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Three Phases of Corporal Tracing Elicited by Electrical Field Stimulation on Rabbit Corpus Cavernosum Smooth Muscle in Penile Perfusion Model
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Three Phases of Corporal Tracing Elicited by Electrical Field Stimulation on Rabbit Corpus Cavernosum Smooth Muscle in Penile Perfusion Model jsm_2178
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Chen Zhao, MD,* Kyung Woo Cho, MD, PhD,† and Jong Kwan Park, MD, PhD* *Department of Urology, Medical School, and Institute for Medical Sciences, Chonbuk National University, and Research Institute and CTC for Medical Device of Chonbuk National University Hospital, Jeonju, Korea; †Department of Physiology, College of Oriental Medicine, Wonkwang Univeristy, Iksan, Korea DOI: 10.1111/j.1743-6109.2010.02178.x
ABSTRACT
Introduction. Nitric oxide (NO) has been shown to mediate electrical field stimulation (EFS)-caused smooth muscle relaxation. It is known that the neural control of penile erection involves adrenergic, cholinergic, and nonadrenergic-non-cholinergic (NANC) neuro-effector systems; however, the effects of EFS on adrenergic and cholinergic nerves are not clear. Aims. To elucidate EFS-induced signal transductions involved in adrenergic, cholinergic, and NANC neuroeffector systems by using an in vitro penile perfusion model. Methods. EFS was performed on penile corpus cavernosum smooth muscle from male New Zealand White rabbits, which was pre-contracted with L-phenylephrine (10 mM). We investigated the penile tracing elicited by EFS on tissues pre-incubated with guanethidine (Guan, 50 mM), tetrodotoxin (TTX, 10 mM), Nw nitro-L-arginine-methyl ester (L-NAME, 1 mM), atropine (50 mM), or eserine (10 mM). Main Outcome Measures. The time-to-peak of each phase, the percentage of relaxation, and the area under the curve (AUC). Results. We discovered an extraordinary phenomenon: three distinct phases elicited by EFS. Phase I was abolished by L-NAME. Phase II was decreased by eserine and Guan, but increased by L-NAME. Phase III was abolished by atropine, but enhanced by eserine and Guan. TTX diminished all three phases. The time to reach the top of phase I was delayed by TTX. The time to attain the peak of phase II was shortened by L-NAME, but delayed by TTX and atropine. The time to reach the top of phase III was shortened by L-NAME, eserine, and Guan. AUC was significantly decreased by L-NAME and TTX. Conclusions. EFS stimulated adrenergic, cholinergic, and NANC neuro-effector systems simultaneously. Phase I was related to the NO pathway. Phase II was multiply affected by self-recovery properties, and adrenergic and cholinergic nerves. Phase III was related to cholinergic nerves. The corporal tracing elicited by EFS was the balanced result of multiple factors. Zhao C, Cho KW, and Park JK. Three phases of corporal tracing elicited by electrical field stimulation on rabbit corpus cavernosum smooth muscle in penile perfusion model. J Sex Med 2011;8:1039–1047. Key Words. Electrical Field Stimulation (EFS); Smooth Muscle Relaxation; In Vitro Penile Perfusion Model; Extraordinary Phenomenon; Corpus Cavernosum Smooth Muscle
Introduction
P
enile erection is a complex neurovascular process that involves relaxation of the corpus cavernosum smooth muscle [1–5]. Neural control of penile erection involves the adrenergic,
© 2011 International Society for Sexual Medicine
cholinergic, and non-adrenergic-non-cholinergic (NANC) neuro-effector systems [6–8]. Adrenergic nerves mediate intracavernous smooth muscle contraction, which maintains the penis in a flaccid state. Cholinergic nerves contribute to smooth muscle relaxation and penile erection through J Sex Med 2011;8:1039–1047
1040 inhibition of adrenergic nerves via inhibitory interneurons and release of nitric oxide (NO) from the endothelium by acetylcholine (Ach) [9]. NO released from the cavernous nerve, as a NANC neurotransmitter, and from the endothelium, is the principal entity mediating relaxation of the cavernosum muscle [10]. The organ chamber strip model with electrical field stimulation (EFS) is a common technique for evaluating the erectile function in isolated cavernosum smooth muscle of humans and rabbits in vitro [11–13]. Many studies have demonstrated that EFS causes rapid, but transient smooth muscle relaxation, and NO has been shown to mediate relaxation in response to nerve stimulation. EFS of isolated strips of rabbit corpus cavernosum promotes the endogenous formation and release of NO and cyclic guanosine monophosphate (GMP) [14,15]. Corporal smooth muscle relaxation in response to EFS in the presence of guanethidine (Guan) and atropine is abolished by tetrodotoxin (TTX) and potassium-induced depolarization, and is markedly inhibited by NG-nitro-L-arginine, NG-amino-L-arginine, oxyhemoglobin, and methylene blue, but is unaffected by indomethacin [16–18]. The stimulation of the nerve endings promotes the release of NO from NANC nerves and it may also stimulate the adrenergic and cholinergic neural systems. We hypothesized that EFS activated these neuro-effector systems simultaneously and the corporal tracing elicited by EFS could reflect the interactions of the systems. We performed EFS in an in vitro penile perfusion model, which has been demonstrated to have a more sensitive response capacity to Ach and EFS compared with the classic penile strip chamber model [19]. The objective of the present study was to elucidate EFS-induced signal transductions. Methods
This study was approved by Institutional Animal Care and Use Committee of Chonbuk National University Hospital and Medical School. The experimental procedures were performed in accordance with the Guiding Principles in the Use and Care of Animals approved by the Council of American Physiological Society.
Tissue Preparation The penile tissues were prepared as previously described [19]. Healthy control male New Zealand White rabbits weighing 2.5–3.0 kg were anestheJ Sex Med 2011;8:1039–1047
Zhao et al. tized with ketamine (50 mg/kg intravenously) plus rumpun (25 mg/kg), and exsanguinated. The entire penis, including the urethra was rapidly excised from the pubic bone. The urethra was dissected free from the penile body. During the preparation, each step was taken cautiously to prevent damage of functional endothelium or overstretching of the tissue.
Measurement of Isometric Tension and Intracavernosum Pressure (ICP) The glans penis was cut out until the corpus cavernosum was exposed to air through a small opening with a diameter of 5 mm. Two small polyethylene tubes (inner diameter, 1.2 mm and outer diameter, 1.7 mm; Natsume, Tokyo, Japan), which contained small internal platinum electrodes, were inserted into the proximal opening of the crura for inflow, and ligated with a purse string silk suture to prevent leakage. The distal cut of the corpus cavernosum was opened to allow flow out of the penis. The distal end was secured with a cotton thread to a holder at the bottom of the chamber. The cannulated penis was mounted vertically in a 50-mL fully humidified organ chamber without buffer outside of the penis. The penis was immediately perfused interstitially through the cannulae with HEPES (4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl) piperazine-N′-(2ethanesulfonic acid)) buffer using a peristaltic pump (0.5 mL/minute). The HEPES buffer contained the following (in mM): NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25; glucose, 10.0; and HEPES 10 with NaOH (pH 7.4). The perfusion solution was oxygenated with 100% O2 and maintained at 36°C. The hollow organ chamber was covered with Parafilm (Pechiney Plastic Packaging Company, Chicago, IL, USA) to maintain the temperature and humidity in the organ chamber at 36°C. The ICP was measured using a pressure transducer (FT03; Grass Telefactor, W. Warwick, RI, USA) connected to the inflow tube cannulated to the crura and recorded using a PowerLab (Software Chart, version 5; AD Instrument, Castle Hill, Australia). After mounting, tissue was equilibrated for 100 minutes with several adjustments of length until a baseline force was stabilized at 10 g. The chamber for penile perfusion had a hole at the bottom to allow collection of the perfusate. Changes in tension were measured with a force transducer. Electrical Field Stimulation The tissue was stimulated by two parallel platinum electrodes bilaterally inserted into the cavernosum
A New Mechanism of Relaxation by Electrical Field Stimulation through the crura. The intrinsic nerves were fieldstimulated electrically (voltage, 30 V; pulse duration, 1 ms; trains of pulses, 10 seconds; train interval, 3–5 minutes; frequency, 0.5–32 Hz) using a Grass stimulator (S88, Grass Telefactor, W. Warwick, RI, USA). The sampling rate (recording speed) of the PowerLab data 400 acquisition system was set to 40/second.
Protocols This study included seven groups according to the different blocker treatments: L-phenylephrine (PhE); Nw nitro-L-arginine-methyl ester (L-NAME); Guan; atropine; eserine; and a combination of L-NAME, Guan, and atropine. In each group, the functional endothelium was first checked by the presence of at least a 50% relaxation in response to Ach (100 nM) in PhE-contracted tissue. Tissue was washed and contracted with PhE (10 mM). When a stable tone was obtained, EFS was performed. The tissue was washed again, then incubated with blocker for 30 minutes (1 mM L-NAME, 50 mM Guan, 10 mM TTX, 50 mM atropine, 10 mM eserine, or a combination of 1 mM L-NAME, 50 mM Guan, and 50 mM atropine), and the EFS-induced response was obtained in the PhE-precontracted tissue. After determination of the EFS-induced response and the stable contraction, sodium nitroprusside (SNP, 10 mM) was added for 5 minutes to determine whether or not guanylate cyclase activity was inhibited [19–21]. Measurements The representative tracings and the definitions of the phases are shown in Figure 1. In the PhE group, phase I is the first relaxation after EFS, phase II is the contraction following phase I, and phase III is the second relaxation following phase II after EFS. The percentage of EFS-induced relaxation, the time-to-peak of each phase, and the area under the curve (AUC) were calculated and analyzed by PowerLab software. The total AUC was divided into two parts (AUC1 and AUC2; Figure 2). Pharmacological Agents and Solutions Acetylcholine atropine hydrochloride, Guan, L-NAME, PhE, eserine (a cholinesterase inhibitor), and SNP were purchased from Sigma-Aldrich (St. Louis, MO, USA). TTX was purchased from Tocris Cookson (Langford, Bristol, UK). All other chemicals were the highest grade available.
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Statistics The submaximal contractile responses induced by PhE were taken as 100%, and all subsequent responses to EFS were expressed as a percentage of the PhE-induced value. The results are expressed as the mean ⫾ standard error (SE), and N represents the number of tissues in each group. The statistical significance of the differences was calculated by one-way analysis of variance (anova), followed by Bonferroni’s multiple comparison test. The responses before and after treatment with blockers were compared by a Student’s paired t-test. A probability value < 0.05 was considered significant.
Results
Changes in EFS Responses The response to EFS was in a Hz strengthdependent manner at low frequencies (<8 Hz) in the penile perfusion model (Figure 3, N = 8). Phase III first presented at 4 Hz and increased in a frequency-dependent manner. The blocker study was performed at a frequency of 8 Hz because the phases presented most clearly. Changes in Percentage of Relaxation Phase I was significantly decreased by TTX and completely abolished by L-NAME (PhE: ICP, 59.73 ⫾ 7.24%; tension, 52.13 ⫾ 6.52%; TTX: ICP, 3.23 ⫾ 1.42%; tension, 5.25 ⫾ 2.11%; P < 0.01 vs. PhE, Figure 4A, N = 8). Phase II was significantly diminished by eserine, TTX, and Guan (PhE: ICP, 40.69 ⫾ 5.88%; tension, 35.96 ⫾ 4.69%; eserine: ICP, 18.17 ⫾ 6.59%; tension, 14.14 ⫾ 5.39%; TTX: ICP, 3.76 ⫾ 1.43%; tension, 3.18 ⫾ 2.68%; Guan: ICP, 10.16 ⫾ 4.19%; tension, 9.27 ⫾ 5.09%; P < 0.01 vs. PhE, respectively, Figure 4B, N = 8). However, preincubation with L-NAME enhanced phase II over the PhE-precontracted level (L-NAME: ICP, 102.96 ⫾ 1.96%; tension, 122.38 ⫾ 4.72%; P < 0.01 vs. PhE, Figure 1C, D). Phase III was completely abolished by atropine and TTX, significantly decreased by L-NAME (PhE: ICP, 40.71 ⫾ 3.69%; tension, 32.71 ⫾ 4.06%; LNAME: ICP, 12.04 ⫾ 4.03%, tension, 16.13 ⫾ 7.08%; P < 0.01 vs. PhE), but enhanced by eserine and Guan (eserine: ICP, 70.68 ⫾ 15.16%; tension, 80.43 ⫾ 14.09%; Guan: ICP, 56.49 ⫾ 5.88%; tension, 53.76 ⫾ 8.49%; P < 0.01 vs. PhE, Figure 4C, N = 8). All the phases were abolished J Sex Med 2011;8:1039–1047
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Figure 1 Representative tracings elicited by EFS at a frequency of 8 Hz, duration of 1 ms, voltage of 30 V, and the definitions of three phases. Left column: ICP; right column: tension. PhE = phenylephrine; L-NAME = Nw nitro-L-argininemethyl ester; TTX = tetrodotoxin; Guan = guanethidine; ICP = intracavernosum pressure; EFS = electrical field stimulation.
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by combination treatment with L-NAME, Guan, and atropine (Figure 5, N = 8).
Figure 2 Total AUC was divided into two parts (AUC 1 and AUC 2). AUC, area under the curve; PhE = phenylephrine; EFS = electrical field stimulation.
Changes in Time-To-Peak The time-to-peak of phase I was significantly delayed by TTX (P < 0.01 vs. PhE, N = 8). The time-to-peak of phase II was shortened by L-NAME, but delayed by TTX and atropine (P < 0.01 vs. PhE, respectively, N = 8). The timeto-peak of phase III was shortened by L-NAME (P < 0.01 vs. PhE, N = 8), eserine (P < 0.05 vs. PhE, N = 8), and Guan (P < 0.01 vs. PhE, N = 8). The time-to-recover to the PhE-precontracted level was shortened by L-NAME (P < 0.01 vs. PhE, N = 8) and TTX (P < 0.01 vs. PhE, N = 8), but delayed by eserine (P < 0.05 vs. PhE, N = 8) and atropine (P < 0.05 vs. PhE, N = 8; Table 1).
Figure 3 EFS-induced changes of ICP and tension on PhE-precontracted cavernosum smooth muscle in penile perfusion model. The intrinsic nerves were field-stimulated electrically at a voltage of 30 V, a pulse duration of 1 ms, trains of pulses of 10 seconds, and a train interval of 3–5 minutes. PhE = phenylephrine; ICP = intracavernosum pressure; EFS = electrical field stimulation.
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Figure 4 Changes in the percentage of relaxation of each phase. (A) Percent of phase I. (B) Percent of phase II. (C) Percent of phase III. The submaximal contractile responses induced by PhE were taken as 100%, and all subsequent responses to EFS were expressed as a percentage of PhE-induced value. PhE = phenylephrine; L-NAME = Nw nitro-L-arginine-methyl ester; TTX = tetrodotoxin; Guan = guanethidine; ICP = intracavernosum pressure. **P < 0.01 vs. PhE.
Figure 5 Representive tracings elicited by EFS on combination treatment cavernosum smooth muscle with L-NAME, Guan, and atropine. L-NAME = Nw nitro-L-arginine-methyl ester; Guan = guanethidine; ICP = intracavernosum pressure; EFS = electrical field stimulation.
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Changes in AUC The total AUC was divided into AUC 1 and 2 (Figure 2). AUC 1 was decreased by L-NAME and TTX (P < 0.01 vs. PhE, respectively, N = 8), but increased by Guan and atropine (P < 0.05 vs. PhE, respectively, N = 8). AUC 2 was decreased by L-NAME (P < 0.01 vs. PhE, N = 8), but increased by eserine and Guan (P < 0.01 vs. PhE, respectively, N = 8). Total AUC was decreased by L-NAME and TTX (P < 0.01 vs. PhE, respectively, N = 8), but increased by eserine and Guan (P < 0.01 vs. PhE, respectively, N = 8; Table 2). Discussion
It is well known that the state of relaxation or contraction of the arteriolar and trabecular smooth muscle determines penile erection or flaccidity [1,2,22]. The NANC neurotransmitter, NO, plays a crucial role in inducing smooth muscle relaxation and penile erection. NO
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A New Mechanism of Relaxation by Electrical Field Stimulation Table 1
Time to reach the top of each phase and recover the PhE pre-contracted level Phase I
Group
N
(s)
PhE L-NAME + PhE Eserine + PhE TTX + PhE Guan + PhE Atropine + PhE
8 8 8 8 8 8
10.5 ⫾ 0.3 — 10.5 ⫾ 0.3 30.6 ⫾ 0.9** 10.5 ⫾ 0.3 10.5 ⫾ 0.3
Phase II
Phase III
Recovery
22.1 ⫾ 0.8 12.5 ⫾ 1** 20 ⫾ 0.5 55.4 ⫾ 2.4** 19.8 ⫾ 0.5 49.5 ⫾ 1.4**
49.4 ⫾ 1.7 29.3 ⫾ 1.5** 35.5 ⫾ 1.5* — 29.5 ⫾ 1.1** —
137 ⫾ 7.1 88.5 ⫾ 2.6** 153.1 ⫾ 2* 55.4 ⫾ 2.4** 140.5 ⫾ 2 127.6 ⫾ 2.2*
*P < 0.05, **P < 0.01. PhE = L-phenylephrine; L-NAME = Nw nitro-L-arginine-methyl ester; TTX = tetrodotoxin; Guan = guanethidine; N is number of experiment.
synthesized by neurogenic and endothelial NO synthases (neurogenic NO synthases [nNOS] and endothelial NO synthases [eNOS]) diffuses into smooth muscle cells and activates the soluble guanylyl cyclase, which catalyses the formation of cyclic guanosine monophosphate (cGMP). Binding of cGMP to cGMP-dependent protein kinases or cGMP-dependent ion channels results in a reduction of intracellular Ca2+ and activation of myosin light chain phosphatases, which causes diminution of smooth muscle contractility and enhances penile erection [23]. Conversely, penile smooth muscle contraction mediated by the tonic signalling of sympathetic nerves maintains baseline flaccidity and causes detumescence [21]. Cholinergic nerves do not dilate or directly constrict smooth muscle, but modulate other neuro-effector systems via inhibitory interneurons and release of NO from the endothelium by Ach [9,20]. EFS has become a widely used technique in evaluating erectile function in vitro since Ignarro and his colleagues [15] demonstrated that EFSinduced relaxation of isolated rabbit corpus cavernosum was mediated by NO generated from NANC inhibitory nerves in 1990. Several years later, a biphasic response to EFS was reported; specifically, stimulation of the nerve endings induced a transient smooth muscle contraction before relaxation on PhE-precontracted rabbit
Table 2 Changes in AUC of EFS elicited tracings in rabbit penile perfusion model Group
N AUC 1
AUC 2
PhE L-NAME + PhE Eserine + PhE TTX + PhE Guan + PhE Atropine + PhE
8 85.9 ⫾ 3.9 227.5 ⫾ 12.4 8 13.8 ⫾ 1.0** 33.3 ⫾ 4.5** 8 98 ⫾ 1.9 402.5 ⫾ 9.5** 8 31.3 ⫾ 3.6** — 8 118.6 ⫾ 2.7* 415.1 ⫾ 10.4** 8 119.9 ⫾ 4.9* —
Total AUC 313.4 ⫾ 14.8 47.1 ⫾ 4.1** 500.5 ⫾ 10.3** 31.3 ⫾ 3.6** 533.6 ⫾ 12.2** 308.8 ⫾ 8.7
*P < 0.05, **P < 0.01. PhE = L-phenylephrine; L-NAME = Nw nitro-L-arginine-methyl TTX = tetrodotoxin; Guan = guanethidine; N = number of experiment.
ester;
corpus cavernosum and the contraction was abolished by the addition of Guan. This study confirmed that the sympathetic system was involved in the EFS pathway [21]. Toda and his colleagues [24] reported a triphasic response to EFS on prostaglandin F2a-precontracted monkey corpus cavernosum; however, they focused on the EFS-NO pathway and the relaxant effect was not affected by a cholinergic antagonist, such as atropine, and the effect of EFS on cholinergic nerves was unclear [24]. In our study, we verified the EFS-NO pathway in an in vitro penile perfusion model. We have demonstrated that this model had a more sensitive response capacity to Ach and EFS than the classic penile strip chamber model in the previous study [19]. We observed the triphasic tracing elicited by EFS at a recording speed of 40/second on the PowerLab data 400 acquisition system. Phase I was abolished by a NOS inhibitor and the relaxation was not inhibited by adrenergic and cholinergic blockers, indicating the NO pathway was the most potent factor in inducing penile smooth muscle relaxation. We think that the NO released by EFS has 2 sources: one is released by NANC nerve as a neurotransmitter, which in charge of the relaxation of phase I; the other one is released by endothelium activated by Ach which in charge of the relaxation of phase III. EFS releases NO and the NO production is significantly inhibited by L-NAME. However, atropine and eserine did not block the EFS induced NO production. In many previous papers, atropine did not block EFS induced relaxation but acetylcholine induced relaxation [19,24]. L-NAME inhibits both nNOS and eNOS, so phase I and phase III are significantly decreased in this study (Figure 4A, C). Phase II was significantly diminished by Guan and eserine, suggesting that the sympathetic system is involved in EFS-induced signal transductions and can be inhibited by cholinergic nerves. Phase III was intensified by the choline esterase inhibitor, J Sex Med 2011;8:1039–1047
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Figure 6 A conceptual framework depicting mechanisms involved in EFS-induced signal transductions. EFS stimulated adrenergic, cholinergic, and NANC neuro-effector systems simultaneously. The NO released by NANC nerve plays the most important role in inducing smooth muscle relaxation. EFS, electrical field stimulation; NANC, nonadrenergicnoncholinergic; L-NAME, Nw nitro-Larginine-methyl ester; nNOS, neurogenic NO synthases; NO, nitric oxide; Ach, acetylcholine; MR, muscarinic receptor; eNOS, endothelial NO synthases; Guan, guanethidine; GC, guanylate cyclase; PLC, phospholipase C; IP3, inositol trisphosphate.
eserine, and abolished by atropine, indicating that the relaxation of phase III was mediated by the cholinergic neuro-effector system (Figure 6). Other factors may also affect the corporal tracing elicited by EFS, such as the self-recovery property of the cavernosum smooth muscle and ion channels in smooth muscle membranes [25–30]; however, the neuronal systems play the most important role. The triphasic response to EFS can help us understand the potential mechanism involved in EFS-induced signal transductions. Conclusions
EFS stimulated adrenergic, cholinergic, and NANC neuro-effector systems simultaneously. The corporal tracing elicited by EFS was the balanced result of multiple factors. Phase I was related to the NO-cyclic GMP pathway. Phase II was affected by self-recovery properties, and adrenergic and cholinergic nerves. Phase III was related to cholinergic nerves.
National University, Jeonju 560-180, Korea. Tel: 82-63250-1510; Fax: 82-63-250-1564; E-mail: rain@ chonbuk.ac.kr Conflict of Interest: None. Statement of Authorship
Category 1 (a) Conception and Design Kyung Woo Cho; Jong Kwan Park (b) Acquisition of Data Chen Zhao (c) Analysis and Interpretation of Data Chen Zhao; Jong Kwan Park
Category 2 (a) Drafting the Article Chen Zhao; Jong Kwan Park (b) Revising It for Intellectual Content Jong Kwan Park
Category 3 (a) Final Approval of the Completed Article Chen Zhao; Jong Kwan Park; Kyung Woo Cho
Acknowledgments
This study was supported by grants from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (90583). Corresponding Author: Jong Kwan Park, MD, Department of Urology, Medical School, Chonbuk J Sex Med 2011;8:1039–1047
References 1 Sáenz de Tejada I. In the physiology of erection, signposts to impotence. Contemp Urol 1992;7:52–68. 2 Andersson KE, Wagner G. Physiology of penile erection. Physiol Rev 1995;75:191–236. 3 Traish AM. Androgens play a pivotal role in maintaining penile tissue architecture and erection: A review. J Androl 2009; 30:363–9.
A New Mechanism of Relaxation by Electrical Field Stimulation 4 Gratzke C, Angulo J, Chitaley K, Dai YT, Kim NN, Paick JS, Simonsen U, Uckert S, Wespes E, Andersson KE, Lue TF, Stief CG. Anatomy, physiology, and pathophysiology of erectile dysfunction. J Sex Med 2010;7:445–75. Erratum in: J Sex Med 2010;7:1316. 5 Ryu JK, Zhang LW, Jin HR, Piao S, Choi MJ, Tuvshintur B, Tumurbaatar M, Shin SH, Han JY, Kim WJ, Suh JK. Derangements in endothelial cell-to-cell junctions involved in the pathogenesis of hypercholesterolemia-induced erectile dysfunction. J Sex Med 2009;6:1893–907. 6 Giuliano F, Rampin O. Neural control of erection. Physiol Behav 2004;83:189–201. 7 Bella AJ, Lue TF. Male sexual dysfunction. In: Tanagho EA, McAninch JW, eds. Smith’s general urology. 17th international edition. Seoul: McGraw-Hill; 2008:589–610. 8 Andersson KE. Erectile physiological and pathophysiological pathways involved in erectile dysfunction. J Urol 2003; 170:13–4. 9 Hedlund P, Ny L, Alm P, Andersson KE. Cholinergic nerves in human corpus cavernosum and spongiosum contain nitric oxide synthase and heme oxygenase. J Urol 2000;164:868–75. 10 Bivalacqua TJ, Liu T, Musicki B, Champion HC, Burnett AL. Endothelial nitric oxide synthase keeps erection regulatory function balance in the penis. Eur Urol 2007;51:1732–40. 11 Giuliano F, Bernabe J, Alexandre L, Niewoehner U, Haning H, Bischoff E. Pro-erectile effect of vardenafil: In vitro experiments in rabbits and in vivo comparison with sildenafil in rats. Eur Urol 2003;44:731–6. 12 Vural IM, Ozturk GS, Sarioglu Y. Functional characterization of nonadrenergic noncholinergic neurotransmitter release via endocannabinoids: An in vitro study in rabbit corpus cavernosum. J Sex Med 2009;6:717–29. 13 Hallén K, Wiklund NP, Gustafsson LE. Inhibitors of phosphodiesterase 5 (PDE 5) inhibit the nerve-induced release of nitric oxide from the rabbit corpus cavernosum. Br J Pharmacol 2007;150:353–60. 14 Rajfer J, Aronson WJ, Bush PA, Dorey FJ, Ignarro LJ. Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. N Engl J Med 1992;326:90–4. 15 Ignarro LJ, Bush PA, Buga GM, Wood KS, Fukuto JM, Rajfer J. Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle. Biochem Biophys Res Commun 1990;170:843–50. 16 Ghalayini IF. Nitric oxide-cyclic GMP pathway with some emphasis on cavernosal contractility. Int J Impot Res 2004; 16:459–69. 17 Kilicarslan H, Bagcivan I, Yildirim MK, Sarac B, Kaya T. Effect of hypothyroidism on the NO/cGMP pathway
18
19
20
21
22 23
24 25
26
27
28
29
30
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of corpus cavernosum in rabbits. J Sex Med 2006;3:830– 7. Kim N, Azadzoi KM, Goldstein I, Saenz de Tejada I. A nitric oxide-like factor mediates nonadrenergic-noncholinergic neurogenic relaxation of penile corpus cavernosum smooth muscle. J Clin Invest 1991;88:112–8. Zhao C, Chae HJ, Kim SH, Cui WS, Lee SW, Jeon JH, Park JK. A new perfusion model for studying erectile function. J Sex Med 2010;7:1419–28. Saenz de Tejada I, Blanco R, Goldstein I, Azadzoi K, de las Morenas A, Krane RJ, Cohen RA. Cholinergic neurotransmission in human corpus cavernosum. I. Responses of isolated tissue. Am J Physiol 1988;254:459–67. Maggi M, Filippi S, Ledda F, Magini A, Forti G. Erectile dysfunction: From biochemical pharmacology to advances in medical therapy. Eur J Endocrinol 2000;143:143–54. Burnett AL. Nitric oxide in the penis: Physiology and pathology. J Urol 1997;157:320–4. Moreland RB, Goldstein I, Kim NN, Traish A. Sildenafil citrate, a selective phosphodiesterase type 5 inhibitor: Research and clinical implications in erectile dysfunction. Trends Endocrinol Metab 1999;10:97–104. Toda N, Ayajiki K, Okamura T. Nitric oxide and penile erectile function. Pharmacol Ther 2005;106:233–66. Simonsen U, Prieto D, Sánez de Tejada I, García-Sacristán A. Involvement of nitric oxide in the non-adrenergic noncholinergic neurotransmission of horse deep penile arteries: Role of charybdotoxin-sensitive K(+)-channels. Br J Pharmacol 1995;116:2582–90. El-Metwally MA, Sharabi FM, Daabees TT, Senbel AM, Mostafa T. Involvement of alpha-receptors and potassium channels in the mechanism of action of sildenafil citrate. Int J Impot Res 2007;19:551–7. Ghasemi M, Sadeghipour H, Dehpour AR. Anandamide improves the impaired nitric oxide-mediated neurogenic relaxation of the corpus cavernosum in diabetic rats: Involvement of cannabinoid CB1 and vanilloid VR1 receptors. BJU Int 2007;100:1385–90. McCloskey C, Cagney V, Large R, Hollywood M, Sergeant G, McHale N, Thornbury K. Voltage-dependent Ca2+ currents contribute to spontaneous Ca2+ waves in rabbit corpus cavernosum myocytes. J Sex Med 2009;6:3019–31. Chung SD, Kuo YC, Liu SP, Chang HC, Yu HJ, Hsieh JT. The role of chloride channels in rat corpus cavernosum: In vivo study. J Sex Med 2009;6:708–16. Sergeant GP, Craven M, Hollywood MA, McHale NG, Thornbury KD. Spontaneous Ca2+ waves in rabbit corpus cavernosum: Modulation by nitric oxide and cGMP. J Sex Med 2009;6:958–66.
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Chen Zhao, MD,* Kyung Woo Cho, MD, PhD,† and Jong Kwan Park, MD, PhD* *Department of Urology, Medical School, and Institute for Medical Sciences, Chonbuk National University, and Research Institute and CTC for Medical Device of Chonbuk National University Hospital, Jeonju, Korea; †Department of Physiology, College of Oriental Medicine, Wonkwang Univeristy, Iksan, Korea DOI: 10.1111/j.1743-6109.2010.02178.x
ABSTRACT
Introduction. Nitric oxide (NO) has been shown to mediate electrical field stimulation (EFS)-caused smooth muscle relaxation. It is known that the neural control of penile erection involves adrenergic, cholinergic, and nonadrenergic-non-cholinergic (NANC) neuro-effector systems; however, the effects of EFS on adrenergic and cholinergic nerves are not clear. Aims. To elucidate EFS-induced signal transductions involved in adrenergic, cholinergic, and NANC neuroeffector systems by using an in vitro penile perfusion model. Methods. EFS was performed on penile corpus cavernosum smooth muscle from male New Zealand White rabbits, which was pre-contracted with L-phenylephrine (10 mM). We investigated the penile tracing elicited by EFS on tissues pre-incubated with guanethidine (Guan, 50 mM), tetrodotoxin (TTX, 10 mM), Nw nitro-L-arginine-methyl ester (L-NAME, 1 mM), atropine (50 mM), or eserine (10 mM). Main Outcome Measures. The time-to-peak of each phase, the percentage of relaxation, and the area under the curve (AUC). Results. We discovered an extraordinary phenomenon: three distinct phases elicited by EFS. Phase I was abolished by L-NAME. Phase II was decreased by eserine and Guan, but increased by L-NAME. Phase III was abolished by atropine, but enhanced by eserine and Guan. TTX diminished all three phases. The time to reach the top of phase I was delayed by TTX. The time to attain the peak of phase II was shortened by L-NAME, but delayed by TTX and atropine. The time to reach the top of phase III was shortened by L-NAME, eserine, and Guan. AUC was significantly decreased by L-NAME and TTX. Conclusions. EFS stimulated adrenergic, cholinergic, and NANC neuro-effector systems simultaneously. Phase I was related to the NO pathway. Phase II was multiply affected by self-recovery properties, and adrenergic and cholinergic nerves. Phase III was related to cholinergic nerves. The corporal tracing elicited by EFS was the balanced result of multiple factors. Zhao C, Cho KW, and Park JK. Three phases of corporal tracing elicited by electrical field stimulation on rabbit corpus cavernosum smooth muscle in penile perfusion model. J Sex Med 2011;8:1039–1047. Key Words. Electrical Field Stimulation (EFS); Smooth Muscle Relaxation; In Vitro Penile Perfusion Model; Extraordinary Phenomenon; Corpus Cavernosum Smooth Muscle
Introduction
P
enile erection is a complex neurovascular process that involves relaxation of the corpus cavernosum smooth muscle [1–5]. Neural control of penile erection involves the adrenergic,
© 2011 International Society for Sexual Medicine
cholinergic, and non-adrenergic-non-cholinergic (NANC) neuro-effector systems [6–8]. Adrenergic nerves mediate intracavernous smooth muscle contraction, which maintains the penis in a flaccid state. Cholinergic nerves contribute to smooth muscle relaxation and penile erection through J Sex Med 2011;8:1039–1047
1040 inhibition of adrenergic nerves via inhibitory interneurons and release of nitric oxide (NO) from the endothelium by acetylcholine (Ach) [9]. NO released from the cavernous nerve, as a NANC neurotransmitter, and from the endothelium, is the principal entity mediating relaxation of the cavernosum muscle [10]. The organ chamber strip model with electrical field stimulation (EFS) is a common technique for evaluating the erectile function in isolated cavernosum smooth muscle of humans and rabbits in vitro [11–13]. Many studies have demonstrated that EFS causes rapid, but transient smooth muscle relaxation, and NO has been shown to mediate relaxation in response to nerve stimulation. EFS of isolated strips of rabbit corpus cavernosum promotes the endogenous formation and release of NO and cyclic guanosine monophosphate (GMP) [14,15]. Corporal smooth muscle relaxation in response to EFS in the presence of guanethidine (Guan) and atropine is abolished by tetrodotoxin (TTX) and potassium-induced depolarization, and is markedly inhibited by NG-nitro-L-arginine, NG-amino-L-arginine, oxyhemoglobin, and methylene blue, but is unaffected by indomethacin [16–18]. The stimulation of the nerve endings promotes the release of NO from NANC nerves and it may also stimulate the adrenergic and cholinergic neural systems. We hypothesized that EFS activated these neuro-effector systems simultaneously and the corporal tracing elicited by EFS could reflect the interactions of the systems. We performed EFS in an in vitro penile perfusion model, which has been demonstrated to have a more sensitive response capacity to Ach and EFS compared with the classic penile strip chamber model [19]. The objective of the present study was to elucidate EFS-induced signal transductions. Methods
This study was approved by Institutional Animal Care and Use Committee of Chonbuk National University Hospital and Medical School. The experimental procedures were performed in accordance with the Guiding Principles in the Use and Care of Animals approved by the Council of American Physiological Society.
Tissue Preparation The penile tissues were prepared as previously described [19]. Healthy control male New Zealand White rabbits weighing 2.5–3.0 kg were anestheJ Sex Med 2011;8:1039–1047
Zhao et al. tized with ketamine (50 mg/kg intravenously) plus rumpun (25 mg/kg), and exsanguinated. The entire penis, including the urethra was rapidly excised from the pubic bone. The urethra was dissected free from the penile body. During the preparation, each step was taken cautiously to prevent damage of functional endothelium or overstretching of the tissue.
Measurement of Isometric Tension and Intracavernosum Pressure (ICP) The glans penis was cut out until the corpus cavernosum was exposed to air through a small opening with a diameter of 5 mm. Two small polyethylene tubes (inner diameter, 1.2 mm and outer diameter, 1.7 mm; Natsume, Tokyo, Japan), which contained small internal platinum electrodes, were inserted into the proximal opening of the crura for inflow, and ligated with a purse string silk suture to prevent leakage. The distal cut of the corpus cavernosum was opened to allow flow out of the penis. The distal end was secured with a cotton thread to a holder at the bottom of the chamber. The cannulated penis was mounted vertically in a 50-mL fully humidified organ chamber without buffer outside of the penis. The penis was immediately perfused interstitially through the cannulae with HEPES (4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl) piperazine-N′-(2ethanesulfonic acid)) buffer using a peristaltic pump (0.5 mL/minute). The HEPES buffer contained the following (in mM): NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25; glucose, 10.0; and HEPES 10 with NaOH (pH 7.4). The perfusion solution was oxygenated with 100% O2 and maintained at 36°C. The hollow organ chamber was covered with Parafilm (Pechiney Plastic Packaging Company, Chicago, IL, USA) to maintain the temperature and humidity in the organ chamber at 36°C. The ICP was measured using a pressure transducer (FT03; Grass Telefactor, W. Warwick, RI, USA) connected to the inflow tube cannulated to the crura and recorded using a PowerLab (Software Chart, version 5; AD Instrument, Castle Hill, Australia). After mounting, tissue was equilibrated for 100 minutes with several adjustments of length until a baseline force was stabilized at 10 g. The chamber for penile perfusion had a hole at the bottom to allow collection of the perfusate. Changes in tension were measured with a force transducer. Electrical Field Stimulation The tissue was stimulated by two parallel platinum electrodes bilaterally inserted into the cavernosum
A New Mechanism of Relaxation by Electrical Field Stimulation through the crura. The intrinsic nerves were fieldstimulated electrically (voltage, 30 V; pulse duration, 1 ms; trains of pulses, 10 seconds; train interval, 3–5 minutes; frequency, 0.5–32 Hz) using a Grass stimulator (S88, Grass Telefactor, W. Warwick, RI, USA). The sampling rate (recording speed) of the PowerLab data 400 acquisition system was set to 40/second.
Protocols This study included seven groups according to the different blocker treatments: L-phenylephrine (PhE); Nw nitro-L-arginine-methyl ester (L-NAME); Guan; atropine; eserine; and a combination of L-NAME, Guan, and atropine. In each group, the functional endothelium was first checked by the presence of at least a 50% relaxation in response to Ach (100 nM) in PhE-contracted tissue. Tissue was washed and contracted with PhE (10 mM). When a stable tone was obtained, EFS was performed. The tissue was washed again, then incubated with blocker for 30 minutes (1 mM L-NAME, 50 mM Guan, 10 mM TTX, 50 mM atropine, 10 mM eserine, or a combination of 1 mM L-NAME, 50 mM Guan, and 50 mM atropine), and the EFS-induced response was obtained in the PhE-precontracted tissue. After determination of the EFS-induced response and the stable contraction, sodium nitroprusside (SNP, 10 mM) was added for 5 minutes to determine whether or not guanylate cyclase activity was inhibited [19–21]. Measurements The representative tracings and the definitions of the phases are shown in Figure 1. In the PhE group, phase I is the first relaxation after EFS, phase II is the contraction following phase I, and phase III is the second relaxation following phase II after EFS. The percentage of EFS-induced relaxation, the time-to-peak of each phase, and the area under the curve (AUC) were calculated and analyzed by PowerLab software. The total AUC was divided into two parts (AUC1 and AUC2; Figure 2). Pharmacological Agents and Solutions Acetylcholine atropine hydrochloride, Guan, L-NAME, PhE, eserine (a cholinesterase inhibitor), and SNP were purchased from Sigma-Aldrich (St. Louis, MO, USA). TTX was purchased from Tocris Cookson (Langford, Bristol, UK). All other chemicals were the highest grade available.
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Statistics The submaximal contractile responses induced by PhE were taken as 100%, and all subsequent responses to EFS were expressed as a percentage of the PhE-induced value. The results are expressed as the mean ⫾ standard error (SE), and N represents the number of tissues in each group. The statistical significance of the differences was calculated by one-way analysis of variance (anova), followed by Bonferroni’s multiple comparison test. The responses before and after treatment with blockers were compared by a Student’s paired t-test. A probability value < 0.05 was considered significant.
Results
Changes in EFS Responses The response to EFS was in a Hz strengthdependent manner at low frequencies (<8 Hz) in the penile perfusion model (Figure 3, N = 8). Phase III first presented at 4 Hz and increased in a frequency-dependent manner. The blocker study was performed at a frequency of 8 Hz because the phases presented most clearly. Changes in Percentage of Relaxation Phase I was significantly decreased by TTX and completely abolished by L-NAME (PhE: ICP, 59.73 ⫾ 7.24%; tension, 52.13 ⫾ 6.52%; TTX: ICP, 3.23 ⫾ 1.42%; tension, 5.25 ⫾ 2.11%; P < 0.01 vs. PhE, Figure 4A, N = 8). Phase II was significantly diminished by eserine, TTX, and Guan (PhE: ICP, 40.69 ⫾ 5.88%; tension, 35.96 ⫾ 4.69%; eserine: ICP, 18.17 ⫾ 6.59%; tension, 14.14 ⫾ 5.39%; TTX: ICP, 3.76 ⫾ 1.43%; tension, 3.18 ⫾ 2.68%; Guan: ICP, 10.16 ⫾ 4.19%; tension, 9.27 ⫾ 5.09%; P < 0.01 vs. PhE, respectively, Figure 4B, N = 8). However, preincubation with L-NAME enhanced phase II over the PhE-precontracted level (L-NAME: ICP, 102.96 ⫾ 1.96%; tension, 122.38 ⫾ 4.72%; P < 0.01 vs. PhE, Figure 1C, D). Phase III was completely abolished by atropine and TTX, significantly decreased by L-NAME (PhE: ICP, 40.71 ⫾ 3.69%; tension, 32.71 ⫾ 4.06%; LNAME: ICP, 12.04 ⫾ 4.03%, tension, 16.13 ⫾ 7.08%; P < 0.01 vs. PhE), but enhanced by eserine and Guan (eserine: ICP, 70.68 ⫾ 15.16%; tension, 80.43 ⫾ 14.09%; Guan: ICP, 56.49 ⫾ 5.88%; tension, 53.76 ⫾ 8.49%; P < 0.01 vs. PhE, Figure 4C, N = 8). All the phases were abolished J Sex Med 2011;8:1039–1047
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Figure 1 Representative tracings elicited by EFS at a frequency of 8 Hz, duration of 1 ms, voltage of 30 V, and the definitions of three phases. Left column: ICP; right column: tension. PhE = phenylephrine; L-NAME = Nw nitro-L-argininemethyl ester; TTX = tetrodotoxin; Guan = guanethidine; ICP = intracavernosum pressure; EFS = electrical field stimulation.
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A New Mechanism of Relaxation by Electrical Field Stimulation
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by combination treatment with L-NAME, Guan, and atropine (Figure 5, N = 8).
Figure 2 Total AUC was divided into two parts (AUC 1 and AUC 2). AUC, area under the curve; PhE = phenylephrine; EFS = electrical field stimulation.
Changes in Time-To-Peak The time-to-peak of phase I was significantly delayed by TTX (P < 0.01 vs. PhE, N = 8). The time-to-peak of phase II was shortened by L-NAME, but delayed by TTX and atropine (P < 0.01 vs. PhE, respectively, N = 8). The timeto-peak of phase III was shortened by L-NAME (P < 0.01 vs. PhE, N = 8), eserine (P < 0.05 vs. PhE, N = 8), and Guan (P < 0.01 vs. PhE, N = 8). The time-to-recover to the PhE-precontracted level was shortened by L-NAME (P < 0.01 vs. PhE, N = 8) and TTX (P < 0.01 vs. PhE, N = 8), but delayed by eserine (P < 0.05 vs. PhE, N = 8) and atropine (P < 0.05 vs. PhE, N = 8; Table 1).
Figure 3 EFS-induced changes of ICP and tension on PhE-precontracted cavernosum smooth muscle in penile perfusion model. The intrinsic nerves were field-stimulated electrically at a voltage of 30 V, a pulse duration of 1 ms, trains of pulses of 10 seconds, and a train interval of 3–5 minutes. PhE = phenylephrine; ICP = intracavernosum pressure; EFS = electrical field stimulation.
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Figure 4 Changes in the percentage of relaxation of each phase. (A) Percent of phase I. (B) Percent of phase II. (C) Percent of phase III. The submaximal contractile responses induced by PhE were taken as 100%, and all subsequent responses to EFS were expressed as a percentage of PhE-induced value. PhE = phenylephrine; L-NAME = Nw nitro-L-arginine-methyl ester; TTX = tetrodotoxin; Guan = guanethidine; ICP = intracavernosum pressure. **P < 0.01 vs. PhE.
Figure 5 Representive tracings elicited by EFS on combination treatment cavernosum smooth muscle with L-NAME, Guan, and atropine. L-NAME = Nw nitro-L-arginine-methyl ester; Guan = guanethidine; ICP = intracavernosum pressure; EFS = electrical field stimulation.
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Changes in AUC The total AUC was divided into AUC 1 and 2 (Figure 2). AUC 1 was decreased by L-NAME and TTX (P < 0.01 vs. PhE, respectively, N = 8), but increased by Guan and atropine (P < 0.05 vs. PhE, respectively, N = 8). AUC 2 was decreased by L-NAME (P < 0.01 vs. PhE, N = 8), but increased by eserine and Guan (P < 0.01 vs. PhE, respectively, N = 8). Total AUC was decreased by L-NAME and TTX (P < 0.01 vs. PhE, respectively, N = 8), but increased by eserine and Guan (P < 0.01 vs. PhE, respectively, N = 8; Table 2). Discussion
It is well known that the state of relaxation or contraction of the arteriolar and trabecular smooth muscle determines penile erection or flaccidity [1,2,22]. The NANC neurotransmitter, NO, plays a crucial role in inducing smooth muscle relaxation and penile erection. NO
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A New Mechanism of Relaxation by Electrical Field Stimulation Table 1
Time to reach the top of each phase and recover the PhE pre-contracted level Phase I
Group
N
(s)
PhE L-NAME + PhE Eserine + PhE TTX + PhE Guan + PhE Atropine + PhE
8 8 8 8 8 8
10.5 ⫾ 0.3 — 10.5 ⫾ 0.3 30.6 ⫾ 0.9** 10.5 ⫾ 0.3 10.5 ⫾ 0.3
Phase II
Phase III
Recovery
22.1 ⫾ 0.8 12.5 ⫾ 1** 20 ⫾ 0.5 55.4 ⫾ 2.4** 19.8 ⫾ 0.5 49.5 ⫾ 1.4**
49.4 ⫾ 1.7 29.3 ⫾ 1.5** 35.5 ⫾ 1.5* — 29.5 ⫾ 1.1** —
137 ⫾ 7.1 88.5 ⫾ 2.6** 153.1 ⫾ 2* 55.4 ⫾ 2.4** 140.5 ⫾ 2 127.6 ⫾ 2.2*
*P < 0.05, **P < 0.01. PhE = L-phenylephrine; L-NAME = Nw nitro-L-arginine-methyl ester; TTX = tetrodotoxin; Guan = guanethidine; N is number of experiment.
synthesized by neurogenic and endothelial NO synthases (neurogenic NO synthases [nNOS] and endothelial NO synthases [eNOS]) diffuses into smooth muscle cells and activates the soluble guanylyl cyclase, which catalyses the formation of cyclic guanosine monophosphate (cGMP). Binding of cGMP to cGMP-dependent protein kinases or cGMP-dependent ion channels results in a reduction of intracellular Ca2+ and activation of myosin light chain phosphatases, which causes diminution of smooth muscle contractility and enhances penile erection [23]. Conversely, penile smooth muscle contraction mediated by the tonic signalling of sympathetic nerves maintains baseline flaccidity and causes detumescence [21]. Cholinergic nerves do not dilate or directly constrict smooth muscle, but modulate other neuro-effector systems via inhibitory interneurons and release of NO from the endothelium by Ach [9,20]. EFS has become a widely used technique in evaluating erectile function in vitro since Ignarro and his colleagues [15] demonstrated that EFSinduced relaxation of isolated rabbit corpus cavernosum was mediated by NO generated from NANC inhibitory nerves in 1990. Several years later, a biphasic response to EFS was reported; specifically, stimulation of the nerve endings induced a transient smooth muscle contraction before relaxation on PhE-precontracted rabbit
Table 2 Changes in AUC of EFS elicited tracings in rabbit penile perfusion model Group
N AUC 1
AUC 2
PhE L-NAME + PhE Eserine + PhE TTX + PhE Guan + PhE Atropine + PhE
8 85.9 ⫾ 3.9 227.5 ⫾ 12.4 8 13.8 ⫾ 1.0** 33.3 ⫾ 4.5** 8 98 ⫾ 1.9 402.5 ⫾ 9.5** 8 31.3 ⫾ 3.6** — 8 118.6 ⫾ 2.7* 415.1 ⫾ 10.4** 8 119.9 ⫾ 4.9* —
Total AUC 313.4 ⫾ 14.8 47.1 ⫾ 4.1** 500.5 ⫾ 10.3** 31.3 ⫾ 3.6** 533.6 ⫾ 12.2** 308.8 ⫾ 8.7
*P < 0.05, **P < 0.01. PhE = L-phenylephrine; L-NAME = Nw nitro-L-arginine-methyl TTX = tetrodotoxin; Guan = guanethidine; N = number of experiment.
ester;
corpus cavernosum and the contraction was abolished by the addition of Guan. This study confirmed that the sympathetic system was involved in the EFS pathway [21]. Toda and his colleagues [24] reported a triphasic response to EFS on prostaglandin F2a-precontracted monkey corpus cavernosum; however, they focused on the EFS-NO pathway and the relaxant effect was not affected by a cholinergic antagonist, such as atropine, and the effect of EFS on cholinergic nerves was unclear [24]. In our study, we verified the EFS-NO pathway in an in vitro penile perfusion model. We have demonstrated that this model had a more sensitive response capacity to Ach and EFS than the classic penile strip chamber model in the previous study [19]. We observed the triphasic tracing elicited by EFS at a recording speed of 40/second on the PowerLab data 400 acquisition system. Phase I was abolished by a NOS inhibitor and the relaxation was not inhibited by adrenergic and cholinergic blockers, indicating the NO pathway was the most potent factor in inducing penile smooth muscle relaxation. We think that the NO released by EFS has 2 sources: one is released by NANC nerve as a neurotransmitter, which in charge of the relaxation of phase I; the other one is released by endothelium activated by Ach which in charge of the relaxation of phase III. EFS releases NO and the NO production is significantly inhibited by L-NAME. However, atropine and eserine did not block the EFS induced NO production. In many previous papers, atropine did not block EFS induced relaxation but acetylcholine induced relaxation [19,24]. L-NAME inhibits both nNOS and eNOS, so phase I and phase III are significantly decreased in this study (Figure 4A, C). Phase II was significantly diminished by Guan and eserine, suggesting that the sympathetic system is involved in EFS-induced signal transductions and can be inhibited by cholinergic nerves. Phase III was intensified by the choline esterase inhibitor, J Sex Med 2011;8:1039–1047
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Figure 6 A conceptual framework depicting mechanisms involved in EFS-induced signal transductions. EFS stimulated adrenergic, cholinergic, and NANC neuro-effector systems simultaneously. The NO released by NANC nerve plays the most important role in inducing smooth muscle relaxation. EFS, electrical field stimulation; NANC, nonadrenergicnoncholinergic; L-NAME, Nw nitro-Larginine-methyl ester; nNOS, neurogenic NO synthases; NO, nitric oxide; Ach, acetylcholine; MR, muscarinic receptor; eNOS, endothelial NO synthases; Guan, guanethidine; GC, guanylate cyclase; PLC, phospholipase C; IP3, inositol trisphosphate.
eserine, and abolished by atropine, indicating that the relaxation of phase III was mediated by the cholinergic neuro-effector system (Figure 6). Other factors may also affect the corporal tracing elicited by EFS, such as the self-recovery property of the cavernosum smooth muscle and ion channels in smooth muscle membranes [25–30]; however, the neuronal systems play the most important role. The triphasic response to EFS can help us understand the potential mechanism involved in EFS-induced signal transductions. Conclusions
EFS stimulated adrenergic, cholinergic, and NANC neuro-effector systems simultaneously. The corporal tracing elicited by EFS was the balanced result of multiple factors. Phase I was related to the NO-cyclic GMP pathway. Phase II was affected by self-recovery properties, and adrenergic and cholinergic nerves. Phase III was related to cholinergic nerves.
National University, Jeonju 560-180, Korea. Tel: 82-63250-1510; Fax: 82-63-250-1564; E-mail: rain@ chonbuk.ac.kr Conflict of Interest: None. Statement of Authorship
Category 1 (a) Conception and Design Kyung Woo Cho; Jong Kwan Park (b) Acquisition of Data Chen Zhao (c) Analysis and Interpretation of Data Chen Zhao; Jong Kwan Park
Category 2 (a) Drafting the Article Chen Zhao; Jong Kwan Park (b) Revising It for Intellectual Content Jong Kwan Park
Category 3 (a) Final Approval of the Completed Article Chen Zhao; Jong Kwan Park; Kyung Woo Cho
Acknowledgments
This study was supported by grants from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (90583). Corresponding Author: Jong Kwan Park, MD, Department of Urology, Medical School, Chonbuk J Sex Med 2011;8:1039–1047
References 1 Sáenz de Tejada I. In the physiology of erection, signposts to impotence. Contemp Urol 1992;7:52–68. 2 Andersson KE, Wagner G. Physiology of penile erection. Physiol Rev 1995;75:191–236. 3 Traish AM. Androgens play a pivotal role in maintaining penile tissue architecture and erection: A review. J Androl 2009; 30:363–9.
A New Mechanism of Relaxation by Electrical Field Stimulation 4 Gratzke C, Angulo J, Chitaley K, Dai YT, Kim NN, Paick JS, Simonsen U, Uckert S, Wespes E, Andersson KE, Lue TF, Stief CG. Anatomy, physiology, and pathophysiology of erectile dysfunction. J Sex Med 2010;7:445–75. Erratum in: J Sex Med 2010;7:1316. 5 Ryu JK, Zhang LW, Jin HR, Piao S, Choi MJ, Tuvshintur B, Tumurbaatar M, Shin SH, Han JY, Kim WJ, Suh JK. Derangements in endothelial cell-to-cell junctions involved in the pathogenesis of hypercholesterolemia-induced erectile dysfunction. J Sex Med 2009;6:1893–907. 6 Giuliano F, Rampin O. Neural control of erection. Physiol Behav 2004;83:189–201. 7 Bella AJ, Lue TF. Male sexual dysfunction. In: Tanagho EA, McAninch JW, eds. Smith’s general urology. 17th international edition. Seoul: McGraw-Hill; 2008:589–610. 8 Andersson KE. Erectile physiological and pathophysiological pathways involved in erectile dysfunction. J Urol 2003; 170:13–4. 9 Hedlund P, Ny L, Alm P, Andersson KE. Cholinergic nerves in human corpus cavernosum and spongiosum contain nitric oxide synthase and heme oxygenase. J Urol 2000;164:868–75. 10 Bivalacqua TJ, Liu T, Musicki B, Champion HC, Burnett AL. Endothelial nitric oxide synthase keeps erection regulatory function balance in the penis. Eur Urol 2007;51:1732–40. 11 Giuliano F, Bernabe J, Alexandre L, Niewoehner U, Haning H, Bischoff E. Pro-erectile effect of vardenafil: In vitro experiments in rabbits and in vivo comparison with sildenafil in rats. Eur Urol 2003;44:731–6. 12 Vural IM, Ozturk GS, Sarioglu Y. Functional characterization of nonadrenergic noncholinergic neurotransmitter release via endocannabinoids: An in vitro study in rabbit corpus cavernosum. J Sex Med 2009;6:717–29. 13 Hallén K, Wiklund NP, Gustafsson LE. Inhibitors of phosphodiesterase 5 (PDE 5) inhibit the nerve-induced release of nitric oxide from the rabbit corpus cavernosum. Br J Pharmacol 2007;150:353–60. 14 Rajfer J, Aronson WJ, Bush PA, Dorey FJ, Ignarro LJ. Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. N Engl J Med 1992;326:90–4. 15 Ignarro LJ, Bush PA, Buga GM, Wood KS, Fukuto JM, Rajfer J. Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle. Biochem Biophys Res Commun 1990;170:843–50. 16 Ghalayini IF. Nitric oxide-cyclic GMP pathway with some emphasis on cavernosal contractility. Int J Impot Res 2004; 16:459–69. 17 Kilicarslan H, Bagcivan I, Yildirim MK, Sarac B, Kaya T. Effect of hypothyroidism on the NO/cGMP pathway
18
19
20
21
22 23
24 25
26
27
28
29
30
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of corpus cavernosum in rabbits. J Sex Med 2006;3:830– 7. Kim N, Azadzoi KM, Goldstein I, Saenz de Tejada I. A nitric oxide-like factor mediates nonadrenergic-noncholinergic neurogenic relaxation of penile corpus cavernosum smooth muscle. J Clin Invest 1991;88:112–8. Zhao C, Chae HJ, Kim SH, Cui WS, Lee SW, Jeon JH, Park JK. A new perfusion model for studying erectile function. J Sex Med 2010;7:1419–28. Saenz de Tejada I, Blanco R, Goldstein I, Azadzoi K, de las Morenas A, Krane RJ, Cohen RA. Cholinergic neurotransmission in human corpus cavernosum. I. Responses of isolated tissue. Am J Physiol 1988;254:459–67. Maggi M, Filippi S, Ledda F, Magini A, Forti G. Erectile dysfunction: From biochemical pharmacology to advances in medical therapy. Eur J Endocrinol 2000;143:143–54. Burnett AL. Nitric oxide in the penis: Physiology and pathology. J Urol 1997;157:320–4. Moreland RB, Goldstein I, Kim NN, Traish A. Sildenafil citrate, a selective phosphodiesterase type 5 inhibitor: Research and clinical implications in erectile dysfunction. Trends Endocrinol Metab 1999;10:97–104. Toda N, Ayajiki K, Okamura T. Nitric oxide and penile erectile function. Pharmacol Ther 2005;106:233–66. Simonsen U, Prieto D, Sánez de Tejada I, García-Sacristán A. Involvement of nitric oxide in the non-adrenergic noncholinergic neurotransmission of horse deep penile arteries: Role of charybdotoxin-sensitive K(+)-channels. Br J Pharmacol 1995;116:2582–90. El-Metwally MA, Sharabi FM, Daabees TT, Senbel AM, Mostafa T. Involvement of alpha-receptors and potassium channels in the mechanism of action of sildenafil citrate. Int J Impot Res 2007;19:551–7. Ghasemi M, Sadeghipour H, Dehpour AR. Anandamide improves the impaired nitric oxide-mediated neurogenic relaxation of the corpus cavernosum in diabetic rats: Involvement of cannabinoid CB1 and vanilloid VR1 receptors. BJU Int 2007;100:1385–90. McCloskey C, Cagney V, Large R, Hollywood M, Sergeant G, McHale N, Thornbury K. Voltage-dependent Ca2+ currents contribute to spontaneous Ca2+ waves in rabbit corpus cavernosum myocytes. J Sex Med 2009;6:3019–31. Chung SD, Kuo YC, Liu SP, Chang HC, Yu HJ, Hsieh JT. The role of chloride channels in rat corpus cavernosum: In vivo study. J Sex Med 2009;6:708–16. Sergeant GP, Craven M, Hollywood MA, McHale NG, Thornbury KD. Spontaneous Ca2+ waves in rabbit corpus cavernosum: Modulation by nitric oxide and cGMP. J Sex Med 2009;6:958–66.
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