International Journal of Impotence Research (2009) 21, 356–361; doi:10.1038/ijir.2009.41; published online 17 September 2009
Yohimbine relaxes the human corpus cavernosum through a non-adrenergic mechanism involving the activation of K+ATP-dependent channels
Correspondence: Professor FC Freitas, Surgery Division Urology, Ceara Federal University, Rua Rua Henriqueta Galeno n° 1000, ap 2103, Fortaleza, Ceara 60.135-420, Brazil. E-mail: [email protected]
Received 16 March 2009; Revised 6 August 2009; Accepted 6 August 2009; Published online 17 September 2009.
The mechanism by which yohimbine relaxes the human corpus cavernosum remains unclear. Using the human corpus cavernosum strips immersed in isometric baths containing Krebs–Henseleit solution, this study investigates the effect of yohimbine on the relaxation of the human corpus cavernosum through nitrergic pathways involving the activation of ATP-dependent potassium channels (KATP). The maximal relaxation induced by yohimbine in the human corpus cavernosum strips pre-contracted with phenylephrine was 100±0% and only 30.5±5.0% when they were pre-contracted with 60-mm potassium (K+) solution. The maximal relaxation induced by yohimbine in phenylephrine pre-contracted tissues was significantly inhibited by tetrodotoxin, 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 7-nitroindazole (43.6, 36.1 and 42.6%, respectively). Neither the combination charybdotoxin–apamin nor tetraethylammonium altered the response of the human corpora cavernosa strips to yohimbine. Nevertheless, glibenclamide decreased the maximum relaxant response to yohimbine by 29.8% (P<0.05; n=12). The results suggest that yohimbine relaxes the human corpus cavernosum by a non-adrenergic, non-cholinergic mechanism, probably activating the nitrergic-soluble guanylate cyclase (NO-sGc) pathway and KATP.
yohimbine, human corpus cavernosum, erectile dysfunction, K+ channels
Approximately 52% of men between 40 and 70 years suffer from erectile dysfunction.1 The condition may worsen under the influence of stress and anxiety, which stimulate post-synaptic α-adrenergic receptor in the corpus cavernosum.2
Inspite of being phosphodiesterase type 5 inhibitors, the only drugs approved for a specific sexual function disorder,3 the yohimbine (an α-antagonist) is also used in the erectile dysfunction treatment.
Although the mechanism is not completely understood, after more than 70 years of usage in the treatment of erectile dysfunction, yohimbine is still currently indicated for this purpose,4 like phentolamine, another α-antagonist used in intracavernosal injection.5 Yohimbine (17α-hydroxy-yohimban-16α-carboxylic acid methyl ester) is an alkaloid extracted from the bark of Pausinystalia yohimbe or from the root of Rauwolfia, both of which are endemic in West-Africa.4 It is a potent selective antagonist of α2-adrenergic receptors, but a weak antagonist of α1-adrenergic receptors.6 It appears to affect the central erectogenic mechanism also, as α2-adrenergic receptors are abundant in the central nervous system.7
Yohimbine also stimulates the nitrergic pathways in both the rabbit and the human corpus cavernosum.4 Similarly, via the same pathway, a medicine known as Prelox (a combination of l-arginine aspartate and Pycnogenol) also improves the erectile function.8
Historically, studies using the human corpus cavernosum have mostly used samples from donors receiving penile prosthesis,4 but the general difficulty in obtaining samples from humans has led most researchers to work with rat and rabbit tissues.
The objective of the present study was to demonstrate that yohimbine relaxes the human corpus cavernosum by a non-adrenergic, non-cholinergic mechanism activating the nitrergic-soluble guanylate cyclase–cyclic GMP (NO-sGC–cGMP) pathway, possibly by the activation of ATP-dependent potassium channels.
Materials and methods
A series of pharmacological experiments were carried out exposing samples of the human corpus cavernosum to yohimbine. The research protocol and the consent form for the donation of fragments of the corpus cavernosum were previously approved by the Human Subjects Research Ethics Committee of the Federal University of Ceará, an institution accredited by the Brazilian Ministry of Health/CONEP.
Fragments of the human corpus cavernosum were collected through xiphoid pubic incision during surgery for organ transplantation. Samples were retrieved from 12 cadaver donors aged 18–31 years with documented brain death from craniocerebral trauma caused by automobile accidents.
After removal of the heart, the liver and kidneys, the corpus cavernosum was located above the pubic symphysis by digital hypodermic approach. The penis was everted cranially to make room for the incision and excision of the desired fragments (5 cm × 2 cm) enveloped by the tunica albuginea. After sample collection the penis was returned to its normal anatomical position with no external sequelae.
Immediately after retrieval, the human corpus cavernosum samples were transported to the laboratory and stored on ice in a closed glass vial containing Krebs–Henseleit solution (KHS) composed of 114.6-mm NaCl, 4.96-mm KCl, 0.58-mm MgSO4, 1.23-mm NaH2PO4, 2.0-mm CaCl2, 25-mm NaHCO3 and 3.6-mmglucose.
Sample processing and mounting
The human corpus cavernosum samples were placed on a petri dish containing Krebs–Henseleit solution gassed with 5% CO2 and 95% O2. Connective tissues (muscle, fat and so on) were removed with a scalpel. Then the tunica albuginea was carefully dissected and the corpus cavernosum muscle was divided in strips measuring 8 mm × 2 mm. The human corpus cavernosum strips were mounted vertically side by side in isometric baths with continuously gassed Krebs–Henseleit solution (pH 7.4; 37 °C; tension 1.0 g). Tissues were allowed to rest for 1 h in order to reach a functional equilibrium with washing at 15-min intervals. Changes in tension (g) were registered with a force transducer (F-60, Narco Bio-Systems, Houston, TX, USA) connected to a four-channel physiograph (DMP-4B, Narco Bio-Systems, Houston, TX, USA) with the filter set at 1 Hz, amplifier sensitivity at 50 mV/cm and paper speed at 0.0025 cm/s. This was coupled to a PANLAB four-channel data acquisition system (ADInstruments, Sidney, Australia) with data analyzed by the software Chart 4.0 (ADInstruments, Sidney, Australia).
Dose–response curves were plotted for different concentrations of yohimbine (10−12–10−4 m) added to the human corpus cavernosum strips pre-contracted with 10-μm phenylephrine (PE) and high potassium depolarizing solution (60-mmK+), with a calcium (Ca++) concentration of 2 mm and a nutritive medium containing 10-μm guanethidine and 10-μm phentolamine.
Concentration values were then registered for EC50 (dose required to induce half the maximum response) and PD2 (the respective co-logarithm).
Next, yohimbine-induced relaxation (10−4 m) was tested separately in the absence and presence of 100-μm tetrodotoxin, a neuronal sodium channel blocker.
In another series of experiments, the same concentration of yohimbine was tested in the absence and presence of 10-μm 7-nitroindazole, 100-μm NG-nitro-l-arginine methyl ester, both blockers of nitric oxide synthase (nNOS and eNOS, respectively), and 10-μm 1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxalin-1-one, a soluble guanylate cyclase inhibitor.
In yet another series of experiments, yohimbine-induced relaxation was tested in the absence and presence of 100-μm tetraethylammonium, a non-selective Ca++-activated voltage-dependent K+ channel inhibitor, 0.1-μm apamin in combination with 1-μm charybdotoxin, both of which are low-, medium- and high-conductance Ca-activated K+ channel blockers, and 10-μm glibenclamide, an ATP-dependent potassium channel blocker.
The results were expressed in average percentages (±standard error) of relaxation in relation to maximum induced contraction.
The statistical significance was tested with analysis of variance followed by the Tukey–Kramer test for multiple comparisons.
PD2 (−logEC50) values were calculated with the software Graph Pad 3.0 (San Diego, CA, USA) and expressed along with their respective 95% confidence intervals.
The addition of yohimbine in vitro produced relaxation in the human corpus cavernosum strips pre-contracted with phenylephrine or with a high-K+depolarizing solution combined with guanethidine and phentolamine, especially in the former case (P<0.05 with 10−5 and 10−4 m yohimbine). The maximal relaxation induced by yohimbine in the human corpus cavernosum strips pre-contracted with phenylephrine was 100±0% and only 30.5±5.0% when tissues were pre-contracted with 60-mm K+ solution (Figure 1).
Relaxation induced by yohimbine (10−12–10−4 m) in the human corpus cavernosum strips pre-contracted with 10-μm phenylephrine (closed dots) or with 60-mm potassium solution (K+; open squares) with a calcium concentration of 2 mm and a nutritive medium containing 10-μmguanethidine and 10-μm phentolamine (*P<0.05 yohimbine–phenylephrine vs vehicle; #P<0.05 yohimbine–phenylephrine vsyohimbine–K+). Vehicle (NaCl 0.9% in distilled water) was added isovolumetrically as a negative control.
NO-sGC pathway evaluation
To investigate the potential involvement of NO-sGC–cGMP pathway, yohimbine-induced relaxation was tested in tissues preincubated for 30 min with 100-μmtetrodotoxin, 10-μm 1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxalin-1-one or 10-μm 7-nitroindazole. The maximal relaxation induced by yohimbine was significantly inhibited by all of these pharmacological blockers. The maximal relaxation was inhibited by 43.6, 36.1 and 42.6%, respectively (Figure 2).
Relaxation induced by yohimbine (10−12–10−4 m) in the human corpus cavernosum strips pre-contracted with 10-μm phenylephrine (closed dots) in tissues preincubated for 30 min with 100-μm tetrodotoxin, 10-μm 1H-[1, 2, 4]Oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ) or 10-μm 7-nitroindazole.
K+ channel blockade
To investigate the role of some K+ channels, the human corpus cavernosum strips were initially pre-contracted with phenylephrine and yohimbine-induced relaxation was probed in tissues that were incubated for a 30-min period with 10-μm glibenclamide, 1-μm charybdotoxin combined with 0.1-μm apamin or 100-μmtetraethylammonium. Neither the combination charybdotoxin–apamin nor tetraethylammonium altered the response of the human corpora cavernosa strips to yohimbine. Nevertheless, glibenclamide decreased the maximum relaxant response to yohimbine by 29.8% (P<0.05; n=12) (Figure 3).
The following three types of α2-adrenergic receptors are known: α2a-adrenergic receptors, α2b-adrenergic receptors and α2c-adrenergic receptors. Although α2b-adrenergic receptor has not been reported in smooth muscle cells, but only in nerves and endothelium, the other two contract human corpus cavernosum muscle fibers when stimulated, or may relax these fibers if inhibited by α2b-adrenergic receptor antagonists, suggesting the latter are physiologically functional in the human corpus cavernosum.9
At present it is known that the yohimbine acts in the penile corpus cavernosum through a non-adrenergic, non-cholinergic mechanism, as demonstrated by other authors.4 Another α2-adrenergic antagonist, the phentolamine, relaxes both the rabbit and the human corpus cavernosum by blocking off α1 and α2-adrenergic receptors, using the nitrergic and K+ channel pathways as well.10, 11 As mentioned before, the yohimbine appears to affect the central erectogenic mechanism, as well, as α2-adrenergic receptors are abundant in the central nervous system.7 Another medication, bremelanotide, a melanocortin agonist of central action, seems to improve the quality of erection in male patients with erectile dysfunction.12
The medicine commonly used for erectile dysfunction, the sildenafil, has its central effect improved when associated to yohimbine.13
Unlike most published studies, using compromised corpus cavernosum tissues donated by penile prosthesis patients,4, 14 in this study we used samples retrieved from cadaver donors during the removal of organs for transplantation. All donors (n=12) had died of craniocerebral trauma and, according to relatives, none had suffered from erectile dysfunction.
Yohimbine loses its relaxation power when the human corpus cavernosum is pre-contracted with a K+ solution. With the sudden change in the extracellular K+concentration, the balance of the transmembrane potential is lost and the ionic current is inverted resulting in the opening of voltage-dependent calcium channels and, subsequently, muscle contracture.11
Matching findings reported by Filippi4 in our study 10−4–m yohimbine was found to induce an adequate level of relaxation in the human corpus cavernosum pre-contracted with phenylephrine. When samples were pre-contracted with a K+solution in combination with guanetidine and phentolamine, thus abolishing the adrenergic pathway, yohimbine induced a lesser degree of relaxation but was still effective, indicating the involvement of another pathway in addition to the known adrenergic pathway, as demonstrated by Silva et al.11 in a study using phentolamine.
When cellular hyperpolarization occurs as a result of the opening of K+ channels, the voltage-dependent calcium channels are closed and intracellular Ca++ levels decrease, leading to relaxation of the smooth muscle.15
Based on this knowledge, the present study was designed to assess the pharmacodynamic profile of yohimbine in the presence of substances acting on the human corpus cavernosum erection mechanism through different pathways.
Thus, tetrodotoxin partially inhibited yohimbine-induced relaxation in the human corpus cavernosum samples, as shown in Figure 2.
The adrenergic pathway involved in the contraction of the human corpus cavernosum smooth muscle is regulated by presynaptic α2-adrenergic receptors. Acting on this pathway, yohimbine increases relaxation in the human corpus cavernosum. For instance, the release of noradrenaline (which acts to keep detumescence) is inhibited by α2-adrenoceptor agonists and facilitated by α2-adrenoceptor antagonists.16 Similarly, in tissues pre-contracted with phenylephrine, yohimbine may also presynaptically increase the release of nitric oxide by counteracting the inhibitory effect of the α2-adrenoceptor. This can be a component of the relaxant effect induced by yohimbine. This hypothesis in corroborated by the inhibition of the maximum relaxant effect is inhibited by tetrodotoxin (a neuronal sodium channel blocker) and 7-nitroindazole a nNOS inhibitor.
Hence, the facilitation of the neurotransmission through nitrergic fibers may be one mechanism by which yohimbine relaxes the human corpora cavenosa strips. This is further reinforced by a similar blockade of the maximal relaxation induced by yohimbine in the presence of 1H-[1, 2, 4]Oxadiazolo[4, 3-a]quinoxalin-1-one, which inhibits the nitric oxide activation of the soluble guanylate cyclase.
Nitric oxide release is known to be the main factor stimulating relaxation of the human corpus cavernosum smooth muscle; thus, stimulation of α2-adrenergic receptors by norepinephrine (or phenylephrine) would have an inhibitory effect. By inhibiting these receptors, yohimbine would allow more nitric oxide to be released and thereby promote penile erection.17
Filippi et al.4 observed similar changes in the presence of NG-nitro- L-arginine methyl ester and 1H-[1, 2, 4]Oxadiazolo[4, 3-a]quinoxalin-1-one, and reported yohimbine to be potentialized when associated with sildenafil, a powerful phosphodiesterase type 5 inhibitor. This finding, further corroborates with the involvement of the NO-sGC–cGMP pathway. Our suggestion is that the source of nitric oxide for human corpus cavernosum relaxation may also be from nitrergic fibers instead of the endothelium, as suggested by Filippi and coworkers.4
On the other hand, charybdotoxin and apamin are known to exert an inhibitory effect on Kca channels. The former efficiently blocks medium and high-conductance channels (maxi-K), whereas the latter acts on low-conductance channels.18
As maxi-K channels have been shown to be stimulated by the intracellular release of cGMP and also has an important role in the relaxation of smooth muscles, they may represent an alternative pathway for the treatment of erectile dysfunction.19, 20
In spite of the importance of maxi-K channels in human corpus cavernosum relaxation and the fact that they are the most abundant K+ channels in these tissues,21 in our experiment yohimbine-induced relaxation was not affected when charybdotoxin and apamin were added (Figure 3). Likewise, no change was observed when yohimbine was combined with tetraethylammonium (Figure 3).
Studies on the effects of pinacidil and levocromakalim have shown the importance of KATP channels in modulating the human corpus cavernosum smooth muscle tone.22
In the present study, when glibenclamide was added, yohimbine-induced relaxation decreased (Figure 3), indicating that yohimbine also acts through KATPpathways. Recently, Silva et al.11 reported similar findings using phentolamine aα1-adrenoceptor blocker. Sildenafil (phosphodiesterase type 5 inhibitor) also relaxes the human corpus cavernosum by KATP channels.23
It would therefore seem that, by acting through KATP channels, yohimbine hyperpolarizes the muscle cell membrane, and thereby reduces Ca++ flow into the cell promoting relaxation in the human corpus cavernosum smooth muscle. This mechanism may be similar to phentolamine, an α1-adrenoceptor blocker, as reported before.11 This can be achieved directly or through a cGMP-dependent pathway.
Through a perhaps little different mechanism, an alkaloid-rich fraction (F3–5) from Aspidosperma ulei (Markgr) relaxes the rabbit corpus cavernosum by directly blocking calcium influx.24
The current findings offer new perspectives on the mechanisms of yohimbine-induced relaxation. In spite of its long history in the treatment of erectile dysfunction,4 little is known about the non-adrenergic, non-cholinergic pathways involved in yohimbine-induced relaxation, and controlled studies have only been undertaken for the past few years.6
This opens the door to many new lines of research, such as investigations based on molecular biology, in search of a more complete pharmacodynamic profile of the drug, along with that of other substances which can shed light on the mechanism of relaxation of the human corpus cavernosum smooth muscle and reveal alternative ways of improving the quality of erection and the quality of life of couples affected by the problem of erectile dysfunction.
In conclusion, yohimbine was shown to relax the corpus cavernosum smooth muscle by way of a non-adrenergic, non-cholinergic mechanism probably through the endothelial and the neuronal nitric oxide release and ATP-dependent potassium channels activation. The facilitation of the nitrergic transmission through α2-adrenoceptor blockade associated to α1-adrenoceptor blockade in higher concentration may explain the direct relaxant response induced by yohimbine in the human corpora cavernosa.
Conflict of interest
The authors declare no conflict of interest.
- Feldman HA, Goldstein I, Hatzichristou DG, Krane RJ, McKinlay JB. Impotence and its medical and psychosocial correlates: results of the Massachusetts Male Aging Study. J Urol 1994; 15: 54–61.
- Andersson KE, Wagner G. Physiology of penile erection. Physiol Rev 1995;75: 191–236. | PubMed | ISI | ChemPort |
- Fallon B. ‘Off-label’ drug use in sexual medicine treatment. Int J Impot Res2007; 20: 127–134. | Article | PubMed
- Filippi S, Luconi M, Granchi S, Natali A, Tozzi P, Forti G et al. Endotheliumdependency of yohimbine-induced corpus cavernosum relaxation. Int J Impot Res 2002; 14: 295–307. | Article | PubMed | ChemPort |
- Traish A, Gupta S, Gallant C, Huang Y-H, Goldstein I. Phentolamine mesylate relaxes penile corpus cavernosum tissue by adrenergic and non-adrenergic mechanisms. Int J Impot Res 1998; 10: 215–223. | Article | PubMed | ChemPort |
- Tam SW, Worcel M, Wylle M. Yoimbine: a clinical review. Pharmacol Ther2001; 91: 215–243. | Article | PubMed | ChemPort |
- Glina S, Martins FG, Damião R. Tratamento oral. In: I Consenso Brasileiro de disfunção erétil (ed). BG cultural. Rio de Janeiro, Brazil, 1998, pp, 63–70.
- Stanislavov R, Nikolova V, Rohdewald P. Improvement of erectile function with Prelox: a randomized, double-blind, placebo-controlled, crossover trial. Int J Impot Res 2007; 20: 173–180. | Article | PubMed | ChemPort |
- Traish AM, Moreland RB, Huang YH, Goldstein I. Expression of functional 2- adrenergic receptor subtypes in human corpus cavernosum and in cultured trabecular smooth muscle cells. Recept Signal Transduct 1997; 7: 55–67. | PubMed | ChemPort |
- Kim NN, Goldstein I, Moreland RB, Traish AM. Alpha-adrenergic receptor blockade by phentolamine increases the eficacy of vasodilators in penile corpus cavernosum. Int J Impot Res 2000; 12(Suppl 1): S26–S36. | Article | PubMed
- Silva LF, Nascimento NR, Fonteles MC, de Nucci G, Moraes ME, Vasconcelos PR et al. Phentolamine relaxes human corpus cavernosum by a non-adrenergic mechanism activating ATP-sensitive K+ channel. Int J Impot Res2005; 17: 27–32. | Article | PubMed | ChemPort |
- Hellstrom WJG. Clinical applications of centrally acting agents in male sexual dysfunction. Int J Impot Res 2008; 20: S17–S23. | Article | PubMed | ChemPort |
- Senbel AM, Mostafa T. Yohimbine enhances the effect of sildenafil on erectile process in rats. Int J Impot Res 2008; 20: 409–417. | Article | PubMed | ChemPort |
- Saenz de Tejada I, Garvey DS, Schroeder JD, Shelekhin TL, Letts G, Fernandez A et al. Design and evaluation of nitrosylated -adrenergic receptor antagonists as potential agents for the treatment of impotence.Pharmacol Exp Ther 1999; 290: 121–128.
- Saenz de Tejada I. Molecular mechanisms for the regulation of penile smooth muscle contractility. Int J Impot Res 2002; 14(Suppl 1): S6–S10. | Article | PubMed
- Molderings GJ, Göthert M, Van Ahlen H, Porst H. Noradrenaline release in human corpus cavernosum and its modulation via presynaptic alpha 2-adrenoceptors. Fundam Clin Pharmacol 1989; 3: 497–504. | Article | PubMed | ChemPort |
- Saenz De Tejada I, Kim NN, Goldstein I, Traish AM. Regulation of pre-synaptic alpha-adrenergic activity in the corpus cavernosum. Int J Impot Res 2000; 12(Suppl 1): S20–S25. | Article | PubMed
- Archer SL. Potassium channels and erectile dysfunction. Vasc Pharmacol2002; 38: 61–71. | Article | ISI | ChemPort |
- Spector M, Rodriguez R, Rosenbaum RS, Wang HZ, Melman A, Christ GJ. 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 Urol2002; 167: 2628–2635. | Article | PubMed | ISI | ChemPort |
- Stumpff F, Strauss O, Boxberger M, Wiederholt M. Characterization of maxi-channels in bovine trabecular meshwork and their activation by cyclic guanosine monophosphate. Invest Ophthalmol Vis Sci 1997; 38: 1883–1892. | PubMed | ChemPort |
- Fanh SF, Brink PR, Melmann A, Christ GJ. An analysis of the Maxi-K+ (Kca) channel in cultured human corporal smooth muscle cells. J Urol 1995; 153: 818–825. | Article | PubMed | ISI | ChemPort |
- Venkateswarlu K, Giraldi A, Zhao W, Wang HZ, Melman A, Spektor M et al. Potassium channels and human corporeal smooth muscle cell tone: diabetes and relaxation of human corpus cavernosum smooth muscle by adenosine thiphosphate sensitive potassium channel operners. J Urol 2002;168: 355–361. | Article | PubMed | ISI | ChemPort |
- El-Metwally MA, Sharabi FM, Daabees TT, Senbel AM, Mostafa T. Involvement of alpha-receptors and potassium channels in the mechanism of action of sildenafil citrate. Int J Impot Res 2007; 19: 551–557. | Article | PubMed | ChemPort |
- Campos AR, Cunha KMA, Santos FA, Silveira ER, Uchoa DEA, Nascimento NRF, Rao VSN. Relaxant effects of an alkaloid-rich fraction from Aspidosperma ulei root bark on isolated rabbit corpus cavernosum. Int J Impot Res 2007; 20: 255–263. | Article | PubMed | ChemPort |