Corpus Cavernosum Smooth Muscle Strips Research Articles

Fast voltage-dependent sodium (NaV ) currents are functionally expressed in mouse corpus cavernosum smooth muscle cells.

Corpus cavernosum smooth muscle (CCSM) exhibits phasic contractions that are coordinated by ion channels. Mouse models are commonly used to study erectile dysfunction, but there are few published electrophysiological studies of mouse CCSM. We describe the voltage-dependent sodium (NaV ) currents in mouse CCSM and investigate their function. We used electrophysiological, pharmacological and immunocytochemical methods to study the NaV currents in isolated CCSM cells from C57BL/6 mice. Tension measurements were carried out using crural sections of the corpus cavernosum in whole tissue. Fast, voltage-dependent, sodium currents in mouse CCSM were induced by depolarising steps. Steady-state activation and inactivation curves revealed a window current between -60 and -30 mV. Two populations of NaV currents, 'TTX-sensitive' and 'TTX-insensitive', were identified. TTX-sensitive currents showed 48% block with the NaV channel subtype-specific blockers ICA-121431 (NaV 1.1-1.3), PF-05089771 (NaV 1.7) and 4,9-anhydro-TTX (NaV 1.6). TTX-insensitive currents were resistant to blockade by A803467, specific for NaV 1.8 channels. Immunocytochemistry confirmed expression of NaV 1.5 and NaV 1.4 in freshly dispersed CCSM cells. Veratridine, a NaV channel activator, reduced time-dependent inactivation of NaV currents and increased duration of evoked action potentials. Veratridine induced phasic contractions in CCSM strips, reversible with TTX and nifedipine but not KB-R7943. There are fast, voltage-dependent, sodium currents in mouse CCSM. Stimulation of these currents increased contractility of CCSM in vitro, suggesting an involvement in detumescence and potentially providing a clinically relevant target in erectile dysfunction. Further work will be necessary to define its role.

MP47-06 EFFECTS OF FESOTERODINE (COMPETITIVE MUSCARINIC RECEPTOR ANTAGONIST) ON FUNCTIONAL RESPONSES OF RAT CORPUS CAVERNOSUM

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2014MP47-06 EFFECTS OF FESOTERODINE (COMPETITIVE MUSCARINIC RECEPTOR ANTAGONIST) ON FUNCTIONAL RESPONSES OF RAT CORPUS CAVERNOSUM Didem Yilmaz, Tamila Akyeva, Sahika Guner, Salih Erpulat Ozis, Ecem Kaya, and Serap Gur Didem YilmazDidem Yilmaz More articles by this author , Tamila AkyevaTamila Akyeva More articles by this author , Sahika GunerSahika Guner More articles by this author , Salih Erpulat OzisSalih Erpulat Ozis More articles by this author , Ecem KayaEcem Kaya More articles by this author , and Serap GurSerap Gur More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1459AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Erectile dysfunction (ED) is strongly associated with overactive bladder (OAB) symptoms. Fesoterodine (Toviaz®, New York, NY: Pfizer, Inc) is competitive muscarinic receptor antagonist which is used for treatment of OAB symptoms. We determined the effects of fesoterodine on functional response of rat corpus cavernosum smooth muscle (CCSM). Methods Adult Sprague-Dawley male rats were used in experiments. In vitro contractile and relaxation responses in the absence and presence of fesoterodine were evaluated using isolated strips of CCSM. Results We provided as a first evidence that fesoterodine induced the marked relaxation (87.2%) after precontraction with phenylephrine (Phe, 10μM) in rat CCSM. N(G)-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase [NOS] inhibitor, 100μM) (88.7%), 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one (ODQ, soluble guanosyl cyclase blocker, 10μM) (66.5%) and both (62.7%) did not suppress the relaxation response of fesoterodine. The relaxation response to acetylcholine (100μM) after preincubation with fesoterodine was increased in CCSM of rats. Preincubation with fesoterodine did not alter electrical field stimulation (nitrergic) induced relaxation. Fesoterodine enhanced remarkably sodium nitroprusside (SNP)-induced relaxation (at 0.01 nM concentration) in CCSM (before 0.5%; after 22.5%, p<0,05). Contractile response to Phe after preincubation with fesoterodine was decreased in CCSM (before 69.2%; after 12.1%, p<0,001). The contractile response to EFS after preincubation with fesoterodine (10μM) in penile tissue was not altered. Conclusions We conclude that the inhibitory action of fesoterodine on isolated rat CCSM is independent upon the endogenous nitric oxide, soluble guanylate cyclase and cyclic guanosine monophosphate system and dependent on corporal smooth muscle components. To the best of our knowledge, this is the first antagonist able to inhibit two different system (cholinergic and adrenergic) in the penile tissue. Thus, inhibition of muscarinic receptors improves OAB in men, either with BPH and ED. Future studies are warranted to investigate effect of fesoterodine on ED. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e520 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Didem Yilmaz More articles by this author Tamila Akyeva More articles by this author Sahika Guner More articles by this author Salih Erpulat Ozis More articles by this author Ecem Kaya More articles by this author Serap Gur More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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 ...

Amplified NO/cGMP‐mediated relaxation and ryanodine receptor‐to‐BKCa channel signalling in corpus cavernosum smooth muscle from phospholamban knockout mice

Relaxation of corpus cavernosum smooth muscle (CCSM) is induced by NO. NO promotes the formation of cGMP, which activates cGMP-dependent protein kinase I (PKGI). The large conductance calcium-activated potassium (BK(Ca) ) channel is regarded as a major target of NO/cGMP signalling; however, the mechanism of BK(Ca) activation remains unclear. The aim of the present study was to determine whether sarcoplasmic reticulum (SR) Ca(2+) load and Ca(2+) release from the SR via ryanodine receptors (RyRs) is important for BK(Ca) channel activation in response to NO/cGMP. In vitro myography was performed on CCSM strips from wild-type and PLB knockout (PLB(-/-)) mice to evaluate contraction and relaxation in response to pharmacological agents and electrical field stimulation (EFS). In CCSM strips from PLB(-/-) mice, a model of increased SR Ca(2+) load, contractile force in response to EFS or phenylephrine (PE) was increased by nearly 100%. EFS of strips precontracted with PE induced transient relaxation in CCSM, an effect that was significantly larger in PLB(-/-) strips. Likewise, the relaxation of PE-induced contraction in response to SNP and cGMP was greater in PLB(-/-) , as demonstrated by a shift in the concentration-response curve towards lower concentrations. Blocking RyRs and BK(Ca) channels diminished the induced relaxations and eliminated the difference between wild-type and PLB(-/-). NO/cGMP activates BK(Ca) channels through RyR-mediated Ca(2+) release. This signalling pathway is responsible for approximately 40% of the NO/cGMP effects and is amplified by increased SR Ca(2+) concentrations.

A Protein Tyrosine Kinase Inhibitor, Imatinib Mesylate (Gleevec), Improves Erectile and Vascular Function Secondary to a Reduction of Hyperglycemia in Diabetic Rats

Erectile dysfunction (ED) afflicts 50% of diabetic men, many of whom experience poor results with phosphodiesterase type 5 inhibitors. The protein tyrosine kinase (PTK) inhibitor imatinib (Gleevec, Novartis Pharmaceuticals, Basel, Switzerland) has therapeutic potential in diabetic men by maintaining β-cell function. To determine if imatinib has a beneficial effect on erectile and vascular function in diabetic rats. Male Sprague-Dawley rats were divided into six groups: (i) control; (ii) imatinib (50mg/kg, daily gavage)-treated control; (iii) diabetic; (iv) preventive imatinib (8 weeks); (v) reversal imatinib (4 weeks untreated diabetes and 4 weeks of treatment); and (vi) insulin (8 weeks)-treated diabetic rats. After 8 weeks, all groups underwent cavernosal nerve stimulation and measurements of intracavernosal pressure (ICP) and mean arterial pressure (MAP). Contractile and relaxation responses were evaluated using isolated strips of corpus cavernosum smooth muscle (CCSM) and aorta. Diabetic rats exhibited a 32% decrease in weight and fivefold increase in blood glucose levels. Imatinib-treated diabetic rats gained weight and partially improved blood glucose levels. Diabetic rats displayed a decrease in ICP/MAP. While maximum electrical field stimulation- and acetylcholine (ACh)-induced relaxations in CCSM strips from the diabetics were reduced, preventive imatinib or insulin treatment normalized ICP/MAP ratios and improved relaxation responses. ACh responses in diabetic aortas were diminished by 50.1% and restored by imatinib. While contractile responses to phenylephrine in diabetic CCSM were not altered, there was a significant enhancement (59.4 %) in the aortic contractile response in diabetic rats, which was restored by imatinib and insulin treatment. In diabetic rats, prolonged therapy with imatinib improves diabetes-related ED and vascular function, which may involve normalization of high glucose levels and restoration of PTK activation. Future studies are needed to elaborate on the actions of imatinib on diabetic vascular complications.

Characterization of Erectile Function in Monocrotaline‐Treated Pulmonary Hypertensive Rats

The aim of this study was to evaluate erectile function in monocrotaline (MCT)-treated rats with pulmonary hypertension (PH). Forty rats were divided into control (n = 20) and MCT-treated (n = 20) groups. Rats were treated with MCT (60 mg/kg subcutaneously) for 3 weeks to induce PH. Mean pulmonary arterial pressure (mPAP), medial hypertrophy index (percentage of wall thickness of pulmonary artery), and right ventricular hypertrophy (ratio of right ventricle [RV] to left ventricle + septum weight) were evaluated. In vivo erectile responses were assessed by measurement of intracavernosal pressure (ICP)/mean arterial pressure and total ICP (area under the curve). In vitro organ bath studies with corpus cavernosum smooth muscle strips were performed under both normoxic (95% O(2)/5% CO(2)) and hypoxic (by changing gas mixture to 95% N(2)/5% CO(2)) conditions. Erectile tissue was processed for immunohistochemistry. The MCT-treated group was associated with an increase in mPAP, medial hypertrophy index, and RV hypertrophy. MCT-induced PH rats had significantly reduced erectile responses compared with controls. Nitrergic, endothelium-dependent relaxations, as well as alpha-adrenergic contractile responses were significantly reduced in the corpus cavernosum of MCT rats. The functional responses during prolonged periods of hypoxia were similar to those observed in MCT-treated tissues. PH rats showed enhanced inducible nitric oxide synthase (NOS) protein localization, but endothelial NOS and neuronal NOS were unchanged. These results suggest changes in cavernosal physiology are caused by MCT acting on the penile tissues and the systemic vasculature.

Alfuzosin attenuates erectile dysfunction in rats with partial bladder outlet obstruction

To determine how partial bladder outlet obstruction (PBOO) in a rat model affects erectile function, and whether an uroselective alpha1-adrenoceptor antagonist, alfuzosin (Sanofi-Aventis, Paris, France) attenuates any erectile dysfunction (ED). Adult male Sprague-Dawley rats (120) were randomized into four groups: 1, sham-operated; 2, alfuzosin-treated; 3, PBOO; and 4, alfuzosin-treated with PBOO. Groups 3 and 4 were subjected to PBOO for 6 weeks by ligation of the urethra, while groups 2 and 4 rats received daily oral alfuzosin (10 mg/day) for 6 weeks. In vivo erectile responses were monitored by evaluating ratios of intracavernosal pressure (ICP)/mean arterial pressure, and total ICP (area under the curve). Organ-bath studies were performed on corpus cavernosum smooth muscle (CCSM) strips. Nitric oxide synthase (NOS) expression was determined immunohistochemically (IHC) for neuronal (n)NOS and by Western blot analysis for endothelial (e) and inducible (i) NOS protein. Rats with PBOO showed lower erectile responses than controls. Maximum electrical field stimulation-mediated and endothelium-dependent acetylcholine-induced relaxations and contractile responses to phenylephrine were significantly reduced in CCSM strips from the PBOO group. The NO donor sodium nitroprusside completely relaxed CCSM from rats in all groups. IHC analyses showed decreased expression of nNOS in PBOO groups compared with controls; by contrast, protein expression of eNOS and iNOS was increased. Alfuzosin-treatment partially attenuated functional and molecular changes in penises of PBOO rats. Rats with PBOO show ED, most likely due to altered NOS expression and NO bioavailability. The alpha-adrenoreceptor antagonist alfuzosin reversed this ED by altering sympathetic tone, increasing NO-induced relaxation and augmenting blood flow in the penis. This study suggests a rationale for further clinical trials using combinations of alpha-adrenoceptor antagonists and phosphodiesterase-5 inhibitors in patients with ED and lower urinary tract symptoms.

Hypercontractility and impaired sildenafil relaxations in the BKCachannel deletion model of erectile dysfunction

Erectile dysfunction (ED) can be elicited by a variety of pathogenic factors, particularly impaired formation of and responsiveness to nitric oxide (NO) and the downstream effectors soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase I (PKGI). One important target of PKGI in smooth muscle is the large-conductance, Ca2+ -activated potassium (BKCa) channel. In our previous report (42), we demonstrated that deletion of the BKCa channel in mice induced force oscillations and led to reduced nerve-evoked relaxations and ED. In the current study, we used this ED model to explore the role of the BKCa channel in the NO/sGC/PKGI pathway. Electrical field stimulation (EFS)-induced contractions of corpus cavernosum smooth muscle strips were significantly enhanced in the absence of BKCa channel function. In strips precontracted with phenylephrine, EFS-induced relaxations were converted to contractions by inhibition of sGC, and this was further enhanced by loss of BK channel function. Sildenafil-induced relaxations were decreased to a similar extent by inhibition of sGC or BKCa channels. At concentrations >1 microM, sildenafil caused relaxations independent of inhibition of sGC or BKCa channels. Sildenafil did not affect the enhanced force oscillations that were induced by the loss of BKCa channel function. Yet, these oscillations could be completely eliminated by blocking L-type voltage-dependent Ca2+ channels (VDCCs). These results suggest that therapeutically relevant concentrations of sildenafil act through cGMP and BKCa channels, and loss of BKCa channel function leads to hypercontractility, which depends on VDCCs and cannot be modified by the cGMP pathway.

Erectile dysfunction in mice lacking the large‐conductance calcium‐activated potassium (BK) channel

Penile erection is dependent on the nitric oxide (NO)/cGMP-dependent protein kinase I (PKGI) pathway. One important target of PKGI in smooth muscle is the large-conductance, calcium-activated potassium (BK) channel, which upon activation hyperpolarizes the smooth muscle cell membrane, causing relaxation. Relaxation of arterial and corpus cavernosum smooth muscle (CCSM) is necessary to increase blood flow into the corpora cavernosa that leads to penile tumescence. We investigated the functional role of BK channels in the corpus cavernosum utilizing a knock-out mouse lacking the Slo gene (Slo-/-) responsible for the pore-forming subunit of the BK channel. Whole-cell currents were recorded from isolated CCSM cells of Slo+/+ and Slo-/- mice. Iberiotoxin-sensitive voltage- and [Ca2+]-activated K+ currents, the latter activated by local transient calcium releases (calcium sparks), were present in Slo+/+ CCSM cells, but absent in Slo-/- cells. CCSM strips from Slo-/- mice demonstrated a four-fold increase in phasic contractions, in the presence of phenylephrine. Nerve-evoked relaxations of precontracted strips were reduced by 50%, both in strips from Slo-/- mice and by blocking BK channels with iberiotoxin in the Slo+/+ strips. Consistent with the in vitro results, in vivo intracavernous pressure exhibited pronounced oscillations in Slo-/- mice, but not in Slo+/+ mice. Furthermore, intracavernous pressure increases to nerve stimulation, in vivo, were reduced by 22% in Slo-/- mice. These results indicate that the BK channel has an important role in erectile function, and loss of the BK channel leads to erectile dysfunction.

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.

Secondhand tobacco smoke impairs neurogenic and endothelium-dependent relaxation of rabbit corpus cavernosum smooth muscle: improvement with chronic oral administration of L-arginine

The first goal of this study was to examine the effect of secondhand smoking on neurogenic, endothelium- and cGMP-dependent relaxant responses of rabbit corpus cavernosum smooth muscle. Our second goal was to determine whether such an effect can be prevented by oral administration of L-arginine. Male New Zealand rabbits were divided into control, chronic passive cigarette smoking and L-arginine treatment groups. Relaxant or contractile responses in isolated corpus cavernosum smooth muscle strips were determined by using in vitro muscle technique. There was no significant difference in the relaxant response of the strips to papaverine, sodium nitroprusside and contractile response to KCl among the groups. Relaxant responses to acetylcholine and electrical field stimulation and contractile response to phenylephrine were significantly decreased in the strips of the smoking group than that of the control group. The impaired relaxations of strips were markedly improved by treatment of L-arginine, but the contractile responses to phenylephrine were not affected. These data indicate that secondhand smoking may impair both neurogenic and endothelium-dependent relaxation of corpus cavernosum smooth muscle, and may contribute to the etiology of impotence. Chronic dietary supplementation with L-arginine offsets the impairment of neurogenic and endothelial relaxation. Therefore, we suggest that secondhand smoking exposure to cigarette produces selective impairment of neurogenic and endothelium-dependent relaxation of corpus cavernosum smooth muscle via a mechanism related to the decreased production and/or availability of nitric oxide.

Effects of Diabetes on Nitric Oxide-Mediated Relaxations in Male Rat Corpus cavernosum Smooth Muscle

Introduction: We evaluated the effects of diabetes on nitric oxide-mediated relaxations and nitric oxide synthase activity in male rat corpus cavernosum smooth muscles. Methods: Eight-week-old male rats were assigned to three groups: control (injected with the vehicle), DM (diabetes mellitus, induced by injection with 65 mg/kg streptozotocin), and TES (testosterone, testosterone supplemented after induction of diabetes). After 8 weeks, corpus cavernosum smooth muscle strips were mounted in an organ bath for isometric tension recordings. Electrical field stimulation (EFS, 2-ms pulse duration, 0.3–20 Hz and 3 s train) was applied to the strips precontracted with 30 µM phenylephrine. The microdialysis probe was inserted into the strip, and Krebs-Henseleit solution was perfused into the probe. The dialysate during EFS was collected, and the amount of NO^–₂/NO^–₃ (NOx) released in the dialysate was measured by the Greiss method. Sodium nitroprusside (0.1 nM to 10 mM) and carbachol (1 nM to 10 mM) were cumulatively added to the strips precontracted with 30 µM phenylephrine. Results: EFS caused frequency-dependent relaxations and NOx releases of the strips. Pretreatment with N^ω-nitro-L-arginine (100 µM) and tetrodotoxin (1 µM) completely inhibited the relaxations and NOx releases. The maximum relaxation was significantly greater in the DM group than in the control or TES group. The release of NOx was significantly greater in the DM group than in the control or TES group. Sodium nitroprusside, the endothelium-independent vasodilator, relaxed the tissues in all three groups. There were no significant differences among control, DM and TES groups in the maximum relaxation to sodium nitroprusside. Conclusion: The present data suggest that diabetes enhances nitric oxide synthase activity and nitric oxide-mediated relaxations in the male rat corpus cavernosum by the reduced testosterone level in the diabetic animals.

Effects of Castration on Nitric Oxide- Mediated Relaxations in Male Rat Corpus cavernosum Smooth Muscle

Background: The effects of castration on nitric oxide- mediated relaxations and nitric oxide synthase activity in male rat corpus cavernosum smooth muscles. Methods: Eight-week-old male rats were assigned to two groups: control (sham operated) and castrated animals. After 8 weeks, corpus cavernosum smooth muscle strips were mounted in an organ bath for isometric tension recordings. Electrical field stimulation (EFS) was applied to the strips precontracted with 30 µM phenylephrine. The microdialysis probe was inserted into the strip, and Krebs-Henseleit solution was perfused into the probe. The dialysate during EFS and cholinergic stimulation was collected, and the amount of NO^–₂/NO^–₃ (NOx) released in the dialysate was measured by the Greiss method. Sodium nitroprusside and carbachol were cumulatively added to the strips precontracted with 30 µM phenylephrine. Results: EFS caused frequency-dependent relaxations and NOx releases in the strips. Pretreatment with N^ω-nitro-L-arginine (100 µM) and tetrodotoxin (1 µM) completely inhibited relaxations and NOx releases. The maximum relaxation in the castration group was significantly greater than that in the control group. The release of NOx was significantly greater in the castration group than in the control group. Sodium nitroprusside relaxed the tissues in both groups similarly. Carbachol failed either to relax the tissue or to increase the amount of NOx production in the tissue. Conclusion: The present data suggest that castration enhances nitric oxide synthase activity and nitric oxide-mediated relaxations in the male rat corpus cavernosum.

The expression of functional postsynaptic alpha2-adrenoceptors in the corpus cavernosum smooth muscle.

1. The purpose of this study was to determine if corpus cavernosum smooth muscle expresses functional postsynaptic alpha2-adrenoceptors (AR). 2. The alpha2-adrenoceptor agonist UK 14,304 elicited concentration-dependent contractions in rabbit corpus cavernosum smooth muscle (CCSM). The half-maximal response occurred at 0.32+/-0.03 microM and the maximum contraction at 10 microM UK 14,304. 3. Pretreatment of CCSM strips with selective alpha2-adrenoceptor antagonists, rauwolscine and RS-15385, produced rightward shifts in the dose-response curves to UK 14,304 (pA2 values 7.1 and 8.5, respectively). In contrast, these antagonists did not alter contraction induced by the alpha1-adrenoceptor agonist phenylephrine (PE) or oxymetazoline. UK 14,304-induced contractions were also inhibited by prazosin (pA2 = 9.08). 4. UK 14,304-induced contractions, unlike those to PE, were highly dependent on the presence of extracellular Ca2+. 5. [3H]-rauwolscine bound to CCSM membranes with high affinity (Kd = 1.5 nM). [3H]-rauwolscine binding was displaced by unlabelled rauwolscine, RS-15385, UK 14,304 and prazosin, but not by PE. 6. UK 14,304 inhibited forskolin and prostaglandin E1 (PGE1)-induced increases in intracellular cyclic AMP concentration in primary cultures of rabbit CCSM cells. 7. These results demonstrate that CCSM expresses Gi-coupled postsynaptic alpha2-adrenoceptors, and activation of these receptors causes contraction of trabecular smooth muscle.

Nitric oxide mediates relaxation in rabbit and human corpus cavernosum smooth muscle.

We investigated in vitro the relaxant effect of exogenous acetylcholine (ACh) and electric-field stimulation (EFS) on rabbit and human corpus cavernosum smooth muscle strips (CC) precontracted with phenylephrine. The effects of EFS and ACh were monitored alone, after muscarinic receptor blockade and after inhibition of nitric oxide (NO) formation with L-N-nitro-arginine (L-NOARG). In rabbit and human CC, both atropine and L-NOARG abolished the relaxant effects of ACh. The relaxant effects of EFS, however, were only slightly reduced by atropine to 97.5 +/- 17.5% in human CC and to 89.0 +/- 6.1% in rabbit CC. L-NOARG further reduced the EFS effects to 0.8 +/- 1.7% in human CC and to 16.2 +/- 8.7% in rabbit CC. In strips obtained from impotent patients with diabetes mellitus, the relaxant effects appeared to be significantly less than in strips from nondiabetic impotent men. Tetrodotoxin blocked the relaxant EFS effects in human and rabbit strips completely. The data indicate the important role of NO in cholinergically induced relaxation of cavernous smooth muscle in rabbits and humans. Our findings support the idea of NO as the nonadrenergic noncholinergic neurotransmitter in penile erection in both species. Rabbit erectile tissue might serve as an in vitro animal model for further investigation.

Hypercholesterolemia Impairs Endothelium-Dependent Relaxation of Rabbit Corpus Cavernosum Smooth Muscle

The majority of cases of impotence are associated with vascular risk factors such as diabetes, hypercholesterolemia, hypertension and smoking. These factors induce impairment of endothelium-dependent relaxation of blood vessels in man and in experimental animals. In this study the effects of hypercholesterolemia on the reactivity of rabbit corpus cavernosum smooth muscle strips to endothelium-dependent and endothelium-independent agents were investigated. New Zealand White rabbits (n = 14) were randomly divided into control and treatment groups. The control group (n = 7) received a regular diet while the treatment group (n = 7) was fed a diet of 0.5% cholesterol and 4% peanut oil for 10 weeks. Animals were then sacrificed and the corporal tissue studied in organ chambers for isometric tension measurement. Tissue was contracted with phenylephrine and concentration-dependent relaxation to acetylcholine, in the presence and absence of indomethacin, and to nitroprusside were examined. Blood level of cholesterol in the cholesterol-fed group was significantly higher compared to the control group. Contractions to phenylephrine were similar in both groups. Hypercholesterolemia, however, inhibited relaxation to acetylcholine but did not alter relaxation to nitroprusside, a cyclic guanosine monophosphate (cGMP)-dependent, direct smooth muscle dilator. Indomethacin enhanced the relaxations to acetylcholine in both control and cholesterol-fed groups but did not correct the difference in the relaxation to acetylcholine between both groups. It is concluded that hypercholesterolemia impairs endothelium-mediated relaxation of rabbit corpus cavernosum smooth muscle. The mechanism for the endothelial dysfunction does not appear to involve alteration in cyclooxygenase products of arachidonate or the cGMP-dependent relaxation of corporal smooth muscle. Impairment of endothelium-dependent relaxation of corporal smooth muscle may contribute to the pathophysiology of impotence associated with hypercholesterolemia in man.