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Ca2+-activated K+ (KCa) channels are involved in the relaxations elicited by sildenafil in penile resistance arteries
European Journal of Pharmacology 531 (2006) 232 – 237 www.elsevier.com/locate/ejphar
Ca 2+ -activated K + (KCa) channels are involved in the relaxations elicited by sildenafil in penile resistance arteries Dolores Prieto ⁎, Luis Rivera, Sara Benedito, Paz Recio, Nuria Villalba, Medardo Hernández, Albino García-Sacristán Departamento de Fisiología, Facultad de Farmacia, Universidad Complutense de Madrid, 28040-Madrid, Spain Received 28 July 2005; received in revised form 21 November 2005; accepted 19 December 2005 Available online 27 January 2006
Abstract The aim of the present study was to evaluate the role of K+ channels in the vasorelaxant effect of the phosphodiesterase 5 inhibitor, sildenafil, in isolated horse penile resistance arteries mounted in microvascular myographs. In phenylephrine-precontracted arteries, sildenafil elicited potent relaxations which were markedly reduced by raising extracellular K+, by the non-selective blocker of Ca2+-activated K+ channels (KCa), tetraethylammonium and by the blocker of large- and intermediate-conductance KCa channels, charybdotoxin. Sildenafil relaxant responses were also reduced by the selective inhibitor of large conductance KCa (BKCa) channels iberiotoxin, but not by the blocker of small conductance KCa channels apamin. The inhibitor of the cGMP-dependent protein kinase (PKG), Rp-8-Br-PET-cGMPS, reduced the relaxations elicited by sildenafil but combined treatment with iberiotoxin and Rp-8-Br-PET-cGMPS did not further inhibit these relaxations, compared to the effect of either blocker alone. Iberiotoxin also shifted to the right the relaxations elicited by both the NO donor, S-nitrosoacetyl-D,L-penicillamine (SNAP) and the adenylate cyclase activator forskolin; treatment with both iberiotoxin and Rp-8-Br-PET-cGMPS did cause an additional inhibition. The present results demonstrate that the relaxant effect of sildenafil and NO in penile resistance arteries is due in part to activation of BKCa channels through a PKG-dependent mechanism. © 2006 Elsevier B.V. All rights reserved. Keywords: Sildenafil; K+ channel; Relaxation; Nitric oxide; Protein kinase G; Penile resistance artery
1. Introduction Stimulation of K+ channels is a powerful mechanism of smooth muscle relaxation and both cyclic guanosine 3′,5′monophosphate (cGMP) and cyclic adenosine 3′,5′-monophosphate (cAMP) induce vasodilatation at least in part by modulation of K+ channel activity (Lincoln et al., 2001; Schubert and Nelson, 2001; Schlossmann et al., 2003). Thus, large conductance Ca2+-activated K+ (BKCa) channels are activated by intracellular Ca 2+ and also by membrane depolarization and are particularly abundant in vascular smooth muscle cells (Nelson and Quayle, 1995; Schubert and Nelson, 2001). Physiological activation of BKCa is an important buffering mechanism to counteract active tension induced by vasoconstrictors and also to maintain basal levels of tone in ⁎ Corresponding author. Tel.: +34 913947193; fax: +34 913947184. E-mail address: [email protected] (D. Prieto). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.033
arterial smooth muscle, since they respond to increases in intracellular Ca2+ by attenuating transmembrane Ca2+ influx via repolarization-induced closure of L-type voltage-dependent Ca2+ channels (Nelson and Quayle, 1995; Schubert and Nelson, 2001). KCa channels play a key role in the regulation of corporal and arterial smooth muscle from the penis (Christ, 2000). Under basal conditions there is enough KCa channel activity to regulate membrane potential (Nelson and Quayle, 1995) and therefore these channels are involved in the maintenance of the resting tone of both corpus cavernosum and penile resistance arteries (Prieto et al., 1998; Simonsen et al., 2002; Spektor et al., 2002). Moreover, KCa channels are downstream mediators of the nitric oxide (NO)/cGMP signalling cascade involved in the relaxation of penile erectile tissue; NO is released from nerves and endothelium upon sexual stimulation and relaxes tonically constricted helicine arteries and corporal smooth muscle to induce penile erection (Andersson and Wagner, 1995; Simonsen
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et al., 1995, 1997, 2002). In penile small arteries, relaxations elicited by both endothelial- and neural-released NO involve activation of charybdotoxin-sensitive KCa channels through a cGMP-dependent mechanism (Prieto et al., 1998; Simonsen et al., 1995, 2002). Furthermore, both apamin- and charybdotoxinsensitive KCa channels are involved in the release and/or actions of the endothelium-derived hyperpolarizing factor (EDHF) of penile resistance arteries (Prieto et al., 1998; Simonsen et al., 1995, 2002). By selectively inhibiting the cGMP-specific phosphodiesterase (PDE) or PDE5, sildenafil citrate compensates for the reduced NO release, cGMP production and impaired penile perfusion in male erectile dysfunction (Goldstein et al., 1998; Moreland et al., 2001). Sildenafil enhances the relaxation and cGMP accumulation elicited by both exogenous and neuralreleased NO in corpus cavernosum (Ballard et al., 1998; Chuang et al., 1998) and is a powerful direct vasodilator of penile arteries (Medina et al., 2000; Simonsen et al., 2001; Prieto et al., in press). Electrophysiological studies have shown that NO increases the activity of KCa channels through a cGMPdependent mechanism in myocytes from human corpus cavernosum and that sildenafil enhances the NO-induced activation of KCa channels (Lee and Kang, 2001). However, no information is available about the role of these channels in the vasodilator action of sildenafil in penile arteries. Therefore, the aim of the present study was to further investigate the mechanisms underlying the direct relaxant effect of sildenafil in isolated penile resistance arteries in relation to the involvement of KCa channels. 2. Methods 2.1. Dissection and mounting Penises from young healthy horses were obtained once a week at the local slaughterhouse immediately after death and placed in cold physiological saline solution (PSS) of the following composition (mmol/l): NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, CaCl2 1.5, ethylenediaminetetraacetic acid (EDTA) 0.027 and glucose 11. Throughout the subsequent dissection the penis was bathed in cold PSS (4 °C) gassed with 5% CO2 / 95% O2 to maintain pH at 7.4. The horse was used as an experimental animal due to the close pharmacological reactivity between equine and human penile resistance arteries (Simonsen et al., 1997, 2002). Penile resistance arteries, which are second- or third-order branches of the deep penile artery, with a normalized lumen diameter of 200–600 μm in the horse, were dissected by carefully removing the adhering trabecular tissue (Prieto et al., 1998). Arterial segments (ca 2 mm long) were mounted as ring preparations on two 40 μm wires in double vascular myographs for isometric tension recording. Arteries were allowed to equilibrate in PSS, 37 °C for about 30 min. The relation between the resting wall tension and the internal circumference was determined for each artery, and from this, the internal circumference L100, corresponding to a transmural pressure of 100 mm Hg for a relaxed vessel in situ was calculated. The arteries were set to the internal circumfer-
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ence L1, given by L1 = 0.9 × L100, since preliminary experiments showed that force development is close to maximal at this internal circumference (Simonsen et al., 1997, 2002). 2.2. Experimental procedure The contractile ability of the vessels was tested at the beginning of each experiment by stimulating them twice with KPSS (equivalent to PSS but NaCl exchanged with KCl on equimolar basis giving a final concentration of 123.7 mM K+). In order to investigate an involvement of K+ channels in sildenafil relaxant responses, concentration–response curves for sildenafil and the NO donor S-nitrosoacetyl-D,L-penicillamine (SNAP) were constructed on arteries precontracted with either phenylephrine or a 25 mM K+ solution by about 40–60% of the KPSS contractile response. The role of KCa channels in the sildenafil-elicited relaxations was evaluated by incubating the arteries with either 1 mM tetraethylammonium (TEA), 30 nM charybdotoxin, 30 nM iberiotoxin or 0.3 μM apamin. In addition the effects of iberiotoxin on sildenafil, SNAP and forskolin responses were also tested in combination with the PKG blocker Rp-8-Br-PET-cGMPS (3 μM). For each treated artery, a control concentration–response-curve was run in parallel. 2.3. Drugs Apamin, charybdotoxin, forskolin, iberiotoxin, phenylephrine hydrochloride, S-nitrosoacetyl-D,L-penicillamine (SNAP) and tetraethylammonium (TEA), were purchased from Sigma (Spain) and β-Phenyl-1, N2-etheno-8-bromoguanosine-3′,5′cyclic mnophosphorothioate, Rp-isomer (Rp-8-Br-PET-cGMPS) from Biolog Life Science Institute (Bremen, Germany). Sildenafil citrate was synthesized by Pfizer (Sandwich, Bristol, Kent, UK). 2.4. Statistical analysis Mechanical responses of the arteries were measured as force and expressed as active wall tension, ΔT, which is the increase in force, ΔF, divided by twice the segment length. Relaxant responses are given as a percentage of the precontraction induced by phenylephrine. Sensitivity to the agonists is expressed in terms of pEC50 = − log(EC50), EC50 being the concentration of the agonist required to give half-maximal relaxation. Results are expressed as means ± S.E.M. and n represents the number of arteries. Statistical differences between means were analyzed by a two-tailed unpaired Student's t test. Means of multiple groups were compared by one-way analysis of variance (ANOVA) followed by a Bonferroni as a posterio test. Probability levels less than 5% were considered significant. 3. Results In order to investigate the role of K+ channels in sildenafilinduced relaxations, the effects of raising extracellular K+ and selective KCa channel blockers were evaluated. In arteries precontracted with 25 mM K+ by 63 ± 5% (n = 6) of the KPSS-
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Fig. 1. Effects of (A) raising extracellular K+ and (B) the nonselective blocker of K+ channels tetraethylammonium (TEA) (1 mM) on the average concentration– relaxation response curves to sildenafil of penile small arteries. Results (mean ± S.E.M. of 6–8 arteries) are expressed as a percentage of the contraction elicited by either 25 mM K+ or phenylephrine (Phe).
responses (Fig. 1B; Table 1). The inhibitor of large-(BKCa) and intermediate-(IKCa) conductance KCa channels charybdotoxin (30 nM) and the selective inhibitor of BKCa channels iberiotoxin (30 nM), both caused similar rightwards displacements of the sildenafil concentration–response curves (Figs. 2, 3A, B; Table 1), whereas the selective blocker of small conductance KCa channels apamin (0.3 μM) did not alter sildenafil-induced relaxations (Fig. 3C, Table 1). The inhibitor of the cGMP-dependent protein kinase (PKG), Rp-8-Br-PET-cGMPS (3 μM), significantly reduced sildenafil relaxant responses, pEC50 and maximal relaxations being 7.51 ± 0.06 and 96 ± 3% (n = 5) and 7.12 ± 0.05 (n = 5, P b 0.001, ANOVA) and 70 ± 8% (n = 5, P b 0.05, ANOVA) in control and treated arteries, respectively. Combined treatment with iberiotoxin and Rp-8-Br-PET-cGMPS did not further inhibited sildenafil-induced relaxations (Fig. 4), pEC50 and maximal relaxation being 7.14 ± 0.15 and 60 ± 4%, respectively (n = 5; P N 0.05 vs. Rp-8-Br-PET-cGMPS-treated arteries, ANOVA). Iberiotoxin also inhibited by 3-fold the relaxations induced by the NO donor SNAP, pEC50 values being 6.34 ± 0.11 and 5.86 ± 0.04 (P b 0.05, ANOVA, n = 5), in the absence and the presence of the blocker, respectively (Fig. 5A). Treatment with iberiotoxin and the PKG inhibitor Rp-8-Br-PET-cGMPS (3 μM) caused a larger inhibition of the SNAP-induced relaxations (Fig.
induced contraction, the relaxant responses of sildenafil were significantly reduced compared to those in arteries precontracted with phenylephrine by 46 ± 11% (n = 6) of the KPSSinduced responses (Fig. 1A; Table 1). Treatment with low concentrations of the non-selective blocker of KCa channels TEA (1 mM) also inhibited the sildenafil concentration– response curves, reducing both sensitivity and maximal Table 1 Effect of raising extracellular K+and KCa channel blockers on the relaxations elicited by sildenafil in penile small arteries Sildenafil
Control K+ (25 mM) Control 1 mM TEA Control 30 nM charybdotoxin Control 30 nM iberiotoxin Control 0.3 μM apamin
pEC50 (− logEC50)
Emax (%)
n
l1(μm)
7.76 ± 0.09 7.46 ± 0.09a 7.74 ± 0.09 6.95 ± 0.32a 7.85 ± 0.05 7.33 ± 0.06c 7.95 ± 0.10 7.42 ± 0.06b 7.82 ± 0.06 7.84 ± 0.12
95.7 ± 1.7 66.7 ± 9.4a 93.2 ± 2.8 74.9 ± 6.2a 93.5 ± 1.2 94.7 ± 0.9 85.0 ± 3.3 76.0 ± 4.8 92.8 ± 1.5 93.0 ± 1.3
6 6 7 7 5 5 4 4 4 4
547 ± 40 502 ± 48 483 ± 51 482 ± 58 350 ± 13 393 ± 11 423 ± 67 507 ± 88 461 ± 97 377 ± 91
Values represent mean ± S.E.M. of the number n of individual arteries. pEC50 is − logEC50, EC50 being the concentration of the agonist giving half maximal relaxation (Emax). Emax is the maximum relaxation expressed as a percentage of the contraction induced phenylephrine. l1 is the effective lumen diameter of penile resistance arteries at which experiments were performed, determined as l1 = L1 π− 1. Significant differences from controls were analyzed by an unpaired ttest. a P b 0.05; b P b 0.01; cP b 0.001. TEA: tetraethylammonium.
Fig. 2. Isometric force recording showing the relaxant effect of sildenafil in 2 penile resistance arteries of 346 μm (upper trace) and 368 μm (lower trace) in control conditions (upper trace) and after blockade of BKCa channels with 30 nM iberiotoxin. The artery was incubated 20 min with iberiotoxin before being precontracted with phenylephrine (Phe). Vertical bars show force in mN and horizontal bar shows time in min.
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Fig. 4. Effect of the BKCa channel blocker iberiotoxin (30 nM), alone or in combination with 3 μM of the PKG inhibitor Rp-8-Br-PET-cGMPS, on the average concentration–relaxation response curves to sildenafil in penile small arteries. Results (mean ± S.E.M. of 4–5 arteries) are expressed as a percentage of the contraction elicited by phenylephrine (Phe).
(P b 0.01 vs control arteries, ns vs iberiotoxin-arteries, ANOVA, n = 4), in control, iberiotoxin-treated and iberiotoxin plus Rp-8Br-PET-cGMPS-treated arteries, respectively). 4. Discussion We have recently reported that sildenafil potently relaxes penile resistance arteries by enhancing the effects of NO released from the endothelium (Prieto et al., in press) and thus
Fig. 3. Effects of blockers of (A) BKCa and IKCa channels, charybdotoxin (30 nM) (B) BKCa channels, iberiotoxin (30 nM) and (C) small-conductance KCa channels, apamin (0.3 μM) on the average concentration–relaxation response curves to sildenafil of penile small arteries. Results (mean ± S.E.M. of 4–6 arteries) are expressed as a percentage of the contraction elicited by phenylephrine (Phe).
5A), pEC50 and maximal responses being 5.39 ± 0.13 (n = 5, P b 0.05 vs iberiotoxin-treated arteries; P b 0.001 vs control, ANOVA) and 64 ± 7%, respectively (P N 0.05 vs iberiotoxintreated arteries; P b 0.01 vs control arteries, ANOVA, n = 5). However, the inhibition caused by the combined treatment of the arteries with iberiotoxin and Rp-8-Br-PET-cGMPS was not significantly different from that induced by the PKG blocker alone (pEC50 5.43 ± 0.05 y Emax 56 ± 5%, P N 0.05 ANOVA, vs Rp-8-Br-PET-cGMPS-treated arteries, n = 5) (Fig. 5A). The concentration–relaxation curves elicited by the adenylate cyclase activator forskolin in penile small arteries were also reduced by 2.2-fold by 30 nM iberiotoxin and this inhibition was not further enhanced by a combined treatment with iberiotoxin and Rp-8-Br-PET-cGMPS (Fig. 5B). Thus, pEC50 values for the forskolin-induced relaxations were 6.98 ± 0.09, 6.63 ± 0.09 (P b 0.05 vs control arteries, ANOVA, n = 4) and 6.59 ± 0.05 09
Fig. 5. Effect of the BKCa channel blocker iberiotoxin (30 nM), alone or in combination with 3 μM of the PKG inhibitor Rp-8-Br-PET-cGMPS, on the average concentration–relaxation response curves to (A) SNAP and (B) forskolin of penile small arteries. Results (mean ± S.E.M. of 4–5 arteries) are expressed as a percentage of the contraction elicited by phenylephrine (Phe).
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activating the NO-cGMP-PKG cascade, which is essential for the relaxation of arterial and corporal smooth muscle during penile erection (Andersson and Wagner, 1995; Simonsen et al., 1995, 2002; Prieto et al., 1998). The results of the present study demonstrate a selective involvement of BKCa channels in the sildenafil-evoked relaxations of penile resistance arteries, thus extending earlier investigations showing that KCa channels are involved in the relaxations elicited by both neural- (Simonsen et al., 1995) and endothelial- (Prieto et al., 1998) derived NO. By inhibiting the cGMP-specific PDE5, sildenafil increases the basal content and relaxant effects of cGMP in penile small arteries (Simonsen et al., 2001; Prieto et al., in press). Cyclic nucleotides relax arterial smooth muscle by activating specific cyclic nucleotide-dependent protein kinases which reduce either the intracellular Ca2+ concentration or the sensitivity of the contractile apparatus to Ca2+ in smooth muscle cells (Schlossmann et al., 2003). PKG can decrease cytosolic Ca2+ by enhancing the activity of BKCa channels and hyperpolarizing the membrane, which inhibits intracellular Ca2+ influx through Ltype voltage-gated channel (Lincoln et al., 2001). The present results show that the relaxant responses elicited by sildenafil, presumably mediated by endothelial-derived NO (Prieto et al., in press), involve activation of KCa channels in penile resistance arteries. Thus, decreasing the K+ gradient across the cell membrane by raising extracellular K+ had a pronounced inhibitory effect on sildenafil-induced relaxations, which suggests that sildenafil may hyperpolarize penile arterial smooth muscle by increasing K+ conductance. ATP-sensitive K+ (KATP) channels do not appear to be involved in the sildenafil relaxant responses, as shown by the lack of effect of the selective blocker of these channels, glibenclamide (Ruiz Rubio et al., 2004a). In the present study, the inhibition caused by a concentration of TEA selective for KCa channels and by the inhibitor of large- and intermediate conductance KCa channels charybdotoxin suggests an involvement of KCa channels in the sildenafil relaxant responses. These data are consistent with earlier studies demonstrating that NO causes relaxation of penile small arteries (Simonsen et al., 1995, 2002; Prieto et al., 1998) and human corporeal smooth muscle (Lee and Kang, 2001) through a cGMP-dependent activation of charybdotoxin-sensitive KCa channels. Moreover, our data would be in agreement with electrophysiological studies showing that sildenafil enhances the NO-induced activation of KCa channels in smooth muscle cells of human corpus cavernosum (Lee and Kang, 2001). In the present study, iberiotoxin antagonized the relaxations elicited by sildenafil while the specific blocker of smallconductance KCa channels, apamin, had no effect, thus indicating that sildenafil selectively activates BKCa channels in penile small arteries. Activation of smooth muscle BKCa channels is an essential mechanism for the NO-mediated regulation of arterial tone in both large and small arteries (Sausbier et al., 2000; Archer, 2002). Accordingly, relaxant responses elicited by the NO donor SNAP were also inhibited by iberiotoxin thus indicating that NO stimulates BKCa channels in penile small arteries and reinforcing the idea of an involvement of NO in sildenafil responses (Prieto et al., in press). The present results are in agreement with a recent report
in rat clitoris showing that the relaxations elicited by both NO donors and sildenafil are inhibited by iberiotoxin and involve activation of BKCa channels (Gragasin et al., 2004). Thus, BKCa channels are ubiquitous in vascular smooth muscle cells and their regulation by NO and cGMP is a common vasodilator mechanism in several arterial beds (Sausbier et al., 2000; Schubert and Nelson, 2001; Archer, 2002) which can be extended to both male and female erectile tissue. Both PKA and PKG are potentially important direct regulators of BKCa channel activity in smooth muscle (Schubert and Nelson, 2001). However, phosphorylation and activation of PKG seems to be the main molecular mechanism underlying the NO-mediated regulation of BKCa channels in arterial smooth muscle (Robertson et al., 1993; Sausbier et al., 2000; Schubert and Nelson, 2001; Archer, 2002). This is firmly supported by the fact that NO does not activate BKCa currents in knockout mice lacking the cGMP kinase (Sausbier et al., 2000). Sildenafil-induced relaxations in penile small arteries are iberiotoxin-sensitive and are inhibited by blockers of both PKG and PKA (Prieto et al., in press), which suggests that either kinase may modulate BKCa channel activity. However, the present results show that the cGMP-mediated relaxations elicited by sildenafil and by the NO donor SNAP were inhibited by both iberiotoxin and Rp-8-CPT-cGMPS, but combined blockade of BK Ca channels and PKG did not further antagonized these relaxant responses. These results therefore suggest that cGMP modulates BKCa channel activity through activation of PKG in penile small arteries and support the importance of the NO/cGMP/PKG pathway in erectile tissues; thus, Hedlund et al. (2000) have shown that PKG knockout mice have erectile dysfunction and impaired reproduction which is not compensated by the cAMP pathway. Although PKG is essential in the NO-elicited relaxation of corpus cavernosum (Hedlund et al., 2000), increasing evidence suggests a cross-talk between cGMP and cAMP signalling pathways in the erectile tissues of the penis (Kim et al., 2000; Stief et al., 2000; Ruiz Rubio et al., 2004b). Thus, we have recently demonstrated that cAMP-elevating agents may cross-activate PKG and that the relaxations elicited by PGE1 and forskolin are inhibited by PKG blockers (Ruiz Rubio et al., 2004b). The inhibition caused by iberiotoxin on forskolin-elicited relaxations of penile resistance arteries extend earlier results showing the involvement of KCa channels in the cAMP-mediated relaxant responses (Ruiz Rubio et al., 2004a) and suggest that cAMPelevating agents may also modulate BKCa channel activity. Since forskolin-evoked vasodilation was not further antagonized by combined treatment with blockers of BKCa channels and PKG compared to either blocker alone, the present results indicate that in addition to cGMP, cAMP may also modulate BKCa channel activity through activation of PKG in penile small arteries. These findings are consistent with biochemical and electrophysiological studies in coronary arteries showing that cAMP-dependent vasodilators increase PKG activity and open BKCa channels by cross-activation of PKG (Jiang et al., 1992; White et al., 2000; Schubert and Nelson, 2001). In conclusion, the present results demonstrate that the relaxations induced by sildenafil and the NO donor SNAP in
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penile small arteries in vitro are due in part to a PKG-mediated activation of BKCa channels. These data confirm the importance of KCa channels in the physiological regulation of the erectile tissue tone and, indirectly, in the treatment of erectile dysfunction. Thus, by increasing the effects of NO, sildenafil enhances the activity of BKCa channels in penile small arteries. In fact, these channels have been recently pointed out as molecular targets for the treatment of erectile dysfunction and intracavernous injection of cDNA for BKCa channels reverses both age(Melman et al., 2003) and diabetes- (Christ et al., 2004) induced erectile dysfunction in vivo. Furthermore, the present data showing that PKG acts downstream as a common mediator of the signalling transduction cascade of both cGMP- and cAMPinduced vasodilatations in penile small arteries support the essential role of PKG in penile erection (Hedlund et al., 2000). Acknowledgements This work was supported by grant n° SAF2002/02923 from the Spanish Ministry of Science and Technology. The authors wish to thank Hugh Tyrell-Gray for expert language editing and Manuel Perales and Francisco Puente for technical assistance. They also thank the Segovia Slaughterhouse for kindly donating horse penises. References Andersson, K.-E., Wagner, G., 1995. Physiology of penile erection. Physiol. Rev. 75, 191–236. Archer, S.L., 2002. Potassium channels and erectile dysfunction. Vascul. Pharmacol. 38, 61–71. Ballard, S.A., Gingell, C.J., Tang, K., Turner, L.A., Price, M.E., Naylor, A.M., 1998. Effects of sildenafil on the relaxation of human corpus cavernosum tissue in vitro and on the activities of cyclic nucleotide phosphodiesterase isozymes. J. Urol. 159, 2164–2171. Christ, G.J., 2000. Gap junctions and ion channels: relevance to erectile dysfunction. Int. J. Impot. Res. 12, S15–S25. Christ, G.J., Day, N., Santizo, C., Sato, Y., Zao, W., Sclafani, T., Bakal, R., Salman, M., Davies, K., Melman, A., 2004. Intracorporal injection of hSlo cDNA restores erectile capacity in STZ-diabetic F-344 rats in vivo. Am. J. Physiol. 87, H1544–H1553. Chuang, A.T., Strauss, J.D., Murphy, R.A., Steers, W.D., 1998. Sildenafil, a type-5 cGMP phosphodiesterase inhibitor, specifically amplifies endogenous cGMP-dependent relaxation in rabbit corpus cavernosum smooth muscle in vitro. J. Urol. 160, 257–261. Goldstein, I., Lue, T.F., Padma-Nathan, H., Rosen, R.C., Steers, W.D., Wicker, P.A., 1998. Oral sildenafil in the treatment of erectile dysfunction. Sildenafil Study Group. N. Engl. J. Med. 338, 1397–1404. Gragasin, F.S., Michealis, E.D., Hogan, A., Moudgil, R., Hashimoto, K., Wu, X., Bonnet, S., Haromy, A., Archer, S.L., 2004. The neurovascular mechanism of clitoral erection: nitric oxide and cGMP-stimulated activation of BKCa channels. FASEB J. 18, 1291–1382. Hedlund, P., Aszodi, A., Pfeifer, A., Alm, P., Hofmann, F., Ahmad, M., Fassler, R., Andersson, K.-E., 2000. Erectile dysfunction in cyclic GMP-dependent kinase I-deficient mice. PNAS 97, 2349–2354. Jiang, H., Colbran, J.L., Francis, S.H., Corbin, J.D., 1992. Direct evidence for cross-activation of cGMP-dependent protein kinase in pig coronary arteries. J. Biol. Chem. 267, 1015–1019. Kim, N.N., Huang, Y., Moreland, R.B., Kwak, S.S., Goldstein, I., Traish, A., 2000. Cross-regulation of intracellular cGMP and cAMP in cultured human corpum cavernosum smooth muscle cells. Mol. Cell Biol. Res. Commun. 4, 10–14.
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Lee, S.W., Kang, T.M., 2001. Effects of nitric oxide on the Ca2+-activated potassium channels in smooth muscle cells of the human corpus cavernosum. Urol. Res. 29, 359–365. Lincoln, M., Dey, N., Sellak, H., 2001. cGMP-dependent protein kinase signalling mechanisms in smooth mucle: from the regulation of tone to gene expression. J. Appl. Physiol. 91, 1421–1430. Medina, P., Segarra, G., Vila, J.M., Doménech, C., Martinez-Leon, J.B., Lluch, S., 2000. Effects of sildenafil on human penile blood vessels. Urology 56, 539–543. Melman, A., Zhao, W., Davies, K.P., Bakal, R., Christ, G.J., 2003. The successful long-term treatment of age related erectile dysfunction with hSlo cDNA in rats in vivo. J. Urol. 170, 285–290. Moreland, R.M., Hsieh, G., Nakane, M., Brioni, J.D., 2001. The biochemical and neurologic basis for the treatment of erectile dysfunction. J. Pharmacol. Exp. Ther. 296, 225–234. Nelson, M.T., Quayle, J.M., 1995. Physiological roles and properties of potassium channels in arterial smooth muscle. Am. J. Physiol. 268, C799–C822. Prieto, D., Simonsen, U., Hernández, M., García-Sacristán, A., 1998. Contribution of K+ channels and ouabain-sensitive mechanisms to the endothelium-dependent relaxations of horse penile small arteries. Br. J. Pharmacol. 123, 1609–1620. Prieto, D., Rivera, L., Recio, P., Hernández, M., García-Sacristán, A., in press. Role of nitric oxide in the relaxations elicited by sildenafil in penile resistance arteries. J. Urol. Robertson, B.E., Schubert, R., Hescheler, J., Nelson, M.T., 1993. cGMPdependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am. J. Physiol. 265, C299–C303. Ruiz Rubio, J.L., Hernández, M., Rivera de los Arcos, L., Benedito, S., Recio, P., García, P., García Sacristan, A., Prieto, D., 2004a. Role of ATP-sensitive K+ channels in the relaxations of penile resistance arteries. Urology 63, 800–805. Ruiz Rubio, J.L., Hernández, M., Rivera de los Arcos, L., Martínez, A.C., García Sacristán, A., Prieto, D., 2004b. Mechanisms of prostaglandin E1 induced relaxation in penile small arteries. J. Urol. 171, 968–973. Sausbier, M., Schubert, R., Voigt, V., Hirneiss, C., Pfeifer, A., Korth, M., Kleppisch, T., Ruth, P., Hofmann, F., 2000. Mechanisms of NO/cGMPdependent vasorelaxation. Circ. Res. 87, 825–830. Schlossmann, J., Fiel, R., Hofmann, F., 2003. Signalling through NO and cGMP-dependent protein kinases. Ann. Med. 35, 21–27. Schubert, R., Nelson, M.T., 2001. Protein kinases: tuners of the BKCa channel in smooth muscle. TIPS 22, 505–512. Simonsen, U., Prieto, D., Sáenz de Tejada, I., García-Sacristán, A., 1995. Involvement of nitric oxide in the non-adrenergic neurotransmission of horse deep penile arteries. Br. J. Pharmacol. 116, 2582–2590. Simonsen, U., Prieto, D., Delgado, J.A., Hernández, M., Resel, L., Sáenz de Tejada, I., García-Sacristán, A., 1997. Nitric oxide is involved in the inhibitory neurotransmission and endothelium-dependent relaxations of human small penile arteries. Clin. Sci. 92, 269–275. Simonsen, U., Contreras, J., García-Sacristán, A., Martínez, A.C., 2001. Effect of sildenafil in non-adrenergic non-cholinergic neurotransmission in bovine penile small arteries. Eur. J. Pharmacol. 412, 155–169. Simonsen, U., García-Sacristán, A., Prieto, D., 2002. Penile arteries and erection. J. Vasc. Res. 39, 283–303. Spektor, M., Rodriguez, R., Rosenbaum, R.S., Wang, H.Z., Melman, A., Christ, G.J., 2002. Potassium channels and human corporeal smooth muscle cell tone: further evidence of the physiological relevance of the maxi-K channel subtype to the regulation of human corporeal smooth muscle tone in vitro. J. Urol. 167, 2628–2635. Stief, C.G., Uckert, S., Becker, A.J., Harringer, W., Truss, M.C., Forssmann, W. G., Jonas, U., 2000. Effects of sildenafil on cAMP and cGMP levels in human cavernous and cardiac tissue. Urology 55, 146–150. White, R.E., Kryman, J.P., El-Mofawy, A.M., Han, G., Carrier, G.O., 2000. cAMP-dependent vasodilators cross-activate cGMP-dependent protein kinase to stimulate BKCa channel activity in coronary artery smooth muscle. Circ. Res. 86, 897–905.
Ca 2+ -activated K + (KCa) channels are involved in the relaxations elicited by sildenafil in penile resistance arteries Dolores Prieto ⁎, Luis Rivera, Sara Benedito, Paz Recio, Nuria Villalba, Medardo Hernández, Albino García-Sacristán Departamento de Fisiología, Facultad de Farmacia, Universidad Complutense de Madrid, 28040-Madrid, Spain Received 28 July 2005; received in revised form 21 November 2005; accepted 19 December 2005 Available online 27 January 2006
Abstract The aim of the present study was to evaluate the role of K+ channels in the vasorelaxant effect of the phosphodiesterase 5 inhibitor, sildenafil, in isolated horse penile resistance arteries mounted in microvascular myographs. In phenylephrine-precontracted arteries, sildenafil elicited potent relaxations which were markedly reduced by raising extracellular K+, by the non-selective blocker of Ca2+-activated K+ channels (KCa), tetraethylammonium and by the blocker of large- and intermediate-conductance KCa channels, charybdotoxin. Sildenafil relaxant responses were also reduced by the selective inhibitor of large conductance KCa (BKCa) channels iberiotoxin, but not by the blocker of small conductance KCa channels apamin. The inhibitor of the cGMP-dependent protein kinase (PKG), Rp-8-Br-PET-cGMPS, reduced the relaxations elicited by sildenafil but combined treatment with iberiotoxin and Rp-8-Br-PET-cGMPS did not further inhibit these relaxations, compared to the effect of either blocker alone. Iberiotoxin also shifted to the right the relaxations elicited by both the NO donor, S-nitrosoacetyl-D,L-penicillamine (SNAP) and the adenylate cyclase activator forskolin; treatment with both iberiotoxin and Rp-8-Br-PET-cGMPS did cause an additional inhibition. The present results demonstrate that the relaxant effect of sildenafil and NO in penile resistance arteries is due in part to activation of BKCa channels through a PKG-dependent mechanism. © 2006 Elsevier B.V. All rights reserved. Keywords: Sildenafil; K+ channel; Relaxation; Nitric oxide; Protein kinase G; Penile resistance artery
1. Introduction Stimulation of K+ channels is a powerful mechanism of smooth muscle relaxation and both cyclic guanosine 3′,5′monophosphate (cGMP) and cyclic adenosine 3′,5′-monophosphate (cAMP) induce vasodilatation at least in part by modulation of K+ channel activity (Lincoln et al., 2001; Schubert and Nelson, 2001; Schlossmann et al., 2003). Thus, large conductance Ca2+-activated K+ (BKCa) channels are activated by intracellular Ca 2+ and also by membrane depolarization and are particularly abundant in vascular smooth muscle cells (Nelson and Quayle, 1995; Schubert and Nelson, 2001). Physiological activation of BKCa is an important buffering mechanism to counteract active tension induced by vasoconstrictors and also to maintain basal levels of tone in ⁎ Corresponding author. Tel.: +34 913947193; fax: +34 913947184. E-mail address: [email protected] (D. Prieto). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.033
arterial smooth muscle, since they respond to increases in intracellular Ca2+ by attenuating transmembrane Ca2+ influx via repolarization-induced closure of L-type voltage-dependent Ca2+ channels (Nelson and Quayle, 1995; Schubert and Nelson, 2001). KCa channels play a key role in the regulation of corporal and arterial smooth muscle from the penis (Christ, 2000). Under basal conditions there is enough KCa channel activity to regulate membrane potential (Nelson and Quayle, 1995) and therefore these channels are involved in the maintenance of the resting tone of both corpus cavernosum and penile resistance arteries (Prieto et al., 1998; Simonsen et al., 2002; Spektor et al., 2002). Moreover, KCa channels are downstream mediators of the nitric oxide (NO)/cGMP signalling cascade involved in the relaxation of penile erectile tissue; NO is released from nerves and endothelium upon sexual stimulation and relaxes tonically constricted helicine arteries and corporal smooth muscle to induce penile erection (Andersson and Wagner, 1995; Simonsen
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et al., 1995, 1997, 2002). In penile small arteries, relaxations elicited by both endothelial- and neural-released NO involve activation of charybdotoxin-sensitive KCa channels through a cGMP-dependent mechanism (Prieto et al., 1998; Simonsen et al., 1995, 2002). Furthermore, both apamin- and charybdotoxinsensitive KCa channels are involved in the release and/or actions of the endothelium-derived hyperpolarizing factor (EDHF) of penile resistance arteries (Prieto et al., 1998; Simonsen et al., 1995, 2002). By selectively inhibiting the cGMP-specific phosphodiesterase (PDE) or PDE5, sildenafil citrate compensates for the reduced NO release, cGMP production and impaired penile perfusion in male erectile dysfunction (Goldstein et al., 1998; Moreland et al., 2001). Sildenafil enhances the relaxation and cGMP accumulation elicited by both exogenous and neuralreleased NO in corpus cavernosum (Ballard et al., 1998; Chuang et al., 1998) and is a powerful direct vasodilator of penile arteries (Medina et al., 2000; Simonsen et al., 2001; Prieto et al., in press). Electrophysiological studies have shown that NO increases the activity of KCa channels through a cGMPdependent mechanism in myocytes from human corpus cavernosum and that sildenafil enhances the NO-induced activation of KCa channels (Lee and Kang, 2001). However, no information is available about the role of these channels in the vasodilator action of sildenafil in penile arteries. Therefore, the aim of the present study was to further investigate the mechanisms underlying the direct relaxant effect of sildenafil in isolated penile resistance arteries in relation to the involvement of KCa channels. 2. Methods 2.1. Dissection and mounting Penises from young healthy horses were obtained once a week at the local slaughterhouse immediately after death and placed in cold physiological saline solution (PSS) of the following composition (mmol/l): NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, CaCl2 1.5, ethylenediaminetetraacetic acid (EDTA) 0.027 and glucose 11. Throughout the subsequent dissection the penis was bathed in cold PSS (4 °C) gassed with 5% CO2 / 95% O2 to maintain pH at 7.4. The horse was used as an experimental animal due to the close pharmacological reactivity between equine and human penile resistance arteries (Simonsen et al., 1997, 2002). Penile resistance arteries, which are second- or third-order branches of the deep penile artery, with a normalized lumen diameter of 200–600 μm in the horse, were dissected by carefully removing the adhering trabecular tissue (Prieto et al., 1998). Arterial segments (ca 2 mm long) were mounted as ring preparations on two 40 μm wires in double vascular myographs for isometric tension recording. Arteries were allowed to equilibrate in PSS, 37 °C for about 30 min. The relation between the resting wall tension and the internal circumference was determined for each artery, and from this, the internal circumference L100, corresponding to a transmural pressure of 100 mm Hg for a relaxed vessel in situ was calculated. The arteries were set to the internal circumfer-
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ence L1, given by L1 = 0.9 × L100, since preliminary experiments showed that force development is close to maximal at this internal circumference (Simonsen et al., 1997, 2002). 2.2. Experimental procedure The contractile ability of the vessels was tested at the beginning of each experiment by stimulating them twice with KPSS (equivalent to PSS but NaCl exchanged with KCl on equimolar basis giving a final concentration of 123.7 mM K+). In order to investigate an involvement of K+ channels in sildenafil relaxant responses, concentration–response curves for sildenafil and the NO donor S-nitrosoacetyl-D,L-penicillamine (SNAP) were constructed on arteries precontracted with either phenylephrine or a 25 mM K+ solution by about 40–60% of the KPSS contractile response. The role of KCa channels in the sildenafil-elicited relaxations was evaluated by incubating the arteries with either 1 mM tetraethylammonium (TEA), 30 nM charybdotoxin, 30 nM iberiotoxin or 0.3 μM apamin. In addition the effects of iberiotoxin on sildenafil, SNAP and forskolin responses were also tested in combination with the PKG blocker Rp-8-Br-PET-cGMPS (3 μM). For each treated artery, a control concentration–response-curve was run in parallel. 2.3. Drugs Apamin, charybdotoxin, forskolin, iberiotoxin, phenylephrine hydrochloride, S-nitrosoacetyl-D,L-penicillamine (SNAP) and tetraethylammonium (TEA), were purchased from Sigma (Spain) and β-Phenyl-1, N2-etheno-8-bromoguanosine-3′,5′cyclic mnophosphorothioate, Rp-isomer (Rp-8-Br-PET-cGMPS) from Biolog Life Science Institute (Bremen, Germany). Sildenafil citrate was synthesized by Pfizer (Sandwich, Bristol, Kent, UK). 2.4. Statistical analysis Mechanical responses of the arteries were measured as force and expressed as active wall tension, ΔT, which is the increase in force, ΔF, divided by twice the segment length. Relaxant responses are given as a percentage of the precontraction induced by phenylephrine. Sensitivity to the agonists is expressed in terms of pEC50 = − log(EC50), EC50 being the concentration of the agonist required to give half-maximal relaxation. Results are expressed as means ± S.E.M. and n represents the number of arteries. Statistical differences between means were analyzed by a two-tailed unpaired Student's t test. Means of multiple groups were compared by one-way analysis of variance (ANOVA) followed by a Bonferroni as a posterio test. Probability levels less than 5% were considered significant. 3. Results In order to investigate the role of K+ channels in sildenafilinduced relaxations, the effects of raising extracellular K+ and selective KCa channel blockers were evaluated. In arteries precontracted with 25 mM K+ by 63 ± 5% (n = 6) of the KPSS-
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Fig. 1. Effects of (A) raising extracellular K+ and (B) the nonselective blocker of K+ channels tetraethylammonium (TEA) (1 mM) on the average concentration– relaxation response curves to sildenafil of penile small arteries. Results (mean ± S.E.M. of 6–8 arteries) are expressed as a percentage of the contraction elicited by either 25 mM K+ or phenylephrine (Phe).
responses (Fig. 1B; Table 1). The inhibitor of large-(BKCa) and intermediate-(IKCa) conductance KCa channels charybdotoxin (30 nM) and the selective inhibitor of BKCa channels iberiotoxin (30 nM), both caused similar rightwards displacements of the sildenafil concentration–response curves (Figs. 2, 3A, B; Table 1), whereas the selective blocker of small conductance KCa channels apamin (0.3 μM) did not alter sildenafil-induced relaxations (Fig. 3C, Table 1). The inhibitor of the cGMP-dependent protein kinase (PKG), Rp-8-Br-PET-cGMPS (3 μM), significantly reduced sildenafil relaxant responses, pEC50 and maximal relaxations being 7.51 ± 0.06 and 96 ± 3% (n = 5) and 7.12 ± 0.05 (n = 5, P b 0.001, ANOVA) and 70 ± 8% (n = 5, P b 0.05, ANOVA) in control and treated arteries, respectively. Combined treatment with iberiotoxin and Rp-8-Br-PET-cGMPS did not further inhibited sildenafil-induced relaxations (Fig. 4), pEC50 and maximal relaxation being 7.14 ± 0.15 and 60 ± 4%, respectively (n = 5; P N 0.05 vs. Rp-8-Br-PET-cGMPS-treated arteries, ANOVA). Iberiotoxin also inhibited by 3-fold the relaxations induced by the NO donor SNAP, pEC50 values being 6.34 ± 0.11 and 5.86 ± 0.04 (P b 0.05, ANOVA, n = 5), in the absence and the presence of the blocker, respectively (Fig. 5A). Treatment with iberiotoxin and the PKG inhibitor Rp-8-Br-PET-cGMPS (3 μM) caused a larger inhibition of the SNAP-induced relaxations (Fig.
induced contraction, the relaxant responses of sildenafil were significantly reduced compared to those in arteries precontracted with phenylephrine by 46 ± 11% (n = 6) of the KPSSinduced responses (Fig. 1A; Table 1). Treatment with low concentrations of the non-selective blocker of KCa channels TEA (1 mM) also inhibited the sildenafil concentration– response curves, reducing both sensitivity and maximal Table 1 Effect of raising extracellular K+and KCa channel blockers on the relaxations elicited by sildenafil in penile small arteries Sildenafil
Control K+ (25 mM) Control 1 mM TEA Control 30 nM charybdotoxin Control 30 nM iberiotoxin Control 0.3 μM apamin
pEC50 (− logEC50)
Emax (%)
n
l1(μm)
7.76 ± 0.09 7.46 ± 0.09a 7.74 ± 0.09 6.95 ± 0.32a 7.85 ± 0.05 7.33 ± 0.06c 7.95 ± 0.10 7.42 ± 0.06b 7.82 ± 0.06 7.84 ± 0.12
95.7 ± 1.7 66.7 ± 9.4a 93.2 ± 2.8 74.9 ± 6.2a 93.5 ± 1.2 94.7 ± 0.9 85.0 ± 3.3 76.0 ± 4.8 92.8 ± 1.5 93.0 ± 1.3
6 6 7 7 5 5 4 4 4 4
547 ± 40 502 ± 48 483 ± 51 482 ± 58 350 ± 13 393 ± 11 423 ± 67 507 ± 88 461 ± 97 377 ± 91
Values represent mean ± S.E.M. of the number n of individual arteries. pEC50 is − logEC50, EC50 being the concentration of the agonist giving half maximal relaxation (Emax). Emax is the maximum relaxation expressed as a percentage of the contraction induced phenylephrine. l1 is the effective lumen diameter of penile resistance arteries at which experiments were performed, determined as l1 = L1 π− 1. Significant differences from controls were analyzed by an unpaired ttest. a P b 0.05; b P b 0.01; cP b 0.001. TEA: tetraethylammonium.
Fig. 2. Isometric force recording showing the relaxant effect of sildenafil in 2 penile resistance arteries of 346 μm (upper trace) and 368 μm (lower trace) in control conditions (upper trace) and after blockade of BKCa channels with 30 nM iberiotoxin. The artery was incubated 20 min with iberiotoxin before being precontracted with phenylephrine (Phe). Vertical bars show force in mN and horizontal bar shows time in min.
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Fig. 4. Effect of the BKCa channel blocker iberiotoxin (30 nM), alone or in combination with 3 μM of the PKG inhibitor Rp-8-Br-PET-cGMPS, on the average concentration–relaxation response curves to sildenafil in penile small arteries. Results (mean ± S.E.M. of 4–5 arteries) are expressed as a percentage of the contraction elicited by phenylephrine (Phe).
(P b 0.01 vs control arteries, ns vs iberiotoxin-arteries, ANOVA, n = 4), in control, iberiotoxin-treated and iberiotoxin plus Rp-8Br-PET-cGMPS-treated arteries, respectively). 4. Discussion We have recently reported that sildenafil potently relaxes penile resistance arteries by enhancing the effects of NO released from the endothelium (Prieto et al., in press) and thus
Fig. 3. Effects of blockers of (A) BKCa and IKCa channels, charybdotoxin (30 nM) (B) BKCa channels, iberiotoxin (30 nM) and (C) small-conductance KCa channels, apamin (0.3 μM) on the average concentration–relaxation response curves to sildenafil of penile small arteries. Results (mean ± S.E.M. of 4–6 arteries) are expressed as a percentage of the contraction elicited by phenylephrine (Phe).
5A), pEC50 and maximal responses being 5.39 ± 0.13 (n = 5, P b 0.05 vs iberiotoxin-treated arteries; P b 0.001 vs control, ANOVA) and 64 ± 7%, respectively (P N 0.05 vs iberiotoxintreated arteries; P b 0.01 vs control arteries, ANOVA, n = 5). However, the inhibition caused by the combined treatment of the arteries with iberiotoxin and Rp-8-Br-PET-cGMPS was not significantly different from that induced by the PKG blocker alone (pEC50 5.43 ± 0.05 y Emax 56 ± 5%, P N 0.05 ANOVA, vs Rp-8-Br-PET-cGMPS-treated arteries, n = 5) (Fig. 5A). The concentration–relaxation curves elicited by the adenylate cyclase activator forskolin in penile small arteries were also reduced by 2.2-fold by 30 nM iberiotoxin and this inhibition was not further enhanced by a combined treatment with iberiotoxin and Rp-8-Br-PET-cGMPS (Fig. 5B). Thus, pEC50 values for the forskolin-induced relaxations were 6.98 ± 0.09, 6.63 ± 0.09 (P b 0.05 vs control arteries, ANOVA, n = 4) and 6.59 ± 0.05 09
Fig. 5. Effect of the BKCa channel blocker iberiotoxin (30 nM), alone or in combination with 3 μM of the PKG inhibitor Rp-8-Br-PET-cGMPS, on the average concentration–relaxation response curves to (A) SNAP and (B) forskolin of penile small arteries. Results (mean ± S.E.M. of 4–5 arteries) are expressed as a percentage of the contraction elicited by phenylephrine (Phe).
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activating the NO-cGMP-PKG cascade, which is essential for the relaxation of arterial and corporal smooth muscle during penile erection (Andersson and Wagner, 1995; Simonsen et al., 1995, 2002; Prieto et al., 1998). The results of the present study demonstrate a selective involvement of BKCa channels in the sildenafil-evoked relaxations of penile resistance arteries, thus extending earlier investigations showing that KCa channels are involved in the relaxations elicited by both neural- (Simonsen et al., 1995) and endothelial- (Prieto et al., 1998) derived NO. By inhibiting the cGMP-specific PDE5, sildenafil increases the basal content and relaxant effects of cGMP in penile small arteries (Simonsen et al., 2001; Prieto et al., in press). Cyclic nucleotides relax arterial smooth muscle by activating specific cyclic nucleotide-dependent protein kinases which reduce either the intracellular Ca2+ concentration or the sensitivity of the contractile apparatus to Ca2+ in smooth muscle cells (Schlossmann et al., 2003). PKG can decrease cytosolic Ca2+ by enhancing the activity of BKCa channels and hyperpolarizing the membrane, which inhibits intracellular Ca2+ influx through Ltype voltage-gated channel (Lincoln et al., 2001). The present results show that the relaxant responses elicited by sildenafil, presumably mediated by endothelial-derived NO (Prieto et al., in press), involve activation of KCa channels in penile resistance arteries. Thus, decreasing the K+ gradient across the cell membrane by raising extracellular K+ had a pronounced inhibitory effect on sildenafil-induced relaxations, which suggests that sildenafil may hyperpolarize penile arterial smooth muscle by increasing K+ conductance. ATP-sensitive K+ (KATP) channels do not appear to be involved in the sildenafil relaxant responses, as shown by the lack of effect of the selective blocker of these channels, glibenclamide (Ruiz Rubio et al., 2004a). In the present study, the inhibition caused by a concentration of TEA selective for KCa channels and by the inhibitor of large- and intermediate conductance KCa channels charybdotoxin suggests an involvement of KCa channels in the sildenafil relaxant responses. These data are consistent with earlier studies demonstrating that NO causes relaxation of penile small arteries (Simonsen et al., 1995, 2002; Prieto et al., 1998) and human corporeal smooth muscle (Lee and Kang, 2001) through a cGMP-dependent activation of charybdotoxin-sensitive KCa channels. Moreover, our data would be in agreement with electrophysiological studies showing that sildenafil enhances the NO-induced activation of KCa channels in smooth muscle cells of human corpus cavernosum (Lee and Kang, 2001). In the present study, iberiotoxin antagonized the relaxations elicited by sildenafil while the specific blocker of smallconductance KCa channels, apamin, had no effect, thus indicating that sildenafil selectively activates BKCa channels in penile small arteries. Activation of smooth muscle BKCa channels is an essential mechanism for the NO-mediated regulation of arterial tone in both large and small arteries (Sausbier et al., 2000; Archer, 2002). Accordingly, relaxant responses elicited by the NO donor SNAP were also inhibited by iberiotoxin thus indicating that NO stimulates BKCa channels in penile small arteries and reinforcing the idea of an involvement of NO in sildenafil responses (Prieto et al., in press). The present results are in agreement with a recent report
in rat clitoris showing that the relaxations elicited by both NO donors and sildenafil are inhibited by iberiotoxin and involve activation of BKCa channels (Gragasin et al., 2004). Thus, BKCa channels are ubiquitous in vascular smooth muscle cells and their regulation by NO and cGMP is a common vasodilator mechanism in several arterial beds (Sausbier et al., 2000; Schubert and Nelson, 2001; Archer, 2002) which can be extended to both male and female erectile tissue. Both PKA and PKG are potentially important direct regulators of BKCa channel activity in smooth muscle (Schubert and Nelson, 2001). However, phosphorylation and activation of PKG seems to be the main molecular mechanism underlying the NO-mediated regulation of BKCa channels in arterial smooth muscle (Robertson et al., 1993; Sausbier et al., 2000; Schubert and Nelson, 2001; Archer, 2002). This is firmly supported by the fact that NO does not activate BKCa currents in knockout mice lacking the cGMP kinase (Sausbier et al., 2000). Sildenafil-induced relaxations in penile small arteries are iberiotoxin-sensitive and are inhibited by blockers of both PKG and PKA (Prieto et al., in press), which suggests that either kinase may modulate BKCa channel activity. However, the present results show that the cGMP-mediated relaxations elicited by sildenafil and by the NO donor SNAP were inhibited by both iberiotoxin and Rp-8-CPT-cGMPS, but combined blockade of BK Ca channels and PKG did not further antagonized these relaxant responses. These results therefore suggest that cGMP modulates BKCa channel activity through activation of PKG in penile small arteries and support the importance of the NO/cGMP/PKG pathway in erectile tissues; thus, Hedlund et al. (2000) have shown that PKG knockout mice have erectile dysfunction and impaired reproduction which is not compensated by the cAMP pathway. Although PKG is essential in the NO-elicited relaxation of corpus cavernosum (Hedlund et al., 2000), increasing evidence suggests a cross-talk between cGMP and cAMP signalling pathways in the erectile tissues of the penis (Kim et al., 2000; Stief et al., 2000; Ruiz Rubio et al., 2004b). Thus, we have recently demonstrated that cAMP-elevating agents may cross-activate PKG and that the relaxations elicited by PGE1 and forskolin are inhibited by PKG blockers (Ruiz Rubio et al., 2004b). The inhibition caused by iberiotoxin on forskolin-elicited relaxations of penile resistance arteries extend earlier results showing the involvement of KCa channels in the cAMP-mediated relaxant responses (Ruiz Rubio et al., 2004a) and suggest that cAMPelevating agents may also modulate BKCa channel activity. Since forskolin-evoked vasodilation was not further antagonized by combined treatment with blockers of BKCa channels and PKG compared to either blocker alone, the present results indicate that in addition to cGMP, cAMP may also modulate BKCa channel activity through activation of PKG in penile small arteries. These findings are consistent with biochemical and electrophysiological studies in coronary arteries showing that cAMP-dependent vasodilators increase PKG activity and open BKCa channels by cross-activation of PKG (Jiang et al., 1992; White et al., 2000; Schubert and Nelson, 2001). In conclusion, the present results demonstrate that the relaxations induced by sildenafil and the NO donor SNAP in
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penile small arteries in vitro are due in part to a PKG-mediated activation of BKCa channels. These data confirm the importance of KCa channels in the physiological regulation of the erectile tissue tone and, indirectly, in the treatment of erectile dysfunction. Thus, by increasing the effects of NO, sildenafil enhances the activity of BKCa channels in penile small arteries. In fact, these channels have been recently pointed out as molecular targets for the treatment of erectile dysfunction and intracavernous injection of cDNA for BKCa channels reverses both age(Melman et al., 2003) and diabetes- (Christ et al., 2004) induced erectile dysfunction in vivo. Furthermore, the present data showing that PKG acts downstream as a common mediator of the signalling transduction cascade of both cGMP- and cAMPinduced vasodilatations in penile small arteries support the essential role of PKG in penile erection (Hedlund et al., 2000). Acknowledgements This work was supported by grant n° SAF2002/02923 from the Spanish Ministry of Science and Technology. The authors wish to thank Hugh Tyrell-Gray for expert language editing and Manuel Perales and Francisco Puente for technical assistance. They also thank the Segovia Slaughterhouse for kindly donating horse penises. References Andersson, K.-E., Wagner, G., 1995. Physiology of penile erection. Physiol. Rev. 75, 191–236. Archer, S.L., 2002. Potassium channels and erectile dysfunction. Vascul. Pharmacol. 38, 61–71. Ballard, S.A., Gingell, C.J., Tang, K., Turner, L.A., Price, M.E., Naylor, A.M., 1998. Effects of sildenafil on the relaxation of human corpus cavernosum tissue in vitro and on the activities of cyclic nucleotide phosphodiesterase isozymes. J. Urol. 159, 2164–2171. Christ, G.J., 2000. Gap junctions and ion channels: relevance to erectile dysfunction. Int. J. Impot. Res. 12, S15–S25. Christ, G.J., Day, N., Santizo, C., Sato, Y., Zao, W., Sclafani, T., Bakal, R., Salman, M., Davies, K., Melman, A., 2004. Intracorporal injection of hSlo cDNA restores erectile capacity in STZ-diabetic F-344 rats in vivo. Am. J. Physiol. 87, H1544–H1553. Chuang, A.T., Strauss, J.D., Murphy, R.A., Steers, W.D., 1998. Sildenafil, a type-5 cGMP phosphodiesterase inhibitor, specifically amplifies endogenous cGMP-dependent relaxation in rabbit corpus cavernosum smooth muscle in vitro. J. Urol. 160, 257–261. Goldstein, I., Lue, T.F., Padma-Nathan, H., Rosen, R.C., Steers, W.D., Wicker, P.A., 1998. Oral sildenafil in the treatment of erectile dysfunction. Sildenafil Study Group. N. Engl. J. Med. 338, 1397–1404. Gragasin, F.S., Michealis, E.D., Hogan, A., Moudgil, R., Hashimoto, K., Wu, X., Bonnet, S., Haromy, A., Archer, S.L., 2004. The neurovascular mechanism of clitoral erection: nitric oxide and cGMP-stimulated activation of BKCa channels. FASEB J. 18, 1291–1382. Hedlund, P., Aszodi, A., Pfeifer, A., Alm, P., Hofmann, F., Ahmad, M., Fassler, R., Andersson, K.-E., 2000. Erectile dysfunction in cyclic GMP-dependent kinase I-deficient mice. PNAS 97, 2349–2354. Jiang, H., Colbran, J.L., Francis, S.H., Corbin, J.D., 1992. Direct evidence for cross-activation of cGMP-dependent protein kinase in pig coronary arteries. J. Biol. Chem. 267, 1015–1019. Kim, N.N., Huang, Y., Moreland, R.B., Kwak, S.S., Goldstein, I., Traish, A., 2000. Cross-regulation of intracellular cGMP and cAMP in cultured human corpum cavernosum smooth muscle cells. Mol. Cell Biol. Res. Commun. 4, 10–14.
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