Corpus Cavernosum Smooth Muscle Cells Research Articles

Abnormal lipid metabolism down-regulates adenosine triphosphate synthase β-subunit protein expression in corpus cavernosum smooth musclein vitroandin vivo

Metabolic syndrome is closely related to erectile dysfunction (ED), and hyperlipidaemia is considered a major risk factor for ED. Adenosine triphosphate (ATP) synthase is believed to play an important role in metabolic syndrome; it has been hypothesised that ATP synthase contributes to ED development. We have verified this hypothesis using primary cultured human corpus cavernosum smooth muscle (HCCSM) cells treated with excessive free fat acid (FFA) and a high-fat diet (HFD) mouse model. Our results showed that high fatty factors could cause lipid accumulation in HCCSM cells, which could result in abnormal lipid metabolism, such as high levels of triglycerides, cholesterol and glucose in the HFD mice. There was a remarkable down-regulation of ATP synthase and p-Akt after in vivo and in vitro excessive FFA treatments. These results indicated that abnormal lipid metabolism could induce ATP synthase down-regulation via the Akt phosphorylation pathway and that ATP synthase may be a target of lipotoxicity in corpus cavernosum smooth muscle cells.

SaRNA Guided iNOS Up-Regulation Improves Erectile Function of Diabetic Rats

Promoter targeted saRNAs mediate sequence specific up-regulation of gene expression. We explored the therapeutic effect of RNA activation mediated iNOS gene activation on improving erectile function in a rat model of diabetes mellitus. An optimal saRNA sequence specific for iNOS promoter was cloned into an adenoviral vector, resulting in AdU6/shiNOS and AdU6/shControl. The corresponding viruses were used to transduce cultured rat cavernous smooth muscle cells. Streptozotocin induced diabetes models were established in rats and used to test the effects of intracavernous delivery of iNOS saRNA viruses on erectile function. iNOS expression in the cavernous smooth muscle cells or penile tissue of treated rats was assessed by reverse transcriptase-polymerase chain reaction and Western blot. Cyclic guanosine monophosphate was analyzed by enzyme-linked immunosorbent assay. Intracavernous pressure in response to cavernous nerve stimulation was measured using a data acquisition system on post-injection days 1, 3, 5, 7, 10 and 14. Adenovirus mediated expression of iNOS saRNA caused sustained up-regulation of iNOS in cavernous smooth muscle cells. Intracavernous injection of AdU6/shiNOS activated iNOS expression in vivo and significantly increased peak intracavernous pressure in streptozotocin induced diabetic rats via nitric oxide/intracellular cyclic guanosine monophosphate activation. Results show that saRNA mediated iNOS over expression in the penis can restore erectile function in streptozocin diabetic rats via the nitric oxide-cyclic guanosine monophosphate pathway.

TRPM4 channels in smooth muscle function

The melastatin (M) transient receptor potential (TRP) channel TRPM4 is selective for monovalent cations and is activated by high levels of intracellular Ca(2+). TRPM4 is broadly distributed and may be involved in numerous functions, including electrical conduction in the heart, respiratory rhythm, immune response, and secretion of insulin by pancreatic β-cells. The significance of TRPM4 in smooth muscle cell function is reviewed here. Several studies indicate that TRPM4 channels are critically important for pressure-induced cerebral arterial myocyte depolarization and myogenic vasoconstriction as well as autoregulation of cerebral blood flow. Regulation of TRPM4 activity in arterial smooth muscle cells is complex and involves release of Ca(2+) from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate receptors and translocation of TRPM4 channels to the plasma membrane in response to protein kinase Cδ. TRPM4 is also present in colonic, urinary bladder, aortic, interlobar pulmonary and renal artery, airway, and corpus cavernosum smooth muscle cells, but its significance and regulation in these tissues is less well characterized.

Abstract 647: Toll-like Receptor 4 (TLR4) Activation Impairs the Nitrergic Relaxation Response in Rat Corpus Cavernosum

Cardiovascular disease etiology of hypertension and atherosclerosis has been shown to involve pattern-recognition receptor activation, including toll-like receptor 4 (TLR4). Indeed, our lab showed that TLR4 activation was implicated in blood pressure regulation, and its blockade lowered blood pressure in spontaneously hypertensive rats. However, whether TLR4 activation plays a role in erectile dysfunction is not known. Therefore, we hypothesized that TLR4 activation using its ligand, lipopolysaccharide (LPS), would increase reactive oxygen species (ROS) and impair corpus cavernosum (CC) relaxation. To test our hypothesis, rat CC smooth muscle cells were incubated for 15 min with LPS (100 ng/mL) and ROS detected with a fluorescent probe. Treatment with LPS resulted in an increase in ROS, compared to baseline (Fig.1). To investigate vascular reactivity to TLR4 activation, we performed myograph studies on CC isolated from 13-15 week old Sprague-Dawley rats, then incubated 3 hours in DMEM with LPS (+LPS) or vehicle (VEH). Concentration response curves to phenyleprine (PE, 10 -9 -10 -4 M) and to non-adrenergic, non-cholinergic (NANC) stimulation were used to investigate CC reactivity. Contractility to PE did not differ between groups, but +LPS-induced relaxation responses decreased significantly to 20-volt electrical field stimulation compared to VEH (8Hz: 16.3 vs 24.9, p< 0.01 and 16 Hz: 21.2 vs 35.1, p< 0.001). In conclusion, treatment with the TLR4 agonist, LPS, decreased NANC relaxation in the CC. Increased ROS generation may result in scavenging and reducing availability of nitric oxide that could impair relaxation in the CC.

Effects of integrin-linked kinase on human corpus cavernosum smooth muscle cell cytoskeletal organisation

We investigated the effects of integrin-linked kinase (ILK) on the in vitro attachment, spreading, migration and microfilament dynamics of human corpus cavernosum smooth muscle cells. ILK small interfering RNA (siRNA) was used to transfect human corpus cavernosum smooth muscle cells; and cell attachment, spreading and migration were assessed. Additionally, microfilament dynamics were evaluated using Alexa Fluor 488 and phalloidin staining. We found that ILK gene knock-down significantly inhibited human corpus cavernosum smooth muscle cell attachment, spreading and migration. Moreover, blocking the expression of ILK disturbed actin cytoskeleton reorganisation and morphology in human corpus cavernosum smooth muscle cells. These results show that the targeting of ILK with siRNA significantly inhibited cell attachment, spreading, migration and microfilament dynamics in human corpus cavernosum smooth muscle cells. These findings indicate that ILK might be a potential therapeutic molecular target for the treatment of erectile dysfunction.

Characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction

Phenotypic modulation from a contractile to a proliferative state within vascular smooth muscle cells has a critical role in the pathogenesis of a variety of cardiovascular diseases. To investigate the characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction, a group of Sprague-Dawley rats (n=30) were induced by intraperitoneal injection of streptozotocin (60 mg kg(-1)) and screened by subcutaneous injection of apomorphine (100 μg kg(-1)) for the measurement and comparison of the penile erections, and then three different groups were defined. Primary corpus cavernosum smooth muscle cells were cultured and passaged. The cavernous tissue segments were subjected to quantitative real-time polymerase chain reaction to determine the expressions of smooth muscle α-actin (SMA), SM myosin heavy chain (SMMHC), smoothelin, calponin and myocardin. Cell contractility in vitro and western blot analysis of SMA and SMMHC in the cavernous tissues and cells were determined. Compared with the control group (n=8) and the diabetes mellitus group (n=5), the expressions of SMA, calponin, SMMHC, smoothelin and myocardin mRNA were decreased in the cavernous tissues in rats of the diabetic erectile dysfunction group (n=15; P=0.001 and 0.02, P=0.014 and 0.012, both P<0.001, P=0.005 and <0.001, P=0.003 and 0.035, respectively). The levels of SMA and SMMHC proteins showed a significant decrease in cavernous tissues and cultured cells in rats of the diabetic erectile dysfunction group. Cells of the diabetic erectile dysfunction group exhibited significantly less contractility compared with those of other groups (P<0.001). Corpus cavernosum SM cell possesses the ability to modulate the phenotype under hyperglycemic conditions, which could have a key role in the pathogenesis of diabetic erectile dysfunction.

The relaxant effect of methanolic extract of yohimbe bark (&amp;lt;i&amp;gt;Pausinystalia yohimbe&amp;lt;/i&amp;gt;) on rabbit corpus cavernosum smooth muscles

Background: Development and maintenance of penile erection requires the relaxation of corpus cavernosum smooth muscle (CCSM) cells and it is essentially mediated by nitric oxide (NO). Pausinystalia yohimbe bark is traditionally used for the treatment of erectile dysfunction (ED). Objectives and Methods: This study aimed to evaluate the effects of the methanolic extract of P. yohimbe bark (10 mg/ml) on isolated rabbit corpus cavernosum smooth muscle (CCSM). Results: Methanolic extract of P. yohimbe bark blocked contractions in CCSM induced by adrenergic agonists, through facilitating NO release from the endothelial cells. Conclusion: The obtained results revealed that, methanolic extract of P. yohimbe bark caused relaxation of CCSM that validates its traditional aphrodisiac property, thus it could be used to initiate and maintain erection in cases of erectile dysfunction.

Up‐regulation of VEGF by Small Activator RNA in Human Corpus Cavernosum Smooth Muscle Cells

Functional failure of smooth muscle cells and endothelial cells in corpus cavernosum contributes to erectile dysfunction (ED) in aging men. Given that vascular endothelial growth factor (VEGF) may improve the function of smooth muscle cells and endothelial cells through different mechanisms, it is thus expected that increasing the expression of VEGF may have beneficial effects on erectile function. The aim of this article is to explore the possibility that VEGF can be induced by ribonucleic acid activation (RNAa) technology, and VEGF induction by RNAa has the potential of treating ED. Primary human corpus cavernosum smooth muscle cells (CCSMCs) were isolated and cultured in vitro. The expression of α-smooth muscle actin was detected by immunohistochemistry to identify CCSMCs. A previously identified VEGF promoter-targeted small activator RNA (saRNA, double-stranded [ds]VEGF-706) and a negative control dsRNA were chemically synthesized. Cultured human CCSMCs were transfected with the saRNAs. The expression of VEGF messenger RNA (mRNA) and protein in transfected CCSMCs was evaluated by real-time polymerase chain reaction (RT-PCR) and Western blotting assay, respectively. Immunofluorescent staining was also used to confirm VEGF protein expression in cultured CCSMCs. The expression of VEGF was assessed by RT quantitative PCR, Western blotting, and immunofluorescence assays. After transfection, RT quantitative PCR analysis showed that the expression of VEGF mRNA was significantly induced in dsVEGF-706 transfected cells compared with cells receiving control treatments (P < 0.05). Consistent with mRNA induction, Western blotting and immunofluorescence analysis showed that VEGF protein expression was also induced by dsVEGF-706. VEGF expression can be activated by RNAa in primary human CCSMCs, suggesting a potential application of RNAa-mediated VEGF activation for the treatment of ED.

Effects of Schisandra chinensis extract on the contractility of corpus cavernosal smooth muscle (CSM) and Ca2+ homeostasis in CSM cells

What's known on the subject? And what does the study add? Schisandra chinensis extract (SCE) has been known to have relaxative effects on penile smooth muscle. A recent study showed that SCE could enhance slidenafil citrate-induced relaxation of penile corpus cavernosum. The current study investigated the mechanism of action of SCE and its constituents on corporal smooth muscle cells. And this study shows that SCE induced relaxation of CSM primarily through an endothelium independent pathway and the relaxation effects of SCE on corporal smooth muscle are, in part, due to the activation of K(+) channels and inhibition of TRPC6 channels, resulting in decreased [Ca(2+)]. • To evaluate the relaxant effects of Schisandra chinensis extract (SCE) on corporal tissue in the penis and to investigate the mechanism of action of SCE and its constituents on corporal smooth muscle (CSM) cells. • The fruit of SC was collected and extracted with ethanol. Six SC lignans (schisandrol A, schisandrol B, schisandrin A, schisandrin B, gomisin N, and schisandrin C) were isolated and purified, and the chemical structures were confirmed by (1)H-nuclear magnetic resonance (NMR) and (13)C-NMR data. • Isolated rabbit CSM strips were mounted in an organ-bath system, and the effects of SCE were evaluated. • To estimate the intracellular Ca(2+) level ([Ca(2+)](i)), we used a Fura-2 fluorescent technique, and a conventional whole-cell patch-clamp technique was used to measure the calcium-sensitive K(+) channels (K(Ca)), inward rectifier K(+) channels (K(IR)), and canonical transient receptor potential cation channel 6 (TRPC6) currents. • SCE induced concentration-dependent relaxation in contracted CSM tissue, and the removal of the endothelium did not significantly affect their relaxation potencies. • In CSM cells, extracellular application of SCE significantly increased whole-cell K(Ca) currents (117.4%) and K(IR) currents (110.0%). These effects were completely abolished by charybdotoxin or BaCl(2). • In contrast, carbachol-induced TRPC6 channel activity was significantly inhibited (87.3%) by SCE in green fluorescent protein-TRPC6 pcDNA transfected HEK 293 cells. [Ca(2+)](i) measurements showed that SCE effectively reduced basal [Ca(2+)](i) in both cell lines (CSM cells and A7r5 cells) and the [Arg8]-vasopressin (AVP)-induced [Ca(2+)](i) increase in A7r5 cells. • Among the six SC lignans, schisandrin A and schisandrin B most effectively attenuated the AVP-induced [Ca(2+)](i) increase. • SCE induced relaxation of CSM that occurred primarily via an endothelium-independent pathway. • The relaxation effects of SCE on CSM were, in part, due to the activation of K(+) channels and inhibition of TRPC6 channels, resulting in decreased [Ca(2+)](i).

Silencing MaxiK Activity in Corporal Smooth Muscle Cells Initiates Compensatory Mechanisms to Maintain Calcium Homeostasis

The MaxiK potassium channel is regulated by voltage and intracellular calcium, and plays a critical role in regulating intracellular calcium concentration ([Ca(2+) ](i)), which is the ultimate determinant of smooth muscle tone. Tight control of corpus cavernosum smooth muscle (CCSM) tone is critically important and misregulation can result in erectile dysfunction. Because of the tight functional linkage of MaxiK and calcium channel activity, the aim of this study was to determine the effects of silencing and pharmacological inhibition of MaxiK on calcium homeostasis and intercellular calcium signaling in CCSM cells. We compared changes in the basal intracellular [Ca(2+) ](i) and parameters defining intercellular calcium wave (ICW) spread in 48 hours MaxiK silenced CCSM cells vs. acute blockade of the channel with iberiotoxin. To analyze changes occurring in gene expression we performed micro-array analysis following MaxiK silencing for 48 hours. Changes in Fura-2 fluorescence intensities were measured to evaluate basal [Ca(2+) ](i) levels and ICW parameters. Microarray analysis of global gene expression was performed. Forty-eight hours after MaxiK silencing the basal [Ca(2+) ](i) , the ICW amplitude and spread among CCSM cells were not markedly different in silenced compared to mock transfected controls, whereas short-term blockade significantly increased basal [Ca(2+) ](i) level and amplified Ca(2+) signaling among CCSM cells. Micro-array analysis showed that several genes within Ca(2+) homeostasis and smooth muscle tone regulation pathways had significantly altered expression. Our results indicate that while short-term blockade of the MaxiK channel is associated with an increase in basal [Ca(2+) ](i), Ca(2+) homeostasis is restored during the 48 hours period following silencing. We hypothesize that the different pathways regulating [Ca(2+) ](i) and CCSM tone are linked through molecular crosstalk and that their coordinated regulation is part of a compensatory mechanism aimed to maintain Ca(2+) homeostasis and CCSM tone.

Vardenafil and Resveratrol Synergistically Enhance the Nitric Oxide/Cyclic Guanosine Monophosphate Pathway in Corpus Cavernosal Smooth Muscle Cells and Its Therapeutic Potential for Erectile Dysfunction in the Streptozotocin‐Induced Diabetic Rat: Preliminary Findings

Phosphodiesterase type 5 (PDE5) inhibitors are very effective agents for erectile dysfunction; however, specific patient populations are hard to treat. The efficacy of PDE5 inhibitors is limited because a minimum amount of nitric oxide (NO) is necessary. Resveratrol, a plant polyphenol, is reported to activate endothelial NO synthase (eNOS) through activation of sirtuin 1. We previously reported that human corpus cavernosal smooth muscle cells (CCSMCs) express eNOS and synthesize cyclic guanosine monophosphate (cGMP) via the NO/cGMP pathway. To investigate the ability of resveratrol and/or vardenafil to increase cGMP in an in vitro model using CCSMCs and to improve erectile function in an in vivo rat model of streptozotocin (STZ)-induced diabetes. CCSMCs were treated with resveratrol and/or vardenafil. Twenty male Sprague-Dawley rats were randomly divided into five groups (N = 4 in each group): age-matched controls, diabetic controls, and diabetic rats treated with resveratrol, vardenafil, or both in combination for the last 4 weeks of an 8-week period of diabetes induction. Intracellular cGMP measurement, intracovernous pressure (ICP)/mean arterial pressure (MAP) ratio, and smooth muscle/collagen ratio. Intracellular cGMP level was elevated by resveratrol treatment in CCSMCs. The combination treatment of resveratrol and vardenafil had a synergistic effect. Diabetic rats showed impairment of erectile function. Treatment with either resveratrol or vardenafil improved ICP/MAP ratio, and combination therapy with resveratrol and vardenafil had a synergistic effect in improvement of ICP/MAP. Treatment with either resveratrol or vardenafil elevated cGMP level in CCSMCs and improved erectile function in STZ-induced diabetic rats. Furthermore, a synergistic effect was observed in vitro and in vivo. Resveratrol or combination therapy of resveratrol and vardenafil can improve erectile function in which NO release is impaired, although further study is needed to confirm the results.

Effect of tankyrase 1 on autophagy in the corpus cavernosum smooth muscle cells from ageing rats with erectile dysfunction and its potential mechanism

This study compared tankyrase 1 expression and autophagy quantity between erectile dysfunction (ED) and non-ED rats' corpus cavernosum smooth muscle cells (CSMCs). This study aslo explored the effect and possible mechanism of tankyrase 1 on autophagy and cell proliferation in ageing ED rats' CSMCs. The intracavernous pressure and mean systemic arterial pressure were measured to investigate erectile function so that eight 24-month-old ED and eight 8-month-old male Wistar rats were chosen respectively. The rat CSMCs were isolated and cultured by enzyme digestion, in which tankyrase 1 expression and autophagy quantity were compared. Tankyrase 1 overexpression was induced with plasmid transfection by Lipofectamine. The effect of tankyrase 1 overexpression on proliferation, autophagy and mTOR pathway in 24-month-old ED rats' CSMCs was measured by the cell growth curve in MTT assay, cell cycle analysis in flow cytometry (FCM), key protein expression in Western blot, autophagy quantity in transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 fluorescence. The primary CSMCs were confirmed by immunofluorescence, and the purity was 99.1% in FCM. Compared with that of 8-month-old rats, tankyrase 1 expression and autophagy quantity significantly decreased in 24-month-old ED rats' primary CSMCs (P < 0.01). Tankyrase 1 overexpression significantly increased the growth rate (P < 0.05) and increased the S phase of cell cycle (P < 0.01). The autophagosome quantity was remarkably increased (P < 0.01), LC3-I/II and Beclin 1 were upregulated (P < 0.01 and P < 0.05), and p-p70S6K (Thr(389)) was downregulated in 24-month-old ED rat CSMCs (P < 0.05). In conclusion, Tankyrase 1 and autophagy decrease in the CSMCs from aging rats with ED, and tankyrase 1 may have a positive effect on proliferation by enhancing autophagy and regulating the mTOR signalling pathway.

875 RESVERATROL, A POLYPHENOLIC SIRT1 ACTIVATOR, IMPROVES ERECTILE FUNCTION IN STREPTOZOTOCIN INDUCED DIABETIC RATS

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2010875 RESVERATROL, A POLYPHENOLIC SIRT1 ACTIVATOR, IMPROVES ERECTILE FUNCTION IN STREPTOZOTOCIN INDUCED DIABETIC RATS Shinichiro Fukuhara, Hidenobu Okuda, Keisuke Yamamoto, Jiro Nakayama, Tetsuya Takao, Yasushi Miyagawa, Akira Tsujimura, Norio Nonomura, and Akihiko Okuyama Shinichiro FukuharaShinichiro Fukuhara More articles by this author , Hidenobu OkudaHidenobu Okuda More articles by this author , Keisuke YamamotoKeisuke Yamamoto More articles by this author , Jiro NakayamaJiro Nakayama More articles by this author , Tetsuya TakaoTetsuya Takao More articles by this author , Yasushi MiyagawaYasushi Miyagawa More articles by this author , Akira TsujimuraAkira Tsujimura More articles by this author , Norio NonomuraNorio Nonomura More articles by this author , and Akihiko OkuyamaAkihiko Okuyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1631AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES SIRT1 is an important enzyme for many cellular processes including cell cycle regulation, fatty acid metabolism, and life span extension. Recently, it has been reported that SIRT1 promotes endothelium-dependent vascular relaxation by activating endothelial nitric oxide synthase (eNOS) in endothelial cells, however its effect on corpus cavernosal smooth muscle relaxation remain poorly defined. Resveratrol is a polyphenolic compound, and has been reported to be an activator of SIRT1. Therefore, we investigated the effects of resveratrol on NO/cGMP pathway in human corpus cavernosal smooth muscle cells (CCSMCs) which were shown to express eNOS and synthesize NO in our previous report, and its therapeutic potential for erectile dysfunction in diabetic rats. We also examined the hypothesis that a combination of SIRT1 activation and phosphodiesterase 5 (PDE5) inhibition has synergistic effect in vitro and vivo. METHODS We measured intraceller cGMP concentration after incubation of human CCSMCs with resveratrol, PDE5 inhibitor (vardenafil), and both. In addition, to eliminate possible nonspecific effects, the cells were also incubated with sirtinol which inhibit SIRT1. Then, to investigate erectile function, four weeks after establishment of diabetes due to streptozotocin injection (50mg/kg i.p), rats treated with either only vehicle, PDE5 inhibitor (vardenafil 0.5m/kg/day p.o), resveratrol (5mg/kg/day p.o) or resveratrol plus PDE5 inhibitor. After four weeks treatment, the intracavernosal pressure (ICP) with electrical stimulation to the cavernous nerve and mean arterial pressure (MAP) were measured. RESULTS The levels of cGMP in human CCSMCs were significantly increased either by administration of PDE5 inhibitor or resveratrol compare with those of controls. A drastic synergistic increase in cGMP was observed when treated with both PDE5 inhibitor and resveratorol simultaneously. Furthermore, the effects of resveratrol on cGMP synthesis were reversed by sirtinol a specific SIRT1 inhibitor. In vivo experiments, while the ICP/MAP was significantly lower in treated with only vehicle rats, ICP/MAP in treated with resveratrol rats showed significantly higher. And more, The ICP/MAP in treated with both agents was higher than all other groups. CONCLUSIONS Resveratorol leads to enhancement of cGMP synthesis in human CCSMSs and improves erectile function in storeptozotocin induced diabetic rats, particularly with PDE5 inhibitor. Our data indicate that resveratrol has therapeutic potential for diabetic erectile dysfunction. Suita, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e342 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shinichiro Fukuhara More articles by this author Hidenobu Okuda More articles by this author Keisuke Yamamoto More articles by this author Jiro Nakayama More articles by this author Tetsuya Takao More articles by this author Yasushi Miyagawa More articles by this author Akira Tsujimura More articles by this author Norio Nonomura More articles by this author Akihiko Okuyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

886 SPHINGOSINE KINASE 1 KNOCK DOWN IMPROVES ERECTILE FUNCTION IN STREPTOZOTOCIN- INDUCED DIABETIC RATS AND DECREASES RHO-KINASE ACTIVITY IN VITRO

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2010886 SPHINGOSINE KINASE 1 KNOCK DOWN IMPROVES ERECTILE FUNCTION IN STREPTOZOTOCIN- INDUCED DIABETIC RATS AND DECREASES RHO-KINASE ACTIVITY IN VITRO Guillermo Villegas, Moses Tar, Arnold Melman, and Michael DiSanto Guillermo VillegasGuillermo Villegas More articles by this author , Moses TarMoses Tar More articles by this author , Arnold MelmanArnold Melman More articles by this author , and Michael DiSantoMichael DiSanto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1642AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that is mainly formed by phosphorylation of sphingosine by sphingosine kinase 1 (SPHK1) and can be reverted to sphingosine by sphingosine-1-phosphate phosphatase 1 (SPP1). Our recent studies show that S1P plays a mayor role in the Ca+2 sensitization pathway and contractility of corpus cavernosum smooth muscle (CCSM). Furthermore we demonstrated that SPHK1 expression is elevated in Type 1 diabetic patients with impotence while SPHK1 overexpression in vivo induces erectile dysfunction (ED) in normal rats and upregulates the expression of Rho-kinase (ROCK). Our goals were a) to asses if knocking down expression of SPHK1 in CCSM of diabetic (DM) rats improves erectile function and therefore confirm the determinant role of S1P in the etiology of DM-induced ED b) To examine if SPHK1 expression modulates smooth muscle calcium sensitization by increasing or decreasing ROCK activity and c) to determine whether ROCK is modulated directly by SPHK1 or by S1P. METHODS 1) SPHK1 mRNA was silenced to prevent the synthesis of S1P by using small hairpin RNA (sh) cloned into a shRNA expression vector (pSiren/SPHK1) 2) Human SPP1 and SPHK1 were cloned into the pVax vector to overexpress these proteins 3) For in vivo studies, 2 month strepotozotocin induced diabetic rats (STZ) with ED were injected in the CC with 100 μg pSiren/SPHK1 or pSiren/Luciferase (MOC) and erectile function assessed via cavernous nerve stimulation-induced increase in intracavernous pressure (ICP) one week post-injection 4) For the in vitro studies rat and human primary CCSM cells were transfected with either pSiren/SPHK1, pVax/SPP1 or pVax/SPHK1. In addition S1P receptors were blocked with suramin or N-ethylmaleimide. Cells were harvested at 48 hours, lysed for protein and RNA extraction and analyzed by real time PCR and Western blot. RESULTS a) pSiren/SPHK1 injected STZ rats showed an improved erectile function especially at lower stimulations compared to MOC b) Transfection of CCSM cells with either SPHK1 shRNA or SPP1 significant decreased ROCK activity and c) Blocking of the S1P receptors also downregulated ROCK activity. CONCLUSIONS We showed that in STZ rats, treatment of CC with SPHK1 shRNA improves erectile function confirming the direct role S1P plays in DM-induced ED. Our results further suggest that S1P mediates Ca+2 sensitization of CCSM cells by modulating ROCK activity and that the mechanism involves S1P and not SPHK1. Bronx, NY© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e346-e347 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Guillermo Villegas More articles by this author Moses Tar More articles by this author Arnold Melman More articles by this author Michael DiSanto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Insulin-like Growth Factor-1 Gene Delivery May Enhance the Proliferation of Human Corpus Cavernosal Smooth Muscle Cells

To investigate the effects of IGF-1 gene transfer in human corpus cavernosal smooth muscle cells (HCCSMCs) ex vivo. Insulin-like growth factor-1 (IGF-1) promotes the proliferation of penile cavernous smooth muscle cells in the rats. A plasmid expressing human IGF-1 was constructed by subcloning hIGF-1 into pcDNA3.1 vector. HCCSMCs were harvested from 3 impotent patients and cultured in vitro. The cultured smooth muscle cells were identified by immunofluorescent staining. RNA was extracted from HCCSMCs, and the gene expression of IGF-1 was determined by reverse transcription-polymerase chain reaction. Cell growth was examined by use of a novel cell proliferation assay based on the bioreduction of the fluorescent dye Alamar blue. The subcloned product was transfected into HCCSMCs. Western blotting and immunoassay were performed 2 days after transfection to evaluate the transfection efficiency. Endogenous IGF-1 mRNA expression was detected by reverse transcription-polymerase chain reaction analysis of total RNA extracted from cultured HCCSMCs. Increased proliferation of HCCSMCs in vitro was observed with exogenous treatment with IGF-1 (100 ng/mL) in a dose-dependent manner. Exogenous IGF-1 gene transfer to cultured HCCSMCs enhanced IGF-1 protein expression compared with the control, and the expression level peaked at 4 days after transfection and decreased slowly thereafter. Secretion of IGF-1 from transfected HCCSMCs induced cellular proliferation. IGF-1 gene transfer into HCCSMCs enhanced cellular proliferation, which was mediated by secretion of IGF-1. Our results suggest that IGF-1 gene therapy may be applied to corpus cavernosum regeneration.

NS11021, a novel opener of large‐conductance Ca2+‐activated K+ channels, enhances erectile responses in rats

Large-conductance Ca(2+)-activated K(+) channels (BK(Ca)), located on the arterial and corporal smooth muscle, are potential targets for treatment of erectile dysfunction (ED). This study investigated whether NS11021 (1-(3,5-Bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea), a novel opener of BK(Ca) channels, relaxes erectile tissue in vitro and enhances erectile responses in intact rats. The effects were compared with sildenafil, an inhibitor of phosphodiesterase type 5. Patch clamp was used to record whole cell current in rat isolated corpus cavernosum smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs). Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from rats and men, and simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension were performed in intracavernous arteries. Erectile response was measured in anaesthetized rats. In patch clamp recordings, NS11021 increased currents sensitive to the selective BK(Ca) channel blocker, iberiotoxin (IbTX) in SMCs, but did not modulate K(+) current in HUVECs. NS11021 reduced [Ca(2+)](i) and tension in penile arteries. IbTX inhibited the vasorelaxation induced by NS11021 and sildenafil in human erectile tissue. NS11021 and sildenafil but not vehicle increased erectile responses in anaesthetized rats, an effect which was abolished after pretreatment with tetraethylammonium. NS11021 leads to relaxation of both intracavernous arteries and corpus cavernosum strips primarily through opening of BK(Ca) channels. It is also effective in facilitating erectile responses in anaesthetized rats. These results suggest a potential for use of BK(Ca) openers in the treatment of ED.

Expression and function of ryanodine receptors in rabbit penile corpus cavernosum smooth muscle cells

This study aimed to investigate the expression and function of ryanodine receptors (RyRs) in rabbit penile corpus cavernosum smooth muscle (CCSM) cells. New Zealand white rabbit CCSM cells were cultured by primary tissue culture and identified by immunofluorescence technique. mRNA of three RyRs subunits in cultured CCSM cells was detected by reverse transcription polymerase chain reaction (RT-PCR). After excitation by noradrenaline, the concentration of Ca(2+) in CCSM cells was detected by laser scanning confocal microscopy. It was found that only the RyRs1 subunit is expressed in CCSM by RT-PCR. The CCSM cells were divided into two groups: Group A, CCSM cells + 10 micromol l(-1) noradrenaline and Group B, CCSM cells + 50 micromol l(-1) procaine 10 micromol l(-1) noradrenaline. Compared with the base level and the level after noradrenaline excitation, fluorescence intensity improved 44.10 +/- 6.01% in the test group A (n = 8) and 32.92 +/- 4.92% (n = 8) in the procaine control group B, respectively. There were statistically significant differences between group A and group B (P < 0.01). It is concluded that RyRs1 subunit is expressed in CCSM cells and may contribute to regulating the cytoplasmic Ca(2+) level.

Spontaneous Ca2+ Waves in Rabbit Corpus Cavernosum: Modulation by Nitric Oxide and cGMP

Detumescent tone and subsequent relaxation by nitric oxide (NO) are essential processes that determine the erectile state of the penis. Despite this, the mechanisms involved are incompletely understood. It is often assumed that the tone is associated with a sustained high cytosolic Ca(2+) level in the corpus cavernosum smooth muscle cells, however, an alternative possibility is that oscillatory Ca(2+) signals regulate tone, and erection occurs as a result of inhibition of Ca(2+) oscillations by NO. The aim of this study is to determine if smooth muscle cells displayed spontaneous Ca(2+) oscillations and, if so, whether these were regulated by NO. Male New Zealand white rabbits were euthanized and smooth muscle cells were isolated by enzymatic dispersal for confocal imaging of intracellular Ca(2+) (using fluo-4AM) and patch clamp recording of spontaneous membrane currents. Thin tissue slices were also loaded with fluo-4AM for live imaging of Ca(2+). Cytosolic Ca(2+) was measured in isolated smooth muscle cells and tissue slices. Results. Isolated rabbit corpus cavernosum smooth muscle cells developed spontaneous Ca(2+) waves that spread at a mean velocity of 65 microm/s. Dual voltage clamp/confocal recordings revealed that each of the Ca(2+) waves was associated with an inward current typical of the Ca(2+)-activated Cl(-) currents developed by these cells. The waves depended on an intact sarcoplasmic reticulum Ca(2+) store, as they were blocked by cyclopiazonic acid (Calbiochem, San Diego, CA, USA) and agents that interfere with ryanodine receptors and IP(3)-mediated Ca(2+) release. The waves were also inhibited by an NO donor (diethylamine NO; Tocris Bioscience, Bristol, Avon, UK), 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1) (Alexis Biochemicals, Bingham, Notts, UK), 8-bromo-cyclic guanosine mono-phosphate (Tocris), and sildenafil (Viagra, Pfizer, Sandwich, Kent, UK). Regular Ca(2+) oscillations were also observed in whole tissue slices where they were clearly seen to precede contraction. This activity was also markedly inhibited by sildenafil, suggesting that it was under NO regulation. These results provide a new basis for understanding detumescent tone in the corpus cavernosum and its inhibition by NO.

A NOVEL CLINICAL ENTITY OF OBSTRUCTIVE AZOOSPERMIA: PHENOTYPIC ANALYSIS OF TESTICULAR GERM CELL-SPECIFIC GENE MEICHROACDIN-DISRUPTED MICE AND GENOTYPIC ANALYSIS OF HUMAN MEICHROACIDIN IN MALE INFERTILE POPULATION

Phosphodiesterase type 5 (PDE5) inhibitors are very effective agents for erectile dysfunction; however, specific patient populations are hard to treat. The efficacy of PDE5 inhibitors is limited because a minimum amount of nitric oxide (NO) is necessary. Resveratrol, a plant polyphenol, is reported to activate endothelial NO synthase (eNOS) through activation of sirtuin 1. We previously reported that human corpus cavernosal smooth muscle cells (CCSMCs) express eNOS and synthesize cyclic guanosine monophosphate (cGMP) via the NO/cGMP pathway.To investigate the ability of resveratrol and/or vardenafil to increase cGMP in an in vitro model using CCSMCs and to improve erectile function in an in vivo rat model of streptozotocin (STZ)‐induced diabetes.CCSMCs were treated with resveratrol and/or vardenafil. Twenty male Sprague‐Dawley rats were randomly divided into five groups (N = 4 in each group): age‐matched controls, diabetic controls, and diabetic rats treated with resveratrol, vardenafil, or both in combination for the last 4 weeks of an 8‐week period of diabetes induction.Intracellular cGMP measurement, intracovernous pressure (ICP)/mean arterial pressure (MAP) ratio, and smooth muscle/collagen ratio.Intracellular cGMP level was elevated by resveratrol treatment in CCSMCs. The combination treatment of resveratrol and vardenafil had a synergistic effect. Diabetic rats showed impairment of erectile function. Treatment with either resveratrol or vardenafil improved ICP/MAP ratio, and combination therapy with resveratrol and vardenafil had a synergistic effect in improvement of ICP/MAP.Treatment with either resveratrol or vardenafil elevated cGMP level in CCSMCs and improved erectile function in STZ‐induced diabetic rats. Furthermore, a synergistic effect was observed in vitro and in vivo. Resveratrol or combination therapy of resveratrol and vardenafil can improve erectile function in which NO release is impaired, although further study is needed to confirm the results. Fukuhara S, Tsujimura A, Okuda H, Yamamoto K, Takao T, Miyagawa Y, Nonomura N, and Okuyama A. Vardenafil and resveratrol synergistically enhance the nitric oxide/cyclic guanosine monophosphate pathway in corpus cavernosal smooth muscle cells and its therapeutic potential for erectile dysfunction in the streptozotocin‐induced diabetic rat: Preliminary findings.

The Ontogenetic Expression Pattern of Type 5 Phosphodiesterase Correlates with Androgen Receptor Expression in Rat Corpora Cavernosa

The mechanisms controlling erection in animals and in humans are mainly age-dependent. However, the ontogenesis of the biochemical machinery of erection is largely unknown. The aim of this article was to study the expression pattern of androgen receptor (AR) and the major cyclic guanosine monophosphate-hydrolyzing enzyme present in the corpora cavernosa, type 5 phosphodiesterase (PDE5), in the rat penis during development. AR and PDE5 expression was tested on ribonucleic acids (RNAs) and proteins extracted from the whole penis or from primary cultures of smooth muscle cells obtained from the corpora cavernosa of 3- (rCC3), 20- (rCC20), and 60- (rCC60) day-old rats. Rat corpus cavernosum cells were characterized by immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of PDE5 and AR messenger RNA (mRNA) and protein have been measured by RT-PCR and Western blot, respectively. A significant increase in PDE5 mRNA expression was observed with RT-PCR from prepuberty to adulthood (0.5 +/- 0.06 vs. 1.6 +/- 0.046 arbitrary units [a.u.]P = 0.049). This age-dependent increase was mirrored by the increase in PDE5 protein expression found when comparing neonatal to adult corpus cavernosum smooth muscle cells (1.5 +/- 0.26 vs. 4.9 +/- 0.59 a.u. P = 0.0038) and the further 1.6-fold increase from rCC20 to rCC60 (4.9 +/- 0.59 vs. 8.0 +/- 0.8 a.u. P = 0.0024). This is the first demonstration of the ontogenetic profile of PDE5 expression in corpus cavernosum smooth muscle. As it has been demonstrated that androgens control PDE5 expression and that PDE5 inhibitors need an optimal androgenic milieu to act perfectly on erection, the expression of AR protein in rat corpus cavernosum cells was then tested by Western blot. A 7.0-fold increase was observed in primary cultured cells from 3 to 60 days old (1.4 +/- 0.38 vs. 9.8 +/- 1.3 a.u. P = 0.0052). The increase in ARs during rat penile development parallels that of PDE5 RNA and protein, thus suggesting a positive effect of androgens on PDE5 expression.