Wayne J.G. Hellstrom, Section of Andrology, Tulane University, Health Sciences Center, Department of Urology, 1430 Tulane Avenue, SL-42, New Orleans, LA 70112, USA. e-mail: email@example.com
Study Type – Aetiology (case control)
Level of Evidence 2b
What's known on the subject? and What does the study add?
In the present study the mechanisms regulating EFS-evoked neurogenic contraction in the human corpus cavernosum (HCC) were investigated. Overall, our data adds to current knowledge that the NO-independent heme dependent activation of sGC and the RhoA/Rho-kinase signaling pathways play an important role in the regulation of neurogenic contractile activity in HCC tissue.
• To investigate the mechanisms of adrenergically mediated smooth muscle contraction in the human corpus cavernosum (HCC) using an organ bath approach.
• Human corpus cavernosum specimens were obtained from patients (aged 59–72 years) with erectile dysfunction (ED), undergoing penile prosthesis implantation surgery.
• Isolated HCC strips (1 × 1 × 6 mm) were suspended in tissue bath chambers for isometric tension recording.
• The effects of various drugs on neurogenic contractions evoked by electrical field stimulation (EFS) were investigated. The drugs included nitric oxide (NO) donors, phosphodiesterase 5 (PDE5) inhibitor, Rho kinase (ROCK) inhibitor, NO-independent stimulator, L-type Ca2+ channel blocker and α-receptor antagonist.
• Pre-incubation with the NO donor sodium nitroprusside (SNP; 104 M) significantly reduced the initial peak increase in tension evoked by EFS (by 71%, P < 0.05). The PDE5 inhibitor sildenafil (10−4 M) reduced the increase in tension by 69%, while a combination of sildenafil and ROCK inhibitor, fasudil, inhibited tension by 81%.
• The EFS-induced contractile response at 80 Hz was decreased by 65% with fasudil and by 70% with isradipine (P < 0.001), while a combination of these drugs decreased the response by 88%. An NO-independent stimulator soluble guanylate cyclase (sGC), BAY 41-8543, significantly reduced the response (by 82%, P < 0.001) Phentolamine, an α-receptor antagonist, nearly eliminated the contractile response (98%, P < 0.001).
• These data suggest that neurogenic contractions are mediated by an increase in Ca(2+) influx via L-type voltage-gated Ca(2+) channels and that an increase in Ca(2+) sensitivity is mediated by the ROCK pathway and the PDE5 enzyme system as well as by the inhibitory NO/sGC/cGMP pathway.
• The neurogenic contractile response in HCC is mediated by several intracellular pathways, including adrenergic receptors, Ca(2+) entry, Ca(2+) sensitization and activation of the PDE5 enzyme. The Rho-kinase (ROCK) inhibitor fasudil, L-type Ca(2+) channel antagonist isradipine, and PDE5 inhibitor sildenafil, as well as a NO-independent stimulator of sGC, had similar inhibitory effects, suggesting parallel mechanisms in the HCC.
The contractile tone of trabecular smooth muscle in the corpus cavernosum (CC) is the ultimate determinant of penile flaccidity . In the flaccid state, human CC (HCC) smooth muscle is tonically contracted by noradrenergic nerves, allowing minimal penile arterial inflow [1,2]. The CC smooth muscle, however, is in the contracted state most of the time, and this is controlled through noradrenaline released from nerve endings, acting on postjunctional α1-adrenoceptors (ARs) [1,3]. The mechanism of noradrenergic contraction is based on the activation of contractile signalling systems and Ca(2+) mobilization, resulting in an increased intracellular Ca(2+) concentration . In addition, distinct regulatory mechanisms mediated by activation of heterotrimeric G protein-coupled receptors are known to augment contraction without increasing [Ca2+]i, a process known as Ca(2+) sensitization. This mechanism involves RhoA, a small, monomeric G protein that activates Rho kinase (ROCK) inhibitor, which phosphorylates the regulatory subunit of myosin light chain phosphatase, leading to sensitization of myofilaments to Ca(2+) [4,5]. The ROCK inhibitor Y-27632 antagonizes noradrenergic contractions in human and rabbit penile CC tissue [6,7] suggesting that the RhoA/ROCK pathway is active in the contracted state of the CC. Jin and Burnett  suggest that the upstream regulation of the RhoA/ROCK pathway in the penis has not been fully delineated. Ca(2+) sensitization induced by phenylephrine, noradrenaline and carbachol is markedly antagonized by ROCK inhibitors .
Previous investigations using CC tissue have shown that blockers of voltage-dependent Ca2+ channels lower CC tone . At least two voltage-dependent calcium channels are expressed in rabbit CC myocytes . One is L-type Ca2+ current, and the other is a putative T-type current. The L-current promotes conversion of local Ca2+ events into global Ca2+ waves, and the putative T-current plays a brief part in this process. Recently, the role of L-type Ca2+ current in causing detumescence tone in the CC has been determined . In addition, the activation of α1-AR implicates both Ca(2+) influx through L-type and receptor-operated Ca(2+) channels and Ca(2+) sensitization processes mediated by protein kinase C, tyrosine kinases and ROCK . Earlier studies using CC muscle strips show that the L-type channel blocker isradipine was effective in relaxing noradrenaline-induced contractions 
Cellek and Moncada  reported that human genitourinary tissues possess dense nitrergic innervation that regulates sympathetic responses [14,15]. Intracavernosal injection of sodium nitrite (NaNO2) produced dose-dependent increases in intracavernosal pressure and decreases in systemic arterial pressure . Sodium nitroprusside (SNP), a nitrovasodilator that induces relaxation, is entirely dependent on cGMP, and produces its biological effects by releasing nitric oxide (NO) .
To date, studies of adrenergic neurogenic contractile responses have focused on the role of nitrergic  and other transmitter systems . The aim of the present study was to elucidate the mechanism involved in electrical field stimulation (EFS)-evoked neurogenic contraction in HCC.
MATERIAL AND METHODS
Cavernosal tissues were obtained according to institutional review board guidelines. Samples were obtained from 23 patients with erectile dysfunction (ED) who were undergoing penile prosthesis implantation and whose mean (sd; range) age was 63.2 (1.47; 59–72) years. Four samples were obtained from patients after radical prostatectomy, 10 samples were obtained from patients with coexistent Peyronie's disease and nine samples were obtained from a patient with only ED. HCC tissue samples were placed in ice-cold Krebs solution and transported immediately to the laboratory for organ bath investigation [20,21].
ISOMETRİC TENSİON MEASUREMENT İN İN VİTRO STUDİES
Strips of HCC tissue (1 × 1 × 6 mm; mean no. of strips 4) were prepared from each cavernosal sample. Strips were suspended in 20-mL organ bath chambers (Radnoti Glass Technology Inc., Monrovia, CA, USA) with one end fixed to a tissue holder and the other secured to a force transducer (FT03; Grass Instruments, Quincy, MA, USA). Cavernosal tissue strips were maintained in Krebs-bicarbonate solution (containing [in mM] NaCl 118.1, KCl 4.7, KH2PO4 1.0, MgSO4 1.0, NaHCO3 25.0, CaCl2 2.5, and glucose 11.1) The organ chamber temperature was maintained at 37 °C via a circulating water bath. Oxygen saturation and a pH of 7.4 were maintained by continuous aeration with a mixture of 95% O2, 5% CO2. After placement in the organ chamber the preparations were allowed to equilibrate for a minimum of 90 min and the bath solution was replaced every 15 min.
After tissue equilibration at optimum isometric resting tension, EFS at 150 V, 1 ms pulse width, trains of stimuli lasting 10 ms at varying frequencies (0–80 Hz) were delivered by a current amplifier and a stimulator (Grass S88, Grass Instruments). In an attempt to study the contractile response elicited only by the adrenergic nerve component of EFS, the tissues were treated with L-N(G)-nitroarginine methyl ester (L-NAME, a non-selective NO synthase  inhibitor, 100 µmol/L) and the muscarinic receptor antagonist atropine (1 µmol/L) for 30 min to eliminate nitrergic and cholinergic influences . The increase in the tension of the muscle in response to EFS was measured in grams.
In organ bath experiments, EFS-induced contractile responses were obtained with stimulus frequencies of 1–80 Hz. In addition, the EFS responses were elicited 15 min after incubation with each of the pharmacological agents. To examine the role of endogenous formation of NO, independent of NO synthase, the EFS response was measured in the absence and presence of NO-donor NaNO2 (100 µM) or SNP (100 µM). Three consective reproducible responses were obtained before the next higher concentration of the drug was applied.
To investigate the role of the ROCK pathway in the response to EFS, HCC tissues were treated with fasudil (10 µM) . Isradipine (10 µM) [12,24] was used to investigate the contribution of voltage-activated Ca(2+) channels to EFS. A combination of low concentrations of fasudil (10 µM) and isradipine (10 µM) was also used to inhibit EFS-induced contractions.
To understand the role of PDE5 enzyme the EFS response was measured in the absence and presence of the PDE5 inhibitor sildenafil (10 µM) . The effect of combined treatment with fasudil and sildenafil on the response to EFS was investigated. An NO-independent stimulator of soluble guanylate cyclase (sGC), BAY 41-8543, (10 µM) was used to inhibit EFS-induced contractile responses . The contribution of α-ARs to the response to EFS or noradrenaline was studied in the presence of the non-selective α-AR antagonist phentolamine (10 µM) .
CHEMİCALS AND SOLUTİONS
Atropine, phentolamine, fasudil, isradipine, sodium nitrite and SNP were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sildenafil citrate was obtained from Pfizer (Viagra, Pfizer Pharmaceuticals, New York, NY, USA) and prepared freshly in physiological saline on the day of each experiment (in 5 mg/mL aqueous stock solution obtained by suspending crushed 50 mg sildenafil tablets in 10 mL distilled water and filtering the solution). BAY 41- 8543 (2-[1-[2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5(4-morpholinyl)-4,6-pyrimidinediamine) was obtained from Dr. Johannes-Peter Stasch of the Institute of Cardiovascular Research, Pharma Research Centre, Bayer AG, Wuppertal, Germany, and was dissolved in Transcutol®/Cremophor EL®/0.9% NaCl solution (10/10/80) .
All values are expressed as mean (sem). Isometric force generation was measured as contraction (g tension/g tissue) in the absence and presence of each of the studied agents. Statistical differences were determined by anova with repeated measures followed by a Bonferroni post-hoc test performed using Prism 4 statistical analysis packages for Windows (GraphPad Software, La Jolla, CA, USA). A P value of <0.05 was considered to indicate statistical significance.
Pretreatment of the HCC strips with the NO donor, NaNO2, reduced slightly, but not significantly, the EFS-induced contractile response at 80 Hz (21%), as compared with the response in control strips (Fig. 1A). Incubating tissues with NO donor, SNP, significantly reduced the response to EFS at 40, 60 and 80 Hz (71%, P < 0.001), as compared with responses in the control strips (Fig. 1B).
After incubation of the HCC tissues with the selective ROCK inhibitor, fasudil, EFS-induced contractile responses were significantly reduced at 20, 40, 60 and 80 Hz (65%, P < 0.001) (Fig. 2A). Incubation of tissues with the selective L-type Ca2+ channel blocker, isradipine, reduced EFS-induced contractile responses significantly at 40, 60 and 80 Hz (71%, P < 0.001), as compared to responses in control strips (Fig. 2B). After incubation of the HCC tissues with fasudil (10−4 M) and isradipine, EFS-induced contractile responses were significantly reduced at all frequencies (88%, P < 0.001) (Fig. 2C).
After incubation of the HCC tissues with sildenafil, EFS-induced contractile responses were significantly reduced at 20, 40, 60 and 80 Hz (69%, P < 0.001) (Fig. 3A). Sildenafil at 10 µM exerted a significant inhibitory effect on contractile responses at 20–60 Hz.
After incubation of the HCC tissues with sildenafil and fasudil, EFS-induced contractile responses were significantly reduced at all frequencies. At 80 Hz frequency, the EFS-induced contractile response was decreased by 81% (P < 0.001 [Fig. 3B]).
After incubation of HCC tissues with NO-independent stimulator of sGC, BAY 41-8543, EFS-induced contractile responses were significantly reduced at 20–80 Hz. The reduction was 82% (P < 0.001) at 80 Hz (Fig. 4A).
Incubation of tissues with the non-selective α-AR blocker, phentolamine, completely blocked EFS-induced contractile responses at 20, 40, 60 and 80 Hz (98%, P < 0.001), as compared with responses in control strips (Fig. 4B).
In the normal flaccid state, noradrenergic nerve activity decreases penile arterial inflow and tonically contracts the smooth muscle of the corpora cavernosum. The principal conclusions from the present study are that the NO-sGC-cGMP- PDE5, and the ROCK pathways and calcium entry play an important role in regulating the adrenergically medicated contractile response to EFS in HCC. Because of the difficulty of obtaining normal HCC, the present results have been interpreted without having true control tissues.
In the present study, we evaluated the inhibitory effect of NO donors, such as sodium nitrite and SNP on neurogenic contractile responses of HCC when Non-adrenergic and non-cholinergic (NANC) neurotransmission was inhibited by L-NAME. The NO donor sodium nitrite had no significant effect on neurogenic contractions in HCC. It has recently been hypothesized that the nitrite anion is an important reservoir for NO and that NaNO2 further enhances relaxation in response to nitrergic stimulation . In other tissue studies, the addition of NaNO2 in rabbit jejunum inhibited the amplitude of spontaneous contractions in a concentration-dependent fashion  and inhibited EFS-induced contractions in human placental blood vessels rings . Lasker et al. have recently shown in the rat that NaNO2 administered intracavernosally increases intracavernosal pressure (ICP), decreases systemic arterial pressure and is 1000-fold less potent than the NO donor SNP. Based on the present data, we suggest that the basal direct neurogenic contractile response was not influenced by nitrite because insufficient amounts of NO were generated in the isolated tissue setting; however, NO donor SNP, which directly releases NO, reduced the contractile response to EFS. A study by Teixeira et al. showed that SNP had little effect on contractile activity elicited by EFS over the full range of the frequency–response curve when their lowest concentration was used. Thus, sodium nitrite and SNP had a different effect on the response to EFS. The present data indicate that more NO than can be generated by the reduction of nitrate to NO in the isolated tissue setting is needed for inhibition of contraction. These data indicate that NO inhibition of EFS-induced contraction occurs and is consistent with the concept that the release to nerve-released norepenephrine is attenuated. Recently, the use of exogenous NO and nitrovasodilators has received considerable attention as a treatment for lung and cardiovascular disorders ; e.g. exogenous NO exerts bronchodilatory effects in bronchial asthma  and NO is used in preterm children to improve lung capacity. Different strategies can be applied for using NO donors for the treatment of ED.
It is known that NO activates sGC and increases the conversion of GTP to cGMP. We have reported that sildenafil negatively modulates sympathetic neurotransmission in HCC. Similarly El-Metwally et al. and Fovaeus et al. observed that sildenafil induced concentration-dependent inhibition of EFS-induced contractions. Sildenafil, 1.5 µM, reduced neurogenic contractions in HCC mediated predominantly by adrenergic activity . Similar findings, showing that sildenafil inhibited neurogenic contraction, have been documented in human penile blood vessels and rabbit CC tissue [35,36]. Furthermore, sildenafil has also been reported to cause a marked increase in plasma NA levels and increase muscle sympathetic nerve activity, both at rest and during stress, in men . Although the effect of sildenafil is known to be mediated through a classic NO/cGMP-dependent pathway, Lau and Adaikan  showed that, in rabbit CC, sildenafil induced direct relaxant activity in associated with inhibition of Ca+ cascade. In the present study, the combination of sildenafil and fasudil enhanced the inhibition of the contractile response to EFS in HCC, suggesting that noradrenergic contraction is modulated by the PDE5 enzyme.
Surprisingly, we showed that a potent NO-independent stimulator of sGC, BAY 41-8543, inhibited EFS-induced the contractile response by 82% at 80 Hz. To date there are no similar findings in the literature, but Badejo et al. showed that i.v. injections of BAY 41-8543, under elevated tone conditions, caused decreases in pulmonary and systemic arterial pressures. The present study suggests that sGC stimulation may be more potent than ROCK or PDE5 activation in modulating the adrenergic response.
Rho-kinase antagonism represents a potential therapeutic option for the treatment of ED in situations where there is elevated noradrenergic tone . In addition to the well established noradrenergic contractile mechanisms in the penis, the important role of increased calcium sensitivity has been established [40,41]. In the present study, we evaluated the in vitro effects of fasudil (H-1077) in mediating the contractile response induced by stimulation of the adrenergic nerves. Fasudil inhibits EFS-induced contractions by 65%, suggesting that the contractile response of EFS increases RhoA/ROCK activity in HCC. These data suggest that vasoconstriction in the penile circulation may be regulated, in part, by the RhoA/Rho-kinase Ca(2+) sensitization pathway [40,42]. The RhoA/ROCK signalling pathway has been reported to have an important role in the maintenance of contractile activity in the rat anococcygeus and retractor penis muscle . In isolated human penile CC strips, EFS-induced contractions were inhibited by Y-27632 in a concentration-dependent manner, suggesting that the ROCK pathway is involved in the noradrenergic contractile response in HCC smooth muscle . In smooth muscle contraction, it is well established that membrane depolarization opens voltage-gated Ca(2+) channels, allowing Ca(2+) entry into the cell. In our previous study, in isolated HCC, relaxation responses to L-arginine were potentiated in the presence of the ROCK inhibitor Y-27632 . Moreover, ROCK inhibitors do not require a functional nitrergic system and could possibly be more effective in patients with complete loss of nitrergic function and who are not responsive to PDE5 inhibitors . Moreover, the finding that sildenafil also attenuated endogenous NA-mediated contractions is important.
Previous work in the guinea pig ileum and human trachea, in which Y-27632 was effective in inhibiting tonic but not ROCK-mediated contractions, suggests that PDE5 inhibitors can be used in the treatment of ED when elevated noradrenergic tone is present .
After neurogenic release, noradrenaline binds to postjuctional α1-AR, initiating a cascade of events leading to increased intracellular calcium and increased smooth muscle contractility. Simultaneous measurement of intracellular free calcium and smooth muscle tone is an approach used to test this hypothesis. The L-type Ca(2+) channel blocker isradipine had an inhibitory effect on contractions induced by EFS, suggesting that Ca(2+) entry through L-type channels is involved in neurogenically mediated contractions in HCC. Calcium channel blockers are effective in relaxing cavernosal smooth muscle and may be used as intracavernous agents for the treatment of ED . In a previous study, human cavernosal cells expressing Ca2+ channels involved in maintaining [Ca2+](i) homeostasis and regulating the NO-cGMP-induced smooth muscle relaxation–contraction responsible for penile erection and flaccidity were investigated . Rabbit cavernosal strips were pre-contracted by EFS and calcium channel blockers were found to be effective in inhibiting contractions induced by EFS . In the present study, isradipine at 10 µM and 100 µM induced 25% and 69% relaxation respectively at 1 Hz, while HCC (10 µM) produced a 71% decrease at 80 Hz. In an earlier study by Kerfoot et al., CC tissue from rabbits were not pretreated with L-NAME (NO synthase inhibitor) and the muscarinic receptor antagonist atropine to eliminate nitrergic and cholinergic components. Thus stimulation frequency, species variation and the concentration of Ca channel blocker used are known to alter adrenergic neurogenic response of CC smooth muscle. Similarly, verapamil, a calcium channel blocker, attenuated electrically induced contraction in rabbit CC .
The adrenergic contraction is inhibited by a non-specific α-AR antagonist. Thus, both the direct contractile effects and the potentiation effect appear to be mediated primarily by the α1-AR. The present data suggest that the neurogenic response is mediated mainly by noradrenaline released from perivascular nerves, as the response was significantly diminished by the α-AR antagonist phentolamine. EFS-evoked contractions were totally blocked by phentolamine at all frequencies of stimulation, indicating that responses are neural in origin and α1-AR in nature. Phentolamine potentiates relaxation and attenuates contraction in response to the endogenous neurotransmitters released during EFS .
This action of phentolamine has been reported previously [33,36] and is attributed to its α1-AR blocking effect. The present study increases our understanding of the balance between nitrergic and sympathetic systems in HCC and shows that any alteration in this system may produce a pathological response. The present data indicate that NA release and binding to postjunctional α1-AR receptors initiate a cascade of events leading to increased intracellular calcium and increased contractility of the CC.
In conclusion, modulation of adrenergic activity is an important mechanism by which the contractile state of the smooth muscle of the CC is regulated. The present results suggest that the NO-independent heme-dependent activation of sGC and the RhoA/ROCK signalling pathways play an important role in the regulation of neurogenic contraction in HCC tissue. In addition, new information about adrenergic contraction of the HCC involving the activation of ROCK, PDE5 and sGC pathways and Ca(2+) channel opening, mediated by α-ARs, may lead to a new understanding of the regulation of penile erection and flaccidity. Combined inhibition of several pathways involved in contraction lead to a new form of therapy for the treatment of ED, particularly in situations where noradrenergic tone is elevated or NO-mediated responses are impaired.