Relaxant effect of diallyl sul
fide on nonpregnant rat uterus:
Involvement of voltage-dependent calcium channels
Oguzhan E. Efe, K. Michael Lux, Selda Emre Aydingöz and Meral Tuncer
Department of Pharmacology, Baskent University Faculty of Medicine, Ankara, TurkeyAbstract
Aim: We aimed to determine the effect and mechanism of action of diallyl sulfide (DAS), an active compo-nent of sulfur-containing foods such as garlic on rat uterine activity.
Methods: Isometric tension changes in longitudinal uterine strips obtained from 20 female Sprague–Dawley rats (250–300 g) in estrus stage of estrous cycle were studied in isolated organ baths containing Krebs– Henseleit solution.
Results: Diallyl sulfide (10−8–10−6M) caused a concentration-dependent relaxation on KCl (60 mM)-induced contractions and inhibited spontaneous peristaltic activity of uterine strips (P < 0.05). None of the following antagonists significantly changed the inhibitory effect of DAS on both KCl-precontracted uterine strips and spontaneous peristaltic activity of the uterus (P > 0.05): nitric oxide synthase inhibitor L-NAME (10−4M), hydrogen sulfide-producing enzymes cystation β synthase and cystation γ-lyase inhibitors, aminooxyacetic acid (10−4M) and propargylglycine (10−3M) and nonselective cyclooxygenase inhibitor indomethacin (10−4M). However, in calcium-free Krebs solution containing high KCl (30 mM), DAS significantly inhibited CaCl2(10−5–10−2M)-induced uterine contractions in a concentration-dependent manner (P < 0.05).
Conclusion: Diallyl sulfide has a relaxing effect on KCl-contracted rat uterus strips and an inhibitory effect on spontaneous uterine activity, possibly by decreasing the calcium influx into the cytoplasm of uterine smooth muscle cells.
Key words:diallyl sulfide, garlic, organ bath, rat, uterus.
Introduction
Uterus is a myogenic organ that can produce regular spontaneous contractions without neural or hormonal stimulation. Spontaneous contractions and relaxations of the nonpregnant uterus are critical for reproduc-tion, while abnormal contractility may lead to signi fi-cant clinical disorders such as dysmenorrhea and endometriosis.1,2
Despite recent advances in the physiology of myometrium, the mechanism of spontaneous contrac-tion and relaxacontrac-tion of the nonpregnant uterus remains unclear.3–5It has been reported that these contractions occur with different mechanisms in different phases
of the menstrual cycle.4,6 In experimental studies, it was suggested that calcium (Ca2+), nitric oxide (NO), G-protein-coupled receptors, phosphodiesterase activ-ity, carbon monoxide and hydrogen sulfide (H2S), which act as a signaling molecule in uterus tissue, play a role in spontaneous contractions and relaxa-tions of the nonpregnant uterus.4,7,8Additionally con-centration of intracellular-free Ca2+ has critical importance in contraction and relaxation of both preg-nant and nonpregpreg-nant uterus.4,9,10 In the light of all this information, further studies are needed to deter-mine the exact mechanisms of spontaneous contrac-tion and relaxacontrac-tion of the nonpregnant uterus and to
Received: March 31 2020. Accepted: July 25 2020.
Correspondence: Dr Selda Emre Aydingöz, Department of Pharmacology, Baskent University Faculty of Medicine, 06530 Baglıca, Etimesgut, Ankara, Turkey. Email: seldaemre71@yahoo.com
develop effective and safe treatment options against clinical conditions with increased myometrial activity. The active organosulfide components of garlic, a medicinal plant belonging to the family of Liliaceae (Allium sativum L.), have been suggested to relax the tissues containing smooth muscle such as gastric fun-dus, mesenteric artery, aorta, trachea, bladder and corpus cavernosum via NO, cyclooxygenase (COX) metabolites and ion channels, by releasing H2S, or by inhibiting L-type Ca2+ channel currents.11–14 How-ever, their effects on uterine tissue, which can produce regular spontaneous contractions, have not been stud-ied so far. On the basis of the relaxing effect of the active components of garlic on tissues containing extra-vascular smooth muscle, it is important to dem-onstrate experimentally their effects on uterine activ-ity to evaluate the potential use of these compounds in the treatment of clinical conditions accompanied by increased myometrial activity.
The aim of this study was to evaluate the effect and mechanism of action of diallyl sulfide (DAS), which is an active ingredient of sulfur-containing foods such as garlic, onion and spring onion, on nonpregnant rat uterine activity. For this purpose, the effect of DAS on spontaneous uterine peristaltic activity as well as on the precontracted uterine tissue was evaluated.
Methods
Experimental protocol and animals
This experimental study was approved by Baskent University Ethical Committee for Experimental Research on Animals (project no: DA 19/21, date September 16, 2019) and carried out in accordance with the Eight Edition of Guide for the Care and Use of Laboratory Animals.15
Twenty female Sprague–Dawley rats (250–300 g) in estrus stage of estrous cycle were used in the study. Rats were housed in a fan-ventilated room with con-stant temperature (25 2C) and relative humidity (32 7%) for 7 days before the experiment in a 12-h light/12-h dark cycle and feed with standard rat food and tap water (ad libitum). In order to confirm that the rats were in estrus stage, vaginal smears were taken with cotton swap soaked with physiological saline and stained with Diff Quick kit. The stage of the cycle was determined by microscopy according to the presence of epithelial tissue, erythrocyte and immature nucleated cells.16 The reason for choosing estrus stage is that the hypothalamopituitary ovarian
axis is functional and animal is in reproductive period in this stage. Additionally, estrogen and progesterone levels are at baseline during estrus stage, which mini-mizes hormonal effect on the studyfindings.16,17
In rats confirmed to be in estrus period, a midline thoraco-abdominal incision was performed under general anesthesia (60 mg/kg, i.m. ketamine and 7 mg/kg, i.m. xylazine) and the whole uterus (uterine horn) was removed into a petri dish containing cold Krebs–Henseleit solution. After cleaning the sur-rounding fat and connective tissues, four to six 3-mm longitudinal uterine strips obtained from each uterus were hanged in 10-mL double jacketed glass organ baths containing Krebs–Henseleit solution ([mM]: NaCl 118.2; KCl 4.7; MgSO4 7; H2O 1.2; CaCl2 2.5; KH2PO4 1.2; NaHCO3 25; glucose 11.1). One end of tissue strips was connected to the isometric force-displacement transducer (FDT05, Commat Ltd.), the other end was fixed vertically to the organ sling. The solution was gassed continuously with a mixture of 95% O2 and 5% CO2 throughout the experiment and maintained at a constant temperature of 37C by means of a thermostatic water circulator (WBC3044, Commat Ltd.). Isometric tension changes in uterine strips were recorded by a computer-mediated physio-logical data acquisition and recording system (BIOPAC MP100, Commat Ltd.). All of the uterine strips in isolated organ baths were washed with fresh solution every 15 min, kept under a resting tension of 0.5 g, and rested for 30 min before the experimental protocols described below.
Effect of DAS on uterine strips contracted with KCl
Diallyl sulfide (10−8–10−6M) produced concentration-dependent relaxations on uterine strips contracted with KCl (60 mM). This KCl concentration was determined to produce the submaximal contraction in the preliminary experiments. In order to deter-mine the mechanism of DAS-induced relaxations, the response of DAS (10−8–10−6M) on uterine strips con-tracted with KCl was obtained after 30 min of incuba-tion with each of the following antagonists in separate uterine strips: NO synthase inhibitor Nω-Nitro-L-argi-nine-methyl-ester-hydrochloride (L-NAME) (10−4M), adenosine triphosphate (ATP)-sensitive potassium channel blocker glibenclamide (10−5M), cystation β synthase inhibitor aminooxyacetic acid (AOAA) (10−4M), cystation γ-lyase inhibitor propargylglycine (PAG) (10−3M), nonselective COX inhibitor indometh-acin (10−4M), and propranolol (nonselective beta
receptor blocker, 10−7M). The experimental protocol for control DAS response and DAS response in the presence of each antagonist was repeated in at least six uterine strips obtained from different rats.
Effect of DAS on spontaneous peristaltic activity of rat uterine strips
In rested uterine strips which showed spontaneous peristaltic activity (n = 6), the effect of increasing con-centrations (10−8–10−6M) of DAS on amplitude of the peristaltic activity was evaluated. The net amplitude of the last peristaltic activity immediately prior to administration of each concentration was measured in milligram. The inhibitory effect of DAS on maximum amplitude was calculated as a percentage. In order to determine the mechanism of this effect, the response of DAS (10−8–10−6M) on spontaneous peristaltic activity of uterine strips was obtained after 30 min of incubation with L-NAME (10−4M), AOAA (10−4M) and PAG (10−3M). Since indomethacin (10−4M), glibenclamide (10−5M) and propranolol (10−7M) inhibited the spontaneous activity of uter-ine strips, they could not be used as antagonists in peristaltic activity experiments. The frequency of peristaltic activity could not be evaluated due to the disappearance of spontaneous uterine activity in a short time and irregularity of peristaltism. The experimental protocol for control DAS response and DAS response in the presence of each antagonist was repeated in at least six uterine strips obtained from different rats.
Role of voltage-dependent calcium channels in the effect of DAS on rat uterine strips
In order to evaluate a possible role of Ca2+channels in the effect of DAS on rat uterus; a separate set of uterine strips (n = 6) were stabilized in Krebs solution
containing EGTA (1 mmol/L) but not Ca2+ ([mM]: NaCl, 118.2; KCl, 4.7; MgSO4, 7; H2O, 1.2; MgCl2, 2.5; KH2PO4, 1.2 NaHCO3, 25; glucose, 11.1). The uterine strips were then contracted by phenylephrine (10−7M) for 90 min to induce the release of Ca2+from the sarcoplasmic reticulum (SR). The uterine strips were then washed and exposed to Ca2+-free Krebs solution containing high KCl (30 mM) to induce depolarization by opening the voltage-operated Ca+2 channels. Then, CaCl2 (10−5–10−2M) was added cumulatively in the absence of DAS and after pre-incubation with DAS (10−7–10−6M) and nifedipine (10−6M). The percentages of contraction induced by CaCl2 (10−5–10−2M) in the absence and presence of DAS and nifedipine were compared.
All of the chemicals and drugs used in the study were obtained from Sigma-Aldrich (Merck).
Statistical analysis
Study data were summarized by descriptive statistics (e.g., mean and standard error of mean). Responses were calculated as a percentage of maximum KCl contraction or maximum spontaneous uterine con-traction. Statistical significance of concentration-dependent effect of DAS was tested by repeated measures analysis of variance (ANOVA). Tukey’s multiple comparison test was used for pairwise com-parison of the effect of each concentration with maxi-mum KCl contraction and spontaneous peristaltic activity amplitude. Two-way ANOVA was used to evaluate the significance of the effect of antagonists on this change and the effect of DAS on CaCl2 induced contractions. DAS concentration causing 50% of maximum relaxant response (IC50) in iso-lated uterus strips was calcuiso-lated in logarithmic scale by using nonlinear regression analysis and given as mean and 95% confidence interval (CI) for
Figure 1 Original recording showing the diallyl sulfide (DAS)-induced concentration-dependent relaxations on KCl-contracted uter-ine strips.
control experiments. Statistical analyses were per-formed using GraphPad Prism (version 5.0). Statisti-cal level of significance was set at P < 0.05.
Results
Effect of DAS on uterine strips contracted with KCl
In the isolated uterine strips, KCl (60 mM) caused a sustained contraction of 3.38 0.3 g (n = 6). Diallyl sul-fide at a concentration range of 10−8–10−6M caused a concentration-dependent relaxation on KCl-induced uterus contraction with an IC50value (−log [M]) of 6.47 (95% CI, 6.51–6.43; R2= 0.8391) (Figure 1). The relaxation responses obtained by DAS in uterine strips were statis-tically significant starting from 2 × 10−7M concentration of DAS (P < 0.0001, repeated measures ANOVA) with-out any interaction of the antagonists – propranolol, PAG, glibenclamide, AOAA, L-NAME and indometha-cin (P > 0.05, two-way ANOVA) (Figure 2). None of the antagonists had a remarkable effect on KCl-induced contractions.
Effect of DAS on spontaneous peristaltic activity of rat uterine strips
The maximum amplitude of the spontaneous peristaltic activity of the uterus in control uterine strips was 2.5 0.3 g (n = 6). Diallyl sulfide (10−8–10−6M) completely inhibited this activity by decreasing the amplitude of peristaltic contractions in a concentration-dependent manner with an IC50value (−log [M]) of 6.74 (95% CI, 6.84–6.64; R2= 0.8391) (Figure 3). The inhibitory effect of DAS on peristaltic activity of uterine strips was statistically significant at 10−7M and higher concentra-tions (P < 0.0001, repeated measures ANOVA) without any interaction of PAG, AOAA and L-NAME (P > 0.05, two-way ANOVA) (Figure 4).
Effect of DAS on CaCl2-induced contractions in the rat uterine strips
In order to determine the effect of DAS on the voltage-dependent calcium channels, CaCl2 (10−5–10−2M) was added cumulatively to induce contraction in rat uterine strips incubated in Ca2+-free Krebs solution containing
Figure 2 Diallyl sulfide (DAS)-induced relaxations on KCl-contracted uterine strips (n = 6). The relaxant effect of DAS was statistically significant for 2 × 10−7M and
higher concentrations (P < 0.0001, repeated measures ANOVA) without any significant interaction of the glibenclamide (10−5M) and propranolol (10−7M) (a); L-NAME (10−4M) and indomethacin (10−4M) (b); and PAG (10−3M) and AOAA (10−4M) (c) (P > 0.05, two-way ANOVA). Vertical deviations indicate the standard error of the mean. Kontrol, Glibenklamid, Pro-pronalol, Kontrol, L-NAME, Indometazin,
high KCl (30 mM). The maximum contraction induced by CaCl2 was 5.4 0.9 g (n = 7). Preincubation with DAS (10−6M) significantly inhibited the contractions induced by CaCl2 at concentration from 3× 10−4 to 10−2M (Emax= 40.9 10.9%, P < 0.001). Similarly, DAS at 3× 10−7M concentration inhibited the contractions induced by 10−3–10−2M CaCl2 (Emax = 70.2 6.7%, P < 0.05). Nifedipine (10−6M), the L-type Ca2+channel blocker, also inhibited the contractions induced by CaCl2 (Emax= 10.2 1.9%, P < 0.001) (Figure 5).
Discussion
DAS, which is an active organosulfide components of sulfur-containing foods such as garlic, onion and spring
onion, produced a significant inhibition on both sponta-neous and KCL-induced uterine contractions with an IC50 value (−log [M]) of 6.74 and 6.47, respectively. Garlic-based organosulfide compounds are known to have a wide variety of beneficial effects on smooth muscle and other tissues.11–14However, relaxant effect on nonpregnant uterus of one of its organosulfide com-ponents was first demonstrated by this study. The results indicate that DAS has a relaxant effect on rat uterus by inhibiting voltage-dependent Ca2+ channels located on cellular membrane.
It has been known that free intracellular Ca2+ con-centration is the key initiating and regulatory factor for uterine contractions.4,9,10 Depolarization of the myometrial cell membrane opens voltage-dependent Ca2+channels leading to inward Ca2+current and the
Figure 3 Original recording showing the diallyl sulfide (DAS)-induced concentration-dependent inhibition on spontane-ous peristaltic activity of the uterine strips.
Figure 4 Diallyl sulfide (DAS)-induced reduction in the amplitude of maximum spontaneous peristaltic activ-ity of the uterine strips (n = 6). The inhibitory effect of DAS on uterine strips was statistically significant for 10−7M and higher concentrations (P < 0.0001, repeated measures ANOVA) without any interaction of the antagonists– PAG (10−3M), AOAA (10−4M) and L-NAME (10−4M) (P > 0.05, two-way ANOVA). Vertical deviations indicate the standard error of the mean.
Control, AOAA, L-NAME, PAG.
Figure 5 The effect of diallyl sulfide (DAS, 10−7–10−6M)
on the cumulative CaCl2(10−5–10−2M)-induced
contrac-tions in rat uterine strips incubated in Ca2+-free high
KCl (30 mmol/L) solution (n = 7). Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test and presented as % contraction induced by maxi-mum CaCl2concentration. Vertical deviations indicate
the standard error of the mean.*P < 0.05, **P < 0.01, and***P < 0.001 versus control CaCl2contractions.
Control, DAS (10−7M), DAS (3× 10−7M), DAS (10−6M), Nifedipine (10−6M)
increase in intracellular Ca2+resulting in activation of myosin light chain kinase by the calcium-calmodulin complex andfinally uterine contractions in both preg-nant and nonpregpreg-nant uterus.5,10,18 This Ca2+ entry occurs almost entirely via L-type Ca2+channels in rat uterus.5,19Intracellular Ca2+may also release from the SR, but its role is limited compared to voltage-dependent Ca2+ channels located on the cell mem-brane.5 In order to determine the role of voltage-dependent Ca2+ channels in the relaxant action of DAS in rat uterus tissue, we compared the concentration-response curve of CaCl2 in Ca2+-free solution containing 30 mM KCl. The results showed that DAS significantly reduced the Ca2+-induced tractions in rat uterine strips exposed to high KCl con-centration. Thesefindings indicated that DAS relaxes rate uterine tissue by decreasing the calcium influx into the cytoplasm of uterine smooth muscle cells by interfering voltage-dependent Ca2+ channels. Our finding was in line with the recent report by Tang et al.14 that showed inhibitory effect of diallyl trisul-fide, another bioactive compound of garlic, on L-type calcium channels causing concentration-dependent relaxation of the rat colon smooth muscle. This pre-sent study showed for thefirst time that diallyl disul-fide we used in this study relaxes the rat uterus by inhibiting the voltage-dependent Ca2+channels.
In addition to voltage-dependent Ca2+ channels located on the uterine cell membrane, intracellular Ca2+ may also release from the SR in a smaller extent.5 Ca2+ can be released from the SR into the cytoplasm via two main channels present on the SR membrane: inositol three phosphate (IP3) channels that are mainly activated by IP3 second messenger and ryanodine receptors which are activated mainly by Ca2+itself.9Although SR has little effect in contrac-tile activity of rat uterus,5,19we evaluated its role in DAS-induced inhibition on rat uterus strips by deplet-ing Ca2+ stores in the SR by phenylephrine-induced contraction. Since, CaCl2-induced concentration-dependent contractions obtained in intracellular and extracellular Ca2+-depleted tissues were inhibited by DAS and nifedipine, L-type calcium channel blocker, we suggest that DAS exerts its inhibitory effect on rat uterine tissue by blocking the voltage-dependent Ca2+ channels located on the cell membrane, not by inter-fering Ca2+release from the SR.
The relaxation of both spontaneous and contracted uterus is mediated predominantly by potassium chan-nels.4,5ATP-dependent potassium channels have been shown to be involved in the relaxin-induced
relaxation of the rat uterus.20Uterus is also relaxed by β-adrenergic receptor agonists via increased cAMP and by indomethacin via inhibition of prostaglandin synthesis.4,5Indeed, indomethacin, glibenclamide and propranolol abolished the spontaneous activity of uterine strips in our study. This expected finding prevented us to evaluate the involvement of these pathways in the inhibitory effect of DAS on spontane-ous activity of uterus. On the other hand, the inhibi-tory activity of DAS on uterine tissue precontracted with KCl is unlikely to involve ATP-dependent potas-sium channels, β-receptor signaling pathway and prostaglandins; since this activity was not abolished in the presence of glibenclamide, propranolol and indomethacin.
NO is a well-known mediator in uterine muscle relaxation.4,21 Since NO synthase inhibitor L-NAME had no significant effect on inhibitory action of DAS on spontaneous uterine peristaltic activity as well as on the precontracted uterine tissue, we suggest that NO has no role in DAS-induced inhibition in rat uter-ine tissue.
Garlic-derived active organosulfide components act as natural H2S donors through cysteine degradation in various tissues.22 It has been suggested that H2S mediates many biological effects of organosulfide components of garlic, such as smooth muscle relaxa-tion, anti-oxidation, cardioprotection, anti-cancer, cytoprotection, anti-inflammation and angiogene-sis.8,20,23 H2S is endogenously produced by either cystathionineβ synthase or cystathionine γ-lyase.24To evaluate the role of H2S in DAS-mediated relaxation in rat uterus strpis, we determined the effect of cystation β synthase inhibitor AOAA and cystation γ-lyase inhibitor PAG on inhibitory effect of DAS on spontaneous uterine activity and its relaxing effect on KCl-contracted rat uterus strips. Inhibitory effect of DAS on rat uterine strips did not change with either AOAA or PAG indicating that H2S has no role in DAS-induced relaxation in rat uterus.
It is important to note that our findings belong to the nonpregnant uterus. Since pregnant uterus is largely different organ compared to nonpregnant uterus, ourfindings cannot be directly extrapolated to pregnant uterus. However, based on our findings, it would be interesting and of clinical value to explore whether DAS has a relaxant effect on the pregnant uterus in further studies.
The main limitation of the presented study is that the findings obtained are completely based on in vitro organ systems; lacking molecular, in vivo, or clinical data. Since
this is an in vitro study performed on rat uterine strips placed in organ baths, ourfindings are independent of regulatory effect of estrogen and progesterone on uterine activities. In order to evaluate the interaction of sex hor-mones and relaxant effect of DAS; in vivo studies, in which hormonal mechanisms are intact, should be per-formed. Furthermore, clinical studies are needed for development of DAS- or similar molecules-based new therapeutic approaches for various contractility disor-ders of uterus. Despite the limitations of in vitro set-up, the present study provides criticalfindings on the effect of DAS on rat uterus, which form the basis for these fur-ther studies. Anofur-ther weakness of this study is that this research is performed with uterus of rat. Since the effect of DAS may differ between the species, to reflect these results into human, further studies including human uterine smooth muscle cell lines would be valuable. Another limitation to note is that the wide variation in the contractility and responsiveness of uterus prepara-tions according to the region of the organ made it diffi-cult to obtain homogeneous results in the experimental process.
In conclusion, our results suggest that DAS has an inhibitory effect on spontaneous uterine activity and a relaxing effect on KCl-contracted rat uterus strips, possibly by decreasing the calcium influx into the cytoplasm of uterine smooth muscle cells. This new knowledge on DAS function on uterine contraction may contribute to the clinical practice by proposing a new therapeutic approach for reproductive diseases such as dysmenorrhea and infertility. Further studies are needed to fully elucidate the mechanism of the relaxing effect of DAS on the uterus and to evaluate the potential use of DAS in the treatment of clinical conditions accompanied by increased myometrial activity.
Acknowledgments
This study was approved by Baskent University Ethi-cal Committee for Experimental Research on Animals (project no: DA 19/21, date September 16, 2019) and supported by Baskent University Research Fund.
Disclosure
None declared.References
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