Human Corpus Cavernosum Smooth Muscle Research Articles

Characterization and functional roles of KCNQ-encoded voltage-gated potassium (Kv7) channels in human corpus cavernosum smooth muscle.

The group of KCNQ-encoded voltage-gated potassium (Kv7) channels includes five family members (Kv7.1-7.5). We examined the molecular expression and functional roles of Kv7 channels in corporal smooth muscle (CSM). Isolated rabbit CSM strips were mounted in an organ bath system to characterize Kv7 channels during CSM relaxation. Intracellular Ca2+ levels were measured in the CSM using the Ca2+ dye Fluo-4AM. The expression of the KCNQ1-5 (the encoding genes for Kv7.1-7.5) and KCNE1-5 subtypes was determined by quantitative real-time PCR. Electrophysiological recordings and an in situ proximity ligation assay (PLA) were also performed. ML213 (a Kv7.2/7.4/7.5 activator) exhibited the most potent relaxation effect. XE911 (a Kv7.1-7.5 blocker) significantly inhibited the relaxation caused by ML213. Removal of the endothelium from the CSM did not affect the relaxation effect of ML213. H-89 (a protein kinase A inhibitor) and ESI-09 (an exchange protein directly activated by cAMP inhibitor) significantly inhibited ML213-induced relaxation (H-89: 31.3%; ESI-09: 52.7%). XE991 significantly increased basal [Ca2+]i in hCSM cells. KCNQ4 (the Kv7.4-encoding gene) and KCNE4 in CSM were the most abundantly expressed subtypes in humans and rats, respectively. KCNQ4 and KCNE4 expression was significantly decreased in diabetes mellitus rats. ML213 significantly increased the outward current amplitude. XE991 inhibited the ML213-induced outward currents. ML213 hyperpolarized the hCSM cell membrane potential. Subsequent addition of XE991 completely reversed the ML213-induced hyperpolarizing effects. A combination of Kv7.4 and Kv7.5 antibodies generated a strong PLA signal. We found that the Kv7.4 channel is a potential target for ED treatment.

Ivabradine, the hyperpolarization-activated cyclic nucleotide-gated channel blocker, elicits relaxation of the human corpus cavernosum: a potential option for erectile dysfunction treatment

Objective To evaluate the effect of the I f channel inhibitor, ivabradine on human corpus cavernosum (HCC) smooth muscle tone. Methods HCC samples were obtained from erectile dysfunction(ED) patients (n = 12) undergoing penile prosthesis surgery. Concentration–response curves for ivabradine were exposed to various inhibitory and stimulatory agents. The relaxant and contractile responses to electrical field stimulation (EFS, 10 Hz and 80 Hz) were examined in the presence or absence of ivabradine (10 μM). HCN3 and HCN4 channel expression and localization were determined by Western blot and immunohistochemical analyses of HCC tissues. Results Increasing ivabradine concentrations dependently reduced the maximal contractile responses of isolated HCC strips induced by KCl (59.5 ± 2.5%) and phenylephrine (84.0 ± 9.8%), which was not affected by nitric oxide synthase and soluble guanylyl cyclase inhibitors after phenylephrine-induced contraction. Nifedipine and tetraethylammonium inhibited the maximum relaxation to ivabradine by 75% and 39.3%, respectively. Fasudil and sildenafil increased the relaxation response to ivabradine without altering the maximum response. Pre-incubation with ivabradine significantly increased relaxant responses to EFS (p < 0.01) and reduced the contractile tension evoked by EFS (72.3%) (p < 0.001). Ivabradine incubation did not affect the expression and localization of HCN3 and HCN4 channels in the HCC smooth muscle cells. Conclusions Ivabradine exhibits a relaxant effect on HCC tissues, which is likely to be attributed to the blocking of L-type Ca2+ channels and the opening of K+ channels, independent of changes in the activation of the nitric oxide/cyclic guanosine monophosphate system. Inhibition of HCN channels localized in cavernosal smooth muscle cells may offer pharmacological benefits for patients with cardiovascular risk factors.

075 Characterization of NAD+ Induced Relaxation Responses in Human Corpus Cavernosum: Age-Related Effects

Nicotinamide adenine dinucleotide (NAD+) is an essential small-molecule cofactor for NAD+-dependent enzymes, such as the sirtuins (SIRT) and important for metabolic redox-reactions. SIRT-stimulation by hydrogen sulfide (H2S) reverses these redox changes, even though SIRT and H2S are part of the same pathway. It is now a consensus that NAD+ levels decline at the cellular and tissue/organ levels during aging and slow the rate of glycolysis, mitochondrial electron transport, and ATP formation in cells. It is important to find out the role of NAD+ on penile corpus cavernosum (CC) from aged-men. We aimed to characterize the effectiveness of NAD+ on human CC smooth muscle tone in baths by evaluating for isometric tension recording in CC from middle-aged and aged-individuals. Human CC obtained from ED patients (n=10) undergoing penile prosthesis surgery were processed for organ bath. After precontraction with phenylephrine (Phe,10μM), concentration-response curves were performed for NAD+ (10nM-100μM). Underlying mechanisms of relaxation were evaluated by inhibitory agents, namely L-NAME [N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase (NOS)], ODQ [1H-(1,2,4) oxadiazolo(4,3-α) quinoxalin-1-one, a soluble guanylyl cyclase inhibitor], and tetraethylammonium (TEA, non- selective K+ channel blocker). The relaxation responses to electrical field stimulation (EFS,10Hz), acetylcholine (ACh,100M), sodium hydrogen sulphur (NaHS,10-4M, a donor of H2S), adenosine (Ado,10-4M) and contractile response to EFS (80Hz) were examined in the presence or absence of NAD+(10μM). Cellular localization and semi-quantification of eNOS, nNOS and VEGF expressions were performed by Western blotting and immunofluorescence. We also performed the primary cell culture of human penile tissue samples from different patients. The cells were incubated in growth media for 2 weeks, characterized for smooth muscle cell protein marker, treated with 500μM of NAD+ either 6 hours or 24 hours, and cell lysate was further quantitated for eNOS, nNOS, VEGF and GAPDH expressions.

Effects of eupatilin on the contractility of corpus cavernosal smooth muscle through nitric oxide-independent pathways.

Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is one of the main compounds present in Artemisia species. Eupatilin has both antioxidative and anti-inflammatory properties and a relaxation effect on vascular contraction regardless of endothelial function. We evaluated the relaxant effects of eupatilin on the corpus cavernosum (CC) of rabbits and the underlying mechanisms of its activity in human corpus cavernosum smooth muscle (CCSM) cells. Isolated rabbit CC strips were mounted in an organ bath system. A conventional whole-cell patch clamp technique was used to measure activation of calcium-sensitive K+ -channel currents in human CCSM cells. The relaxation effect of eupatilin was evaluated by cumulative addition (10-5 m ~ 3×10-4 m) to CC strips precontracted with 10-5 m phenylephrine. Western blotting analysis was performed to measure myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17-kDa (CPI-17) expression and to evaluate the effect of eupatilin on the RhoA/Rho-kinase pathway. Eupatilin effectively relaxed the phenylephrine-induced tone in the rabbit CC strips in a concentration-dependent manner with an estimated EC50 value of 1.2±1.6×10-4 m (n=8, p<0.05). Iberiotoxin and tetraethylammonium significantly reduced the relaxation effect (n=8, p<0.001 and p=0.003, respectively). Removal of the endothelium or the presence of L-NAME or indomethacin did not affect the relaxation effect of eupatilin. In CCSM cells, the extracellular application of eupatilin 10-4 m significantly increased the outward currents, and the eupatilin-stimulated currents were significantly attenuated by treatment with 10-7 m iberiotoxin (n=13, p<0.05). Eupatilin reduced the phosphorylation level of MYPT1 at Thr853 of MLCP and CPI-17 at Thr38. Eupatilin-induced relaxation of the CCSM cells via NO-independent pathways. The relaxation effects of eupatilin on CCSM cells were partially due to activation of BKCa channels and inhibition of RhoA/Rho-kinase.

Imatinib Mesylate (Gleevec) Induces Human Corpus Cavernosum Relaxation by Inhibiting Receptor Tyrosine Kinases (RTKs): Identification of New RTK Targets

To evaluate the effect of the tyrosine kinase inhibitor imatinib mesylate (Gleevec) on human corpus cavernosum (HCC) smooth muscle tone. HCC were obtained from 18 erectile dysfunction (ED) patients undergoing penile prosthesis surgery. The effects of imatinib in HCC strips were investigated in the presence of various inhibitors. The human phosphoreceptor protein tyrosine kinase (PTK) array (Proteome Profiler Array) detected changes in receptor phosphorylation before and after imatinib. Immunohistochemistry was used to localize phosphorylated c-kit (CD117/stem cell factor) in HCC smooth muscle cells. Phenylephrine-induced contraction in HCC was significantly inhibited by imatinib (97.7% ± 2.3%). l-nitro-arginine methyl ester (l-NAME) or guanylyl cyclase inhibitor [1H-1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) alone did not reverse the effect of imatinib, but suppressed this response in combination (18.0% ± 0.6%). The K(+) channel blockers (apamin and tetraethyl ammonium) decreased the imatinib-induced relaxation by 64% and 51%, respectively. PTK microarray analysis of 42 different phospho-receptor tyrosine kinases showed 14 were clearly activated in HCC. Imatinib treatment significantly inhibited phosphorylation of PTKs. A high level of CD117/c-kit-positive immunostaining was detected in untreated HCC smooth muscle, but not in treated HCC. Imatinib caused HCC smooth muscle relaxation invitro mediated by nitric oxide/guanosine monophosphate signaling, involving the large-conductance Ca(2+)-activated K(+)-channels (BK(Ca)) or by inhibiting the upregulated PTK pathway. These results suggest that imatinib may also benefit erectile dysfunction patients who are not responsive to phosphodiesterase-5 inhibitors.

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.

Synergistic Effects of BAY 60-4552 and Vardenafil on Relaxation of Corpus Cavernosum Tissue of Patients with Erectile Dysfunction and Clinical Phosphodiesterase Type 5 Inhibitor Failure

Overall efficacy rates of phosphodiesterase type 5 inhibitors (PDE5-i) for erectile dysfunction (ED) are 60-70%. PDE5-i treatment failures currently have to resort to invasive treatment options for restoration of erectile function. AIMS.: To assess changes in the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase (PKG) pathway in human corpus cavernosum (HCC) of PDE5-i nonresponders compared with healthy controls. To evaluate the effects of BAY 60-4552, a stimulator of soluble guanylate cyclase (sGC), and vardenafil on relaxation of HCC strips from PDE5-i nonresponders. mRNA expression, morphological localization of the NO/cGMP/PKG pathway, and relaxant capacity of both compounds alone or combined. Analysis of variance, t-test or Mann-Whitney test based upon number of groups and normality of data. HCC tissues were harvested after consent from individuals undergoing penile prosthesis implantation (patients) and potent patients undergoing transurethral surgery (healthy controls, needle biopsy). HCC tissues of patients were compared with those of healthy controls for the expression of mRNA coding for PDE5A, eNOS, PKGα1, PKG2, sGCα1, sGCα2, sGCβ1, sGCβ2, α-smooth muscle actin (aSMA) and β-actin by quantitative polymerase chain reaction (qPCR). The respective proteins were localized using immunofluorescence. Tissue strips of patients were precontracted with phenylepinephrine followed by incubation with 1 μM of either vardenafil or BAY 60-4552, or both simultaneously. The main targets in the NO/cGMP/sGC pathway were downregulated in PDE5-i nonresponders. The pathway was morphologically located to HCC smooth muscle, of which the overall content was preserved in ED patients based on aSMA expression. BAY 60-4552 and vardenafil have synergistic effects on relaxation of HCC of PDE5-i nonresponders. The main limitation is the small amount of control tissue precluding functional testing on these samples. Despite downregulation of the NO/cGMP/PKG pathway, combining BAY 60-4552 with vardenafil significantly enhanced relaxation HCC strips of PDE5-i nonresponders.

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.

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.

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.

Expression and distribution of key enzymes of the cyclic GMP signaling in the human clitoris: relation to phosphodiesterase type 5 (PDE5)

The clitoris contributes to the normal female sexual response cycle. A significance of cyclic guanosine monophosphate (GMP) has been assumed in the control of clitoral vascular smooth muscle. As only a few investigations on the physiology of the vascular and non-vascular clitoral tissue have been carried out, knowledge on the mechanisms controlling this particular female genital organ is still vague. It has been suggested that human clitoral corpus cavernosum smooth muscle is regulated by nitric oxide (NO)/cyclic GMP and related key enzymes, such as NO synthases (NOSs) and the phosphodiesterase type 5 (PDE5). The present study evaluated in the human clitoris, by means of immunohistochemistry, the expression and distribution of key enzymes of the cyclic GMP pathway, such as the endothelial NOS, PDE2, PDE11 and cyclic GMP-dependent protein kinase type I (cGKI) in relation to the PDE5. Immunohistochemistry revealed the presence of PDE2, PDE5 and cGKI in the smooth muscle wall of blood vessels transversing the supepithelial and stromal space. Immunosignals specific for PDE2 were also identified in interstitial-like cells located in the basal epithelial layer. Staining for PDE11A was observed in single nerve trunks located in the clitoral stroma. The results are in favor of a role of the cyclic GMP signaling in the control of clitoral blood flow. It seems likely that PDE2 and PDE11 are also involved in the mechanism of local (neuro)transmission in the clitoris.

1130 STREPTOZOTOCIN-INDUCED TYPE 1 DIABETES IN RAT ALTERS SMOOTH MUSCLE MYOSIN ISOFORM COMPOSITION TO A MORE CONTRACTILE PHENOTYPE AND INCREASES SENSITIVITY TO THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research1 Apr 20111130 STREPTOZOTOCIN-INDUCED TYPE 1 DIABETES IN RAT ALTERS SMOOTH MUSCLE MYOSIN ISOFORM COMPOSITION TO A MORE CONTRACTILE PHENOTYPE AND INCREASES SENSITIVITY TO THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN Xinhua Zhang, Arnold Melman, and Michael DiSanto Xinhua ZhangXinhua Zhang Camden, NJ More articles by this author , Arnold MelmanArnold Melman Bronx, NY More articles by this author , and Michael DiSantoMichael DiSanto Camden, NJ More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.727AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and the 17 kDa essential light chains (LC17) exist as several different isoforms with distinct contractile properties, produced via alternative splicing mechanisms. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed both rat and human corpus cavernosum smooth muscle (CCSM)(J Sexual Med, 2009). In this study, we determine if diabetes alters SMM expression, alternative splicing and/or functional activities including sensitivity to BLEB. METHODS Fisher 344 male streptozotocin (STZ)-induced diabetic rats were kept for 2 months. Functional activities were then tested in vivo by intracavernous pressure (ICP) recording and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin (a transcriptional regulator of SMM) and embryonic SMM (SMemb) expression by Real-time RT-PCR. RESULTS Average blood glucose levels of STZ rats were 407 vs 129.5 mg/dl in rats injected with saline (SHAM). STZ rats showed severe erectile dysfunction (ED) confirmed by decreased ICP response to electrical stimulation of cavernous nerve. For STZ rats, LC17a and SM-B isoforms (both associated with increased contractility and faster shortening velocity) and total SMM and myocardin expression increased while LC17b and SM-A isoforms (both associated with decreased contractility and slower shortening velocity) were decreased with SM1/2 SMM MHC isoforms and SMemb unchanged. Interestingly, BLEB was more effective in relaxing STZ CCSM both in vitro and in vivo. CONCLUSIONS We show for the first time to our knowledge a diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype is further supported by total SMM expression increase. Moreover, we provide novel data that the change in CCSM phenotype is associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED with specificity to the faster type myosin isoforms found in the diabetic rat CCSM. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e454 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xinhua Zhang Camden, NJ More articles by this author Arnold Melman Bronx, NY More articles by this author Michael DiSanto Camden, NJ More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

1124 PATHOPHYSIOLOGY OF PRIAPISM WITH VARIED DURATION: A NOVEL RAT MODEL STUDY USING THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research1 Apr 20111124 PATHOPHYSIOLOGY OF PRIAPISM WITH VARIED DURATION: A NOVEL RAT MODEL STUDY USING THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN Xinhua Zhang, Arnold Melman, and Michael DiSanto Xinhua ZhangXinhua Zhang Camden, NJ More articles by this author , Arnold MelmanArnold Melman Bronx, NY More articles by this author , and Michael DiSantoMichael DiSanto Camden, NJ More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.721AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Priapism is an important but understudied and underfunded disease that is highly prevalent in the sickle cell population. The exact molecular basis for priapism remains unclear. Dog and rat models involving pharmacologically induced (e.g. papaverine) and vacuum-induced ischemia have been developed but do not fully recapitulate the human condition. Recently, transgenic mice have contributed to understanding this disorder, but weaknesses include low percent priapism, cost and unknown initiation/duration. Last year, we reported that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed rat and human corpus cavernosum smooth muscle (CCSM) (J Sexual Med, 2009). Here, we validate a novel, extended duration BLEB-induced rat priapism model in which the penis can maintain erection for up to 4 days. METHODS BLEB was intracavernously injected (ICI) into adult rats. After 2h or 4h under anesthesia or 4d or 1wk in awake rats, intracavernous pressure (ICP) recording and histology (Hematoxylin & Eosin or Masson's Trichrome staining) were performed. Expression of molecules in major priapism-linked or well known CCSM contractile pathways was determined by Real-time RT-PCR. RESULTS ICI of 1.25 μmols BLEB induced priapism validated by visual full erection and ICP/MAP (∼0.75) up to 4d. At 2h & 4h, CC vasocongestion and ICP can almost be returned to baseline by flushing. Endothelial nitric oxide synthase (eNOS) expression decreased while phosphodiesterase V (PDE5), Rho-kinase alpha (ROKα), endothelin receptor A (ETA) and SM myosin heavy chain (SMMHC) increased. In contrast, 4d rat ICP is refractory to flushing exhibiting CC thrombosis and early fibrosis. eNOS, nNOS and endothelin receptor B (ETB) increased while PDE5, RhoA, ROKα, ETA, SMMHC and adrenergic receptors α1c & α1d decreased. At 1wk, there was severe fibroplasia preventing ICP recording and CC atrophy with SMMHC 10-fold decreased, TGFβ1 2.5-fold increased, NOS and ET receptors unchanged, ROKα/β and α1c decreased. Hypoxia inducible factor (HIF-1α) decreased at 4h but increased 2- and 4.5-fold at 4d and 1wk. CONCLUSIONS We established a novel, easily induced, long duration BLEB-induced rat priapism model. Importantly, this model showed CCSM contractile molecules upregulated and relaxation molecules downregulated with ICP reversible in the early compensated stage while these pathways were opposite (contraction decreased and relaxation increased with ICP irreversible) in the later decompensated stage with eventual severe fibrosis and atrophy. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e452 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xinhua Zhang Camden, NJ More articles by this author Arnold Melman Bronx, NY More articles by this author Michael DiSanto Camden, NJ More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Experimental model of human corpus cavernosum smooth muscle relaxation

To describe a technique for en bloc harvesting of the corpus cavernosum, cavernous artery and urethra from transplant organ donors and contraction-relaxation experiments with corpus cavernosum smooth muscle. The corpus cavernosum was dissected to the point of attachment with the crus penis. A 3 cm segment (corpus cavernosum and urethra) was isolated and placed in ice-cold sterile transportation buffer. Under magnification, the cavernous artery was dissected. Thus, 2 cm fragments of cavernous artery and corpus cavernosum were obtained. Strips measuring 3 x 3 x 8 mm(3) were then mounted vertically in an isolated organ bath device. Contractions were measured isometrically with a Narco-Biosystems force displacement transducer (model F-60, Narco-Biosystems, Houston, TX, USA) and recorded on a 4-channel Narco-Biosystems desk model polygraph. Phenylephrine (1 microM) was used to induce tonic contractions in the corpus cavernosum (3-5 g tension) and cavernous artery (0.5-1 g tension) until reaching a plateau. After precontraction, smooth muscle relaxants were used to produce relaxation-response curves (10(-12) M to 10(-4) M). Sodium nitroprusside was used as a relaxation control. The harvesting technique and the smooth muscle contraction-relaxation model described in this study were shown to be useful instruments in the search for new drugs for the treatment of human erectile dysfunction.

Contractility of Diabetic Human Corpus Cavernosum Smooth Muscle in Response to Serotonin Mediated via Rho-Kinase

Background/Aims: Serotonin (5-hydroxytryptamine, 5-HT)-induced contraction and the involvement of RhoA/Rho-kinase pathway in the 5-HT-induced contraction was investigated isometrically in vitro in both diabetic and nondiabetic human corpus cavernosum (HCC) tissues. Methods: HCC tissues were obtained from 12 patients. The response to 5-HT (10^–9 to 10^–5 mol/l) was studied in isolated HCC tissues in the absence and in the presence of a Rho-kinase inhibitor (Y-27632). Results: Preincubation with Y-27632 attenuated maximum contractions induced by 5-HT in tissues of both nondiabetics and diabetics. When diabetic and nondiabetic groups were compared, no significant difference was seen in 5-HT-induced contraction alone, but in the presence of Y-27632, 5-HT-induced contraction was significantly higher in the diabetic group. Conclusion: These results suggest that the Rho-kinase-mediated pathway plays an important role for 5-HT-induced contraction in diabetic corpus cavernosum tissues.

Enhanced Thromboxane Receptor-Mediated Responses and Impaired Endothelium-Dependent Relaxation in Human Corpus Cavernosum from Diabetic Impotent Men: Role of Protein Kinase C Activity

We have evaluated the influence of protein kinase C (PKC) activity on penile smooth muscle tone in tissues from diabetic and nondiabetic men with erectile dysfunction. Human corpus cavernosum (HCC) strips were obtained from impotent diabetic and nondiabetic men at the time of penile prosthesis implantation and studied in organ chambers. Contractility responses to a prostanoid precursor, to prostanoids, and to the endothelium-dependent vasodilator acetylcholine were studied. Arachidonic acid (AA; 100 microM) caused cyclooxygenase-dependent relaxation of HCC. This relaxation was impaired in diabetic tissues and normalized by blocking thromboxane (TP) receptors with 20 nM [1S-[1alpha,2alpha(Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ29548). Diabetes did not affect prostaglandin (PG)E(1)-induced relaxation, but it reduced relaxation induced by the PGE(1) metabolite PGE(0). This effect was related to an interaction of PGE(0) with TP receptors. Diabetic tissues had reduced endothelium-dependent relaxation, which was partially improved by SQ29548 and completely normalized by the PKC inhibitor 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X; 1 microM). In HCC from nondiabetic patients, treatment with the PKC activator phorbol-12,13-dibutyrate (0.3 microM) significantly attenuated endothelium-dependent relaxation, an effect prevented by coadministration of GF109203X. Tissues from diabetic patients had enhanced sensitivity to the contractile effects of the TP receptor agonist 9,11-dideoxy-9alpha,11alpha-epoxymethano PGF(2alpha) (U46619) (EC(50) = 0.65 +/- 0.42 and 6.01 +/- 2.28 nM in diabetic and nondiabetic patients, respectively). Inhibition of PKC with 1 microM GF109203X, prevented diabetes-induced hypersensitivity to U46619-induced contractions (EC(50) = 8.55 +/- 3.12 microM). Overactivity of PKC in diabetes is responsible for enhanced contraction and reduced endothelium-dependent relaxation of HCC smooth muscle. Such alterations can result in erectile dysfunction.

Corpus Cavernosum from Men with Vasculogenic Impotence Is Partially Resistant to Adenosine Relaxation due to Endothelial A2BReceptor Dysfunction

Although adenosine has been implicated in penile erection in human males, the receptor subtype responsible for adenosine regulation of human corpus cavernosum (HCC) smooth muscle tone is still a matter of debate. Using selective adenosine agonists and antagonists, we aimed at characterizing the adenosine receptors mediating relaxation of precontracted (with 1 microM phenylephrine) HCC strips. HCC specimens were collected from control subjects (organ donors) and from patients with severe vasculogenic erectile dysfunction (ED). In control subjects, adenosine and 5'-N-ethyl-carboxamide adenosine (NECA) fully relaxed HCC. The selective A(2A) receptor agonist 2-[4-(2-p-carboxy ethyl)phenylamino]-5'-N-ethylcarboxamido adenosine (CGS21680C) produced only a partial relaxation (30-50%) of HCC, which could be further enhanced by simultaneous application of 100 microM NECA. The selective A(2B) receptor antagonist N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-il)phenoxy] acetamida (MRS1706) (10 nM) attenuated NECA-induced relaxation without affecting CGS21680C action. The A(2A) receptor antagonist 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol (ZM241385) (50 nM) consistently reduced the actions of both agonists. In contrast to CGS21680C, NECA-induced relaxation was attenuated when endothelial production of NO and prostanoids was reduced by 100 microM N(G)-nitro-l-arginine and 10 microM indomethacin, respectively. HCC strips from patients with vasculogenic ED were partially resistant to NECA but kept relaxation to CGS21680C; the remaining effect was sensitive to blockade of A(2A) receptors with 50 nM ZM241385. Data suggest that adenosine regulates HCC smooth muscle tone through the activation of two receptor populations, CGS21680C-sensitive (A(2A)) and -insensitive (A(2B)) receptors, located on smooth muscle fibers and on endothelial cells, respectively. Endothelial dysfunction may be correlated with a loss of adenosine A(2B) receptor activity in penile vessels from men with vasculogenic ED.

Effect of Doxazosin With and Without Rho-Kinase Inhibitor on Human Corpus Cavernosum Smooth Muscle in the Presence of Bladder Outlet Obstruction

We investigated the relationship of adrenergic responses in corpus cavernosum tissues in the presence of BOO using the alpha1-adrenergic receptor antagonist doxazosin (Pfizer, New York, New York) and the rho-kinase inhibitor Y-27632 (Calbiochem, San Diego, California). CCSM tissue was obtained from patients who underwent penile prosthesis implantation. Patients were divided into 2 groups according to the presence of BOO. The submaximal (EC80) concentration of phenylephrine (Sigma Chemical Co., St. Louis, Missouri) was calculated by evaluating adrenergic activity responses with cumulatively applied phenylephrine. After achieving a stable contraction plateau test compounds were put in an organ bath. The relaxant potencies of doxazosin and Y-27632 were expressed as the percent of inhibition of the contraction plateau induced EC80 concentration of phenylephrine. Relaxation responses in the 2 groups were compared. At the highest dose of increasing concentrations phenylephrine generated 70% more contraction response in the BOO positive group than in the BOO negative group. Doxazosin and Y-27632 caused concentration dependent relaxation in CCSM precontracted by phenylephrine. With doxazosin significantly higher relaxation responses were attained in the BOO positive group in terms of log IC50 and the maximal relaxation response (p = 0.0353 and 0.0003, respectively). Maximum relaxation responses following Y-27632 administration were significantly higher in the BOO positive group. The contractility of human corpus cavernosum is increased in the presence of BOO. Doxazosin and Y-27632 generate effective CCSM relaxation in the presence of BOO. Doxazosin and Y-27632 may be the alternatives for the treatment of erectile dysfunction associated with BPH.

Effects of C-peptide on expression of eNOS and iNOS in human cavernosal smooth muscle cells

Objectives To investigate the role of C-peptide alone or in conjunction with insulin on the expression of nitric oxide synthase (NOS) in human corpus cavernosum smooth muscle cells (HCSMCs). Erectile dysfunction, among diabetic patients, is a significant health problem. The specific causes of erectile dysfunction are unknown. It has been suggested that impairment of penile relaxation is related to a reduction of penile NOS. Plasma levels of C-peptide and insulin are decreased in individuals with type 1 diabetes and late-stage type 2 diabetes. Methods Primary cultures were initiated from explants of HCSMCs. Confluent cells at passages 2 to 4 were assigned to one of four groups with the following incubation conditions: (a) 27 mM glucose, (b) 27 mM glucose and insulin, (c) 27 mM glucose and human recombinant (hr)C-peptide, and (d) 27 mM glucose, insulin, and hrC-peptide. After 24 hours, total RNA and protein were extracted from cells and subjected to reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. Intracellular Ca 2+ was examined under the four conditions, using the Fura 2 method. Results The least expression of endothelial NOS (eNOS) and inducible NOS (iNOS) in HCSMCs was observed in cells exposed to 27 mM glucose alone. Increased expression of eNOS and iNOS was found after treatment with insulin or hrC-peptide alone, and the maximal expression of eNOS and iNOS was detected in HCSMCs exposed to both insulin and hrC-peptide. Western blot analyses using eNOS and iNOS antibodies confirmed the RNA data. These effects are likely mediated by the insulin-induced and/or C-peptide-induced increase in intracellular Ca 2+. Conclusions Our results demonstrated that C-peptide, in the presence of insulin, increases the expression of iNOS and eNOS in HCSMCs. These results suggest that C-peptide, especially in conjunction with insulin, may have beneficial effects on cavernosal smooth muscle relaxation.

1429: Upregulation of Rho-Kinase in a Human Corpus Cavernosum Smooth Muscle Cell Line in Response to Hypoxia

1429: Upregulation of Rho-Kinase in a Human Corpus Cavernosum Smooth Muscle Cell Line in Response to Hypoxia