Introduction

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Intracavernosal prostaglandin E₁ (PGE₁) has been extensively used as a therapeutic agent for the treatment of erectile dysfunction (Buvat et al., 1996, 1998; Linet and Ogring, 1996). Although some success has been obtained, a considerable number of patients (about 30-40%) have not responded to treatment with PGE₁ (Porst, 1996). The reason for the lack of response to PGE₁ in refractory patients is not known, but could depend on many factors (number of receptors, transduction mechanisms, metabolism, tissue structure, etc.). Indeed, it has not been demonstrated that the efficacy in the clinical response to PGE₁ correlates with the relaxation of human penile smooth muscle (arterial and trabecular) to this drug.

PGE₁ promotes relaxation of penile arterial smooth muscle via prostacyclin (IP) receptors and of trabecular smooth muscle through interaction with prostaglandin E (EP) receptors, with some preliminary data supporting a key role for the EP₂ receptor subtype (Andersson and Wagner, 1995; Sáenz de Tejada et al., 1998). IP and EP receptors, coupled to G-proteins, increase intracellular cAMP levels through adenylyl cyclase stimulation (Andersson and Wagner, 1995). Penile smooth muscle relaxation; however, is not only mediated by the cAMP pathway but also by the nitric oxide (NO)-cGMP pathway, which plays a fundamental role in the relaxation of human penile smooth muscle (Kim et al., 1991; Rajfer et al., 1992) and penile erection (Burnett et al., 1992; Trigo-Rocha et al., 1993). Activation of the cGMP pathway is brought about by the endogenous generation of NO from lacunar and vascular endothelia and nitrergic nerves. The existence of NO donors allows for pharmacological activation of the cGMP pathway and facilitates penile erection (Truss et al., 1994; Martínez-Piñeiro et al., 1998). S-Nitrosothiols are NO donor molecules, some of which are present in human plasma and tissues in physiological conditions and one of these compounds, S-nitrosoglutathione (SNO-Glu) has been reported to induce vascular (MacAllister et al., 1995) and penile trabecular (Gupta et al., 1995) smooth muscle relaxation. In addition, promising, although preliminary clinical data have shown that intracavernosal coadministration of PGE₁ and the NO donor linsidomine chlorhydrate (SIN-1) produces better erectile responses than those obtained with either compound individually (Tordjman, 1993). The mechanism behind such possible clinical benefit has not been determined.

The aim of the present study was to analyze the relaxant responses to PGE₁ in human corpus cavernosum and penile resistance arteries from impotent patients and to determine whether the clinical response to PGE₁ would depend, at least in part, on the degree of penile smooth muscle relaxation induced by this prostanoid. We also sought to evaluate possible synergistic effects of combining PGE₁ and SNO-Glu to cause relaxation of penile smooth muscle.

	Experimental Procedures

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Human Corpus Cavernosum Tissues. Human corpus cavernosum specimens were obtained from impotent men at the time of penile prosthesis insertion. All experimental protocols were approved by the local ethics committee. Tissues were maintained at 4-6°C in M-400 solution (composition per 100 ml: 4.19 g of mannitol, 0.205 g of KH₂PO₄, 0.97 g of K₂HPO₄·3H₂O, 0.112 g of KCl, 0.084 g of NaHCO₃, pH 7.4) until used, which ranged between 2 and 16 h after extraction (Simonsen et al., 1997).

Vascular Reactivity of Resistance Penile Arteries. Penile small arteries, helicine arteries (lumen diameter 150-400 µm), which are the terminal branches of deep penile arteries, were dissected by carefully removing the adhering trabecular tissue, and arterial ring segments (2 mm in length) were subsequently mounted on two 40-µm wires on microvascular Halpern-Mulvany myographs (J.P. Trading, Aarhus, Denmark) for isometric tension recordings. The vessels were allowed to equilibrate for 30 min in physiological salt solution (PSS) of the following composition: 119 mM NaCl, 4.6 mM KCl, 1.5 mM CaCl₂, 1.2 mM MgCl₂, 24. 9 mM NaHCO₃, 11 mM glucose, 1.2 mM KH₂PO₄, and 0.027 mM EDTA at 37°C continuously bubbled with 95% O₂, 5% CO₂ to maintain a pH of 7.4. Passive tension and internal circumference of vascular segments when relaxed in situ under a transmural pressure of 100 mm Hg (L₁₀₀), were determined. The arteries were then set to an internal circumference equivalent to 90% of L₁₀₀, at which the force development was close to maximal (Mulvany and Halpern, 1977). The preparations were then exposed to 125 mM K⁺ (equimolar substitution of NaCl for KCl in PSS) and the contractile response was measured. The arteries were contracted with 1 µM norepinephrine and relaxation responses were evaluated by cumulative additions of compounds to the chambers. Relaxant responses are expressed as percentage of total relaxation (loss in tone) induced by the addition of 0.1 mM papaverine HCl to the chambers at the end of the experiment. All data are expressed as mean ± S.E.

Organ Chamber Studies. Strips of corpus cavernosum tissue (3 × 3 × 7 mm) were immersed in 8-ml organ chambers containing PSS, maintained at 37°C, and aerated with 95% O₂, 5% CO₂, pH 7.4. Each tissue strip was incrementally stretched to optimal isometric tension, as determined by the maximal contractile response to 1 µM phenylephrine (Kim et al., 1991; Azadzoi et al., 1992). Relaxant responses were evaluated by adding increasing cumulative concentrations of compounds to strips contracted with 0.5 µM phenylephrine. Relaxation responses are expressed as percentage of total relaxation (loss in tone) induced by the addition of 0.1 mM papaverine HCl to the chambers at the end of the experiment. All data are expressed as mean ± S.E. In studies using a combination of relaxant compounds, a molar proportion of 1:100, PGE₁:SNO-Glu, was chosen based on the EC₅₀ value for penile smooth muscle relaxation for each compound individually.

Measurement of Cyclic Nucleotides in Human Corpus Cavernosum Tissue. Corpus cavernosum strips were immersed in 8-ml organ chambers containing PSS, maintained at 37°C, and aerated with 95% O₂, 5% CO₂, pH of 7.4. Each tissue strip was incrementally stretched to optimal isometric tension, as determined by the maximal contractile response to 1 µM phenylephrine. Then each tissue was treated with the phosphodiesterase inhibitors zaprinast (30 µM) and 3-isobutyl-1-methylxanthine (100 µM) and allowed to incubate for 15 min, after which time tissues were treated with drug or vehicle. Tissues were allowed to incubate for another 30 min and then immediately frozen in liquid nitrogen and stored at 80°C until extraction for the cyclic nucleotide assay. Tissues were extracted by homogenization in 6% trichloroacetic acid followed by ether (H₂O-saturated) extraction and lyophilization. Cyclic nucleotides were determined by enzyme-linked immunosorbent assay using a kit from Cayman Chemical Co. (Ann Arbor, MI).

Protein Determinations. Protein were determined using the Bio-Rad Protein Assay kit microtiter plate assay procedure (Bio-Rad, Hercules, CA) with BSA as the standard.

Drugs and Materials. Phenylephrine, norepinephrine (arterenol), and zaprinast were obtained from Sigma Chemical Co. (St. Louis, MO). 3-Isobuytl-1-methylxanthine was obtained from Research Biochemicals International (Natick, MA). PGE₁--cyclodextrine was kindly provided by Schwarz-Pharma (Monheim, Germany). SNO-Glu was kindly provided by Nitromed Inc. (Bedford, MA). Drugs were dissolved in distilled water at the time of the experiment.

Statistical Evaluation. A total of 57 patients was included in the study of individual responses to PGE₁. We obtained penile resistance arteries from 22 of them and trabecular tissue from 39 (both arteries and cavernosal strips were collected from four patients). Individual EC₅₀ (concentration required to obtain the half-maximum response induced by the compound) and maximum relaxations to PGE₁ were calculated.

	Results

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Evaluation of Individual Responses to PGE₁ in Human Penile Smooth Muscle. Relaxations induced by PGE₁ were individually studied in strips of corpus cavernosum from 39 impotent patients and in penile resistance arteries from 22 patients (Fig. 1). Notable variability was observed with respect to the relaxant responses produced by PGE₁ in trabecular smooth muscle from different patients, resulting in a range of several orders of magnitude in individual EC₅₀ values (mean 0.226 µM, standard deviation 0.418, range from 0.01 to 10 µM) and variability in maximal relaxation (mean 83.5%, standard deviation 12.0, range from <60 to 100%). Similar results were obtained when PGE₁-induced relaxations were evaluated in individual penile resistance arteries, showing variability in EC₅₀ values (mean 0.896 µM, standard deviation 1.097, range from 0.01 to 10 µM), and more noteworthy in maximal relaxation (mean 73.3%, standard deviation 23.2, range from 50 to 100%).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Individualized responses to PGE1 (1 nM to 10 µM) in human corpus cavernosum strips (top) contracted with phenylephrine (1 µM) and human penile resistance arteries (bottom) contracted with norepinephrine (1 µM) from 39 and 22 impotent patients, respectively. Data are expressed as the percentage of total relaxation induced by 0.1 mM papaverine.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   EC50 values for PGE1 (top) and maximum relaxation responses to PGE1 (bottom) obtained in human corpus cavernosum strips contracted with phenylephrine (1 µM) (left) and human resistance penile arteries contracted with norepinephrine (1 µM) (right). The tissues were divided into two groups depending on the patients clinical erectile response (poor, n = 20 and n = 14; partial or complete, n = 19 and n = 8 for trabecular tissue strips and penile arteries, respectively) to intracavernosal injection of alprostadil (PGE1). EC50 is defined as the concentration of PGE1 (in µM) required to obtain half of the maximum response to this drug. Maximum relaxation data are expressed as the percentage of total relaxation induced by 0.1 mM papaverine. *P < .05 and **P < .01 between groups by two-tailed Student's t test.

Relaxant Responses Induced by the Combination of SNO-Glu and PGE₁ on Human Penile Smooth Muscle. The nitrosothiol SNO-Glu produced consistent relaxations of human corpus cavernosum strips and penile resistance arteries in a concentration-dependent manner. Administration of SNO-Glu always elicited a near-to-full relaxation of both penile smooth muscle preparations even when tissue from the same patient responded poorly to PGE₁. Representative examples of this observation are shown in Fig. 3. The coadministration of PGE₁ and SNO-Glu in a 1:100 proportion (PGE₁:SNO-Glu) caused concentration-dependent relaxation of both trabecular and arterial smooth muscle.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Tracings of concentration-response curves to PGE1 (0.1 nM to 10 µM) and SNO-Glu (10 nM to 1 mM) in human corpus cavernosum strips contracted with 1 µM phenylephrine (PE) (top) and penile resistance arteries contracted with 1 µM norepinephrine (NE) (bottom). Note the poor response to PGE1 and full relaxation to SNO-Glu. Concentrations are expressed as the log of molarity. Concentration-response curves to PGE1 and SNO-Glu were performed in parallel tissues from the same patient.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Concentration-response curves to PGE1 (0.1 nM to 10 µM) and to the combination of PGE1 (0.1 nM to 10 µM) and SNO-Glu (PGE1/SNO-Glu, in 1:100 M ratio) in human corpus cavernosum strips contracted with phenylephrine (1 µM) (top) and human penile resistance arteries contracted with norepinephrine (1 µM) (bottom). The concentration-response curve obtained with the combination of PGE1 and SNO-Glu is plotted only as PGE1 concentrations. Data are expressed as mean ± S.E. of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. Concentration-response curves to PGE1 and to the combination of PGE1 and SNO-Glu were carried out in parallel tissues from the same patients. *, two-factor ANOVA indicates significant differences (P < .01) between the concentration-response curve obtained with the combination of PGE1 and SNO-Glu and that elicited by PGE1 alone.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Concentration-response curves to SNO-Glu (10 nM to 1 mM) and to the combination of PGE1 and SNO-Glu (10 nM to 1 mM; PGE1/SNO-Glu, in 1:100 M ratio) in human corpus cavernosum strips contracted with phenylephrine (1 µM) (top) and human penile resistance arteries contracted with norepinephrine (1 µM) (bottom). The concentration-response curve obtained with the combination of PGE1 and SNO-Glu is plotted only as SNO-Glu concentrations. Data are expressed as mean ± S.E. of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. Concentration-response curves to SNO-Glu and to the combination of PGE1 and SNO-Glu were carried out in parallel tissues from the same patients. *, two-factor ANOVA indicates significant differences (P < .01) between the concentration-response curve obtained with the combination of PGE1 and SNO-Glu and that elicited by SNO-Glu alone in human corpus cavernosum strips.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Analysis of synergism of the relaxations elicited by the combination of PGE1 and SNO-Glu in human corpus cavernosum strips (top) and penile resistance arteries (bottom). Starting from the responses obtained with the two compounds individually, the theoretical concentrations of the drugs in the combination required to achieve several specified levels of relaxation, when additive effects are assumed, were calculated and compared with those experimentally determined. Data are expressed as mean ± S.E. of the concentrations of PGE1 (in nM) present in the combination (concentration of SNO-Glu is 100-fold higher). n indicates the number of patients from whom the tissues were collected for the determinations. Synergistic effects were observed in human trabecular smooth muscle (*P < .05 versus theoretical additive effects by two-factor ANOVA) but not in penile resistance arteries.

Effect of the Combination of PGE₁ and SNO-Glu on Cyclic Nucleotide Accumulation in Corpus Cavernosum Tissue. Tissue levels of cGMP were not significantly altered by incubation with PGE₁ (1 µM), but a remarkable increase was observed when treating the tissues with SNO-Glu (100 µM). Incubation of trabecular tissues with the combination of PGE₁ and SNO-Glu induced a significant increase in cGMP content compared with control (Fig. 7, top).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   cGMP (top) and cAMP (bottom) tissue content of human corpus cavernosum after exposure to PGE1 (1 µM), SNO-Glu (100 µM), or both. Data are expressed as mean ± S.E. of picomoles of cGMP or cAMP per milligram of tissue protein content. n indicates the number of patients from whom the tissues were collected for the determinations. *P < .05, **P < .01 versus control. (Student-Newman-Keuls post hoc test).

	Discussion

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In clinical trials, response rates in the range of 40 to 70% to intracavernosal injection of PGE₁ have been reported in patients suffering from erectile dysfunction (Buvat et al., 1996, 1998; Linet and Ogring, 1996; Porst, 1996). This observation, supported by extensive clinical experience, presents intracavernous injection of PGE₁ as an efficacious treatment for impotence. However, a considerable number of patients do not respond to such treatment for unknown reasons. Furthermore, the dose of PGE₁ required to achieve satisfactory erections is markedly variable, ranging from 0.5 to 20 µg (Linet and Ogring, 1996), with some patients needing doses as high as 40 µg to obtain responses (Porst, 1996).

Analysis of in vitro responses to PGE₁ in penile arterial and trabecular tissues from impotent patients shows a great degree of variability in the relaxation of human penile smooth muscle to this prostanoid. The variability of PGE₁-induced relaxations was observed in human penile resistance arteries as well as corpus cavernosum strips and it affected both the sensitivity to PGE₁, obtaining EC₅₀ values separated by several orders of magnitude, and the maximum relaxation, which varied, especially in penile arteries, from less than 50% to complete relaxation (100%). It is interesting to note that the in vitro response to PGE₁ in human penile tissues correlated with the clinical response to PGE₁ in the patients from whom the tissue was obtained. Our results show that poor responses to intracavernosal alprostadil are associated with diminished maximum response and increased EC₅₀ of the relaxations to PGE₁ in vitro. These data suggest that a lack of clinical response to PGE₁ may be, in many cases, due to the inability of this prostanoid to relax penile arterial and/or trabecular smooth muscle sufficiently. The mechanisms underlying reduced penile smooth muscle relaxation to PGE₁ are currently unknown.

The rationale for the use of a combination of PGE₁ with other drugs, for the treatment of erectile dysfunction, is to increase the number of responding patients and to diminish the dose of PGE₁ required to achieve adequate responses because this prostanoid may cause penile pain. To date, the most widely used combination therapy includes PGE₁, papaverine, and phentolamine, which has been shown to improve response rates (Padma-Nathan, 1990; McMahon, 1991; von Heyden et al., 1993).

In the present study, we have investigated the possibility of combining the administration of PGE₁, which activates the cAMP pathway, with the activation of the NO/cGMP pathway, which plays a prominent role in the physiological mechanism of penile erection (Kim et al., 1991; Burnett et al., 1992; Rajfer et al., 1992; Trigo-Rocha et al., 1993). Pharmacological stimulation of cGMP production in penile smooth muscle can be achieved with NO donors, which are compounds that release NO but are more stable, allowing for their use as therapeutic drugs. These NO donors have been reported to relax penile smooth muscle (Sáenz de Tejada et al., 1989; Bush et al., 1992) and have been evaluated for the treatment of erectile dysfunction as a potential alternative treatment to PGE₁ (Truss et al., 1994; Martínez-Piñeiro et al., 1998).

Our results show that SNO-Glu, a nitrosothiol present in plasma, is an effective relaxant of human penile resistance arteries and human trabecular smooth muscle. When the tissues, primarily arteries but also trabecular smooth muscle, relaxed poorly to PGE₁, SNO-Glu alone was able to induce a strong response (near full relaxation) in those tissues. The combination with SNO-Glu improved the relaxations to PGE₁ in human penile arteries and in trabecular smooth muscle, causing a shift to the left of the PGE₁ concentration-response curve and a larger maximum relaxation compared with PGE₁ alone. Although in penile arteries the combination of PGE₁ and SNO-Glu did not improve the relaxation response to SNO-Glu alone, this combination did relax human trabecular smooth muscle more efficiently than SNO-Glu or PGE₁ alone. Furthermore, a synergistic interaction between the two compounds was observed when given in combination to relax trabecular smooth muscle, as shown in the analysis of synergism using the isobole method (Berenbaum, 1989). The constructed theoretical curve for the existence of additive effects between PGE₁ and SNO-Glu predicted significantly higher concentrations of both drugs than those experimentally determined to produce the same level of relaxation, confirming the existence of synergism. Based on these results, we propose that the combination of PGE₁ and SNO-Glu has significant advantages for the relaxation of penile trabecular smooth muscle compared with the separate administration of these drugs.

Improved relaxant properties of the combination of PGE₁ and SNO-Glu are probably due to the ability of this treatment to trigger the activity of the two essential pathways responsible of penile smooth muscle relaxation (i.e., cAMP and cGMP pathways). The activation of both pathways was demonstrated in our experiments by the accumulation of both cyclic nucleotides in human cavernosal tissue after treatment with the combination. Potentiation of the NO/cGMP pathway with oral sildenafil together with local PGE₁ is used on occasions in the clinical management of patients who are partial responders to PGE₁.

The existence of synergism between NO and cAMP is not well recognized in human penile smooth muscle. Furthermore, other investigators have tested the effect of combining two other molecules, vasoactive intestinal polypeptide and SIN-1, stimulants of cAMP and cGMP, respectively, in human corpus cavernosum and cavernous artery, reporting a lack of synergistic interaction (Hempelmann et al., 1995). The difference between the results of Hempelmann et al. (1995) and those of our study may be due to the specificity of stimulating different receptor pathways. The combination of PGE₁ with SIN-1 has been shown to be more effective to produce erections than PGE₁ or SIN-1 individually, when evaluated in a clinical trial involving 50 impotent patients (Tordjman, 1993). Our study provides new information that may explain the mechanism underlying this clinical finding. The synergistic interaction of PGE₁ and SNO-Glu was specific to trabecular smooth muscle because it was not observed in penile arteries, despite the fact that these two tissues are anatomically related, and functionally key to the erectile process.

In summary, the present study characterizes the responses to PGE₁ in human trabecular smooth muscle and penile resistance arteries, which both show large variability in response to PGE₁. We found a correlation of the in vitro response with the clinical erectile response that had not been previously reported. These results may explain why some patients respond and others do not to intracavernosal PGE₁. This study also demonstrates that SNO-Glu consistently relaxes penile smooth muscle whether it relaxes well or not to PGE₁. This suggests that the clinical response to PGE₁ may be limited in some patients by the specific lack of response of penile smooth muscle to this prostanoid, while maintaining the ability to relax in response to agents that activate alternative relaxant pathways. We also found that a combination of PGE₁ and SNO-Glu has a synergistic interaction to relax penile trabecular smooth muscle. Such a combination may have significant therapeutic advantages in the treatment of male erectile dysfunction.