Pharmacology Journal

It is widely accepted that release of nitric oxide (NO) from nonadrenergic, noncholinergic nerves during sexual stimulation plays an important role in penile smooth muscle relaxation and erection. Sildenafil is the first orally active drug approved by the food and drug administration to treat male erectile dysfunction. Sildenafil inhibits cGMP specific type 5 phosphodiesterase, the predominant phosphodiesterase isozyme present in penile smooth muscle. Sildenafil augments penile erection by preventing the inactivation of cGMP and promoting its accumulation in the corpus cavernosum. However, the interaction of Sildenafil with organic nitrates causes marked hypotension and is contraindicated in patients taking nitrates. Therefore there is a clinical need for oral agent(s) that are devoid of serious side-effects to treat male erectile dysfunction.

Phentolamine mesylate, an α-adrenergic antagonist had been marketed to treat hypertension associated with pheochromocytoma. Intracavernosal injection of phentolamine either alone or in combination with other vasoactive agents began to be used to treat male erectile dysfunction in 1983. The intracavernosal injection of drugs for the treatment of erectile dysfunction did not achieve widespread popularity because of the problems associated with the self-administration of drugs directly into the penis. More recently, oral formulations of phentolamine mesylate (Vasomax^®) were developed for erectile dysfunction. Several recently published clinical studies suggested that Vasomax^® significantly improved erectile dysfunction compared to the palcebo in patients with minimal erectile dysfunction.

Phentolamine was thought to relax penile smooth muscle by inhibiting α₁- and α₂-adrenergic receptors on the corpus cavernosum. However, recent reports speculated that phentolamine mesylate may also relax rabbit corpus cavernosum by a non-adrenergic mechanism by activating NO synthase. We reasoned that if pentolamine mesylate relaxes rabbit corpus cavernosum by activating NO synthase, it should augment electrical-field stimulation-evoked relaxation of nonadrenergic, noncholinergic nerves of the penile smooth muscle. Therefore, we assessed the nonadrenergic, noncholinergic relaxant effects of phentolamine mesylate in the rabbit corpus cavernosum. This in vitro preparation is exclusively utilized to assess the role of nitric oxide system in the relaxation of both rabbit and human penile smooth muscle tissue.

Guidelines

Animal experimentation in this study was conducted in accordance with the NIH guidelines for the care and use of laboratory animals and the Animal Welfare Act in an AAALAC-accredited program.

Rabbit corpus cavernosum tissues

The method was previously described in detail. Briefly, male New Zealand white rabbits (3–3.5 kg) were sedated with a mixture of ketamine (60 mg/kg) and xylazine (8 mg/kg), im and were anaesthetised with sodium pentobarbital (15.0 mg/kg, iv) through the marginal ear vein. The rabbit penises were removed en bloc and the corpus cavernosum was dissected free from the surrounding tunica albuginea.

Tissue bath studies

One end of the corpus cavernosum was tied to the electrode and the other end of the corpus cavernosum was tied to the force displacement transducer (FT03; Grass Instruments, Quincy, MA, USA) connected to a Grass polygraph. The strips were immersed in 25 mL organ bath chambers containing a physiological salt solution with the following composition: NaCl 118 m M; NaHC0₃ 25 m M; KCl 4.7 m M; KH₂P0₄ 1.2 m M; MgS0₄ 1.2 m M; glucose 11 m M; CaCl₂ 21.5 m M, maintained at 37 °C and continuously aerated with 95% 0₂ and 5% C0₂. Optimal length–tension curves were obtained by gradual stretching and contracting of the tissues with phenylephrine (10 μ M). The tissues were considered to have reached optimal isometric tension if two consecutive contractions remained within 10% of each other. The integrity of endothelium was tested by recording the relaxation responses to methacholine (10 μ M). The tissues were then equilibrated in a physiological salt solution containing indomethacin (5 μ M), atropine (1 μ M) and guanethidine (5 μ M) to produce cyclooxygenase, cholinergic and adenergic blockade, respectively. An hour after the addition of inhibitors, the tissues were subjected to electrical field stimulation (EFS). EFS was achieved with the help of two parallel platinum electrodes on either side of the strips that were connected to a current amplifier and Grass S 48 stimulator. Each tissue was stimulated at 10 V, 0.5 msec pulse duration, for 10 s at frequencies ranging from 0.5 to 16 Hz. Two control EFS stimulations were performed in tissues contracted submaximally with phenylephrine (3 μ M).

Characterization of EFS-induced relaxation rabbit corpus cavernosum in vitro

After obtaining the baseline EFS frequency responses as mentioned above, the tissues were treated with vehicle, NO synthase inhibitor N^G-nitro- L-arginine (L-NAME; 30 and 100 μ M), the guanylate cyclase inhibitor 1H-1,2,4 oxadiazolo[4,3,-a] quinoxalin-1-one (ODQ; 0.3–3.0 μ M), or tetrodotoxin, a sodium channel blocker (0.3 μ M), and incubated for 30 min. At the end of 30 min, the tissues were contracted with phenylephrine (3 μ M). After the contractile response to phenylephrine was stabilised, the tissues were subjected to EFS and the percentage relaxation was calculated.

Effects of phentolamine on EFS-induced relaxation of rabbit corpus cavernosum

The rabbit corpus cavernosum strips were mounted in the organ bath chambers and EFS frequency responses were obtained in the buffer containing atropine, guanethidine and indomethacin as outlined above. The tissues were incubated with vehicle or phentolamine mesylate (30 and 100 n M) for 30 min. At the end of 20 min, all tissues were contracted with endothelin-1 (ET-1; 30 n M) and after the contraction peaked (10 min), the tissues were subjected to EFS.

In a separate group of cavernosal strips, the role of α₁- and α₂-adrenergic receptors on phentolamine mesylate-induced relaxations to EFS was investigated. The strips were mounted in tissue baths and electrical field stimulation frequency response curves were generated as described above. The tissues were incubated with prazosin and yohimbine (30 μ M each) and 30 min later phentolamine mesylate (100 n M) or vehicle was added. Twenty minutes later, the tissues were challenged with ET-1 and electrical field stimulations were performed.

Efffect of L-NAME on phentolamine mesylate-mediated relaxation of rabbit corpus cavernosum

The rabbit cavernosal strips were mounted in organ bath chambers, equilibrated and EFS-stimulation responses were generated as already described. L-NAME (30 μ M) or vehicle was added to the tissue baths and incubated for 30 min. The tissues were then treated with phentolamine (100 n M) and 20 min later, the tissues were contracted with ET-1 (30 n M) and subjected to EFS.

Chemicals

L-NAME, ODQ, tetrodotoxin, phenylephrine, ET-1, prazosin and yohimbine were purchased from Sigma Chemical Co (St. Louis, MO, USA). Phentolamine mesylate was obtained from RBI Reseach Biochemicals International, Natick, MA, USA.

Statistical analysis

Relaxation was calculated as a percentage of phenylephrine or ET-1 induced contraction. The data were expressed as mean ± SEM. The data were analysed by paired t-test in the same tissues and by unpaired t-test when the responses in different tissues were compared. A P-value < 0.05 was considered significant.

Filed under: Pharmacology Review Articles