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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
luDepartment of Pharmacology, Çukurova University Medical School, Adana, Turkey (C.G., H.S.B., O.K., E.K.K.); and Department of Integrative Biology and Pharmacology, The University of Texas at Houston Medical School, Houston, Texas (A.Y.K., F.M.)
Received August 15, 2005; accepted October 26, 2005.
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-nitro-L-arginine did not alter the relaxations to FPTO or sodium nitroprusside, whereas soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) suppressed relaxation to FPTO and sodium nitroprusside. Exogenously added thiols L-cysteine or dithiothreitol inhibited the relaxant responses to FPTO but not to sodium nitroprusside, whereas glutathione did not influence the responses to both agents. Thiol alkylation agent N-ethylmaleimide significantly enhanced FPTO-induced relaxation, and thiol-modifying agent diamide inhibited relaxation to FPTO. The potentiating effect of N-ethylmaleimide was neutralized by coadministration of N-ethylmaleimide with glutathione, L-cysteine, dithiothreitol, or ODQ. N-Ethylmaleimide but not diamide significantly inhibited relaxation induced by sodium nitroprusside. FPTO potently suppressed contraction to electrical field stimulation, which was prevented by glutathione or L-cysteine. In addition, FPTO did not affect relaxation produced by electrical field stimulation in phenylephrine-precontracted tissue. Our results show that FPTO can relax mouse corpus cavernosum and that the relaxant activity of this agent is thiol- and soluble guanylate cyclase-dependent. This effect could be potentiated by N-ethylmaleimide. FPTO does not potentiate nitrergic relaxation induced by electrical field stimulation.
). Nitric oxide is a potent activator of soluble guanylate cyclase (sGC), which catalyzes the biosynthesis of cGMP. cGMP causes a trabecular smooth muscle relaxation in the penis, facilitating blood flow into the cavernous tissue and thus eliciting an erection (Saenz de Tejada, 1992).
Certain conditions, including aging and vascular and metabolic diseases, can result in the impaired synthesis, release, or bioavailability of NO in the autonomic nerves or endothelium in penile tissue (Saenz de Tejada, 2004). This results in erectile dysfunction due to insufficient relaxation of cavernous smooth muscle and dilation of penile arteries. Certain prodrugs, which can release nitric oxide or nitric oxide-like species under physiological conditions (nitric oxide donors), have been widely used in the therapy of erectile dysfunction. Recently, compounds that potentiate nitric oxide-cGMP signaling and reduce the breakdown of cGMP have been shown to be effective in improving erectile function (Saenz de Tejada, 2004). However, it must be noted that particular lesions in the mechanisms involving endogenous nitric oxide can reduce efficacy of these substances as well. Therefore, some new drugs, such as YC-1 and BAY41-2272, which activate soluble guanylate cyclase via a nitric oxide-independent mechanism, have been investigated in human and rabbit corpus cavernosum in vitro (Friebe and Koesling, 1998; Kalsi et al., 2003). Recently, 4,7-dimethyl-1,2,5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide (FPTO) was synthesized and shown to stimulate soluble guanylate cyclase in a nitric oxide-independent mechanism (Kots et al., 2000). Because the molecule of FPTO comprises a pyridazine di-N-oxide ring condensed with a furoxan moiety, this drug can also generate nitric oxide and nitric oxide-like species, such as nitroxyl and S-nitrosoglutathione, either spontaneously or thiol-dependently (Calvino et al., 1992; Feelisch et al., 1992; Medana et al., 1994; Ferioli et al., 1995). In addition, it was shown that FPTO is an efficient smooth muscle relaxant and vasodilator in rat aortic rings (Kots et al., 2000).
These properties of FPTO suggest that this activator of sGC might relax smooth muscle of corpus cavernosum and can be potentially used for the treatment of erectile dysfunctions, which have been attributed in part to a lack of endogenous nitric oxide production (Saenz de Tejada, 2004). Mouse corpus cavernosum has been used previously for in vivo and in vitro studies of erectile mechanisms (Burnett et al., 1996; Göçmen et al., 1997, 1998; Sezen and Burnett, 2000). Therefore, in the present study, we demonstrated that FPTO is a very potent relaxant in the cavernous tissue. We further analyzed the effect of FPTO on the tone and nitrergic relaxation responses of isolated mouse corpus cavernosum and compared them to those of a known nitric oxide donor, sodium nitroprusside. Previously, it was shown that thiols and thiol-modifying compounds can influence FPTO-dependent activation of sGC in a crude extract of rat lung but not using a highly purified enzyme preparation or intact tissue (Kots et al., 2000). Because it is known that redox state of endogenous thiol groups in corpus cavernosum plays an important role in vascular reactivity (Göçmen et al., 1997, 1998; Mateo and de Artinano, 2000), we also examined the effect of FPTO in the absence or presence of N-ethylmaleimide (nonspecific thiol-alkylating agent), diamide (thiol-modifying agent), cysteine, glutathione, and dithiothreitol (thiol-specific reducing agent) (Murphy et al., 1991; Campbell et al., 1996; Garcia-Pascual et al., 1999, 2000; Resim et al., 2002). To further investigate the mechanism of FPTO-dependent relaxation, we compared the effects of thiols and thiol-modifying substances on FPTO- and sodium nitroprusside-induced activation of highly purified sGC and in organ bath experiments as well as production of nitric oxide from FPTO. The results indicate that FPTO thiol-dependently induces relaxation of cavernous tissue by a dual mechanism involving direct activation of sGC without intermediacy of NO and indirect activation of the enzyme mediated by NO.
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). Immediately prior to the experiment, the electrode was calibrated with NaNO2-KI system as recommended by the manufacturer. Signal from the sensor was recorded with inNO software version 1.9 at 1-s increments. Incubation medium contained 50 mM sodium phosphate buffer, pH 7.4, 10 µM FPTO, and other additions as indicated. Samples were incubated aerobically under constant stirring in a final volume of 1 ml. Baseline was recorded for 3 to 5 min, and then FPTO solution was added to the sample. The signal was then allowed to stabilize for 1 to 2 min, and glutathione, L-cysteine, and dithiothreitol were added.
Assay of Soluble Guanylate Cyclase Activity. Human recombinant soluble guanylate cyclase was expressed in Sf9 insect cells and purified as described previously (Martin et al., 2003). The enzyme (4-20 µg/ml; 200-500 ng/50 µl sample) was incubated in the presence of 5 mM MgCl2, 1 mM GTP, and 1 mg/ml bovine serum albumin in 50 mM triethanolamine-HCl buffer, pH 7.6, for 10 min at 37°C with various thiols and activators. Reaction was stopped by boiling for 2 min, and amount of cGMP formed was determined by radioimmunoassay (Brooker et al., 1979).
To assess inhibition of the enzyme by N-ethylmaleimide and diamide, soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) was treated with 25 µM N-ethylmaleimide or 100 µM diamide for 30 min at 4°C in 50 mM triethanolamine-HCl buffer, pH 7.6, containing 1 mg/ml bovine serum albumin and various thiols. MgCl2 and GTP then were added to final concentrations of 5 and 1 mM, respectively. Samples were incubated for 10 min at 37°C, and the amount of cGMP was measured as above.
Organ Bath Experiments. Male albino mice weighing 30 to 35 g were killed by cervical dislocation. Penises were removed and placed in a Petri dish containing Krebs' solution (119 mM NaCl, 4.6 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 15 mM NaHCO3, 1.2 mM NaHPO4, and 11 mM glucose). Corpus cavernosum was prepared according to the previously described method (Göçmen et al., 1997). The preparations were mounted under a 0.2-g tension in a 5-ml organ bath maintained at 37°C and containing Krebs' solution aerated with 95% O2 and 5% CO2. The tissue was allowed to equilibrate for 1 h. During this period, the preparation was washed with fresh Krebs' solution at 15-min intervals. The responses were recorded on a polygraph (Gemini 7070; Ugo Basile, Comerio VA, Italy) via an isotonic transducer (Gemini 7006; Ugo Basile).
After the equilibration period, the tissue was treated with 5 µM phenylephrine. The active tone reached a stable level within 5 min; at the end of this period, FPTO or sodium nitroprusside was cumulatively added to the organ bath at 0.5 to 10 µM (FPTO) or 0.05 to 1 µM (sodium nitroprusside). Thus, the first series of responses was obtained. After a 30-min washout period, the second series of responses were recorded in a similar manner. In some experiments, after the first series of responses were recorded, the tissue was incubated in a medium containing nitric-oxide synthase inhibitor N-nitro-L-arginine (100 µM), glutathione (1 mM), or L-cysteine (1 mM); selective soluble guanylate cyclase inhibitor ODQ (2 µM), N-ethylmaleimide (25 µM), or diamide (100 µM); thiol-specific reducing agent dithiothreitol (1 mM); nitric oxide binding agent hydroxocobalamin (100 µM); or superoxide-generating agent pyrogallol (100 µM). In some experiments, N-ethylmaleimide (25 µM) was applied to the tissue in combination with glutathione (1 mM), L-cysteine (1 mM), ODQ (2 µM), dithiothreitol (1 mM), or N-nitro-L-arginine (100 µM). Diamide (100 µM) was applied in combination with glutathione or L-cysteine. In preliminary experiments, we have studied a broad range of concentration of N-ethylmaleimide (1-100 µM) and diamide (10-200 µM). However, it was found that N-ethylmaleimide at concentrations over 25 µM and diamide at concentrations over 100 µM dramatically reduced the phenylephrine-dependent contractions. Thus, the use of high concentrations of N-ethylmaleimide or diamide seems to alter the contractile response of the tissue and would make it impossible to analyze the relaxant responses. Hence, 25 µM N-ethylmaleimide and 100 µM diamide were the highest concentrations of these drugs that do not affect the phenylephrine-induced tonus. At these concentrations, we obtained reproducible and effective responses to relaxing agents. All incubations continued for 30 min, and then cumulative concentration-response curves for FPTO or sodium nitroprusside were recorded. In another group of experiments, the tissue was contracted with electrical field stimulation delivered for 30 s as square waves (2, 4, 8, and 16 Hz; 30 V; 0.5-ms duration) by a Grass S88 stimulator (Grass, West Warwick, RI) via two parallel platinum electrodes embedded in Plexiglas for 30 s at each frequency. The effect of FPTO (1 µM) or sodium nitroprusside (1 µM) and the combination of these drugs with glutathione (1 mM) or L-cysteine (1 mM) on the contraction elicited by electrical field stimulation (2-16 Hz; 30 V; 0.5 ms) in N-nitro-L-arginine (100 µM) containing medium was studied to evaluate the effect of FPTO on neurogenic contractions. In some of these separate experiments, we examined the combination of FPTO with N-ethylmaleimide (25 µM) or diamide (100 µM) on the contractions to electrical field stimulation.
In another experimental group, we studied the effect of FPTO (1 and 5 µM), ODQ (2 µM), or N-nitro-L-arginine (100 µM) on relaxation induced by electrical field stimulation (2-16 Hz; 30 V; 0.5 ms) in the tissue precontracted with phenylephrine (5 µM) to evaluate a potentiating action of FPTO on nitrergic relaxations. In experiments in which electrical field stimulation-induced relaxations were studied, atropine (0.2 µM) and guanethidine (1 µM) were always present in the bathing medium to obtain nonadrenergic-noncholinergic conditions (Göçmen et al., 1997).
Drugs and Solutions. All drugs, with the exception of FPTO, were obtained from Sigma-Aldrich (St. Louis, MO). FPTO was a generous gift of Drs. N. Makhova and I. Ovchinnikov (Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia). Stock solutions of N-nitro-L-arginine (N-5501), glutathione (G-6529), L-cysteine (C-7880), N-ethylmaleimide (E-3876), diamide (D-3648), dithiothreitol (D-5545), hydroxocobalamin (H-7126), pyrogallol (P-0381), phenylephrine (P-6126), and sodium nitroprusside (S-0501), with the exception of FPTO, were dissolved in distilled water. FPTO and ODQ were dissolved in dimethyl sulfoxide (D-5879) (final concentration in the bath medium was 0.1%).
Statistics. Relaxations were calculated as percentage peak reduction of contraction elicited by phenylephrine (mean ± S.E.). All of the data were evaluated with the Bonferroni corrected t test that was used in analysis of variance. P values of less than 0.05 were considered significant.
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), the formation of nitric oxide from FPTO has been shown only by indirect methods. To obtain additional confirmation that FPTO is indeed a thiol-dependent nitric oxide donor, generation of nitric oxide from FPTO was evaluated by direct polarographic method using nitric oxide sensor amiNO-2000. The data of Fig. 1 indicate that FPTO does not generate nitric oxide in the absence of reducing agents. In the presence of thiols, FPTO rapidly decomposes and generates a high amount of nitric oxide after the addition of cysteine or glutathione. However, nitric oxide formation was not detected in the presence of dithiothreitol. FPTO did not react with 25 µM N-ethylmaleimide under similar conditions as determined in a separate experiment (data not shown). It should be noted that sodium nitroprusside under similar conditions failed to generate detectable level of nitric oxide.
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To further characterize the mechanism of FPTO-induced stimulation of soluble guanylate cyclase, the effect of this substance was studied in a highly purified preparation of the human enzyme isolated in the absence or presence of thiols. The data shown in Table 1 indicate that soluble guanylate cyclase was efficiently activated by FPTO in the presence of cysteine and glutathione, but dithiothreitol caused a considerable inhibition on soluble guanylate cyclase activation by FPTO (Table 1). FPTO was also able to stimulate cGMP synthesis by soluble guanylate cyclase in the absence of added thiols, thus suggesting a possibility of direct activation of the enzyme by FPTO not involving intermediate formation of nitric oxide. However, sodium nitroprusside stimulation of soluble guanylate cyclase activity was not sensitive to thiols and was not suppressed by dithiothreitol (Table 1).
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Results presented in Table 2 indicate that thiol-modifying agents N-ethylmaleimide and diamide can suppress sodium nitroprusside-mediated activation of soluble guanylate cyclase. However, only N-ethylmaleimide but not diamide inhibited stimulation of the enzyme by FPTO. The inhibitory effect of N-ethylmaleimide and diamide on sodium nitroprusside-stimulated activity was abolished in the presence of thiol-containing reagents glutathione, cysteine, and dithiothreitol. Inhibition of FPTO-stimulated soluble guanylate cyclase by N-ethylmaleimide was effectively neutralized by glutathione and cysteine. Dithiothreitol on its own inhibited the enzyme stimulation by FPTO.
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Relaxant Effects of FPTO and Sodium Nitroprusside in Mouse Corpus Cavernosum. FPTO (0.5-10 µM) or sodium nitroprusside (0.05-1 µM) relaxed mouse corpus cavernosum in a concentration-dependent manner (Fig. 2). FPTO-induced relaxation was relatively slow to develop, whereas sodium nitroprusside-induced relaxation developed very rapidly. However, the onset of the relaxation induced by FPTO became much more rapid after the addition of 25 µM N-ethylmaleimide into the bath (Fig. 2a).
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-Nitro-L-arginine and ODQ on Relaxation to FPTO and Sodium Nitroprusside. N-Nitro-L-arginine (100 µM) did not alter the relaxation to FPTO (0.5-10 µM) and sodium nitroprusside (0.05-1 µM), whereas 2 µM ODQ significantly decreased the relaxation to the low concentrations of FPTO or sodium nitroprusside (Table 3). Thus, both FPTO and sodium nitroprusside are apparently endothelium-independent vasorelaxants directly stimulating smooth muscle soluble guanylate cyclase in the cavernosal tissue.
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Effect of Thiols and Thiol Modulators on Relaxation to FPTO. Glutathione (1 mM) slightly enhanced the response to FPTO, but this increase was not statistically significant (Fig. 3). On the other hand, another thiol, L-cysteine (1 mM), significantly inhibited the relaxation evoked by low concentrations of FPTO (Fig. 3). In addition, a thiol-specific reducing agent dithiothreitol (1 mM) caused significant inhibition of relaxation induced by FPTO (Fig. 3).
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Thiol-alkylating agent N-ethylmaleimide (25 µM) had no influence on phenylephrine-induced contraction as was determined in a separate set of experiments. However, N-ethylmaleimide significantly enhanced FPTO-induced relaxation (Figs. 2a and 4). The enhancement was especially pronounced at low concentrations of FPTO. This potentiating effect of N-ethylmaleimide was reversible after washout of the agent (data not shown) and was not detected when N-ethylmaleimide was added in combination with glutathione (1 mM), L-cysteine (1 mM), dithiothreitol (1 mM), or ODQ (2 µM) (Fig. 4; the data of the combination of diamide with dithiothreitol was not shown). In addition, the potentiating effect of N-ethylmaleimide (25 µM) on relaxation to FPTO was observed in the medium containing 100 µM L-nitroarginine (Table 3). However, another thiol-alkylating agent, diamide (100 µM), caused significant inhibition of relaxations to FPTO (Fig. 5). Inhibition of FPTO-stimulated relaxation by diamide was not detected when the tissue was treated with the combination of this drug with glutathione (1 mM) or L-cysteine (1 mM) (Fig. 5).
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Effect of Thiols and Thiol Modulators on Relaxation to Sodium Nitroprusside. Glutathione (1 mM), L-cysteine (1 mM), or dithiothreitol (1 mM) did not influence the responses to sodium nitroprusside (0.05-1 µM) (data not shown). N-Ethylmaleimide (25 µM), but not diamide (100 µM), caused significant inhibition of relaxation induced by sodium nitroprusside (0.05-1 µM) (Figs. 2b and 6). However, this inhibition was not detected when N-ethylmaleimide was used in combination with glutathione (1 mM) or L-cysteine (1 mM) (Fig. 6).
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Effect of Hydroxocobalamin or Pyrogallol on Relaxation to FPTO and Sodium Nitroprusside. Incubation of the tissue with pyrogallol (100 µM) or hydroxocobalamin (100 µM) did not significantly affect relaxations evoked by FPTO (0.5-10 µM) (Table 3). On the other hand, hydroxocobalamin (100 µM) caused significant inhibition of relaxation to low concentrations of sodium nitroprusside (0.05-0.5 µM) and pyrogallol (100 µM) had no effect on sodium nitroprusside-induced relaxation (Table 3).
Effect of FPTO and Sodium Nitroprusside on Contraction Induced by Electrical Field Stimulation. FPTO (1 µM) or sodium nitroprusside (1 µM) significantly inhibited contraction to electrical field stimulation (2-16 Hz) (Figs. 7 and 8A; Table 4). The inhibitor effect of FPTO on the contraction to electrical field stimulation was not detected when FPTO was coadministered with glutathione (1 mM) or L-cysteine (1 mM) (Fig. 7). On the other hand, these exogenous thiols did not influence the sodium nitroprusside-induced inhibition of contractile responses evoked by electrical field stimulation (Table 4). In addition, the combination of FPTO (1 µM) with N-ethylmaleimide (25 µM) or diamide (100 µM) exhibited more pronounced inhibition of the contraction to electrical field stimulation (data not shown). L-Cysteine (1 mM) or glutathione (1 mM) did not affect the neurogenic contraction, whereas N-ethylmaleimide (25 µM) or diamide (100 µM) significantly inhibited neurogenic contraction (data not shown).
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Effect of FPTO on Relaxations Induced by Electrical Field Stimulation. Electrical field stimulation (2-16 Hz; 30 V; 0.5 ms) elicited reproducible frequency-dependent relaxation responses (Fig. 8B). These relaxations were completely inhibited by N-nitro-L-arginine (100 µM) and ODQ (2 µM) (data not shown). Two concentrations (1 and 5 µM) of FPTO were chosen to study the effect of the drug on these nitrergic responses. These concentrations did not affect the duration or magnitude of relaxation responses at all frequencies (2-16 Hz) in the tissue precontracted with phenylephrine (Fig. 8B; as percentage relaxation, 14.5 + 0.7, 25.9 + 1.3, 43.1 + 1.3, and 45.1 + 0.9 at 2, 4, 8 and 16 Hz, respectively, for control; and 14.4 + 0.8, 27.8 + 1.2, 41.1 + 0.7, and 44.8 + 1.2 at 2, 4, 8, and 16 Hz, respectively, for 5 µM FPTO, respectively).
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). All of these species can account for FPTO-dependent activation of soluble guanylate cyclase and be responsible for potent vasodilatory activity of FPTO. It was also shown that FPTO can induce stimulation of sGC apparently via a direct binding to the heme moiety of sGC. However, what mechanisms are important for FPTO-dependent relaxation remains largely unknown.
The results of the present study suggest that FPTO induces relaxation of the mouse corpus cavernosum and that the relaxant activity of FPTO is mediated predominantly by stimulation of soluble guanylate cyclase, similar to the effect of FPTO in rat aortic rings (Kots et al., 2000). Our data show that this relaxation can be partially attributed to the generation of nitric oxide from FPTO and also to direct activation of soluble guanylate cyclase by FPTO via a nitric oxide-independent mechanism and that this relaxant effect of FPTO can be regulated by endogenous thiols in the tissue as well as exogenous thiols added to the organ bath. The interesting finding of the present study is that N-ethylmaleimide potentiated the relaxations to FPTO.
FPTO caused a relaxation resembling the effect of sodium nitroprusside, and FPTO action was developing slowly (Fig. 2). In the present study, the formation of nitric oxide from FPTO in the presence of cysteine and glutathione was confirmed by direct polarographic assay with nitric oxide electrode (Fig. 1). However, the ineffectiveness of N-nitro-L-arginine, a nitric oxide synthase inhibitor, on responses to FPTO suggests that an endogenous L-arginine/nitric oxide pathway is of minor importance in the mechanism of FPTO-induced relaxation. In addition, guanylate cyclase inhibitor ODQ could inhibit the relaxant responses to both FPTO and sodium nitroprusside. This finding suggests that the vasorelaxant activity of FPTO is mainly due to stimulation of soluble guanylate cyclase in the cavernous smooth muscle cells and is endothelium-independent.
Activation of purified soluble guanylate cyclase by FPTO was not substantially influenced by cysteine or glutathione but was suppressed by dithiothreitol (Table 1). However, suppression of relaxant activity of FPTO by cysteine as well as dithiothreitol may indicate that formation of nitric oxide from FPTO is of little or no importance for relaxation of the tissue. It seems that most of the effects of FPTO are due to direct nitric oxide-independent interaction of FPTO with soluble guanylate cyclase, which enhances cGMP synthesis in the tissue. Likewise, it was previously suggested that FPTO elicited vasorelaxations in the rat aortic rings via activation of soluble guanylate cyclase and that nitric oxide is not apparently essential for the suggested mechanism of soluble guanylate cyclase activation at high concentrations of FPTO (Kots et al., 2000).
Hydroxocobalamin, a nitric oxide binding agent (Rajanayagam et al., 1993; Göçmen et al., 1998), and pyrogallol, a superoxide-generating agent (Gillespie and Sheng, 1990) and putative indirect nitric oxide scavenger (Göçmen et al., 1998), did not affect relaxation to FPTO (Table 2), thus suggesting that direct NO-independent sGC activation by FPTO plays a critical role in relaxant activity of the substance. In mouse corpus cavernosum, a possible role of S-nitrosoglutathione in the action of FPTO may be of low importance because hydroxocobalamin markedly inhibited the relaxant effect of S-nitrosoglutathione in this tissue (Göçmen et al., 1998). On the other hand, L-cysteine significantly decreased the relaxant responses to FPTO. Under certain specific conditions, L-cysteine can act as a nitroxyl scavenger (Ellis et al., 2000) and it was suggested that nitroxyl can induce direct activation of sGC (Wanstall et al., 2001). In addition, it was shown that FPTO can generate nitroxyl; hence, nitroxyl may contribute to the relaxant activity of FPTO in the rat aorta (Kots et al., 2000). In addition, the inhibitory effect of L-cysteine on relaxation to FPTO in the mouse cavernous tissue can be due to, at least in part, a possible nitroxyl-mediated effect of FPTO.
In the present study, thiol modulators N-ethylmaleimide and diamide exhibited different effects on the relaxant responses to FPTO. The interesting finding was that N-ethylmaleimide potentiated the relaxations induced by FPTO. Apparently, the potentiating effect of N-ethylmaleimide is mediated by soluble guanylate cyclase because ODQ could prevent this potentiating effect. Because N-ethylmaleimide can modify some reactive endogenous thiols in the tissue, it can considerably potentiate soluble guanylate cyclase stimulation by FPTO. However, stimulation of purified enzyme by FPTO was inhibited by N-ethylmaleimide (Table 2). The fact that glutathione or L-cysteine abolished the potentiating effect of N-ethylmaleimide may be due to inhibited interaction of N-ethylmaleimide with endogenous thiol groups of soluble guanylate cyclase (Ignarro, 1989; Murphy et al., 1991). For example, it is tempting to speculate that treatment of tissue with N-ethylmaleimide may inactivate the most reactive endogenous thiols that might otherwise contribute to decomposition of FPTO. This can increase the stability of FPTO in the tissue and enhance its activation of cGMP synthesis, again suggesting that direct nitric oxide-independent stimulation of the enzyme by FPTO substantially contributes to the pharmacological activity of this substance.
Surprisingly, another thiol-modifying agent diamide considerably suppressed FPTO-stimulated relaxation. It should be noted that diamide was used at substantially higher concentrations compared with N-ethylmaleimide (100 versus 25 µM, respectively). In addition, N-ethylmaleimide induces alkylation of thiols, whereas diamide mostly causes oxidation of sulfhydryl groups. On the other hand, diamide failed to inhibit FPTO-mediated activation of purified soluble guanylate cyclase. Therefore, it is possible that diamide may influence some other important intracellular components required for FPTO-induced relaxation. Additional studies are needed to clarify this phenomenon.
The mechanism of sodium nitroprusside-induced relaxation is clearly different. Glutathione, L-cysteine, or dithiothreitol did not affect the relaxant response to sodium nitroprusside (Table 3) as well as activation of purified soluble guanylate cyclase by sodium nitroprusside (Table 1). In addition, N-ethylmaleimide significantly inhibited the relaxations to sodium nitroprusside, whereas diamide had no significant effect. When the level of thiols in the tissue drops below some critical point, nitric oxide becomes highly susceptible to oxidation/degradation; thus, its effective concentration is decreased along with soluble guanylate cyclase activation/cGMP synthesis/relaxation (Ignarro et al., 1981; Aleryani et al., 1999; Garcia-Pascual, 1999, 2000). Reversal of the effect of N-ethylmaleimide in the presence of exogenously added glutathione and L-cysteine may confirm this suggestion.
In the present study, we also investigated the effect of FPTO and sodium nitroprusside on the neurogenic contractions induced by electrical field stimulation. FPTO or sodium nitroprusside inhibited the neurogenic contractions in a dose-dependent manner in the medium containing N-nitro-L-arginine, a nitric-oxide synthase inhibitor. FPTO and sodium nitroprusside caused a significant inhibition on the contractions. Administration of N-nitro-L-arginine resulted in electrical field stimulation-induced contraction beginning immediately after initiation of the stimulus and increased the magnitude and duration of the contraction in the corpus cavernosum tissue (Cellek and Moncada, 1997). It has been suggested that the lack of contractile response during stimulation is the result of release of nitric oxide, which also modulates the magnitude of the contraction (Li and Rand, 1989; Kasakov et al., 1995; Cellek and Moncada, 1997). In addition, it was shown that treatment with ODQ enhanced the nerve-evoked contractions, whereas zaprinast, a cGMP-phosphodiesterase inhibitor, decreased the contractile response to electrical field stimulation (Cellek and Moncada, 1997). In this tissue, FPTO may affect the contractile mechanism of the tissue by generating some nitric oxide-like species, thus activating soluble guanylate cyclase. Apart from this, FPTO may stimulate soluble guanylate cyclase directly via a nitric oxide-independent mechanism in the tissue similar to the mechanisms of relaxation discussed above. On the other hand, the combination of FPTO with glutathione and L-cysteine did not cause any inhibition of contraction to electrical field stimulation. These exogenous thiols had no influence on inhibitory effect of sodium nitroprusside, suggesting that their effect can be due to an interaction with certain endogenous thiols. In addition, N-ethylmaleimide or diamide potentiated the inhibition of neurogenic contraction induced by FPTO. These results further support the concept that FPTO interacts with endogenous thiols involved in the neurogenic contractile mechanisms in the cavernous tissue. Interestingly, FPTO had no significant effect on the electrical field stimulation-induced nitrergic relaxations that were completely inhibited by ODQ or N-nitro-L-arginine in this tissue (Rajfer et al., 1992; Göçmen et al., 1997). This result may suggest that FPTO does not synergize with endogenous nitric oxide in the cavernous tissue.
Our results show that FPTO can relax mouse corpus cavernosum and that the relaxant activity of this agent is thiol- and soluble guanylate cyclase-dependent. This effect could be potentiated by N-ethylmaleimide. The mechanism of the potentiating effect of N-ethylmaleimide on the relaxant responses of FPTO may be complex and apparently involves nitric oxide-independent activation of soluble guanylate cyclase and modification of endogenous thiols in the tissue. Therefore, FPTO may be a novel way of treating male erectile dysfunction in patients with impaired endogenous nitric oxide production.
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ABBREVIATIONS: sGC, soluble guanylate cyclase; NO, nitric oxide; YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole; BAY41-2272, 5-cy-clopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-yl amine; FPTO, 4,7-dimethyl-1,2,5-oxadiazolo[3,4-d]pyridazine 1,5, 6-trioxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; SNP, sodium nitroprusside.
Address correspondence to: Dr. Cemil Göçmen, Department of Pharmacology, Çukurova University Medical School, TR-01330 Adana, Turkey. E-mail: cgocmen{at}cu.edu.tr
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