Misoprostol Induces Relaxation of Human Corpus Cavernosum Smooth Muscle: Comparison to Prostaglandin E1

Home , Misoprostol, Prostaglandin, Prostaglandin E, Prostaglandin E1, Thromboxane A2

Misoprostol induces relaxation of human corpus cavernosum smooth muscle: comparison to prostaglandin E1

RB Moreland1, NN Kim1, A Nehra2, BG Parulkar3 and A Traish1,4*

1Department of Urology, Boston University School of Medicine, Boston, MA 02118, USA; 2Department of Urology, Mayo Clinic and Foundation, Rochester, MN 55905, USA; 3Department of Urology, University of Massachusetts Medical Center, Worcester, MA 01604, USA; and 4Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA

Prostaglandin E1 (PGE1) relaxes trabecular smooth muscle by interacting with speci®c G-protein coupled receptors on human corpus cavernosum smooth muscle and increasing intracellular synthesis of cAMP. Misoprostol (CytotecTM), is an oral prostaglandin E analogue. The purpose of this study was to compare the functional activity of misoprostol with PGE1 in human corpus cavernosum and cultured human corpus cavernosum smooth muscle cells. Misoprostol, misoprostol free acid or PGE1 induced dose-dependent relaxations in strips of human corpus cavernosum. At concentrations greater than 1076 M, tissue recontraction was observed with all three agents. This was abrogated by pretreatment with the thromboxane A2 receptor antagonist SQ29,548. From these observations, we conclude that misoprostol is activated by human corpus cavernosum in situ and relaxes phenylephrine-precontrated tissue strips in vitro. This relaxation response is mediated by the increased cAMP synthesis by these agents. International Journal of Impotence Research (2000) 12, 107±110.

Keywords: misoprostol; cAMP; PGE1; EP receptor; TP receptor; vascular smooth muscle; corpus cavernosum; erectile dysfunction

Introduction been conducted using misoprostol as an abortifac- tant7 and in treatment of peripheral vascular disease.8 CaverjectTM (PGE1 for intracavernosal injection) was Prostanoids are autocoids synthesized primarily the ®rst Food and Drug Administration approved from arachadonic acid and include PGD2, PGE2, pharmacotherapeutic for the treatment of erectile PGF2a, prostacyclin and thromboxane A (reviewed dysfunction.1 The labile nature of prostaglandins, in by Pierce et al).9 Of these lipid vasoactive sub- part, has dictated administration of these agents stances, PGD2, PGE2 and prostacyclin can induce primarily intracavernosally; although intraurethral smooth muscle relaxation, depending on the recep- means have been introduced (MUSETM).2 Attempts tors present.9 Prostaglandin E is the major relaxatory have been made to produce more stable formula- prostanoid in corpus cavernosum smooth muscle.10 tions of PGE. These include PGE1=alpha cyclodex- The proposed mechanism of vasodilation of smooth trin complex (EDEXTM=ViridalTM)3 and topical muscle by PGE1 is that PGE1 binds to G-protein PGE1 cream (TopiglanTM).4 The development of a coupled receptors, increasing cAMP synthesis and stable, PGE1 agonist which could be administered eventually resulting in the lowering of intracellular orally may be of value in the treatment of erectile calcium levels.11 The end result is a change in dysfunction either as a single medication or in myosin light chain phosphorylation=dephos- combination with other agents which act through phorylation favoring smooth muscle relaxation.11 different pharmacologic pathways. Misoprostol, an While misoprostol is an effective agent in the gastric orally administered PGE (EP) receptor agonist, was mucosa in the prevention of ulcers in the presence ®rst reported to inhibit gastric acid secretion in of nonsteroidal, anti-in¯ammatory drugs,6 it re- humans5 and has been used successfully in the mains to be demonstrated if this compound is treatment of gastric ulcers.6 Clinical trials have also ef®cacious in the relaxation of penile corpus cavernosum smooth muscle. The goals of this study were to compare misoprostol activity with PGE1 in *Correspondence: A Traish, Room W 607A, 700 Albany Street, Boston MA, 02118, USA. E-mail: [email protected] inducing relaxation in human corpus cavernosum Received 18 May 1999; accepted in revised form and in inducing cAMP synthesis in human corpus 19 October 1999 cavernosum smooth muscle cells. Misoprostol relaxation of human corpus cavernosum RB Moreland et al 108 Methods

Dose responses of misoprostol and PGE1 in human corpus cavernosum

These studies were approved by the Institutional Review Boards at Boston University Medical Center, the Mayo Clinic and Foundation and the University of Massachusetts Medical Center. Human corpus cavernosum was obtained from six patients under- going penile prosthesis insertion at time of surgery. Tissues were stretched to reach isometric tension and maximal contractile responses to 1 mM phenylephrine determined as previously described.12,13 In these experiments, both misoprostol and the misoprostol free acid were tested. We recorded relaxation dose responses to misoprostol (1079±1075 M, Biomol, Plymouth Meeting, PA), misoprostol free acid (1079±1075 M, Biomol, Plymouth Meeting, PA), and PGE1 (1079±1075 M, Cayman Chemical, Ann Arbor, MI). Dose responses were also determined in the presence of 10 mM SQ29548 (Cayman Chemical, Ann Arbor, MI), a thromboxane A2 receptor antagonist.14

Misoprostol and PGE1 stimulation of human corpus cavernosum smooth muscle cell cAMP synthesis

Human corpus cavernosum smooth muscle cells from four different patients were cultured as previously reported.15 Cells at passage 2±3 were grown to con¯uence in six-well plates. Media was aspirated and replaced with fresh media. Cells were treated with 10 mM 3-isobutyl-1-methylxanthine (IBMX) for 15 min and then treated for 5 min with either misoprostol free acid (0.1, 1 and 10 mM) or PGE1 (0.1, 1 and 10 mM). The reaction was termi- nated by quickly aspirating the media and adding 1 ml of ice cold 1 N perchloric acid. The cells were scraped and processed for cAMP with a commercially available radioimmunoassay (Biome- dical technologies, Stoughton, MA), as described previously.12 Figure 1 (A) Effects of misoprostol on relaxation of phenylephrine-precontracted human corpus cavernosum. Dose response of misoprostol on phenylephrine-precontracted human corpus cavernosum. Data represent treatment in the absence (triangles) or Results presence (squares) of 10 mM SQ29548. n 6. Error bars represent standard error of the mean. (B) Effects of misoprostol free acid on relaxation of phenylephrine-precontracted human corpus cavernosum. Dose res- Misoprostol, misoprostol free acid and PGE1 in- ponse of misoprostol free acid on phenylephrine-precontracted duced dose-dependent relaxations in phenylephrine human corpus cavernosum. Data represent treatment in the pre-contracted human corpus cavernosum in organ absence (triangles) or presence (squares) of 10 mM SQ29548. baths in vitro (Figure 1). The order of potency was n 6. Error bars represent standard error of the mean. C) Effects of PGE1 on relaxation of phenylephrine-precon- PGE1 > misoprostol misoprostol free acid. Prosta- tracted human corpus cavernosum. Data represent treatment in 76 glandin E1 at concentrations above 10 M induced the absence (triangles) or presence (squares) of 10 mM SQ29548. dose-dependent contractions in human corpus n 6. Error bars represent standard error of the mean.

International Journal of Impotence Research Misoprostol relaxation of human corpus cavernosum RB Moreland et al 109 cavernosum (Figure 1C). This effect was much more the lung.18 Hence, the ef®cacy of oral PGE1 may be pronounced in misoprostol-treated corpus caverno- complicated by rapid metabolism or from side sum (Figure 1A and B), with the ®nal contraction effects such as increased gastrointestinal mobility of the tissue exceeding that observed with PGE1, due to activity of PGE (EP) receptors in the small particularly for misoprostol free acid. Pretreatment bowel.19,20 A recent trial of oral PGE1 at 30 mg three of smooth muscle strips with the thromboxane A2 times a day noted few side effects but while receptor antagonist SQ29548 (10 mM) before the dose tumescence developed, no rigid erections were responses abrogated misoprostol (Figure 1A) or observed. The authors noted a signi®cant improve- PGE1-induced recontraction of human corpus ca- ment in nocturnal penile tumescence with oral vernosum (Figure 1C). Further, the inclusion of PGE1 administration.21 SQ29548 enhanced the relaxation of phenylephrine In this study we examined the effects of miso- precontracted human corpus cavernosum to increas- prostol on inducing relaxation of phenylephrine ing doses of misoprostol and misoprostol free acid precontracted human corpus cavernosum in vitro (Figure 1B) to the extent observed with PGE1 in the and increasing cAMP synthesis in human corpus presence of SQ29548. cavernosum smooth muscle cells in culture. Mis- Treatment of human corpus cavernosum smooth oprostol is a methyl ester which requires hydrolysis muscle cells in culture with PGE1 or misoprostol for activity with PGE (EP) receptors. Misoprostol is free acid resulted in a dose-dependent increase activated by human corpus cavernosum in situ and in cAMP synthesis (Figure 2). Prostaglandin E1 relaxes phenylephrine pre-contracted human corpus was more effective than misoprostol free acid in cavernosum (Figure 1A). There was no signi®cant inducing cAMP synthesis. difference between the relaxation responses pro- duced by misoprostol free acid (activated) and misoprostol (Figures 1A and B). This was attributed to hydrolysis of misoprostol by corpus cavernosum Discussion tissue during experimentation. The inclusion of the thromboxane receptor antagonist improved the ef®cacy of misoprostol-induced relaxation such that Pharmacotherapy of erectile dysfunction began with it was indistinguishable from that induced by PGE1. injectable vasoactive substances and has progressed Both PGE1 and activated misoprostol (free acid) to the use of oral, systemic agents such as sildena®l induce cAMP synthesis in cultured human corpus citrate.16 There is a need for new pharmacother- cavernosum smooth muscle cells although PGE1 apeutics which can be administered orally with shows a greater response than misoprostol. The minimal side effects but ef®cacious in a wider increased synthesis of cAMP corroborates that spectrum of patients including those with an misoprostol exerts it effects via EP2 and=or EP4 impaired nitric oxide system (for example, diabetics receptors on human corpus cavernosum smooth and radical prostatectomy patients, see ref. 16). muscle cells.12,14 While injectable PGE1 is reported to be effective in a While misoprostol induces smooth muscle re- wide spectrum of patients,17 its use is hampered by laxation and may be a useful pharmacotherapeutic the route of administration. Oral pharmacotherapy agent, its ef®cacy as an oral agent must be assessed by its systemic nature, is subject to increased side in clinical trials. The EC50 from this study appears to effects. Prostaglandins are rapidly metabolised by be comparable to that of PGE1 ( 5 6 1077 M). The need for such high concentrations may preclude the use of this agent due to potential hypotensive, gastrointestinal or other side effects. The success of CytotecTM (misoprostol) in the treatment of gastric ulcers6 may be due to the route of administration and achieving optimal concentrations in the stom- ach. In conclusion, while misoprostol may be an effective agent in relaxing human corpus cavernosum in vitro, it remains to be demonstrated clinically if this agent will be effective orally in the treatment of erectile dysfunction with minimal side effects.

Figure 2 Effects on cAMP synthesis in human corpus caverno- Acknowledgements sum smooth muscle cells by PGE1 and misoprostol free acid. Dosage dependent effects of PGE1 (0.1±10 mM) or misoprostol free acid (0.1±10 mM) on cAMP synthesis in cultured human corpus cavernosum smooth muscle cells. n 6). Error bars represent The authors acknowledge the excellent technical standard error of the mean. assistance of Yue-hua Huang and Erika Line. We are

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International Journal of Impotence Research