉和海绵体内插管连续监测平均动脉压(MAP)和海绵体内压(ICP)。利用电刺激海绵体神经,记录ICP/MAP比值变化。利用免疫组化和Western印迹方法分析ROCK2、HO-2、eNOS在阴茎海绵体中的表达变化。结果:SHR组在利用电刺激海绵体神经后ICP/MAP比值升高不明显(P>0.05),海绵体组织中ROCK2蛋白表达水平升高显著(P=0.017),HO-2表达水平则降低显著(P=0.006)。HO-2主要位于阴茎海绵体的平滑肌细胞及神经细胞内,eNOS则主要位于阴茎海绵体血管内皮细胞,两者在SHR组表达明显降低。结论:NOS/NO、HO/CO、RhoA/Rho激酶与SHRED有关,并且可能互相影响。     

Lack of endothelial nitric oxide synthase promotes endothelin-induced hypertension: lessons from endothelin-1 transgenic/endothelial nitric oxide synthase knockout mice

Endothelin-1 (ET-1) is one of the most potent biologic vasoconstrictors. Nevertheless, transgenic mice that overexpress ET-1 exhibit normal BP. It was hypothesized that vascular effects of ET-1 may be antagonized by an increase of the endothelial counterpart of ET-1, nitric oxide (NO), which is produced by the endothelial NO synthase (eNOS). Therefore, cross-bred animals of ET transgenic mice (ET+/+) and eNOS knockout (eNOS-/-) mice and were generated, and BP and endothelial function were evaluated in these animals. Endothelium-dependent and -independent vascular function was assessed as relaxation/contraction of isolated preconstricted aortic rings. The tissue ET and NO system was determined in aortic rings by quantitative real-time PCR and Western blotting. Systolic BP was similar in ET+/+ and wild-type (WT) mice but was significantly elevated in eNOS-/- mice (117 +/- 4 mmHg versus 94 +/- 6 mmHg in WT mice; P < 0.001) and even more elevated in ET+/+ eNOS-/- cross-bred mice (130 +/- 4 mmHg; P < 0.05 versus eNOS-/-). Maximum endothelium-dependent relaxation was enhanced in ET+/+ mice (103 +/- 6 versus 87 +/- 4% of preconstriction in WT littermates; P < 0.05) and was completely blunted in eNOS-/- (-3 +/- 4%) and ET+/+ eNOS-/- mice (-4 +/- 4%), respectively. Endothelium-independent relaxation was comparable among all groups. Quantitative real-time PCR as well as Western blotting revealed an upregulation of the aortic ET(A) and ET(B) receptors in ET+/+ eNOS-/-, whereas eNOS was absent in aortic rings of eNOS-/- and ET+/+ eNOS-/- mice. ET-1 aortic tissue concentrations were similar in WT mice and ET+/+ eNOS-/- mice most probably as a result of an enhanced clearance of ET-1 by the upregulated ET(B) receptor. These data show for the first time that in transgenic mice that overexpress human ET-1, additional knockout of eNOS results in a further enhancement of BP as compared with eNOS-/- mice. The human ET+/+ eNOS-/- mice therefore represent a novel model of hypertension as a result of an imbalance between the vascular ET-1 and NO systems.     

Soluble Epoxide Hydrolase Inhibition Exhibits Antihypertensive Actions Independently of Nitric Oxide in Mice with Renovascular Hypertension

Objective: The present study was performed to examine whether the blood pressure (BP)-lowering effects of soluble epoxide hydrolase (sEH) inhibition in two-kidney, one-clip (2K1C) Goldblatt hypertension are nitric oxide (NO) dependent. Methods: Mice lacking the endothelial NO synthase (eNOS) gene (eNOS-/-) and their wild-type controls (eNOS+/+) underwent clipping of one renal artery. BP was monitored by radiotelemetry and the treatment with the sEH inhibitor cis-4-[4-(3-adamantan-1-yl-ureido)cyclohex-yloxy]-benzoic acid (c-AUCB) was initiated on day 25 after clipping and lasted for 14 days. Renal concentrations of epoxyeicosatrienoic acids (EETs) and their inactive metabolite dihydroxyeicosatrienoic acids (DHETs) were measured in the nonclipped kidney. Renal NO synthase (NOS) activity was determined by measuring the rate of formation of L-[(14)C]citruline from L-[(14)C]arginine. Results: Treatment with the sEH inhibitor elicited similar BP decreases that were associated with increases in daily sodium excretion in 2K1C eNOS+/+ as well as 2K1C eNOS-/- mice. In addition, treatment with the sEH inhibitor increased the ratio of EETs/DHETs in the nonclipped kidney of 2K1C eNOS+/+ as well as 2K1C eNOS-/- mice. Treatment with the sEH inhibitor did not alter renal NOS activity in any of the experimental groups. Conclusions: Collectively, our present data suggest that the BP-lowering effects of chronic sEH inhibition in 2K1C mice are mainly associated with normalization of the reduced availability of biologically active EETs in the nonclipped kidney and their direct natriuretic actions.     

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。     

Vasoconstriction and vasodilation in erectile physiology

Recent studies have demonstrated that vasoconstriction in the erectile vasculature of the penis is mediated in part by RhoA/Rho-kinase signaling. However, this constrictor activity must be overcome to permit the vasodilation essential for erection. We hypothesize that the primary action of nitric oxide and other agents that cause penile erection is inhibition of the RhoA/Rho-kinase pathway, thereby allowing vasodilation and erection. This hypothesis, as well as experiments using hypogonadal and hypertensive animal models, are discussed in terms of the potential clinical value of Rho-kinase inhibitors for the treatment of erectile dysfunction.     

Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance

Studies from our laboratory using acute pharmacologic blockade of nitric oxide synthase (NOS) activity have suggested that nitric oxide (NO) has an important role in regulating carbohydrate metabolism. We now report on insulin sensitivity in mice with targeted disruptions in endothelial NOS (eNOS) and neuronal NOS (nNOS) genes compared with their wild-type (WT) counterparts. Mice underwent hyperinsulinemic-euglycemic clamp studies after a 24-h fast, during an insulin infusion of 20 mU x kg(-1) x min(-1). Glucose levels were measured at baseline and every 10 min during the clamp. Insulin levels were measured at baseline and at the end of the clamp study. Glucose infusion rates (GIRs) during the last 30 min of the clamp study were in a steady state. Tritiated glucose infusion was used to measure rates of endogenous glucose output (EGO) both at baseline and during steady-state euglycemia. Glucose disposal rates (GDRs) were computed from the GIR and EGO. Fasting and steady-state glucose and insulin levels were comparable in the 3 groups of mice. No differences in fasting EGO were noted between the groups. GIR was significantly reduced (37%, P = 0.001) in the eNOS knockout (KO) mice compared with the WT mice, with values for the nNOS mice being intermediate. EGO was completely suppressed in the nNOS and WT mice during insulin infusion, but not in the eNOS mice. Even so, the eNOS mice displayed significantly reduced whole-body GDRs compared with those of the WT mice (82.67+/-10.77 vs. 103.67+/-3.47 mg x kg(-1) x min(-1), P = 0.03). eNOS KO mice are insulin resistant at the level of the liver and peripheral tissues, whereas the nNOS KO mice are insulin resistant only in the latter. These data indicate that NO plays a role in modulating insulin sensitivity and carbohydrate metabolism and that the eNOS isoform may play a dominant role relative to nNOS.     

Gene transfer of endothelial nitric oxide synthase to pulmonary allografts: impact on acute rejection

Experiments were designed to study whether overexpression of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS) affects acute rejection. Allogenic, orthotopic single-lung transplantation was performed after transbronchial adenoviral-mediated gene transfer (3 × 10⁸ pfu) of either of eNOS or β-galactosidase to donor lungs of rats (n = 6 each). No immunosuppression was used. After 4 days, transplanted lungs were prepared for enzyme activity, cGMP and histology. Calcium-dependent NOS activity, reflecting eNOS, was greater in eNOS-transduced lungs (587 ± 97 vs 2.1 ± 1.4 pmol/mg protein per h, P <0.001). In contrast, calcium-independent NOS activity, reflecting iNOS, was comparable. Concentrations of cGMP were higher in eNOS-transduced lungs (13.2 ± 2.3 vs 4.9 ± 0.5 pmol/mg protein). Positive immunostaining for eNOS was present in pneumocytes only in eNOS-transduced lungs. No difference in histological grade of rejection was observed. eNOS gene transfer to pulmonary allografts results in a functionally active transgene product and increased NO production. Increasing NO from eNOS does not affect histogically identified acute rejection.     

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.     

Activation of vascular protein kinase C-beta inhibits Akt-dependent endothelial nitric oxide synthase function in obesity-associated insulin resistance

Activation of protein kinase C (PKC) in vascular tissue is associated with endothelial dysfunction and insulin resistance. However, the effect of vascular PKC activation on insulin-stimulated endothelial nitric oxide (NO) synthase (eNOS) regulation has not been characterized in obesity-associated insulin resistance. Diacylglycerol (DAG) concentration and PKC activity were increased in the aorta of Zucker fatty compared with Zucker lean rats. Insulin-stimulated increases in Akt phosphorylation and cGMP concentration (a measure of NO bioavailability) after euglycemic-hyperinsulinemic clamp were blunted in the aorta of fatty compared with lean rats but were partly normalized after 2 weeks of treatment with the PKCbeta inhibitor ruboxistaurin (LY333531). In endothelial cell culture, overexpression of PKCbeta1 and -beta2, but not PKCalpha, -delta, or -zeta, decreased insulin-stimulated Akt phosphorylation and eNOS expression. Overexpression of PKCbeta1 and -beta2, but not PKCalpha or -delta, also decreased Akt phosphorylation stimulated by vascular endothelial growth factor (VEGF). In microvessels isolated from transgenic mice overexpressing PKCbeta2 only in vascular cells, Akt phosphorylation stimulated by insulin was decreased compared with wild-type mice. Thus, activation of PKCbeta in endothelial cells and vascular tissue inhibits Akt activation by insulin and VEGF, inhibits Akt-dependent eNOS regulation by insulin, and causes endothelial dysfunction in obesity-associated insulin resistance.     

Nitric oxide inhibits stretch-induced MAPK activation in mesangial cells through RhoA inactivation

Glomerular capillary hypertension is an important determinant of glomerulosclerosis in rats with subtotal renal ablation. Dietary supplementation with L-arginine increases renal nitric oxide (NO) production and limits glomerular injury in this model, and early benefits are seen without altered glomerular capillary pressure. In an in vitro model of hemodynamically mediated signaling, the authors have reported that subjecting MC to cyclic stretch/relaxation activates the mitogen-activated protein kinase p42/44 (Erk) cascade and that NO and cyclic GMP abrogate stretch-induced Erk activation by inducing actin cytoskeletal disassembly. The actin cytoskeleton is regulated by the Rho family of GTPases, including RhoA; therefore, the authors examined the role of RhoA in stretch-induced Erk activation and as an NO target. In primary rat MC subjected to cyclic mechanical strain, RhoA activity was maximally increased (2.4-fold) after 1 min of stretch, and Erk activation temporally followed. The Rho-kinase inhibitor Y-27632 attenuated Erk activation in a dose-dependent manner and prevented stretch-induced actin stress fiber formation. The NO donors S-nitroso-N-acetylpenicillamine and cGMP both inhibited stretch-induced RhoA and Erk activation and stress fiber formation. Infection of MC with the RhoA mutant RhoA-Ala188, which is resistant to NO-dependent phosphorylation, abrogated the effects of NO and cGMP on stretch-induced Erk activation and stress fiber formation. The authors conclude that the early activation of RhoA is essential for stretch-induced actin stress fiber formation and Erk activation in MC, events which are prevented by NO and cGMP through their action on RhoA. Inhibition of RhoA may thus be a new approach to the prevention of hemodynamically mediated glomerular injury.     

The role of nitric oxide in the mechanical repression of RANKL in bone stromal cells

Both mechanical loading and nitric oxide (NO) have positive influences on bone mass. NO production is induced by mechanical strain via upregulation of eNOS mRNA and protein, the predominant NOS in adult bone. At the same time, strain causes decreased expression of RANKL, a factor critical for osteoclastogenesis. In this study, we harvested primary stromal cells from wild-type (WT) and eNOS(-/-) mice to test whether induction of NO by mechanical strain was necessary for transducing mechanical inhibition of RANKL. We found that strain inhibition of RANKL expression was prevented by NOS inhibitors (L-NAME and L-NMMA) in WT stromal cells. Surprisingly, stromal cells from eNOS(-/-) mice showed significant mechanical repression of RANKL expression (p<0.05). Mechanical strain still increased NO production in the absence of eNOS, and was abolished by SMTC, a specific nNOS inhibitor. nNOS mRNA and protein expression were increased by strain in eNOS(-/-) but not in WT cells, revealing that nNOS was mechanically sensitive. When NO synthesis was blocked with either SMTC or siRNA targeting nNOS in eNOS(-/-) cells however, strain still was able to suppress RANKL expression by 34%. This indicated that strain suppression of RANKL can also occur through non-NO dependent pathways. While our results confirm the importance of NO in the mechanical control of skeletal remodeling, they also suggest alternative signaling pathways by which mechanical force can produce anti-catabolic effects on the skeleton.     

The ups and downs of Rho-kinase and penile erection: upstream regulators and downstream substrates of rho-kinase and their potential role in the erectile response

In the absence of arousal stimuli, the activity of the Rho-kinase-mediated signaling pathway promotes vasoconstriction of the cavernosal arterioles and sinuses, keeping the penis in the nonerect state. Upon sexual arousal or during nocturnal tumescence, nitric oxide (NO), released from nonadrenergic/noncholinergic nerves or from local endothelial cells, induces cavernosal vasodilation, resulting in an elevation in blood flow and intracavernosal pressure to initiate the erectile response. Although NO is thought to be the principal stimulator of penile erection, the signaling mechanism(s) of NO-mediated cavernosal vasodilation is unknown. In this article, we will consider the novel hypothesis that NO induces penile erection through the inhibition of endogenous Rho-kinase-mediated vasoconstriction. Additionally, we will look downstream of Rho-kinase, introducing a potential role for various substrates in the mechanism of Rho-kinase-mediated constriction in the cavernosal vasculature.     

Effect of endothelial cell-based iNOS gene transfer on cavernosal eNOS expression and mouse erectile responses

Inducible nitric oxide synthase (iNOS) gene transfer is reported to augment erectile responses in rats, although it is also shown to impair vasorelaxation in cerebral arteries. We investigated the effect of endothelial cell-based iNOS gene transfer on endothelial NOS (eNOS) expression and mouse erectile responses. Human coronary artery endothelial cells (EC) transduced with empty vector (control) or iNOS were grown in culture and transplanted into the corpus cavernosum of severe combined immunodeficient mice. Endothelial NOS expression was compared in control and iNOS-transduced cells grown in the presence or absence of a selective iNOS inhibitor, L-N6- (1-iminoethyl) lysine hydrochloride (L-NIL). At 3-5 days after cell transplantation, we recorded intracorporal pressure (ICP) responses to cavernosal nerve stimulation and measured cavernosal total NO and eNOS protein expression. In this study, EC transduced with iNOS produced significantly more NO than controls but exhibited a twofold downregulation of eNOS protein and mRNA. This effect was reversed by L-NIL. In vivo, the cell-based gene transfer of iNOS led to significantly increased ICP responses, compared to mice transplanted with control ECs. Consistent with the in vitro data, cavernosal lysates had significantly reduced eNOS expression. In conclusion, EC gene transfer of iNOS downregulates EC expression of eNOS by an NOS-dependent mechanism. In the cavernosum of mice transplanted with Inos-transduced EC, nerve-stimulated erectile responses were augmented by the short-term gene transfer. However, our findings suggest that iNOS gene transfer may have deleterious effects on endothelial function if used as a treatment for erectile dysfunction.     

Molecular mechanisms of penile erection

The penis physiological states of flaccidity or erection, result from the contraction or relaxation, respectively, of smooth muscle cells in the corpora cavernosa (CSMCs). They result from the interaction of various inter and intracellular molecular signaling pathways. During the more usual state of flaccidity seems to predominate a tonic sympathetic activity, releasing noradrenaline (NA) and other agonists that generate contractile signals in the CSMCs, with the likely cooperation of endothelium-derived messengers. Through activation of membrane receptors in the CSMCs they raise the intracellular messengers inositol triphosphate (IP3) and diacylglycerol (DAG). This results in a transient increase in cytosolic calcium concentration [Ca2+]i that starts the contractile response which is further sustained by the parallel agonist-induced activation of a "calcium sensitizing" mechanism involving the RhoA/Rho-kinase pathway. Overexpression of the latter might contribute to several vascular disorders as hypertension, vasospasm or erectile dysfunction. On sexual stimulation the cavernous nerves release nitric oxide (NO) that starts the erectile response. They also release acetylcholine that stimulates the endothelium to generate a more sustained release of NO. NO diffuses into CSMCs and increases their intracellular levels of cyclic guanosin monophosphate (cGMP) which decreases [Ca2+]i and deactivates the calcium sensitizing mechanism, thus relaxing CSMCs. This main physiological pathway for CSMCs relaxation is helped by the cyclic adenosin monophosphate (cAMP) pathway activated by various intercellular messengers from neural or paracrine sources, including prostaglandins E (PGE). Different phosphodiesterase enzymes (PDEs) inactivate the cyclic nucleotides thereby limiting their erectogenic action. Indeed the pharmacological inhibition of PDEs, especially the cGMP-specific PDE5, greatly enhances the erectile responses. There are crosstalk mechanisms between the cGMP and cAMP signaling pathways that offer additional possibilities for the pharmacotherapy of erectile dysfunction.     

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.     

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.     

Isoflurane-induced Cerebral Hyperemia in Neuronal Nitric Oxide Synthase Gene Deficient Mice

Background: Nitric oxide (NO) has been reported to play an important role in isoflurane-induced cerebral hyperemia in vivo. In the brain, there are two constitutive isoforms of NO synthase (NOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Recently, the mutant mouse deficient in nNOS gene expression (nNOS knockout) has been developed. The present study was designed to examine the role of the two constitutive NOS isoforms in cerebral blood flow (CBF) response to isoflurane using this nNOS knockout mouse.

Methods: Regional CBF (rCBF) in the cerebral cortex was measured with laser-Doppler flowmetry in wild-type mice (129/SV or C57BL/6) and nNOS knockout mice during stepwise increases in the inspired concentration of isoflurane from 0.6 vol% to 1.2, 1.8, and 2.4 vol%. Subsequently, a NOS inhibitor, Nomega -nitro-L-arginine (L-NNA), was administered intravenously (20 mg/kg), and 45 min later, the rCBF response to isoflurane was tested again. In separate groups of wild-type mice and the knockout mice, the inactive enantiomer, Nomega -nitro-D-arginine (D-NNA) was administered intravenously in place of L-NNA. Brain NOS activity was measured with radio-labeled L-arginine to L-citrulline conversion after treatment with L-NNA and D-NNA.

Results: Isoflurane produced dose-dependent increases in rCBF by 25 +/- 3%, 74 +/- 10%, and 108 +/- 14% (SEM) in 129/SV mice and by 32 +/- 2%, 71 +/- 3%, and 96 +/- 7% in C57BL/6 mice at 1.2, 1.8, and 2.4 vol%, respectively. These increases were attenuated at every anesthetic concentration by L-NNA but not by D-NNA. Brain NOS activity was decreased by 92 +/- 2% with L-NNA compared with D-NNA. In nNOS knockout mice, isoflurane increased rCBF by 67 +/- 8%, 88 +/- 12%, and 112 +/- 18% at 1.2, 1.8, and 2.4 vol%, respectively. The increase in rCBF at 1.2 vol% was significantly greater in the nNOS knockout mice than that in the wild-type mice. Administration of L-NNA in the knockout mice attenuated the rCBF response to isoflurane at 1.2 and 1.8 vol% but had no effect on the response at 2.4 vol%.