Research Paper
The expression, localization and function of a7 nicotinic acetylcholine receptor in rat corpus cavernosum Hedyeh Faghir-Ghanesefata,b, Nastaran Rahimia,b, Fatemeh Yarmohammadia,b, Tahmineh Mokhtaric, Ali Reza Abdollahid, Shahram Ejtemaei Mehra,b and Ahmad R. Dehpoura,b a
Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran, bExperimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran, cDepartment of Anatomy, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran and dDepartment of Pathology, Imam Hospital, Tehran University of Medical Sciences, Tehran, Iran
Keywords a7 nicotinic acetylcholine receptor; corpus cavernosum; nitric oxide; non-adrenergic/ non-cholinergic; rat Correspondence Prof. Ahmad Reza Dehpour, Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, P.O. Box 13145-784, Iran. E-mails:
[email protected];
[email protected] Received February 20, 2017 Accepted July 26, 2017 doi: 10.1111/jphp.12806
Abstract Objectives Alpha7 nicotinic acetylcholine receptor (a7-nAChR), an emerging pharmacological target for a variety of medical conditions, is expressed in the most mammalian tissues with different effects. So, this study was designed to investigate the expression, localization and effect of a7-nAChR in rat corpus cavernosum (CC). Methods & Key findings Reverse transcription polymerase chain reaction (RTPCR) revealed that a7-nAChR was expressed in rat CC and double immunofluorescence studies demonstrated the presence of a7-nAChR in corporal neurons. The rat CC segments were mounted in organ bath chambers and contracted with phenylephrine (0.1 lM -300 lM) to investigate the relaxation effect of electrical field stimulation (EFS,10 Hz) assessed in the presence of guanethidine (adrenergic blocker, 5 lM) and atropine (muscarinic cholinergic blocker, 1 lM) to obtain non-adrenergic non-cholinergic (NANC) response. Cumulative administration of nicotine significantly potentiated the EFS-induced NANC relaxation (-log EC50 = 7.5 0.057). Whereas, the potentiated NANC relaxation of nicotine was significantly inhibited with different concentrations of methyllycaconitine citrate (a7-nAChR antagonist, P < 0.05) in preincubated strips. L-NAME (nonspecific nitric oxide synthase inhibitor, 1 lM) completely blocked the neurogenic relaxation induced by EFS plus nicotine. Conclusion To conclude a7-nAChR is expressed in rat CC and modulates the neurogenic relaxation response to nicotine.
Introduction Relaxation of corpus cavernosum (CC) is critical to induce and maintain the penile erection.[1] The dilation in the cavernosal arterioles and sinuses results in increased blood flow, raising intracavernosal pressure to culminate in erection.[2] It has been demonstrated the neural control of erection via cavernosal nerve stimulation involves adrenergic, cholinergic and non-adrenergic non-cholinergic (NANC) transmitters.[3] Several studies have shown that NANC transmission is the major event leading to relaxation of corporal smooth muscle.[2] NANC neurons can be stimulated experimentally by electrical field stimulation (EFS) or by administration of a variety of agonists such as nicotine 1754
which is a non-specific nicotinic acetylcholine receptors (nAChRs) ligand.[4,5] Neuronal AChRs belong to superfamily of neurotransmitter-gated ion channels that mediate rapid intracellular communication[6] and play key roles in synaptic transmission through the peripheral and central nervous system.[7] Ozturk Fincan et al.l in 2010 reported that nAChR subtypes including alpha3-beta4, alpha4-beta2 and alpha7 involved in relaxation of rabbit corpus cavernosum.[4] The functions of a7 nicotinic acetylcholine receptor (a7-nAChR) have been widely expressed in the mammalian central nervous system and also different peripheral tissues.[8,9] Moreover, a7 receptor is the predominant nAChR subtype on gastrointestinal projecting neurons in the dorsal motor vagal
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nucleus.[4] The presynaptic a7-nAChR mediates nicotineinduced vasodilation via nitric oxide (NO) in porcine basilar arteries.[10] Also, nicotine stimulates nicotinic receptors in nerve terminals and liberates NO or NO-like substance (s) as neurotransmitters in middle cerebral arteries of the cat.[11] Synthesis and release of NO and the consequences of NO binding to soluble guanylyl cyclase are essential steps in the erectile process.[12] As a7-nAChRs are expressed widely in mammalian tissues, pharmacological modulation of these receptors may have unwanted effects on peripheral tissues such as CC. This study was designed to investigate the expression, the presence and role of a7-nAChR in relaxation of rat corpus cavernosum.
Materials and Methods Chemical The following chemicals were used in the preparation of Krebs-bicarbonate solution: Sodium chloride, potassium chloride, potassium dihydrogen phosphate, magnesium sulphate, Sodium bicarbonate, calcium chloride and glucose which were purchased from Merck Chemical Company. The following drugs were used: phenylephrine hydrochloride, N (x)-nitro L-arginine methyl ester (L-NAME), guanethidine sulphate, atropine sulphate, nicotine hydrogen tartrate, methyllycaconitine citrate (MLA) and microtubuleassociated protein 2 (MAP2 antibody) which were purchased from Sigma Chemical Company (Sigma, St. Louis, MO, USA). All drugs were freshly dissolved daily in distilled water.
Animals Male Sprague Dawley rats, weighing 200–250 g, were purchased from Experimental Animal Unit (Department of Pharmacology, Tehran University of Medical Sciences). The animals were housed in a room with controlled temperature (22 2°C) and humidity (approximately 60%– 80%) with a 12 : 12-h light/dark cycle (lights on at 06:00 a.m.). They had free access to water and food. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (1996, published by National Academy Press, 2101 Constitution Ave. NW, Washington, DC 20055, USA) and approved by the appropriate animal care review committee at the Department of Pharmacology, Tehran University of Medical Sciences.
Gene expression of a7-nAChR in corpus cavernosum The CC and liver samples were isolated from four normal and intact rats. Samples were kept at 80 °C until RNA
extraction. Total RNA was extracted using RNeasy fibrous tissue mini kit (Qiagen, Germany) following the manufacturer’s instructions. First-strand cDNA was generated using reverse transcriptase, and PCR were performed using selective forward and reverse primer for b-actin (housekeeping gene) and a7-nAChR. The sequences of primers are presented as below: Rat a7-nAChR, forward: 50 -CCTGGCCAGTGTGGAG0 3 , reverse: 50 -TAAGCAAAGTCTTTGGACAC-30 (gene accession number: NM_012832.3, product size: 414 bp). Rat b-actin, forward: 50 -AGAGGGAAATCGTGCGTGACA30 , reverse: 50 - ACATCTGCTGGAAGGTGGACA-30 (gene accession number: NM_031144, product size: 453 bp). To amplify the cDNA, PCR included incubation at 95 °C for 5 min, followed by 40 cycles of thermal cycling (60 s at 95 °C, 60 s at 60 °C and 60 s at 72 °C). The final cycle was followed by a 5-min extension step at 72 °C. PCR products were subsequently electrophoresed on 1% agarose gel and visualized by UV lamp.
Localization of a7-nAChR in corpus cavernosum Penile mid-shaft tissue was isolated and cut through frozen section (Leica CM3050S Cryostat, GMI, Inc.) and processed for immunofluorescence staining. Double immunofluorescence technique was used for localization of a7-nAChR in rat CC and microtubule-associated protein 2 (MAP2 antibody) as a specific factor for nerve. Double immunofluorescence was performed on frozen sections (6 lm). The primary antibodies were rabbit anti-rat a7nAChR antibody (1:300 dilution, Abcam, MA, USA) and MAP2 antibody (1:300 dilution, Abcam, MA, USA), the second antibody was Goat Anti-Rabbit IgG H&L (FITC) (1:500 dilution, Abcam, MA, USA) for a7-nAChR antibody and Goat Anti-Mouse IgG H&L (Alexa Fluorâ 647) (1:500 dilution, Abcam, MA, USA) for MAP-2 antibody. Sections were photographed by Olympus bx40 fluorescence microscope with a DP27 digital camera.
Experiments The male rats were sacrificed by cervical dislocation. Penises were removed and promptly placed in a Petri dish containing 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); maintained at 37 °C and bubbled with a mixture of 95% O2 and 5% CO2. The CC was separated by cutting the fibrous septum in penis. It was mounted in 25-ml organ chambers with one end tied to an electrode holder and the other to a wire connected to a force transducer (Letica Scientific Instruments, Barcelona, Spain) and was recorded using a PowerLab system
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(ADInstrument, Australia). The chambers contained Krebs-bicarbonate solution (pH 7.4) at 37 °C equilibrated with 95% O2 and 5% CO2. The segments were allowed to equilibrate resting tension of 0.5 g for 60 min based on previous study.[13] In a pilot study, the concentration–response curves to phenylephrine (0.1 lM to 300 lM) were obtained by the cumulative addition of phenylephrine to the chamber. The EC50 value for phenylephrine was evaluated 10 lM and used for following experiments. After 5 min, EFS was applied via two parallel platinum electrodes on either side of the corpus strips. Electrical field stimulation (150 V, 3 ms duration at a frequency of 10 Hz, every 120 s) was used, atropine (1 lM, to produce muscarinic cholinergic blockade) and guanethidine (5 lM, to produce adrenergic blockade) were always present in the bathing medium to obtain NANC conditions.[13] Four experimental groups were used in this study; each group consisted of eight rats. In all the experiments, each strip was separated from one animal. Group I: Tissue + phenylephrine (10 lM) + EFS + Nicotine (0.001 lM 100 lM). After administration of phenylephrine (10 lM) and achieving to a plateau of contraction (5 min), EFS was delivered. After getting the suitable records (about 5 min), different concentrations of nicotine (0.001 lM 100 lM) were administered to the organ bath, and the EC50 value of nicotine relaxation was evaluated using GraphPad Prism7 software. Group II: Tissue + phenylephrine (10 lM) + EFS+MLA (1 nM) + Nicotine (0.001 lM 100 lM). In this group, NANC relaxation was induced by EFS soon after phenylephrine (10 lM) caused contraction and reached a plateau. The tissue was then incubated with MLA (1 nM) for 30 min. The experiment was then terminated following cumulative addition of nicotine (0.001 lM 100 lM) to the chamber. Group III: Tissue + phenylephrine (10 lM) + EFS + MLA (10 nM) + Nicotine (0.001 lM 100 lM). Group IV: Tissue + phenylephrine (10 lM) + EFS + MLA (100 nM) + Nicotine (0.001 lM 100 lM). For confirming the involvement of NANC pathway in the experiment, L-NAME was used. A single concentration of L-NAME (1 lM)[13] was added to the chamber following EFS induction and incubation of the tissue with 0.3 lM concentration of nicotine for 5 min.
Haematoxylin and eosin (H&E) staining The penis was carefully isolated, and CC was dissected. Then, it was immediately fixed in 10% formalin phosphatebuffered solution before paraffin embedding. The tissue was stained with H&E. 1756
Statistical analysis Data were expressed as the mean SEM percentage of contraction and relaxation of CC. EC50 values were calculated for each curve using a non-linear regression method (GraphPad, Prism7). Groups were compared statistically using general linear models of analysis of variance (ANOVA) followed by post hoc analysis with the Bonferroni test. P values of < 0.05 were considered to be statistically significant.
Results Detection of a7-nAChR in corporal tissue RT-PCR was used to detect the expression of a7-nAChR gene in rat corporal tissue. Figure 1a shows the results of RT-PCR for a7-nAChR and b-actin (housekeeping). bactin gene was expressed in all samples of rat. a7-nAChR was detectable in the CC and expressed with corresponding PCR product of ~ 500 bp (Figure 1a). The CC was shown in H&E staining to define the situation of this tissue in the penal stem (Figure 1b). Double immunofluorescence technique was employed for tissue localization of a7-nAChR protein in CC using anti-a7-nAChR antibody (green). MAP-2 antibody was used for detection of nerve in CC (red). As shown in Figure 1c, the two fluorescent modalities were merged using software and this photograph indicates the localization of a7-nAChR on nerve in CC tissue.
Effects of phenylephrine on rat corpus cavernosum strips Figure 2 shows the contraction of CC induced by phenylephrine in the organ bath. Cumulative administration of phenylephrine (0.1 lM 300 lM) caused concentrationdependent contractions in strips of rat CC (Log EC50 was 5 0.31, 95% CI = 5.92 to 4.21). The contraction of CC started in the concentration of 0.3 lM and then gradually increased in a concentration-dependent manner till it reached a plateau in 100 ml.
Effect of electrical field stimulation (EFS) on rat corpus cavernosum strips CC strips, precontracted with phenylephrine, were relaxed by EFS at 150 V, 3 ms duration at a frequency of 10 Hz, every 120 s in the presence of guanethidine (blocker of adrenergic) and atropine (blocker of muscarinic cholinergic) (Figure 3). Relaxation of corpus cavernosal segments by EFS was due to non-adrenergic/ non-cholinergic (NANC) nerves of corpus segments as
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Figure 1 Expression and localization of b-actin and a7-nAChR in rat corporal tissue. (a) Analysis of RT-PCR products on agarose gel for detection of b-actin and a7-nAChR in rat corporal tissue. RNA was isolated and analysed by RT-PCR, (b) H&E staining of corpus cavernosum (409), (c) localization of a7-nAChR using a7-nAChR antibody in corporal tissue and detection of corporal nerve using microtubule-associated protein 2 (MAP2 antibody). The second antibody of FITC was used for a7-nAChR antibody (green) and Alexa Fluorâ 647 for MAP-2 antibody (red), two photographs were merged to show the localization of a7-nAChR on nerve (9400). [Color figure can be viewed at wileyonlinelibrary.com]
they were incubated in guanethidine (5 lM) and atropine (1 lM).
Effects of nicotine on neurogenic relaxation induced by EFS in rat corpus cavernosum strips Figure 4 shows the effect of nicotine (a non-specific agonist of a7-nAChR) on the relaxation response of CC induced by EFS (150 V, 3 ms duration at a frequency of 10 Hz, every 120 s). Cumulative administration of nicotine (0.001 lM 100 lM) increased the EFS-induced NANC relaxation responses significantly compared to peaks before addition of nicotine (Figure 3b, P < 0.001). The relaxation response was started at concentration of 0.003 lM then it gradually increased and -log EC50 of relaxation induced by nicotine was 7.5 0.057, 95% CI = 8.11 to 7.18 (Figure 3).
Figure 2 Concentration-dependent response curve to phenylephrine in isolated corpus cavernosum muscles of rat. Administration of phenylephrine (0.1 lM-300 lM) caused concentration-dependent contractions in strips of corpus cavernosum in the organ bath. This group consisted of eight rats.
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Figure 3 The power laboratory records for confirming the different agents’ effects on isolated rat corpus cavernosum. (a) The effect of methyllycaconitine citrate (MLA) and nicotine on NANC relaxation of isolated corpus cavernosum in response to electrical field stimulation (150 V, 3 ms duration at a frequency of 10 Hz, every 120 s) in cavernosal tissue precontracted with phenylephrine (PE). MLA inhibited the nicotine-induced EFS-evoked neurogenic relaxations responses. This group consisted of eight rats. (b) The effect of nicotine and L-NAME on NANC relaxation of isolated corpus cavernosum in response to electrical field stimulation (150 V, 3 ms duration at a frequency of 10 Hz, every 120 s) in cavernosal tissue precontracted with phenylephrine (PE). Nicotine increased the EFS-induced NANC relaxation responses. Relaxation responses induced by EFS (10 Hz) and potentiation of this response by nicotine inhibited by L-NAME. This group consisted of eight rats.
Effects of methyllycaconitine citrate on nicotine-potentiated EFS-evoked transient neurogenic relaxations Figure 4 shows the inhibitory effect of methyllycaconitine citrate (MLA) on nicotine-potentiated response of EFS (150 V, 3 ms duration at a frequency of 10 Hz, every 120 s) in the CC. Addition of different concentrations of MLA (1 nM, 10 nM and 100 nM) inhibited the potentiated neurogenic relaxations response by nicotine in the organ bath, significantly (P < 0.05) compared with nicotine group (Figure 3a). When nicotine was applied in the presence of MLA, the relaxation inhibited up to 25%. This suggests that the antagonist of a7-nAChR inhibited the relaxation of nicotine up to 75% (Figure 4). The -logEC50 of nicotine was 6.5 0.0174, 95% CI = 7.18 to 6.16 in the presence of MLA.
Effects of L-NAME on nicotine-potentiated EFS-evoked transient neurogenic relaxations Figure 5 shows the effects of non-specific nitric oxide synthase (NOS) inhibitor (L-NAME) on the relaxation 1758
response of CC induced by nicotine. Relaxation responses induced by EFS (10 Hz) plus nicotine was completely inhibited by addition of one concentration of L-NAME (1 lM) in the organ chamber in the presence of nicotine (P < 0.001, Figure 3b).
Discussion This study was designed to assess the presence and impact of a7 subtype of nicotinic acetylcholine receptor in the relaxation of corpus cavernosum. The expression of a7nAChR was demonstrated by RT-PCR, and its localization was found to be corporal nerves based on immunohistochemistry study. In addition, after a complete blockade of adrenergic transmission with guanethidine and muscarinic receptors blockade with atropine, nicotine (a non-specific agonist of a7-nAChR) could increase neurogenic relaxation induced by EFS, a response which was inhibited by incubation with methyllycaconitine citrate (MLA, a specific antagonist of a7-nAChR). L-NAME (non-specific nitric oxide synthase inhibitor) completely blocked the neurogenic relaxation induced by EFS plus nicotine. Regarding our
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Figure 4 concentration–response curve to nicotine in isolated rat corpus cavernosum strips. Nicotine increased concentration dependently the relaxation responses induced by EFS in cavernosal strips. Methyllycaconitine citrate (MLA) inhibited the relaxation response induced by electrical field stimulation (150 V, 3 ms duration at a frequency of 10 Hz, every 120 s) with nicotine in strips of corpus cavernosum tissues precontracted with phenylephrine. Each group consisted of eight rats (**P < 0.01 & ***P < 0.001 significant levels for MLA with concentration of 10 and 1 nM compared with nicotine group, ###P < 0.001 & ####P < 0.0001 significant levels for MLA with concentration of 100 nM compared with nicotine group).
result, we supposed that a7-nAChR expressed in rat corpus cavernosum nerves, also it modulated its neurogenic relaxation. In previous studies, nicotine was carried out for induction of relaxation in smooth muscle of different organs such as gastric fundus, internal mammary artery, bladder myometrium and also CC of rabbit by increasing the EFS.[14–16] Fincan et al. (2010) reported that there was no significant difference between the effects of two concentrations of nicotine (3 9 105M and 104 M.) that used for relaxation of CC strips.[4] According to our response to nicotine, a concentration of nicotine as an effective relaxation mediator (one concentration of 0.3 lM) was selected to evaluate the effects of antagonists in this investigation due to EFS-induced NANC relaxation records. As a clinical point, inducing and maintaining penile erection is associated with the relaxation of smooth muscle in the CC.[17,18] Furthermore, it is documented that NANC transmission plays a critical role in the relaxation of corporal smooth muscle.[2] In this study, pretreatment with L-NAME completely blocked the EFS-induced relaxation potentiated by nicotine which supported the role of NO as a mediator of neurogenic relaxation response of the muscle. Different factors are exogenously involved in penile relaxation and contraction, but the balance between noradrenergic and nitrergic systems is so important.[17,19,20] Nitric oxide, a well-characterized neurotransmitter in the central and peripheral nervous,[21,22] plays an important role as a NANC transmitter
Figure 5 Response to nicotine and L-NAME in isolated rat corpus cavernosum strips. Nicotine produced relaxation responses in cavernosal strips. L-NAME inhibited the relaxation response induced by electrical field stimulation (150 V, 3 ms duration at a frequency of 10 Hz, every 120 s) with nicotine in strips of corpus cavernosum tissues precontracted with phenylephrine. ***Significant difference (P < 0.001) compared to vehicle; ###significant difference (P < 0.001) compared to nicotine (0.3 lM). This group consisted of eight rats.
which mediates the relaxation responses in many tissues in the genitourinary system including of clitoral and penile CC through a cGMP-dependent pathway.[23,24] Nitric oxide is generated in these nerves by activation of the neuronal nitric oxide synthase (nNOS) and diffuses into the smooth muscle, leads to relaxation.[24] Toda et al. (2005) stated that NANC transmitters involve in penile erection by cavernosal nerve and physiological mediators as NO.[25] A preferential antagonist of a7-nAChRs, MLA, was used to characterize a7-nAChR subtype involvement in the alternation of the nicotine-potentiated response of EFS. MLA with three concentrations (each concentration in separated experimental) was investigated in the presence of different concentrations of nicotine. The MLA partially inhibited the neurorelaxation response to nicotine on EFS in which the most effective concentration was related to 100 nM. These results confirm the function of a7-nAChRs in CC tissue in the rat. MLA has markedly multiple affinities for a7-AChRs in brain and muscle.[26,27] Yum et al. (1996) found that MLA was a potent antagonist for a7-AChRs at central nicotinic receptors located on Edinger–Westphal neurons, producing nearly complete functional blockade of nicotinic responses at 10 nM.[26] MLA has been considered as a highly selective nicotinic antagonist characterized with approximately 1000-fold affinity for a7-nAChR.[28] It appears that MLA loses its selectivity for a7-AChRs at concentrations higher than 1 lM.[29] a7-nAChR has been shown to belong to the evolutionarily oldest group of nAChRs.[30] This receptor has been detected in nerve terminals of rat arterial system and porcine brain basilar arteries[10,31] that induced neurogenic relaxation in mammalian smooth muscles. It has been
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demonstrated that a7-nAChR can modulate the effect of nicotine on neurogenic relaxation of rat CC.[32] Subsequently, in our study, the expression of a7-nAChR was recorded by RT-PCR and its localization was found to be corporal nerves based on double immunofluorescence technique.
Declarations Conflict of interest The Authors declare that they have no conflict of interests to disclose.
Conclusions
Acknowledgements
It can be concluded that a7-nAChR is expressed and localized in nerves of rat corpus cavernosum and it modulates the neurogenic relaxation response to nicotine.
This study was part of a M.S. thesis supported by Tehran University of Medical Sciences (grant NO: 93-02-3025784).
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