International Journal of Impotence Research (2007) 19, 551–557 & 2007 Nature Publishing Group All rights reserved 0955-9930/07 $30.00 www.nature.com/ijir
Involvement of a-receptors and potassium channels in the mechanism of action of sildenafil citrate MA El-Metwally1, FM Sharabi1, TT Daabees1, AM Senbel1 and T Mostafa2 1 Department of Pharmacology, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt and 2Department of Andrology& Sexology, Faculty of Medicine, Cairo University, Cairo, Egypt
Modulation of the adrenergic activity and interfering with channels such as potassium channels may affect relaxation and contraction of the corpus cavernosum. Sildenafil is a selective phosphodiesterase-5 inhibitor, proven effective in treating erectile dysfunction.In this study, the effect of sildenafil citrate on a-receptors modulation and potassium channels was tested. The direct relaxant effect of sildenafil citrate was studied by measuring changes in isometric tension in isolated strips of rabbit corpus cavernosum and rat aortic ring precontracted with phenylephrine or KCl compared to that of diazoxide in the presence and absence of tetraethylammonium. The inhibitory effect of sildenafil on electrical field stimulation-induced contraction of rabbit corpus cavernosum and rat anococcygeus muscle was also studied compared to that of phentolamine. Muscle relaxant effect of sildenafil (1 109–1 106 M on phenylephrine-precontracted rabbit corpus cavernosum strips was not attenuated by NG-nitro-L-arginine (3 105 M). Cumulative addition of sildenafil (1 109–1 106 M) and phentolamine (1 109–1 106 M) to the organ bath dose-dependently inhibited electrical field stimulation-induced contraction of rabbit corpus cavernosum and rat anococcygeus muscle, with almost similar EC50 values. Sildenafil (1 107 M) also inhibited phenylephrine-induced contraction of rat aortic rings by 39.8373.01%. In addition, tetraethylammonium (1 103 M) significantly attenuated the muscle relaxant effect of sildenafil (1 109–1 106 M) on phenylephrine-precontracted strips of rabbit corpus cavernosum.Sildenafil citrate is capable of producing cavernosal smooth muscle relaxation by an additional mechanism that may involve a-receptors and potassium channel opening. International Journal of Impotence Research (2007) 19, 551–557; doi:10.1038/sj.ijir.3901590; published online 2 August 2007 Keywords: erectile dysfunction; phosphodiesterase-5 inhibitors; sildenafil citrate; corpus cavernosum; a-receptors; potassium channels
Introduction Modulation of the adrenergic activity seems to be one of the most important means by which the contractile state of the smooth muscle of the corpus cavernosum and the penile vasculature is influenced.1 Activation of the a-adrenoceptors is postulated to be involved in the suppression of erectile activity and the mediation of the contracted penile corpus cavernosal tone in the flaccid state. An increased a-adrenoceptor-mediated tone in the
Correspondence: Dr T Mostafa, Department of Andrology and Sexology, Faculty of Medicine, Cairo University, Kasr Al Aini str., Cairo 11562, Egypt. E-mail: [email protected]
Received 30 October 2006; revised 28 March 2007; accepted 12 April 2007; published online 2 August 2007
trabecular smooth muscle of the corpus cavernosum has been associated with erectile dysfunction in old patients.2 This a-adrenoceptor-mediated activity is regulated by a 1-adrenoceptors and prejunctional a 2-adrenoceptors in the adrenergic nerve terminals and the post-junctional receptors found on corpus cavernosum smooth muscle cells. Blockade of these a-adrenoceptors might decrease the contractile function of the corpus cavernosum smooth muscle cells and facilitate erection by allowing endogenous vasoactive mediators, such as nitric oxide (NO) and prostaglandins, to produce their vasodilatory properties in an unopposed manner.3 Both phenylephrine (selective a 1-adrenoceptor agonist) and clonidine (selective a 2-adrenoceptor agonist) contracted corpus cavernosum tissue, suggesting their involvement.3 Because corporeal smooth muscle contains several potassium channels, it is expected that drugs
Involvement of a-receptors and potassium channels MA El-Metwally et al 552
interfering with these channels may affect relaxation and contraction in this tissue. Potassium channel openers such as pinacidil, nicorandil and cromakalim are believed to relax various types of smooth muscle cells and participate in the treatment of various urological disorders.4 Hyperpolarization with subsequent reduction of calcium influx through voltage-operated calcium channels necessary for activating contraction may account for the relaxant effect of potassium channel openers.5 Introduction of sildenafil citrate (Viagra), in 1998, has been an advancement in treating erectile dysfunction (ED).6 Sildenafil is an orally administered selective inhibitor of phosphodiesterase type 5. It was found to be effective in improving erectile process and enabling successful sexual intercourse in men with broad-spectrum ED cases.7 It has been claimed that previous production of cyclic guanosine monophosphate by NO is required for sildenafil activity.8 Thus sildenafil promotes penile erection only in response to sexual stimulation, which is responsible for the production of NO from nonadrenergic, non-cholinergic cavernosal nerves.9,10 The aim of this study was to assess the involvement of a-receptors modulation and potassium channels in the mechanism of sildenafil citrate action.
Materials and methods This work was performed after both IRB and IACUC approval. Isolated rabbit corpus cavernosum smooth muscle Adult sexually mature male New Zealand white rabbits (2.5–3.0 kg weight) were used. Rabbits were killed by exsanguinations and their penises were excised rapidly and placed in Krebs solution I (in mM: NaCl: 118, KCl: 4.6, MgSO4 7H2O: 1.2, KH2PO4: 1.6, CaCl2 2H2O: 1.2, NaHCO3: 24.0, glucose: 11) at 41C.11 The corpora were dissected and subsequently studied in organ chambers. Tissue baths containing Krebs solution were kept at 371C and constantly bubbled with 95% O2 and 5% CO2. One end of each strip was attached to a fixed pin at the bottom of the organ bath. The other end was attached to a force displacement transducer (Grass FT-03) connected to a Grass polygraph (Model 7D). The initial resting tension was 2 g. The preparation was left to equilibrate for 90 min. To test the modulation of electrical field stimulation-induced contraction, frequency-dependent contractions of the electrically stimulated erectile tissue were first recorded. Contractions were evoked by stimulation by square wave pulses of supramaximal voltage, 0.8 ms at 20 Hz provided in trains of 10 s duration. To study the inhibiting effect of sildenafil and phentolamine on the response to electrical field International Journal of Impotence Research
stimulation, drugs were added cumulatively to the organ bath. Three consecutive reproducible responses were required before the next higher concentration of drugs was applied. The increase in the tension of the muscle in response to electrical field stimulation was calculated in grams. The degree of inhibition induced by prazosin, sildenafil or phentolamine was expressed as percent inhibition of the contraction elicited just before the drug application. In smooth muscle relaxation experiments, each muscle was submaximally contracted with phenylephrine (3 106 M) or KCl (2 102 M). After the contractile response was stabilized, relaxation responses to sildenafil were recorded in a cumulative fashion. The relaxation responses were expressed as percent of submaximal contraction produced by phenylephrine or KCl. Control organ chamber was similarly contracted and appropriate concentrations of the drug vehicle were added. For characterizing sildenafil-induced muscle relaxation, contraction with phenylephrine and relaxation responses were obtained after incubation with NO synthase inhibitor (NG-nitro-L-arginine) for 10 min, a time and a concentration proven effective in inhibiting NO synthase in previous studies.12 The isolated field-stimulated rat anococcygeus muscle Male albino rats (200–250 g) were used. The muscle was prepared according to the method of Gillespie.13 The preparation was left to equilibrate under 1 g tension for 30 min, during which bathing Krebs solution II (in mM: NaCl: 118.1, KCl: 4.7, MgSO4 7 H2O: 1.2, KH2PO4: 1.0, CaCl2 2H2O: 2.5, NaHCO3: 25.0, glucose: 11.1) was replaced every 10 min. To test the modulation of electrical field simulation-induced contraction, the preparations were stimulated by square wave pulses of supramaximal voltage, 0.8 ms pulse duration at 20 Hz provided in trains of 10-s duration. The rat isolated aortic ring preparation Male albino rats (250–300 g) were used. Isolation of the rat aorta was carried out according to Nagao et al.14 and Fahim et al.15 Thoracic aortae were dissected out and transferred to a Petri dish containing Krebs solution II. The aorta was trimmed free of connective tissue and cut into ring segments 2–3 mm in length. Care was taken to avoid any damage to the endothelium. Aortic rings with and without endothelium were mounted in a 25 ml organ bath containing Krebs solution II by means of two stainless steel wire hooks inserted through the lumen of the ring. One of the hooks was anchored to a stationary pin at the bottom of the organ bath and the other was connected to an isometric force displacement transducer that was
Involvement of a-receptors and potassium channels MA El-Metwally et al
Drugs used The following drugs were used: sildenafil citrate (Viagra Pfizer, New York, NY, USA), diazoxide, phenylephrine hydrochloride, NG-nitro-L-arginine, potassium chloride, phentolamine mesylate, prazosin hydrochloride, tetraethylammonium (SigmaAldrich, St Louis, MO, USA). All drugs were dissolved in distilled water, except prazosin, which was prepared by levigation with glycerol and completed to volume by dextrose 5%, and diazoxide, which was dissolved in dimethylsulfoxide. Statistics Results were expressed as mean7s.e.m. The Student’s t-test was used for the analysis of unpaired data. For multiple comparison, one-way analysis of variance or F test followed by Student Newman– Keul’s post-test was utilized (statistical significance was set at 0.05 level). The potencies of sildenafil and phentolamine were expressed as EC50. EC50 values were determined by regression analysis of the linear portion (approximately 15–85% range) of the concentration–response curve in individual tissues, using a computer software program Graph PAD Instat (Version 2.01), Steve Whetzel, Parke-Davis 930762 A, Graph PAD. n throughout the manuscript represents the number of strips from different animals.
Results Effect of NG-nitro-L-arginine on sildenafil-induced muscle relaxation Rabbit corpus cavernosum strips were first incubated with NG-nitro-L-arginine (3 105 M) contracted with phenylephrine and sildenafil was then added in a cumulative fashion. NG-nitro-L-arginine slightly reduced sildenafil-induced relaxation. Results were insignificant (n ¼ 5; Figure 1). Effect of prazosin, phentolamine and sildenafil on electrical field stimulation-induced contractions of rabbit corpus cavernosum Electrical field stimulation of cavernosal strips at resting tension generated contractile response of 0.6270.04 g at 20 Hz (Figure 2). The adrenergic nature of electrical field stimulation-induced contractions was first confirmed: addition of prazosin (5 108 M) to the bath solution significantly
% Inhibition of PE-induced tone
connected to the polygraph for recording isometric contractions of the aorta. The tissue was aerated with a mixture of 95% O2 and 5% CO2 and kept at 371C. An optimum resting tension of 1 g was placed on the tissue and an equilibrium period of 2 h was allowed before the start of the experiment, with the bath fluid being replaced every 20 min.
In absence of L-NNA In presence of L-NNA
60 40 20 0 1x10-9 1x10-8 1x10-7 1x10-6 Concentration of sildenafil (M)
Figure 1 Effect of NG-nitro-L-arginine (L-NNA, 3 105 M) on sildenafil-induced relaxation of phenylephrine-induced tone in the rabbit corpus cavernosum. Responses are expressed as mean7s.e.m. (n ¼ 5).
inhibited the contractile response to electrical field stimulation to 0.0970.03 g (n ¼ 6, 86.2675.20% inhibition). Cumulative addition of sildenafil produced concentration(1 109–1 106 M) dependent inhibition of electrical field stimulation-induced contractions with EC50 value of 0.07470.006 mM (Figure 2). Similarly, cumulative addition of phentolamine (1 109–1 106 M) inhibited electrical field stimulation-induced contractions dose-dependently with EC50 0.1170.003 mM (Figures 2 and 3). Effect of prazosin, phentolamine and sildenafil on electrical field stimulation-induced contractions of rat anococcygeus muscle At resting tension, the rat anococcygeus muscle responded to electrical field stimulation (20 Hz, 0.8 ms duration, for 10 s at supramaximal voltage) with fast high-amplitude contractions of 0.9970.07 g tension (n ¼ 7). The effect of the antagonist was evaluated after a contact period of 10 min. Addition of prazosin (5 108 M) to the bath solution significantly inhibited the contractile response to electrical field stimulation to 0.10570.016 g (n ¼ 7), with corresponding inhibition amounting to 89.1671.41%. Cumulative addition of sildenafil (1 109–1 106 M) to the bath solution produced concentration-dependent inhibition of the electrical field stimulation-induced contractions with EC50 7.4070.51 nM. Cumulative addition of phentolamine (1 109–1 106 M) inhibited the electrical field stimulation-induced contraction concentrationdependently with EC50 value 8.2271.35 nM (Figure 4).
Effect of sildenafil on phenylephrine-induced contractions in the rat aortic rings Addition of phenylephrine (3 106 M) to the bath solution resulted in contraction of rat aortic rings showing a tension of 0.9770.034 g (n ¼ 9). International Journal of Impotence Research
Involvement of a-receptors and potassium channels MA El-Metwally et al 554
Sildenafil 1x10-9 M
0.5 gm 1 min
Phentolamine 1x10-9 M
Sildenafil 1x10-9 M
1x10-6 M 0.5 gm 1 min
Phentolamine 1x10-9 M
Figure 2 Representative tracing showing the effect of sildenafil (1 109–1 106 M) and phentolamine (1 109–1 106 M) on electrical field stimulation (20 Hz, 0.8 ms duration, supramaximal voltage)-induced contractions in the rabbit corpus cavernosum (I, upper panel) and rat anoccocygeus muscle (II, lower panel).
Sildenafil Phentolamine *
Phentolamine * * 60
80 % Inhibition of contraction
% Inhibition of contraction
Concentration (M) Figure 3 Effect of sildenafil (1 109–1 106 M) and phentolamine (1 109–1 106 M) on electrical field stimulation-induced contractions in the rabbit corpus cavernosum. Contractions were elicited by electrical field stimulation at frequency 20 Hz. Responses are expressed as mean7s.e.m. (n ¼ 7). *Significant difference from the control group at Po0.05.
International Journal of Impotence Research
1x10-8 1x10-7 Concentration (M)
Figure 4 Effect of sildenafil (1 109–1 106 M) and phentolamine (1 109–1 106 M) on electrical field stimulation-induced contractions in the rat anococcygeus muscle. Contractions were performed by electrical field stimulation at frequency 20 Hz. Responses are expressed as mean7s.e.m. (n ¼ 7). *Significant difference from the control group at Po0.05.
Involvement of a-receptors and potassium channels MA El-Metwally et al 555
After sildenafil * % Relaxation
Tension in g
60 * 40 * 20
Figure 5 Effect of sildenafil (1 10 M) on the phenylephrine (3 106 M)-induced contraction of rat aortic rings. Responses are expressed as mean7s.e.m. (n ¼ 5). *Significant difference from control at Po0.05. 7
Incubation of aortic rings with sildenafil (1 10 M) for 10 min caused significant inhibition of phenylephrine-induced contraction to 0.58470.06 g (n ¼ 5) with a corresponding inhibition of 39.8373.01% (Figure 5). Relaxant effect of sildenafil on KCl-induced tone in the rabbit corpus cavernosum A submaximal concentration of KCl (2 102 M) was used to contract rabbit corpus cavernosum strips. Cumulative concentrations of sildenafil (1 109–1 106 M) reduced the KCl-induced tension significantly to a maximum of 60.9779.77% (n ¼ 6; Figure 6). Effect of tetraethylammonium on sildenafil-induced relaxation of rabbit corpus cavernosum Concentration-response curve of a potassium channel opener was constructed by cumulative addition of diazoxide (5 106–1 104 M) to PE-precontracted rabbit corpus cavernosum strips. Significant smooth muscle relaxation was observed at all concentrations reaching the maximum response amounting to 90.9373.76% (n ¼ 5). Incubation of rabbit corpus cavernosum strips with tetraethylammonium (1 103 M) for 10 min shifted the dose– response curve of diazoxide to the right indicating an efficient blockade of potassium channels. The same concentration of tetraethylammonium was able to abolish the muscle relaxant effect of 1 109 M sildenafil and significantly attenuated the effect of all other concentrations (Figure 7).
Discussion In the present study, sildenafil induced muscle relaxation of precontracted rabbit corpus caverno-
1x10-8 1x10-7 Concentration (M)
Figure 6 Effect of sildenafil (1 109–1 106 M) on potassium chloride (2 102 M)-precontracted strips of rabbit corpus cavernosum. Responses are expressed as mean7s.e.m. (n ¼ 6). * Significant difference from control at Po0.05.
sum strips. This effect was insensitive to NO synthase inhibitor NG-nitro-L-arginine activity that failed to block or antagonize sildenafil-induced relaxation significantly. This raised the possibility that mechanisms other than modulation of NO pathway may be involved in sildenafil-induced action. Based on this observation, the action of sildenafil on a-receptors was tested. The electrical field stimulation-induced contractions protocol was designed and employed to test the efficacy of sildenafil in inhibiting the neuronal signals responsible for maintaining the adrenergically induced tone of cavernosal smooth muscle. This protocol was previously used by Fovaeus et al.16 The results were then compared to that of phentolamine. Electrical field stimulation elicited a contractile response that was almost completely abolished by the a 1adrenoceptor blocker prazosin. These findings support the view that the contraction of this tissue, which is necessary for keeping the penis in a flaccid state, is produced mainly by a-receptor stimulation. This is consistent with the results of previous reports.2,16–18 Saenz De Tejada18 stated that the blockade of contractions induced by electrical field stimulation by prazosin but not by equimolar concentrations of rauwolscine, an a 2-adrenoceptor blocker, confirmed that the contraction of human corpus cavernosum smooth muscle induced by adrenergic nerve stimulation is elicited via postjunctional a 1-adrenoceptors. In the present study, a similar mechanism was found to exist in the rabbit corpora. International Journal of Impotence Research
Involvement of a-receptors and potassium channels MA El-Metwally et al 556
40 * * * 0
5x10-6 1x10-5 5x10-5 1x10-4 Concentration of diazoxide (M)
60 40 * 20 0
1x10-9 1x10-8 1x10-7 1x10-6 Concentration of sildenafil (M)
Figure 7 Effect of tetraethylammonium (1 103 M) on diazoxide (5 106–1 104 M)-induced relaxation and sildenafil (1 109– 1 106 M)-induced relaxation of the rabbit corpus cavernosum. Tone was elevated by 3 106 M phenylephrine. Responses are expressed as mean7s.e.m. (n ¼ 5). *Significant difference from control at Po0.05.
Both sildenafil and phentolamine were able to significantly attenuate electrical field stimulationinduced contractions of rabbit corpus cavernosum dose dependently, with respective EC50 values of 0.07470.006 and 0.1170.003 mM, respectively. This action of phentolamine has been reported previously,16,19 and is logically explained by its a 1-adrenoceptor-blocking effect. Consequently, the finding that sildenafil also attenuated endogenous norepinephrine-mediated contractions, in a potency close to that of phentolamine, raised the possibility that it may contribute to rabbit corpus cavernosum relaxation by a mechanism that involves a-adrenoceptor blockade. In a similar study examining the effect of phentolamine and sildenafil on isolated rabbit corpus cavernosum, phentolamine effectively counteracted a1-adrenoceptor-mediated contractions. Although the relaxations induced by phentolamine were independent of nitric oxide, those induced by sildenafil were partly sensitive to L-NAME (100 mM).19 Indeed, in the current study, NG-nitro-Larginine (30 mM) slightly reduced sildenafil-induced relaxation, but no significant levels were attained. This could be explained by the use of the more potent NO synthase inhibitor. For confirmation, a similar protocol has been conducted on the rat anococcygeus muscle. The preparation responded to electrical field stimulation with fast high-amplitude contractions that were rapidly relaxed to the prestimulation level at its cessation, in accordance with previous results.20 Sildenafil and phentolamine attenuated these contractions with almost similar potencies, which raised the possibility of a shared mechanism of action. In addition, sildenafil at a dose of 1 107 M was able to reduce the phenylephrine-induced contractions of the rat aortic rings by about 40%. In a similar experiment, Fovaeus et al.16 showed that verapamil, a calcium channel blocker, attenuated the electrically induced contraction in the rabbit International Journal of Impotence Research
corpus cavernosum and the norepinephrine-induced contraction. They concluded that verapamil at high doses may possess a-adrenoceptor-blocking effects, in supporting the conclusions of previous reports.21,22 A combination of these findings showed that sildenafil is capable of blocking endogenous norepinephrine- and exogenous phenylephrine-induced smooth muscle contractions. It is thus capable of producing cavernosal smooth muscle relaxation by a mechanism involving a-receptor blockade. The relaxant effect of sildenafil on phenylephrine precontracted rabbit corpus cavernosum strips was not blocked significantly in the presence of NG- nitro-L-arginine. In rabbit corpus cavernosum precontracted with KCl, sildenafil caused concentration-dependent relaxation. Based on the two above-mentioned observations, and on the previous report that sildenafil-induced relaxations were partly sensitive to L-NAME,19 an additional nonadrenergic, NO-independent mechanism for sildenafil-induced corpus cavernosum relaxation was expected. Therefore, the modulation of potassium channels as a possible additional mechanism of the relaxing action of sildenafil was investigated. Potassium channel activation is followed by a reduced opening of voltage-dependent calcium channels and a reduced tension.23,24 The in vitro study of Holmiquist et al.25 showed that potassium channel opening offers a principle for producing relaxation of erectile tissue. They showed that potassium channel openers, such as pinacidil and cromakalim, were able to relax rabbit corpus cavernosum. The relaxant effect of sildenafil on the phenylephrine-precontracted corpus cavernosum strips resistant to NO synthase inhibitor was attenuated by tetraethylammonium, a nonspecific potassium channel blocker,26 in the same dose proven effective in attenuating diazoxide relaxation. Diazoxide is a known opener of ATP-sensitive potassium channels, which has been tested in different tissues of various species.27
Involvement of a-receptors and potassium channels MA El-Metwally et al
The above results were consolidated by studies of Medina et al.,28 showing that sildenafil-induced inhibition of the electrically evoked contractions in the human vas deferens was not modified by the inhibitor of guanylate cyclase 1H-[[1,2,4]oxadiazolin] quinoxaline-1-one but was abolished by tetraethylammonium. They concluded that the action of sildenafil is not related to the accumulation of cyclic guanosine monophosphate but is probably due to activation of prejunctional large-conductance potassium-activated channels. The fact that sildenafil may activate potassium channels has also been confirmed in vitro using penile resistance arteries and in vivo.29,30 In conclusion, sildenafil is capable of producing cavernosal smooth muscle relaxation by a mechanism involving a-receptors. In addition, potassium channel opening may be a possible mechanism contributing to sildenafil muscle relaxant effect on rabbit corpus cavernosum.
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