Resveratrol Stimulates Hydrogen Sulfide (H2S) Formation to Relax Murine Corpus Cavernosum

2004

ORIGINAL RESEARCH Resveratrol Stimulates Hydrogen Sulfide (H2S) Formation to Relax Murine Corpus Cavernosum Gunay Yetik-Anacak, PhD,* Mehmet V. Dereli, BS Pharm,* Gulnur Sevin, PhD,* Ozge Ozzayım, BS Pharm,* Yasemin Erac, PhD,* and Asif Ahmed, PhD† *Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir, Turkey; †Aston Medical Research Institute, Aston Medical School, Aston University, Birmingham, UK DOI: 10.1111/jsm.12993

ABSTRACT

Introduction. Resveratrol (RVT) found in red wine protects against erectile dysfunction and relaxes penile tissue (corpus cavernosum) via a nitric oxide (NO) independent pathway. However, the mechanism remains to be elucidated. Hydrogen sulfide (H2S) is a potent vasodilator and neuromodulator generated in corpus cavernosum. Aims. We investigated whether RVT caused the relaxation of mice corpus cavernosum (MCC) through H2S. Methods. H2S formation is measured by methylene blue assay and vascular reactivity experiments have been performed by DMT strip myograph in CD1 MCC strips. Main Outcome Measures. Endothelial NO synthase (eNOS) inhibitor Nω-Nitro-L-arginine (L-NNA, 0.1 mM) or H2S inhibitor aminooxyacetic acid (AOAA, 2 mM) which inhibits both cystathionine-β-synthase (CBS) and cystathionine-gamma-lyase (CSE) enzyme or combination of AOAA with PAG (CSE inhibitor) has been used in the presence/absence of RVT (0.1 mM, 30 min) to elucidate the role of NO or H2S pathways on the effects of RVT in MCC. Concentration-dependent relaxations to RVT, L-cysteine, sodium hydrogen sulfide (NaHS) and acetylcholine (ACh) were studied. Results. Exposure of murine corpus cavernosum to RVT increased both basal and L-cysteine-stimulated H2S formation. Both of these effects were reversed by AOAA but not by L-NNA. RVT caused concentration-dependent relaxation of MCC and that RVT-induced relaxation was significantly inhibited by AOAA or AOAA + PAG but not by L-NNA. L-cysteine caused concentration-dependent relaxations, which are inhibited by AOAA or AOAA + PAG significantly. Incubation of MCC with RVT significantly increased L-cysteine-induced relaxation, and this effect was inhibited by AOAA + PAG. However, RVT did not alter the effect of exogenous H2S (NaHS) or ACh-induced relaxations. Conclusions. These results demonstrate that RVT-induced relaxation is at least partly dependent on H2S formation and acts independent of eNOS pathway. In phosphodiesterase 5 inhibitor (PDE-5i) nonresponder population, combination therapy with RVT may reverse erectile dysfunction via stimulating endogenous H2S formation. Yetik-Anacak G, Dereli MV, Sevin G, Ozzayım O, Erac Y, and Ahmed A. Resveratrol stimulates hydrogen sulfide (H2S) formation to relax murine corpus cavernosum. J Sex Med 2015;12:2004–2012. Key Words. Corpus Cavernosum; Resveratrol; Phosphodiesterase 5 Inhibitor; Hydrogen Sulfide; Erectile Dysfunction

J Sex Med 2015;12:2004–2012

© 2015 International Society for Sexual Medicine

Resveratrol Relaxes Mice Penis via Hydrogen Sulfide Introduction

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rectile dysfunction (ED) is an important indicator of poor health, which reduces quality of life. In United States alone, ED affects over 30 million men, and the prevalence of ED increases with age [1]. Normal penile erection is under the control of multiple factors and signaling pathways. Nitric oxide (NO) is known to regulate penile erection [2,3], and phosphodiesterase 5 inhibitors (PDE-5i) have significantly improved the quality of many men’s sexual life with an efficacy about 70% [4], although it is significantly lower in difficult-to-treat subpopulations (e.g., diabetes mellitus, radical prostatectomy), and there are remaining over 30% of patients who are classified as PDE-5i nonresponders [5]. The “French Paradox” of reduced cardiovascular events despite a high cholesterol-containing diet in French population has been atrributed to the protective effect of resveratrol (RVT) in red wine [6]. RVT has phytotherapeutic potential with antioxidant, calorie-restricting, anti-aging, cardioprotective effects [7]. Recent studies showed RVT can induce vasorelaxant effect in penile tissue [8–10] and appears to have beneficial effects in ED induced by hypertension, hypercholesterolemia, and diabetes [8,11,12]. Although RVT has been shown to induce relaxation of mice corpus cavernosum (MCC) independent of the NO pathway [13], the exact mechanism remains unknown. A vasodilator and neuromodulator H2S is generated by the enzymes cystathionine-gamma-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopyruvate sulphurtransferase (MPST) enzymes in corpus cavernosum [14–16]. We therefore investigated whether the vasodilator effect of RVT in MCC could be due to H2S release.

Methods

Animals The present study was approved by Institutional Review Board (7.201.2014.0002) as well as Animal Care and Use Committee of Ege University (February 26, 2014, number 2014-018), in agreement with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals. All experiments were conducted on 10–12 weeks of age male CD1 mice (n = 65) obtained from Breeding Center of Experimental Animals in Ege University (ARGEFAR). The

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animals were stunned by inhalation of CO2, sacrificed by decapitation, and exsanguinated.

Drugs and Treatments In some experiments, isolated MCC strips or homogenates were incubated with NOS inhibitor Nω-Nitro-L-arginine (L-NNA, 0.1 mM, 30 minutes) or CBS and CSE inhibitor (AOAA, 2 mM, 30 minutes) or combination of AOAA with CSE inhibitor (AOAA 2 mM + PAG 10 mM, 30 minutes) before vehicle or RVT exposure (0.1 mM, 30 minutes). RVTs were acquired from Santa Crus Biotechnology, Inc. (Santa Cruz, CA, USA). Unless otherwise stated, all other chemicals were obtained from Sigma. Isolation of Penile Tissue and Treatments The penis was excised at its base with removal of the glans penis and connective and adventitial tissues along the shaft. Two individual MCC strips (1 × 1 × 10 mm) from one animal were separated, mounted in 5-mL organ bath of strip myograph for isometric force recording (Danish Myograph Technology, Aarhus, Denmark) coupled to a PowerLab 8/SP data acquisition system (Chart 5.0 software; ADInstruments, Colorado Springs, CO, USA), and bathed in carboxygenated (95% O2; 5% CO2) modified Krebs–Ringer solution NaCl, 130 mM; NaHCO3, 14.9 mM; dextrose, 5.5 mM; KCl, 4.7 mM; KH2PO4, 1.18 mM; MgSO47H2O, 1.17 mM; and CaCl22H2O, 1.6 mM at 37°C. Tissues were allowed to equilibrate for 90 min under a resting tension of 5 mN. Experiments were done in strips with endothelium as confirmed by relaxation more than 50% to ACh (1 μM) after contraction with phenylephrine (Phe, 10 μM). One concentration–response curve was obtained in each MCC. In the later series of experiments, relaxant responses to RVT (50–100–200–400 μM), ACh (10−9–10−4 M), exogenous H2S donor NaHS (10−6–3.10−2 M), or endogenous H2S donor L-cysteine (10−5–3.10−5 M) were obtained in Pheprecontracted MCC. Measurement of H2S Level by Methylene Blue Assay To assess the activity of CBS and CSE in MCC, H2S determination was evaluated according to Stipanuk and Beck [17] in the presence/absence of AOAA. Since the MCC is small, at least two pair of MCC was combined, homogenized with lysis buffer containing potassium phosphate buffer (100 mM, pH 7.4), sodium orthovanadate (10 mM), and proteases inhibitors. Protein concentration was determined using the Bradford J Sex Med 2015;12:2004–2012

2006 assay. Homogenate samples were added to a reaction mixture (total volume 500 μl) containing pyridoxal- 5′-phosphate (2 mM, 20 μl) and saline (20 μl) or L-cysteine (10 mM, 20 μl) to measure basal and stimulated H2S generation. The reaction was carried out at 37°C for 40 minutes Then, ZnAc2 (1%, 250 μl) was added followed by trichloroacetic acid (10%, 250 μl) incubation to trap H2S and to precipitate proteins. Subsequently, N-dimethyl-p-phenylendiamine-sulphate (20 mM) in 7.2 M HCl and iron chloride (FeCl3, 30 mM) in HCl (1.2 M) was added, and the optical absorbance of the resulting solution was measured after 10 minutes at a wavelength of 650 nm. All samples were assayed in duplicate, and H2S concentration was calculated against a calibration curve of NaHS (3.9–250 μM). Data were calculated as nanomoles per milligram of protein.

Statistics Data are expressed as % relaxation of the Pheinduced tone. Concentration–response curves were fitted by sigmoid curves using the least squares method to calculate EC50. All calculations were determined using a standard statistical software package (Prism5, Graphpad, San Diego, CA, USA). Significance was accepted at P < 0.05. The data were computed as means ± SEM and evaluated statistically using the two-tailed unpaired t-test or two-way anova with Bonferroni post hoc test, when appropriate. N is the number of tissues used. Results

Effect of RVT on H2S Formation in MCC In the absence (basal condition) or presence of L-cysteine, H2S formation is significantly increased by RVT and inhibited by AOAA in MCC as measured by methylene blue assay (Figure 1, P < 0.001, unpaired t-test, n = 10–15). Under basal conditions, RVT caused almost fivefold increase in H2S level, and this is inhibited by AOAA. L-cysteine, the substrate of CBS and CSE, caused a marked increase in H2S formation, which is inhibited by AOAA. In the presence of L-cysteine, RVTs continue to increase H2S formation almost twofold compared with control with L-cysteine. Inhibition of RVT-induced H2S formation by AOAA suggests that RVT induces endogenous H2S formation (Figure 1, P < 0.001, unpaired t-test, n = 10–15). The inhibition rate of H2S synJ Sex Med 2015;12:2004–2012

Yetik-Anacak et al. thesis of RVT-treated group by AOAA (25% inhibition) was similar to the control one in the presence of L-cysteine (21% inhibition). We further investigated whether NO is involved in RVT-induced H2S formation in MCC, since RVT induces NO production and NO could activate H2S generation in aorta. However, in our study, eNOS inhibition by L-NNA (0.1 mM, 30 min) in the presence of L-cysteine did not alter or cause a further inhibition in RVT-induced H2S formation suggesting that H2S formation in MCC is not dependent on eNOS pathway in MCC.

The Role of H2S Formation on Relaxant Effect of RVT To elucidate the role of H2S as a possible mechanism behind the beneficial effect of RVT in erectile function in MCC, relaxation to cumulative concentrations of RVT was tested with or without AOAA or AOAA + PAG. RVT relaxed MCC concentration dependently (50–400 μM), and H2S synthesis inhibitor AOAA or AOAA + PAG significantly inhibited it (Figure 2, P < 0.01, two-way anova, Bonferroni post hoc test, n = 8 and 5, respectively). This inhibition was apparent on last two concentrations of AOAA and at all concentrations of AOAA + PAG. pD2 values were decreased by AOAA or AOAA + PAG (2.877 ± 0.138; 2.888 ± 0,361, respectively) compared with control (3.524 ± 0.1070, n = 12) (P < 0.001, unpaired t-test). However, L-NNA did not change either pD2 values (3.589 ± 0.091) or relaxation response to RVT, parallel to H2S assay data (P = 0.704, unpaired t-test and P > 0.05, two-way anova, n = 6). RVT-induced relaxation in the presence of L-NNA was significantly different than relaxations with AOAA (P < 0.01, n = 8) or AOAA + PAG (P < 0.001, n = 5; two-way anova, compared with L-NNA, n = 6). These data suggest that H2S pathway, but not NOS pathway, play important role in RVT-induced relaxation. The Effect of RVT on L-Cysteine-Induced Relaxation L-cysteine relaxes MCC in a concentrationdependent manner, and this relaxation was inhibited by H2S synthesis inhibitors AOAA or AOAA + PAG significantly at all concentrations (two way anova, P < 0.01 and P < 0.001, n = 5 and 4, respectively). To investigate the role of RVT on endogenous H2S formation-dependent relaxation, we have performed L-cysteine concentration response curve in the presence of RVT incubation. RVT incubation increased L-cysteine-induced

Resveratrol Relaxes Mice Penis via Hydrogen Sulfide

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Figure 1 Effects of resveratrol on H2S formation in MCC. RVT (0.1 mM, 30 minutes) increased H2S formation in the basal (without L-cysteine) or L-cysteine-stimulated conditions (with L-cysteine) in MCC, and this effect was reversed by H2S synthesis inhibitor (AOAA, 2 mM) but was not by NOS inhibitor (L-NNA, 0.1 mM). L-NNA did not cause a further inhibition obtained by AOAA as well. H2S levels in penile tissue coincides with that of NaHS standards (3.9–62.5 μM) ***P < 0.001 compared with control in basal (without L-cysteine) or in stimulated condition (with L-cysteine), +++P < 0.001 compared with RVT in basal or stimulated condition, # # # P < 0.001 control in basal vs. stimulated condition n = 10–15, unpaired t-test. §§§ P < 0.001 NaHS standards compared with blank, unpaired t-test

relaxation significantly (Figure 3, P < 0.05, twoway anova, n = 20 and 9). Although there is a slight tendency to increase pD2 values by RVT, it did not reach statistical significance (P = 0.0810, unpaired t-test, 3.392 ± 0.130 vs. 3.55 ± 0.211; control vs. RVT, n = 20 and 9, respectively). H2S inhibitors did not change pD2 values (AOAA: 3.378 ± 0.2746, PAG + AOAA: 3.251 ± 0.3726). Further RVT-induced augmentation of L-cysteine-induced relaxations was decreased by addition of H2S synthesis inhibitors AOAA + PAG significantly compared with RVT (P < 0.05, two way anova, n = 3–9, respectively).

The Effect of RVT on NaHS-Dependent Relaxation To investigate if RVT alter downstream mechanisms of H2S induced vasorelaxation in MCC, a concentration–response curve to NaHS (1 μM– 3 mM) was performed on Phe-precontracted tissues. RVT incubation did not alter exogenous

H2S-induced relaxation (Figure 4, P > 0.05, two way anova, Bonferroni post hoc test, n = 7). These data suggest that the vasodilator effect of RVT in MCC was related with endogenous H2S formation rather than downstream mechanisms of H2S/cGMP pathway. The pD2 values were not significantly different in the presence of RVT compared with control (P = 0.7929, unpaired t-test, 3.337 ± 0.140 vs. 3.379 ± 0.069, control vs. RVT, n = 7).

The Effect of RVT on ACh-Induced Relaxation Meng et al. [18] reported that H2S donors could increase eNOS expression in rat corpus cavernosum. As RVT can restore impaired activation of eNOS in aorta [19] and our data showed that RVT induced H2S formation, we investigated if RVT induced eNOS via H2S formation in MCC. There was no significant difference on pD2 values (P = 0.5241, unpaired t-test, 6.891 ± 0.118 J Sex Med 2015;12:2004–2012

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Figure 2 The role of H2S on the relaxation response induced by resveratrol. H2S inhibition by AOAA (2 mM) or AOAA + PAG significantly inhibited RVT-concentration– response curve in MCC. NOS inhibitor L-NNA (0.1 mM) did not alter RVT-DRC. **P < 0.01, ***P < 0.001 two-way ANOVA, Bonferroni post hoc test, AOAA compared with control. *** P < 0.001 two-way ANOVA, AOAA + PAG compared with control. +++ P < 0.001, ψ ψ ψ P < 0.001, two-way ANOVA, AOAA, or AOAA + PAG compared with L-NNA, respectively. Control (Cont) n = 12, AOAA n = 8, AOAA + PAG n = 5, L-NNA n = 6

vs. 6.785 ± 0.1087, n = 6, control vs. RVT, respectively) or relaxations response to ACh in RVTtreated MCC compared with control (Figure 5, P > 0.05, two-way anova, Bonferroni post hoc test, n = 6).

Yetik-Anacak et al.

Figure 3 The effect of resveratrol on L-cysteine-induced relaxation. RVT (0.1 mM, 30 minutes) significantly increased endogenous H2S dependent L-cysteine-induced relaxations in MCC. ** P < 0.01, *** P < 0.001 two-way ANOVA, compared with control, + P < 0.05, two-way ANOVA compared with RVT. Control (Cont) n = 20, resveratrol (RVT) n = 9. AOAA n = 5, PAG + AOAA n = 4, RVT + PAG + AOAA n = 3

Discussion

Here, we show that RVT induces both basal and L-cysteine-stimulated H2S formation in mice penile tissues through CBS and/or CSE. An earlier study reported an increased CBS activity in liver of mice fed with red wine polyphenolic compounds [20] and supports our data that RVT-induced H2S formation was dependent on inducing CBS and/or CSE. It has been shown that NO and H2S are mutually dependent on each other for angiogenesis and vascular relaxation [21], and RVT induces eNOS activity in endothelial cell [22]. However, we found that RVT-induced H2S formation in penile tissues was not depending on NOS activation and was due to a direct effect on H2S producing enzymes CBS and/or CSE, since it is inhibited by AOAA but not by NOS inhibitor L-NNA. The inhibition rate of L-cysteine-induced H2S formation by AOAA in MCC (26%) was similar in human corpus cavernosum [23] or pig urethra [24] or human bladder [25] (approximately 21%, 24%, and 18%, respectively). J Sex Med 2015;12:2004–2012

Figure 4 The effect of resveratrol on NaHS-induced relaxation in MCC. RVT (0.1 mM, 30 minutes) did not change exogenous H2S-dependent relaxations in MCC. P > 0.05, two-way ANOVA, compared with control, n = 7.

Resveratrol Relaxes Mice Penis via Hydrogen Sulfide

Figure 5 The effect of resveratrol on ACh-induced relaxation in MCC. RVT (0.1 mM, 30 minutes) did not change NO-dependent relaxations in MCC. P > 0.05, two-way ANOVA, compared with control, n = 6

RVT causes concentration-dependent and NOindependent relaxation in aorta [7] as well as in MCC [13]. However, exact mechanism of RVT-induced relaxation in MCC was not verified [13]. We found that RVT relaxes MCC at least partly through H2S, since RVT-induced relaxation and H2S formation were inhibited by H2S synthesis inhibitor(s). The inhibition of H2S formation (Figure 1), RVT-induced relaxation (Figure 2), and L-cysteine-induced relaxations (Figure 3) by AOAA or AOAA + PAG was of similar magnitude, being reduced by approximately 25%. Addition of PAG—a specific CSE inhibitor—to AOAA did not cause further inhibition as reported in rat aorta [26], suggesting that CSE was already inhibited by AOAA as well as CBS. Inhibition of endogenous H2S relaxation in MCC was twofold lower compared with human CC [23] or rat aorta [26], suggesting a pronounced role of MPST in MCC. However, the contribution of the third enzyme MPST to RVT-induced H2S synthesis and relaxation could not be determined, since there are no specific inhibitors of MPST. Although Boyden et al. showed that eNOS, sGC, potassium channels, cyclooxygenase, cyclic adenosine monophosphate pathways have been found to not contribute to RVT-induced relaxation in MCC, contribution of other possible mechanisms could not be ruled out. Our study suggests that L-cysteine induces endogenous H2S formation and relaxes corpus

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cavernosum in mice as in man [23]. Inhibition of L-cysteine-induced relaxations by H2S inhibitors AOAA or AOAA + PAG confirms presence of endogenous H2S-dependent relaxations in MCC. Further, we noted that RVT increases endogenous H2S-dependent relaxation in MCC, which was prevented by H2S synthesis inhibitors. We conclude that RVT increases endogenous H2S formation and thereby L-cysteine-induced endogenous H2S-dependent relaxation without effecting downstream mechanisms of H2S pathway to relax MCC, since exogenous NaHS-induced relaxations were not altered by RVT. The relaxation mechanisms of H2S may differ from tissue to tissue. Recently, it has been shown that NO-induced TRPA1 and CGRP activation is involved in H2S-induced relaxation in mesenteric artery but not in rat aorta [27]. Other pathways such as PDE inhibition, PKG, and EDHF also enrolled in H2S-induced relaxation [16]. However, the contribution of these pathways to RVT and/or H2S-induced relaxation in penile tissue needs further investigations. Although RVT activates eNOS and restores impaired relaxation to NO in aorta [19,28,29], in line with our studies, a recent paper also found ACh-induced relaxation was not increased by RVT in MCC [13]. This may be due to tissuespecific effect of RVT on eNOS activation as suggested by Muller and colleagues, since L-NAME inhibited RVT-induced relaxation in aorta but not in pulmonary artery [30] or due to prevailing pathological condition as RVT restores impaired ACh-induced relaxation in hypercholesterolemic rabbit corpus cavernosum [8,11,12] or spontaneous hypertensive rat aorta [31]. We should note that this study is a principle study, which demonstrates the pharmacological intervention potential of RVT. The concentration that we used in our study (100 μM) is far from plasma concentration (∼5.6 μM) that was reported to be reached by 2 g/day intake of natural RVT [32]. The bioavailability of naturally occurring RVT is poor. However, there are RVT analogues or approaches to delay metabolism that may increase plasma concentrations and/or tissue distribution of RVT up to 1000-fold as observed by piperine [33]. Besides, evaluation of RVT in plasma may not be an accurate indicator of RVT absorption and exposure, because RVT does accumulate in other tissues such as heart, liver, and skeletal muscle, but there is no information about distribution to penile tissue yet. On the other hand, further studies are required to test if the J Sex Med 2015;12:2004–2012

2010 lower concentrations/doses of RVT cause beneficial effect in ED. Nevertheless, there are few studies showing beneficial effects of RVT (5 mg/ kg/day for 4 or 8 week) in vivo [11,34] on ICP/ MAP ratio in diabetic ED where NO pathway is blunted as well as in vitro (100 μM) on CGMP level [34]. In our study, we showed that H2S may be responsible in part for the erectile effect of RVT. Knowing that H2S has ability to inhibit cGMP breakdown by PDE [21] and activate SIRT [35], it should be tested if H2S formation may responsible for the possible beneficial effect of RVT in ED. Conclusion

Our study reveals RVT induces relaxation of penile tissue at least partly through endogenous H2S formation via activation of the CBS and/or CSE pathway and is independent of NOS pathway. H2S may offer a potential therapeutic option for ED in patients who do not respond to the established PDE-5i therapy, in particular men with ED associated with endothelial dysfunction such as metabolic syndrome [36] or diabetes [37,38] where the severity of ED was reported to be higher [38]. Since H2S is released from mainly smooth muscle cell and does not require endothelial function, a combination therapy with PDE-5i may provide an alternative therapeutic strategy for PDE-5 inhibitor nonresponders [37] where endothelium and NOS/cGMP-dependent pathway are compromised. However, the bioavailability of RVT is poor, and the concentration of RVT used here based on the literature is relatively high (100 μM). Thus, future studies for new formulations of RVT to increase its bioavailability are required to translate these results to clinic. Current findings identify RVT acting via the H2S pathway to offer another option for potential therapy for nonresponders where NO production is compromised. Acknowledgments

The authors would like to thank the financial support by Turkish Scientific Research Council TUBITAK for the grant #114s448 and #109s453 as project involved in COST action BM1005 (European Network on Gasotransmitters) as well as Turkish Academia Young investigator award program; TUBA-Gebip (to G. Y. A.). We also noted that some equipment in FABALpharmaceutical research laboratory of Ege University Faculty of Pharmacy has been used in this study. J Sex Med 2015;12:2004–2012

Yetik-Anacak et al. Corresponding Author: Gunay Yetik-Anacak, PhD, Department of Pharmacology, Faculty of Pharmacy, Ege University, Bornova, Izmir 35100, Turkey. Tel: +9023-2388-5266; Fax: +9023-2388-4687; E-mail: [email protected] Conflict of Interest: The author(s) report no conflicts of interest.

Statement of Authorship

Category 1 (a) Conception and Design Gunay Yetik-Anacak (b) Acquisition of Data Mehmet V. Dereli; Gulnur Sevin; Ozge Ozzayım (c) Analysis and Interpretation of Data Gunay Yetik-Anacak; Mehmet V. Dereli; Yasemin Erac

Category 2 (a) Drafting the Article Gunay Yetik-Anacak; Asif Ahmed (b) Revising It for Intellectual Content Gunay Yetik-Anacak; Asif Ahmed

Category 3 (a) Final Approval of the Completed Article Gunay Yetik-Anacak; Asif Ahmed; Gulnur Sevin; Yasemin Erac; Mehmet V. Dereli; Ozge Ozzayım

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2012 Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Figure S1 The effect of precontraction level on L-cysteineinduced relaxation. The effect of RVT on l-cysteine relaxation compared with 40% less precontracted control strips was tested,

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Yetik-Anacak et al. since RVT caused a 40% decrease in precontraction level. L-cysteine relaxation in control groups either precontracted 40% less or not was not significantly different from each other (P > 0.05, two-way anova, n = 6–20). Relaxation to L-cysteine was increased significantly in RVT-treated strips compared with either less precontracted controls or regular controls. ***P < 0.001, twoway anova, compared with RVT, control n = 20, control—less precontracted, RVT n = 9)