Corpus Cavernosum Smooth Muscle Tone Research Articles

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.

AB302. SPR-29 Sphingosine-1-phosphate in vitro and in vivo modulates corpus cavernosum smooth muscle tone

ObjectiveThe bioactive lipid sphingosine-1-phosphate (S1P) regulates smooth muscle (SM) contractility predominantly via three G protein-coupled receptors. The S1P1 receptor is associated with nitric oxide-mediated SM relaxation while S1P2 & S1P3 receptors are linked to SM contraction via activation of the Rho-kinase (ROK) pathway. The objective of this study was to determine the role of S1P in the modulation of corpus cavernosum (CC) SM (CCSM) tone.MethodsHuman and rat samples were used. Plasma S1P levels were detected by high-performance liquid chromatography. The expression of S1P1-3 receptors and sphingosine kinase-1 (SphK1) was determined by real-time RT-PCR and western blot. In vitro organ bath contractility and in vivo intracavernous pressure (ICP) measurements were performed. Results were expressed as mean ± SEM for n experiments. Statistical analysis was performed using either the Student’s t-test (when two sample treatments were being compared) or using ANOVA when multiple means were compared. P<0.05 was considered significant.ResultsPlasma S1P levels were determined to be ~200 nanomolar. Human and rat CC both express SphK1 and all S1P1-3 receptors. Exogenous S1P and the S1P receptor agonist FTY720-P contracted while antagonist JTE-013 relaxed CCSM in vitro. Meanwhile, force produced by S1P and agonists could be totally reversed by ROK inhibitor. Also, intracavernous injection of FTY720-P inhibited ICP rise induced by submaximal electrical stimulation of cavernous nerve, while JTE-013 alone induced ICP increase in vivo. Finally, SphK1 siRNA knocked down rat CC SphK1 by 80% and ICP were significantly potentiated.ConclusionsIn conclusion, we provide novel data that S1P, possibly coupling S1P2 and S1P3 receptors via RhoA/ROK pathway, mediates CCSM in vitro and in vivo. Antagonizing S1P or its receptors induces CCSM relaxation and proerectile effects. Thus, we provide the first clear evidences that the S1P system is another key contractile regulatory system in CC and thus this pathway is a potential therapeutic target for the treatment of priapism and erectile dysfunction.Funding Source(s)NIH R01 DK077116 to ME DiSanto

1006 IMPAIRMENT OF SENSORY INNERVATION IN THE BLADDER NECK BY OUTLET OBSTRUCTION CONTRIBUTES TO THE DEVELOPMENT OF ERECTILE DYSFUNCTION

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 20121006 IMPAIRMENT OF SENSORY INNERVATION IN THE BLADDER NECK BY OUTLET OBSTRUCTION CONTRIBUTES TO THE DEVELOPMENT OF ERECTILE DYSFUNCTION Qi Lei, Joseph Hypolite, Shaohua Chang, Robert Seftel, Samuel Chacko, Alan Wein, Allen Seftel, and Anna Malykhina Qi LeiQi Lei Glenolden, PA More articles by this author , Joseph HypoliteJoseph Hypolite Glenolden, PA More articles by this author , Shaohua ChangShaohua Chang Camden, NJ More articles by this author , Robert SeftelRobert Seftel Glenolden, PA More articles by this author , Samuel ChackoSamuel Chacko Glenolden, PA More articles by this author , Alan WeinAlan Wein Philadelphia, PA More articles by this author , Allen SeftelAllen Seftel Camden, NJ More articles by this author , and Anna MalykhinaAnna Malykhina Glenolden, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1108AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Benign prostatic hyperplasia (BPH) and erectile dysfunction (ED) are common problems in aging males worldwide. Clinical evidence of impaired sensation in ED suggests a neurogenic component of ED development, especially when ED is co-morbid with lower urinary tract symptoms. The objective of this work was to evaluate the role of the damage to bladder neck nerves during partial bladder outlet obstruction (PBOO) on sensory innervation of the corpus cavernosum (CC) and CC smooth muscle (CCSM) function using an animal model of PBOO. METHODS Four groups of adult male Sprague Dawley rats (N=32) were included in the study: sham operated (control), PBOO surgery, sham and PBOO groups with injected retrograde tracers. PBOO was surgically induced by a partial ligation of the bladder neck during laparotomy. Retrograde labeling technique was used to label L6-S2 dorsal root ganglion (DRG) neurons which innervate the urinary bladder and CC. The contractility and relaxation of the CC smooth muscle strips was studied in vitro in response to different stimuli. RESULTS Urinary bladders from animals with PBOO weighted 308.75±61.83 mg (N=13, 2 weeks PBOO) when compared to sham group (126.60±12.80 mg, N=13, p≤0.05). L6-S1 DRG ganglia were removed bilaterally and sectioned to count bladder and CC labeled sensory neurons. The obtained results showed that L6-S2 DRG ganglia receive input from both the urinary bladder and CC and 2-3% of all labeled neurons were double labeled from both organs suggesting some overlap in sensory innervation. The contractile response of CC muscle strips to electric stimulation was significantly reduced in PBOO group (2.223±0.3262 g/mg, p≤0.05) when compared to control (3.6367±0.3577 g/mg). Force generation of CCSM to 125 mM KCl was also significantly reduced in the PBOO group (2.61±0.35 g/mg vs 4.94±0.91 g/mg in control, p≤0.05). After CCSM was pre-contracted with phenylephrine, relaxation induced by nitroprusside was significantly diminished in PBOO group (2.76±0.29 g/mg vs 3.66±0.27 g/mg in the control, p≤0.05). There was also a significant decrease in n-NOS expression in PBOO animals when compared to the sham group (47%, N=4, p≤0.05). CONCLUSIONS PBOO leads to the impairment of bladder neck innervation followed by an increase in CCSM tone and a down-regulation of nNOS expression in the CC smooth muscle. These results provide evidence that nerve damage in PBOO may contribute to the development of ED causing secondary changes in the contraction/relaxation mechanisms of CCSM. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e409 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qi Lei Glenolden, PA More articles by this author Joseph Hypolite Glenolden, PA More articles by this author Shaohua Chang Camden, NJ More articles by this author Robert Seftel Glenolden, PA More articles by this author Samuel Chacko Glenolden, PA More articles by this author Alan Wein Philadelphia, PA More articles by this author Allen Seftel Camden, NJ More articles by this author Anna Malykhina Glenolden, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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.

Update on Corpus Cavernosum Smooth Muscle Contractile Pathways in Erectile Function: A Role for Testosterone?

Normal erectile function (EF) involves a coordinated relaxation of the arteries that supply the penis and the corpus cavernosum smooth muscle (CCSM), resulting in expansion of the sinusoids and increased intracavernous pressure. But the CCSM spends the majority of its time in the contracted state which is mediated by norepinephrine released from nerve endings and other vasoconstrictors like endothelins released from the endothelium. These agents cause smooth muscle myosin (SMM) phosphorylation by elevating intracellular calcium. When calcium returns to basal levels, the calcium sensitivity increases and prevents myosin dephosphorylation, which involves the RhoA/Rho-kinase (ROK) mechanism, thus maintaining force. Although mounting evidences demonstrate that androgens have a major influence on EF that is not just centrally mediated, this notion remains quite controversial. To summarize the current knowledge on CCSM contractile pathways, the role they play in modulating EF, and the influence of androgens. The article reviews the literature and contains some previously unpublished data on CCSM contraction signaling including the role that androgens are known to play in modulating these pathways. Data from peer-reviewed publications and previously unpublished observations. In addition to downregulation of many pro-erectile molecular mechanisms, decreased testosterone (T) levels upregulate CCSM contractility, including hyperresponsiveness to α-adrenergic agonists, increased SMM phosphorylation, alteration of SMM isoform composition, activation of RhoA/ROK signaling and modulation of sphingosine-1-phosphate regulation of CCSM tone. Decreased T levels upregulate CCSM contractile signaling. Meanwhile, it downregulates CCSM relaxation pathways synergizing to produce erectile dysfunction (ED). Although some urologists and researchers are still skeptical of the influence of androgens on penile erection, understanding these molecular control mechanisms as well as the influence that androgens have on these pathways should provide new evidence supporting the roles of androgens in EF and enhance the discovery of novel targets for drug development to treat ED.

INCREASED CORPUS CAVERNOSUM SMOOTH MUSCLE TONE ASSOCIATED WITH PARTIAL BLADDER OUTLET OBSTRUCTION IS MEDIATED VIA NEUROFILAMENT 200 AND APOPTOSIS

You have accessJournal of Urology1 Apr 2009INCREASED CORPUS CAVERNOSUM SMOOTH MUSCLE TONE ASSOCIATED WITH PARTIAL BLADDER OUTLET OBSTRUCTION IS MEDIATED VIA NEUROFILAMENT 200 AND APOPTOSIS Ki Ho Kim, Jung Dam Kim, and Young Jin Seo Ki Ho KimKi Ho Kim More articles by this author , Jung Dam KimJung Dam Kim More articles by this author , and Young Jin SeoYoung Jin Seo More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(09)60695-9AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "INCREASED CORPUS CAVERNOSUM SMOOTH MUSCLE TONE ASSOCIATED WITH PARTIAL BLADDER OUTLET OBSTRUCTION IS MEDIATED VIA NEUROFILAMENT 200 AND APOPTOSIS." The Journal of Urology, 181(4S), p. 244 © 2009 by American Urological AssociationFiguresReferencesRelatedDetails Volume 181Issue 4SApril 2009Page: 244 Advertisement Copyright & Permissions© 2009 by American Urological AssociationMetricsAuthor Information Ki Ho Kim More articles by this author Jung Dam Kim More articles by this author Young Jin Seo More articles by this author Expand All Advertisement PDF downloadLoading ...

Interaction between spontaneous and neurally mediated regulation of smooth muscle tone in the rabbit corpus cavernosum

Interaction between spontaneous and neurally mediated regulation of tone in the corpus cavernosum smooth muscle (CCSM) of the rabbit was investigated. Changes in isometric muscle tension, intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were recorded. CCSM developed spontaneous contractions, transient increases in [Ca2+]i (Ca2+ transients) and depolarizations. This spontaneous activity was abolished by blocking L-type Ca2+ channels (nicardipine, 1 mum), sarcoplasmic reticulum Ca2+ pump activity (cyclopiazonic acid, 10 microm), Ca2(+)-activated Cl- channels (niflumic acid, 10 mum) or cyclooxygenase-2 (COX-2; NS-398, 1 microm). Transmural nerve stimulation initiated either alpha-adrenergic contractions or nitrergic relaxations of CCSM depending on the level of muscle tone. NS-398 suppressed nerve-evoked contractions by about 70% but caused only a 40% reduction in the corresponding Ca2+ transient. Blocking nitric oxide synthase with N(omega)-nitro-l-arginine (LNA, 100 microm) reinforced nerve-evoked Ca2+ transients by about 150%, whilst increasing the corresponding Ca2+ transients by only 20%. In CCSM preparations that had been pre-contracted with either noradrenaline (0.3 microm) or prostaglandin F(2alpha) (0.1 microm), nerve stimulation inhibited about 70% of the contraction and caused only a 20% decrease in [Ca2+]i. Fluorescent immunohistochemistry with COX-2 antibodies and the reverse transcriptase-polymerase chain reaction (RT-PCR) method showed that the enzyme and its mRNA were highly expressed in the CCSM. These results suggest that spontaneously produced prostaglandins (PGs) not only contribute to the generation of spontaneous contractions but also facilitate nerve-evoked contractions. Conversely, spontaneously released nitric oxide (NO) suppresses excitation. Thus, interaction between spontaneous and neurally mediated regulation of CCSM tone may be fundamental to maintaining the muscle contractility. In addition, both PGs and NO appear to alter CCSM tone with only small changes in [Ca2+]i.

Increased corpus cavernosum smooth muscle tone associated with partial bladder outlet obstruction is mediated via Rho-kinase

Numerous studies have now demonstrated that lower urinary tract symptoms (LUTS) are associated with erectile dysfunction (ED) in men independent of age or general health. Because one-third of men over the age of 50 will develop LUTS and a recent study showed ED in 62% of patients presenting for LUTS, the importance of determining the mechanistic link between these two pathologies is clear. Using a rabbit model of partial bladder outlet obstruction (PBOO), a primary cause of LUTS, we have identified an increased basal corpus cavernosum smooth muscle (CCSM) tone associated with an elevated level of smooth muscle myosin (SMM) phosphorylation in PBOO compared with sham-operated control rabbits (sham). Results from in vitro kinase and phosphatase assays using purified smooth muscle myosin showed increased kinase and decreased phosphatase activities in cellular extracts from corpora cavernosa isolated from PBOO compared with sham rabbits. Increased Rho-kinase expression in the CCSM of PBOO rabbits was suggested by the observations that Rho-kinase inhibitors attenuated the increased kinase activity and were less effective in relaxing CCSM strips from PBOO vs. sham rabbits. This hypothesis was then confirmed by RT-PCR and Western blotting, which demonstrated increased expression of both isoforms of Rho-kinase (ROKalpha and ROKbeta). Increased SMM basal phosphorylation (necessary for SM contraction) in the CCSM of PBOO rabbits, mediated via an increase in Rho-kinase expression/activity, would be expected to make the CCSM more difficult to relax (necessary for erection), which suggests that the RhoA/Rho-kinase pathway as being involved in the mechanism for LUTS-associated ED.

Increased contractility of diabetic rabbit corpora smooth muscle in response to endothelin is mediated via Rho-kinase beta.

Corpus cavernosum smooth muscle (CCSM) from rabbits made diabetic for 6 months as a result of alloxan injection exhibited increased sensitivity (3vs 9 nM EC(50)) and generated 20-50% greater force to endothelin-1 (ET-1) compared to CCSM from normal rabbits. In contrast, the force produced by the CCSM in response to KCl and phenylephrine was not significantly altered in diabetic CCSM. The increased ET-1 sensitivity is associated with a two to three-fold upregulation of ET receptor A at both mRNA and protein levels in diabetic CCSM. ET-1-induced CCSM contraction is largely dependent upon Rho-kinase (ROK), since it is almost completely blocked by Y-27632 (a highly selective ROK inhibitor). Furthermore, expression of ROKbeta isoform is selectively upregulated in CCSM from diabetic rabbits. Thus, an increased CCSM tone, modulated by sensitization of the endothelin-mediated contractile pathway via ROK, may be a key component of the molecular mechanism of diabetes-induced erectile dysfunction.

Comparison of endothelin-1-mediated tissue tension and calcium mobilization effects in isolated rabbit corpus cavernosum

Objectives To directly compare and contrast the effects of endothelin-1 (ET-1) and adrenoreceptor agonists norepinephrine and phenylephrine on eliciting calcium influx in primary rabbit corpus cavernosum cells and their ability to elicit tissue contractions. The potent vasoconstrictor peptide ET-1 and the alpha-adrenoreceptor agonists are important modulators of smooth muscle tone in the penile corpus cavernosum. However, the mechanisms involved in maintaining smooth muscle tone and contraction are not clearly understood. Methods Intracellular calcium mobilization was measured in cultured corpus cavernosum smooth muscle cells using calcium-sensing dyes in conjunction with a fluorometric imaging plate reader. Tissue tension studies on rabbit corpus cavernosum were conducted using organ chambers. Results ET-1 at concentrations as low as 10 nM was sufficient to induce a transient increase of intracellular calcium in these cells. In contrast, concentrations of 1 mM and greater of norepinephrine and phenylephrine were required to elicit comparable calcium fluxes in cavernosum cells. Tissue bath studies indicated that ET-1 is a potent stimulator of corpus cavernosum smooth muscle contraction, with concentrations as low as 10 nM sufficient to initiate contraction. Conclusions The potency of ET-1 in producing contraction on tissue strips and inducing calcium flux suggests that ET-1 might be an important mediator for modulating and maintaining corpus cavernosum smooth muscle tone. Therefore, additional exploration of the role of endothelins and their receptors in the tumescence and detumescence states of the penis would be extremely valuable.

Cyclic AMP regulates mRNA expression of alpha-1d and alpha-2a adrenergic receptors in cultured human corpus cavernosum smooth muscle cells

While the physiological effects of contractile (e.g. norepinephrine) and relaxatory (e.g. PGE1, forskolin) agents on corpus cavernosum smooth muscle tone have been characterized, the regulation of alpha adrenergic receptor mRNA expression in erectile tissue remains to be investigated. The goal of this study was to investigate the modulation of alpha-1 and alpha-2 adrenergic receptor mRNA expression in cultured human corpus cavernosum smooth muscle cells in response to increased intracellular cAMP induced by prostaglandin E1 and forskolin. Human corpus cavernosum smooth muscle cells were incubated for 24 h with or without PGE1 (5.7 µM), forskolin (10 µM) or an admixture of both. Total RNA was prepared from the cultures. Expression of alpha-1d adrenergic receptor, alpha-2a adrenergic receptor and m2 muscarinic acetylcholine receptor was determined by RNase protection assays. Loading was normalized by RNase protection of the housekeeping gene, cyclophilin A. The relative abundance of mRNAs was quantitated by scanning densitometry. Treatment of human corpus cavernosum smooth muscle cells with PGE1 or forskolin resulted in decreased mRNA expression of alpha-1d and alpha-2a adrenergic receptors and m2 muscarinic acetylcholine receptor when compared to untreated cells. Combinations of PGE1 and forskolin produced a more pronounced decrease in mRNA than either agent alone. PGE1 and forskolin increased intracellular levels of cAMP in human corpus cavernosum smooth muscle cells and combinations of both agents produced a more pronounced increase in cAMP synthesis. These results suggest that cAMP modulates the expression of alpha adrenergic receptors, one of the principal contractile receptor systems in the corpora cavernosa. These observations further support the concept that erectile function is a balance between contractile and relaxatory processes, which in turn regulate structure and function of the corpora cavernosa. International Journal of Impotence Research (2000) 12, Suppl 1, S41-S47

SILDENAFIL INHIBITS PHOSPHODIESTERASE TYPE 5 IN HUMAN CLITORAL CORPUS CAVERNOSUM SMOOTH MUSCLE

Abstract Phosphodiesterases play an important physiological role by regulating the intracellular levels of cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of phosphodiesterase (PDE) type 5 (EC 3.1.4.35, 3′,5′-cyclic GMP phosphodiesterase) by a novel, high-affinity, selective PDE type 5 inhibitor, sildenafil, in intact cells and in soluble extracts of human clitoral corpus cavernosum smooth muscle cells. Sildenafil inhibited cGMP hydrolysis in the crude extract (Ki= 7.2 ± 2.7) and in partially purified preparations (Ki= 9 nM) in a competitive manner, as determined by Dixon plots. Sildenafil was a more effective PDE type 5 inhibitor than zaprinast (Ki= 400.0 ± 76.4 nM, crude extracts; 250 nM, partially purified). Stimulation of intracellular cGMP synthesis by the nitric oxide donor sodium nitroprusside resulted in a 3.3- and 2.9-fold increase in cGMP concentration in the presence of sildenafil or zaprinast, respectively, compared to sodium nitroprusside treatment alone in intact cells at physiological temperatures. These observations suggest that human clitoral corpus cavernosum smooth muscle tone may be regulated by the synthesis and release of nitric oxide and that this pathway is dependent on PDE type 5 activity.

Sildenafil Inhibits Phosphodiesterase Type 5 in Human Clitoral Corpus Cavernosum Smooth Muscle

Phosphodiesterases play an important physiological role by regulating the intracellular levels of cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of phosphodiesterase (PDE) type 5 (EC 3.1.4.35, 3′,5′-cyclic GMP phosphodiesterase) by a novel, high-affinity, selective PDE type 5 inhibitor, sildenafil, in intact cells and in soluble extracts of human clitoral corpus cavernosum smooth muscle cells. Sildenafil inhibited cGMP hydrolysis in the crude extract (Ki= 7.2 ± 2.7) and in partially purified preparations (Ki= 9 nM) in a competitive manner, as determined by Dixon plots. Sildenafil was a more effective PDE type 5 inhibitor than zaprinast (Ki= 400.0 ± 76.4 nM, crude extracts; 250 nM, partially purified). Stimulation of intracellular cGMP synthesis by the nitric oxide donor sodium nitroprusside resulted in a 3.3- and 2.9-fold increase in cGMP concentration in the presence of sildenafil or zaprinast, respectively, compared to sodium nitroprusside treatment alone in intact cells at physiological temperatures. These observations suggest that human clitoral corpus cavernosum smooth muscle tone may be regulated by the synthesis and release of nitric oxide and that this pathway is dependent on PDE type 5 activity.

Renin Angiotensin System in Rabbit Corpus Cavernosum: Functional Characterization of Angiotensin II Receptors

Purpose The regulation of the corporal smooth muscle tone is important in the process of penile erection. Although specific angiotensin (ANG) II binding to and effects of ANG II on some reproductive structures have been studied, the presence of the renin-angiotensin system has not yet been defined in the corpus cavernosum. ANG II is formed from ANG I by angiotensin I-converting enzyme (ACE). ANG II and ANG I produce contractions in vascular smooth muscles. Two subtypes of ANG II receptors (AT1 and AT2) have been characterized. The purpose of the present experiments was to determine whether the renin-angiotensin system regulates rabbit corpus cavernosum smooth muscle tone. Materials and Methods A strip of rabbit corpus cavernosum was mounted in an organ chamber to measure the isometric tension. The specific binding for 125 I-ANG II was characterized by in vitro autoradiography. Results ANG II and ANG I, precursor of ANG II, contracted corpus cavernosum smooth muscle dose-dependently, but the response of smooth muscle to ANG I was 10-fold less than that to ANG II. Contractile responses of smooth muscle to ANG II and ANG I were blocked by Dup 753, a specific inhibitor of ANG II type 1 receptor, but not by PD 123,319, a specific inhibitor of ANG II type 2 receptor. The effect of ANG I was attenuated by captopril, an inhibitor of ACE. Specific binding sites for 125 I-ANG II were found in the corpus cavernosum. The dissociation constant (K d 2) was 5.32 +/− 1.65 nM. and maximum binding capacity (B max) was 305.72 +/− 85.24 amol/mm. 2 Specific binding of 125 I-ANG II was displaced by Dup 753 (10 sup −6 M) but not by PD 123,319 (10 sup −5 M). The inhibitory constant (K i) for Dup 753 was 8.09 +/− 2.51 nM. Conclusion The present results suggest that the renin-angiotensin system is involved in the regulation of corpus cavernosum smooth muscle tone of rabbit and the ANG II receptor subtype AT1 is important in the regulation of penile erection.

Investigative Urology: Modulation of Vasoactive Intestinal Polypeptide (VIP)-Mediated Relaxation by Nitric Oxide and Prostanoids in the Rabbit Corpus Cavernosum

The polypeptide VIP has been proposed as an inhibitory nonadrenergic, noncholinergic (NANC) neurotransmitter involved in the relaxation of cavernosal smooth muscle during erection. However, the specific mechanism(s) by which VIP exerts its effect is unknown. To determine whether VIP is involved in NANC-mediated cavernosal relaxation, strips of corpus cavernosum from New Zealand White rabbits were hung in tissue baths; atropine and guanethidine were added to block cholinergic and adrenergic neurotransmission, respectively. The tissue was then submaximally precontracted with norepinephrine (NE) and relaxed by electrical field stimulation (EFS; 10 V square waves, 0.5 msec. duration, 10 second trains at 5, 15 and 40 Hz) before and after preincubation with the VIP antagonist, VIP 10-28. To evaluate the role of nitric oxide (NO) and prostaglandins on the relaxant effect of VIP in the corpus cavernosum, the strips were contracted with NE and subsequently relaxed with cumulative doses of VIP (10(-10) to 10(-6) M.). After VIP dose-response curves were obtained, the strips were preincubated with N omega-nitro-L-arginine (NOARG, an inhibitor of nitric oxide synthase, 10(-4) M.) and the VIP dose-response curve was repeated. Indomethacin (10(-5) M.) was added to one-half of the NOARG treated strips to inhibit prostaglandin synthesis. VIP 10-28 inhibited EFS-induced relaxation (p < 0.05) and produced dose-dependent relaxation, which was inhibited by NOARG (p < 0.05). In contrast, the VIP-induced relaxation was more pronounced in the presence of indomethacin (p < 0.05). Moreover, indomethacin substantially reversed the NOARG inhibition of VIP relaxation (p < 0.05). These results suggest that VIP is a mediator of NANC cavernosal relaxation and that NO synthesis is involved in VIP-induced NANC relaxation in the corpus cavernosum. In addition, the presence of vasoconstrictive prostanoids modulates this VIP-mediated NANC relaxation. Therefore, VIP appears to contribute to NANC neurally mediated cavernosal relaxation, and its mechanisms of relaxation are dependent on prostanoid and involve the generation of NO.

Endothelium-derived nitric oxide and cyclooxygenase products modulate corpus cavernosum smooth muscle tone.

Relaxation of penile corpus cavernosum smooth muscle is controlled by nerve and endothelium derived substances. In this study, endothelium-dependent relaxation of corporal smooth muscle was characterized and the role of arachidonic acid products of cyclooxygenase in endothelium-dependent relaxation was examined. Endothelium removal from rabbit corpora was performed by infusion with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate and was confirmed by transmission electron microscopy. Strips of human and rabbit corporal tissues were studied in the organ chambers for isometric tension measurement. The accumulation of cyclic guanosine monophosphate (cGMP) and the release of eicosanoids from corporal tissue was measured by radioimmunoassay and correlated to smooth muscle relaxation. Our study showed that relaxation of corpus cavernosum tissue to acetylcholine, bradykinin and substance P was endothelium-dependent; potentiated by indomethacin; and inhibited by NG-monomethyl-L-arginine, methylene blue or LY83583. Relaxation to papaverine and sodium nitroprusside was endothelium-independent, and unaffected by NG-monomethyl-L-arginine. Relaxation to vasoactive intestinal polypeptide was partially endothelium-dependent; potentiated by indomethacin; attenuated by NG-monomethyl-L-arginine or methylene blue. The tissue level of cGMP was enhanced by acetylcholine and nitric oxide. Methylene blue inhibited both basal and drug-stimulated levels of cGMP. The release of eicosanoids was enhanced by acetylcholine and blocked by indomethacin. In conclusion, nitric oxide or a closely related substance accounts for the activity of endothelium-derived relaxing factor in the corporal tissue. Inhibition of the release of eicosanoids potentiates the relaxing effect of nitric oxide. Nitric oxide increases tissue cGMP which appears to modulate corporal smooth muscle relaxation.

Cholinergic neurotransmission in human corpus cavernosum. I. Responses of isolated tissue

To investigate the role of cholinergic neurotransmission in erection, human penile corpus cavernosum tissue was studied. Electrical stimulation of strips of corpus cavernosum suspended in an organ chamber induced contractions and relaxations that were blocked by tetrodotoxin. The contractions also were blocked by bretylium and prazosin. Norepinephrine and phenylephrine contracted the isolated strips and this response was prevented by prazosin. Electrical stimulation-induced relaxations were enhanced by bretylium and physostigmine and partially blocked by atropine. The effect of atropine, but not that of physostigmine, was prevented by bretylium. Corporal strips contracted with norepinephrine relaxed to acetylcholine; this effect was blocked by atropine and enhanced by physostigmine. Strips lacking endothelium contracted normally with norepinephrine, but the relaxation caused by acetylcholine was virtually abolished. Thus endothelium lining the lacunar spaces within human corpus cavernosum is required for the relaxation caused by exogenous acetylcholine. There may be three neurotransmitter effector systems that control corpus cavernosum smooth muscle tone: adrenergic (excitatory), cholinergic (inhibitory), and nonadrenergic noncholinergic (inhibitory). Cholinergic nerves do not dilate or constrict directly the smooth muscle but may modulate other neuroeffector systems and/or the endothelium.