Original Papers
Effects of Dehydroeffusol on Spasmogen-Induced Contractile Responses of Rat Intestinal Smooth Muscles
Authors
Fan Di 1, Haifeng Zhai 2, Peng Li 1, Jianmei Huang 1
Affiliations
1 2
Key words " dehydroeffusol l " rat jejunum l " spasmolytic l " smooth muscle l " Juncus effusus l " Juncuaceae l
received revised accepted
January 13, 2014 May 5, 2014 June 23, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382901 Published online August 4, 2014 Planta Med 2014; 80: 978–983 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Jianmei Huang School of Chinese Materia Medica Beijing University of Chinese Medicine 6#, Wangjing Zhonghuan Nanlu, Chaoyang District Beijing 100102 China Phone: + 86 10 84 73 86 19 Fax: + 86 10 84 73 86 11 [email protected] Correspondence Prof. Haifeng Zhai National Institute on Drug Dependence Peking University 38#, Xueyuan Road, Haidian District Beijing 100191 China Phone: + 86 10 82 80 13 43 Fax: + 86 10 62 03 26 24 [email protected]
Di F et al. Effects of Dehydroeffusol …
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China National Institute on Drug Dependence, Peking University, Beijing, China
Abstract !
Dehydroeffusol is a naturally occurring phenanthrene isolated from Juncus effusus. In the context of screening new drugs against gastrointestinal spasms, we investigated its effects on isolated rat jejunum in vitro. Dehydroeffusol (30–90 µM) slightly and transiently enhanced contractions in a concentration-dependent manner but significantly inhibited the contractions induced by KCl (100 mM), (±)-Bay-K8644 (5 µM), pilocarpine (90 µM), and histamine (100 µM). These results
Introduction !
DHE, a phenanthrene isolated from the aerial parts of Juncus effusus L. (Juncuaceae), shows anxiolytic and sedative effects in mice [1, 2]. No studies have reported any additional biological effects of DHE. Phenanthrenes from the Juncus genus share their medicinal uses and biological activities, including, but not limited to, anticancer, antimicrobial, spasmolytic, anti-allergic, anti-inflammatory, antiplatelet aggregation, and anti-al" Fig. 1), gal effects [3]. Some phenanthrenes (l isolated from plants outside the Juncus genus, have been found to possess spasmolytic activities [4–6]. Gastrointestinal spasm is a frequently occurring disorder [7]. With the hope of extending the potential uses of DHE to the treatment of intestinal spasm, this study examined its spasmolytic effects and attempted to elucidate the possible mechanisms underlying them.
Results !
Five in vitro experiments were performed. In each experiment, isolated rat jejunum smooth muscle tubular segments were kept in a tissue bath, and DHE was added separately or together with the
Planta Med 2014; 80: 978–983
show that dehydroeffusol may antagonize the spasmogenic activity of various agents, and therefore, could be a promising agent in the treatment of spasms. Its potential spasmolytic mechanism is also discussed.
Abbreviations !
DHE: L-NAME:
dehydroeffusol NG-nitro-L-arginine methyl ester
spasmogens. The time course of muscle contraction was automatically recorded and analyzed by a computer-controlled system. " Fig. 2) was designed to test the Experiment 1 (l effects of DHE on naïve smooth muscle and the effects of DHE pre-treatment on KCl-induced tonic contraction. The L-type Ca2+ channel blocker nifedipine was used as positive control. The average effects of DHE (10, 30, and 90 µM) and nifedipine (10 µM) on the tension of the isolated jejunum are " Fig. 2 A. DHE, but not nifedipine, proplotted in l duced a slight and transient contraction 60–300 s after administration. Quantified contractile tension 2 min after DHE or nifedipine addition was significantly different among the treatment " Fig. 2 B; F [4, 25] = 6.39; p = 0.001). Tengroups (l sion in the 90 µM DHE group was significantly higher than that in the vehicle group (p < 0.05). After the jejunum was treated with DHE or nifedipine for 10 min, subsequent stimulation with KCl (100 mM) was performed. KCl produced an instant increase of contractile tension in the DHE, nifedipine, and naïve (vehicle) groups. The tension peaks in the DHE and nifedipine groups were attenuated. Quantification of the muscle tension 10 s after KCl administration differed significantly " Fig. 2 C; F [4, 25] = among treatment groups (l 35.80, p < 0.001). Post-hoc tests revealed that nife-
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dipine inhibited KCl-triggered contractions (p < 0.001) and that DHE inhibited KCl-induced contractions in a dose-dependent manner (p < 0.001). " Fig. 3) was designed to test the effects of DHE Experiment 2 (l and nifedipine on jejunums pre-stimulated with KCl. The average effects of DHE (10, 30, and 90 µM) and nifedipine (10 µM) on the " Fig. 3 A. On the DHE- and nicontractile tension are plotted in l fedipine-naïve jejunums, KCl (100 mM) triggered tonic contractions. Two min after the addition of KCl, either DHE or nifedipine was added. It was found that KCl-induced contractions were attenuated by nifedipine and DHE. Nifedipine caused an instant decline in contractile tension, while DHE was characterized by a gradual decrease. Quantification of contractile tension 5 min after nifedipine or DHE addition showed significant differences " Fig. 3 B: F [4, 25] = 18.16; among the treatment groups (l p < 0.001). Post-hoc tests revealed that nifedipine and DHE (30 and 90 µM) significantly inhibited KCl-induced jejunum contractions (p < 0.05). " Fig. 4) was designed to test the effects of DHE Experiment 3 (l and nifedipine on the contraction of jejunums stimulated by (±)-Bay-K8644, an L-type Ca2+ channel activator. The representative effects of (±)-Bay-K8644 (5 µM) on DHE- and nifedipine" Fig. 4 A. Addition of (±)-Baynaïve jejunums are plotted in l K8644 to the bath induced sustained and strong rhythmic contractile responses, a phenomenon consistent with previously reported results [8]. After confirmation of (±)-Bay-K8644-induced contraction (between 1 and 5 min), either nifedipine (10 µM) or DHE (30 µM) was added to the bath. The representative effects " Fig. 4 B and C, respectively. of nifedipine and DHE are plotted in l It was found that nifedipine and DHE reduced the frequency and average amplitude of the rhythmic contractions. Quantification of contractile tension 5 min after DHE or nifedipine addition showed significant differences among treatment groups
Fig. 2 Effects of dehydroeffusol and nifedipine on spontaneous and KClinduced contractions of rat jejunum. The average tension (A), tension quantification (B) at B time (2 min after DHE addition), and tension quantification (C) at C time (10 s after KCl addition) for each group are graphed. Nif: nifedipine at 10 µM; DE_10, DE_30, and DE_90: DHE at 10, 30, and 90 µM, respectively. Data are expressed as mean ± SEM, n = 6 for each group. * P < 0.05; *** p < 0.001 vs. vehicle control.
" Fig. 4 D; F [2, 15] = 11.04; p = 0.001). The contractile tensions (l of jejunum muscles after nifedipine and DHE treatment were significantly lower than that in the group treated only with (±)-BayK8644 (p < 0.001). These effects were not due to the potential toxicity of DHE or nifedipine, because the addition of KCl (100 mM) induced characteristic contractions at the end of the test. " Fig. 5) was designed to test the effects of preExperiment 4 (l treatment with DHE or nifedipine on jejunum contraction in-
Di F et al. Effects of Dehydroeffusol …
Planta Med 2014; 80: 978–983
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Fig. 1 Chemical structure of dehydroeffusol and its related phenanthrenes (1–5) with spasmolytic activity.
Original Papers
Fig. 3 Effects of dehydroeffusol and nifedipine on KCl-induced contractions of the rat jejunum. The average tension (A) and the tension quantification (B) at B time (5 min after DHE addition) for each group are graphed. Nif: nifedipine at 10 µM; DE_10, DE_30, and DE_90: DHE at 10, 30, and 90 µM, respectively. Data are expressed as mean ± SEM, n = 6 for each group. * P < 0.05; *** p < 0.001 vs. vehicle control.
duced by pilocarpine, a non-selective muscarinic acetylcholine receptor agonist. In the absence of DHE or nifedipine pretreatment, the addition of pilocarpine (90 µM) to the bath induced an instant contraction characterized by high amplitude and rhythm, " Fig. 5 A. When the tissue bath as representatively plotted in l was pretreated with nifedipine (10 µM) or DHE (30 µM) for 10 min, the addition of pilocarpine to the bath did not produce the same tonic and rhythmic contractions, as representatively " Fig. 5 B and C, respectively. Quantification of contracplotted in l tile tension 10 s after pilocarpine addition (the peak effect of pilocarpine) showed significant differences among treatment " Fig. 5 D; F [2, 15] = 77.65; p < 0.001). The contractile groups (l tension in groups treated with nifedipine and DHE was significantly lower than that in the groups only treated with pilocarpine (p < 0.001). " Fig. 6) was designed to test the effects of preExperiment 5 (l treatment with DHE or nifedipine on jejunum contraction induced by histamine. In the absence of DHE or nifedipine pretreatment, the addition of histamine (100 µM) to the bath induced a transient contractile response, as representatively plotted in " Fig. 6 A. When pretreated with nifedipine (10 µM) or DHE l (30 µM) for 10 min, histamine did not produce any contractions, " Fig. 6 B and C, respectively. Quanas representatively plotted in l Di F et al. Effects of Dehydroeffusol …
Planta Med 2014; 80: 978–983
Fig. 4 Effects of dehydroeffusol and nifedipine on (±)-Bay K8644-induced contractions of the rat jejunum. The representative tensions in vehicle (A), nifedipine (B), and DHE (C) groups are plotted, and the tension quantifications for each group (D) at D time (5 min after DHE addition) are graphed. Nif: nifedipine at 10 µM; DE_30: DHE at 30 µM. Data are expressed as mean ± SEM, n = 6 for each group; *** p < 0.001 vs. vehicle control.
tification of contractile tension 25 s after histamine addition (the peak effect of histamine) showed significant differences among " Fig. 6 D; F [2, 15] = 15.47; p < 0.001). the treatment groups (l Post-hoc tests revealed that nifedipine and DHE inhibited histamine-induced contractions (p < 0.001).
Discussion !
DHE is a phenanthrene [1]. Previous publications have reported " Fig. 1) the spasmolytic effects of five phenanthrenes (1–5, in l with similar molecular structures to DHE [4–6]. In this study, we characterized the spasmolytic effects of DHE on the contraction of isolated rat jejunums induced by KCl, (±)-Bay-K8644, pilocarpine, and histamine. DHE displayed a dose-dependent inhibitory effect on the KCl-induced contractile response when administered either before or after KCl stimulation. Furthermore, DHE (30 µM) showed inhibitory effects on the contractile responses induced by (±)-Bay-K8644, pilocarpine, and histamine. Unex-
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Fig. 5 Effects of dehydroeffusol and nifedipine on pilocarpine-induced contraction of rat jejunum. The representative contractile tensions in vehicle (A), nifedipine (B), and DHE (C) groups are plotted, and the tension quantifications (D) for each group at D time (10 s after pilocarpine addition) are graphed. Nif: nifedipine at 10 µM; DE_30: DHE at 30 µM. Data are expressed as mean ± SEM, n = 6 for each group; *** p < 0.001 vs. vehicle control.
Fig. 6 Effects of dehydroeffusol and nifedipine on histamine-induced contraction of the rat jejunum. The representative contractile tensions in vehicle (A), nifedipine (B), and DHE (C) groups are plotted, and the tension quantifications (D) for each group at D time (25 s after histamine addition) are graphed. Nif: nifedipine at 10 µM; DE_30: DHE at 30 µM. Data are expressed as mean ± SEM, n = 6 for each group; *** p < 0.001 vs. vehicle control.
pectedly, in the absence of spasmogens, a high dose of DHE (90 µM) produced a transient, slight but significant increase in contraction. Since DHE together with phenanthrenes 1–5 represent a new kind of spasmolytic compounds, their mechanisms of action require addressing. Phenanthrene 1 antagonized contractions of rat ileum induced by histamine and BaCl2 [6]. Phenanthrene 2 inhibited spontaneous contractions of the guinea pig ileum [5]. Phenanthrenes 3, 4, and 5 inhibited contractions of the rat ileum induced by histamine, BaCl2, and L-NAME [4]. To date, the effects of six spasmolytic phenanthrenes have been tested on six spasmogens: KCl, (±)-Bay-K8644, BaCl2, pilocarpine, L-NAME, and histamine. The contraction of gastrointestinal smooth muscle is largely facilitated via a Ca2+-dependent pathway, which includes some key intermediaries such as extracellular Ca2+ entry or intercellular Ca2+ release into the cytoplasm, bonding of Ca2+ to calmodulin, activation of myosin light chain kinase, phosphorylation of myosin light chain, and hydrolysis of adenosine triphosphate
[9, 10]. The molecular signaling of many diverse spasmogens coincides with the rise of Ca2+ in muscle cells. KCl [11], Ba2+ (a K+ channel inhibitor) [12], pilocarpine (an agonist of muscarinic acetylcholine receptor) [13], and L-NAME (an inhibitor of nitric oxide synthase) [14] induce membrane depolarization, voltage-sensitive Ca2+ channel opening, and Ca2+ influx, whilst (±)-Bay-K8644 raises the opening probability of Ca2+ channels [15]. Histamine acts on H1-receptors to facilitate Ca2+ release from endoplasmic reticulum [16]. With common sense, it is unreasonable to expect that phenanthrenes act concurrently as enhancers specific for K+ channel opening, muscarinic, histamine, and nitric oxide signaling, although the involvement of neuronal nitric oxide release is suggested as a mechanism [6, 17]. It is acceptable to conclude, therefore, that phenanthrenes, including DHE, block Ca2+ channels and reduce intracellular Ca2+. However, there are still no supporting data on intracellular free Ca2+ in phenanthrene-treated muscle cells. Therefore, the possibility that phenanthrenes inhibit calmodulin, myosin light chain kinase, or other downstream
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Original Papers
Original Papers
molecules cannot be excluded. In the case of DHE, the pharmacological mechanism is complicated, in that DHE induced a slight but obvious contractile response in the spasmogen-naïve rat jejunum. We would like to propose a hypothesis that DHE may have a weak agonistic activity on Ca2+ channels at high concentrations, working as an antagonist with partial agonist properties [18]. In conclusion, the results of the present investigation provide pharmacological evidence that DHE can modulate the contraction of intestinal muscle, attenuating the tonic contraction induced by various nonspecific and specific spasmogens. These findings suggest that DHE may be used as therapeutics for intestinal spasms. The spasmolytic effects of DHE appear to act through a mechanism of decreasing the intracellular Ca2+ rise or inhibiting contractile mechanisms downstream from the Ca2+ signaling cascade. However, DHE is lightly spasmogenic at high doses. More studies should be done before clinical use of DHE as an anti-spasm medicine.
Materials and Methods !
Animals and drugs Male Sprague–Dawley rats (250–350 g) were used. They were housed under routine environmental conditions (temperature: 23–25 °C; humidity: 40–55%; 12-h light/dark cycle). The rats were provided unrestricted access to food and water. However, the day before the surgical procedures, the animals were fasted overnight but provided free access to water. Animal experiments were conducted in accordance with animal welfare regulations and guidelines, and the protocol was approved (Feb 24, 2012; No. LA2012–34) by the Biomedical Ethics Committee at Peking University Health Science Center, China. DHE (purity > 98%) was obtained from J. effusus as previously described [2]. Nifedipine (purity ≥ 98 %), (±)-Bay-K8644, histamine, and pilocarpine were obtained from Sigma-Aldrich Co. LLC. All other chemical reagents used in the study were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. DHE, (±)-Bay-K8644, and nifedipine were dissolved in DMSO, while other drugs were dissolved in Tyrodeʼs solution and added to the bath at appropriate volumes to obtain the predetermined concentrations. DMSO (0.3 % v/v) was used as the vehicle for DHE and was confirmed not to affect the contractile responses of the isolated tissues at its final concentration [19]. Nifedipine (end concentration of 10 µM) was used as the positive control.
Tissue preparation and recording of contractile tension The rats were killed by cervical dislocation. The abdomen was opened with a midline incision, and the jejunum was carefully removed and placed into Tyrodeʼs solution [20]. The composition of Tyrodeʼs solution was as follows: NaCl (136.9 mM), KCl (2.7 mM), CaCl2 (1.8 mM), MgCl2 (1.1 mM), NaHCO3 (11.9 mM), NaH2PO4 (0.4 mM), and glucose (5.5 mM). The pH of the solution was 7.4. The isolated jejunum was intraluminally washed, and remnants of the surrounding tissues were removed. Tubular segments (length, 8–10 mm) were cut from the jejunum and mounted horizontally in an organ bath containing 10 mL of Tyrodeʼs solution, with the help of two L-shaped holders. The organ bath was maintained at 37 °C and was constantly bubbled with air. The upper side of the tissue preparation was connected to a JH-2 force-displacement transducer [21]. The tissue was allowed to equilibrate for 30 min, and the bathing solution was changed every 15 min [22]. After equilibrium was attained, the tissue was given an ini-
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tial load of 0.5–0.7 g, and tissue responses were recorded for at least 10 min to obtain a control graph. Isometric forces of transverse muscle contractions were monitored using a Muscular Biosystem Software (developed by the National Institute on Drug Dependence, Peking University). Five experiments were performed with four spasmogenic reagents: KCl, (±)-Bay-K8644, pilocarpine, and histamine. Each experiment was performed using jejunum segments obtained from six different rats, and the order of segment sampling was altered among treatment groups to avoid any effects that may be related to segment localization along the intestine. A modified method, according to previous studies on KCl and nifedipine, was employed in experiments 1 and 2 [23]. In experiment 1, 30 µL of DHE was added to attain varying end concentrations (10, 30, and 90 µM). Ten minutes later, 100 mM KCl was added to induce jejunum contractions. Muscle tension was quantified 2 min after DHE addition and 10 s after KCl addition. In experiment 2, 100 mM KCl was first added to induce tetanic contractions. After 2.5 min, DHE was administered to discrete tissue baths to achieve varying end concentrations (10, 30, and 90 µM). Muscle tension was quantified 5 min after DHE addition. In experiment 3, the usage of (±)-Bay-K8644 was derived from a previous study [24]. The effect of DHE was explored with (±)-BayK8644, a 1,4-dihydropyridine Ca2+ channel activator. The tissue preparation was suspended in the bath solution containing 5 µM (±)-Bay-K8644. After confirming strong rhythmic contractions for at least 1 min, 30 µM DHE was added to the bath. Muscle tension was quantified 3 min after DHE addition. Following a period of 10 min, KCl (100 mM) was added to the bath to confirm the viability of the tissue. In experiments 4 and 5, the doses of pilocarpine [25] and histamine [26] were adopted from previous publications. The effects of DHE on pilocarpine-induced and histamine-induced contractions were studied. At 10 min after DHE (30 µM) addition, pilocarpine (90 µM) or histamine (100 µM) was added. Muscle tension was quantified 10 s after pilocarpine addition in experiment 4 and 25 s after histamine addition in experiment 5.
Statistical analysis The absolute tension force data were normalized with that of the average for the vehicle control in each experiment. The average of each treatment group (in experiments 1 and 2) or the representative tension progress (in experiments 3, 4, and 5) was used to determine the tension curves. Data in column figures are expressed as mean ± SEM of six separate samples from different rats. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc comparison tests. The differences were considered significant at p values of < 0.5. The statistical software used was OriginPro 9 from OriginLab Corporation.
Acknowledgements !
This project was sponsored in part by the National Science Foundation of China (No. 81173541, 81271473) and National Innovative Experiment Program for Undergraduate Students (MOE) of China (No. 101002625).
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Conflict of Interest !
The authors declare no conflict of interest.
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Effects of Dehydroeffusol on Spasmogen-Induced Contractile Responses of Rat Intestinal Smooth Muscles
Authors
Fan Di 1, Haifeng Zhai 2, Peng Li 1, Jianmei Huang 1
Affiliations
1 2
Key words " dehydroeffusol l " rat jejunum l " spasmolytic l " smooth muscle l " Juncus effusus l " Juncuaceae l
received revised accepted
January 13, 2014 May 5, 2014 June 23, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382901 Published online August 4, 2014 Planta Med 2014; 80: 978–983 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Jianmei Huang School of Chinese Materia Medica Beijing University of Chinese Medicine 6#, Wangjing Zhonghuan Nanlu, Chaoyang District Beijing 100102 China Phone: + 86 10 84 73 86 19 Fax: + 86 10 84 73 86 11 [email protected] Correspondence Prof. Haifeng Zhai National Institute on Drug Dependence Peking University 38#, Xueyuan Road, Haidian District Beijing 100191 China Phone: + 86 10 82 80 13 43 Fax: + 86 10 62 03 26 24 [email protected]
Di F et al. Effects of Dehydroeffusol …
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China National Institute on Drug Dependence, Peking University, Beijing, China
Abstract !
Dehydroeffusol is a naturally occurring phenanthrene isolated from Juncus effusus. In the context of screening new drugs against gastrointestinal spasms, we investigated its effects on isolated rat jejunum in vitro. Dehydroeffusol (30–90 µM) slightly and transiently enhanced contractions in a concentration-dependent manner but significantly inhibited the contractions induced by KCl (100 mM), (±)-Bay-K8644 (5 µM), pilocarpine (90 µM), and histamine (100 µM). These results
Introduction !
DHE, a phenanthrene isolated from the aerial parts of Juncus effusus L. (Juncuaceae), shows anxiolytic and sedative effects in mice [1, 2]. No studies have reported any additional biological effects of DHE. Phenanthrenes from the Juncus genus share their medicinal uses and biological activities, including, but not limited to, anticancer, antimicrobial, spasmolytic, anti-allergic, anti-inflammatory, antiplatelet aggregation, and anti-al" Fig. 1), gal effects [3]. Some phenanthrenes (l isolated from plants outside the Juncus genus, have been found to possess spasmolytic activities [4–6]. Gastrointestinal spasm is a frequently occurring disorder [7]. With the hope of extending the potential uses of DHE to the treatment of intestinal spasm, this study examined its spasmolytic effects and attempted to elucidate the possible mechanisms underlying them.
Results !
Five in vitro experiments were performed. In each experiment, isolated rat jejunum smooth muscle tubular segments were kept in a tissue bath, and DHE was added separately or together with the
Planta Med 2014; 80: 978–983
show that dehydroeffusol may antagonize the spasmogenic activity of various agents, and therefore, could be a promising agent in the treatment of spasms. Its potential spasmolytic mechanism is also discussed.
Abbreviations !
DHE: L-NAME:
dehydroeffusol NG-nitro-L-arginine methyl ester
spasmogens. The time course of muscle contraction was automatically recorded and analyzed by a computer-controlled system. " Fig. 2) was designed to test the Experiment 1 (l effects of DHE on naïve smooth muscle and the effects of DHE pre-treatment on KCl-induced tonic contraction. The L-type Ca2+ channel blocker nifedipine was used as positive control. The average effects of DHE (10, 30, and 90 µM) and nifedipine (10 µM) on the tension of the isolated jejunum are " Fig. 2 A. DHE, but not nifedipine, proplotted in l duced a slight and transient contraction 60–300 s after administration. Quantified contractile tension 2 min after DHE or nifedipine addition was significantly different among the treatment " Fig. 2 B; F [4, 25] = 6.39; p = 0.001). Tengroups (l sion in the 90 µM DHE group was significantly higher than that in the vehicle group (p < 0.05). After the jejunum was treated with DHE or nifedipine for 10 min, subsequent stimulation with KCl (100 mM) was performed. KCl produced an instant increase of contractile tension in the DHE, nifedipine, and naïve (vehicle) groups. The tension peaks in the DHE and nifedipine groups were attenuated. Quantification of the muscle tension 10 s after KCl administration differed significantly " Fig. 2 C; F [4, 25] = among treatment groups (l 35.80, p < 0.001). Post-hoc tests revealed that nife-
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dipine inhibited KCl-triggered contractions (p < 0.001) and that DHE inhibited KCl-induced contractions in a dose-dependent manner (p < 0.001). " Fig. 3) was designed to test the effects of DHE Experiment 2 (l and nifedipine on jejunums pre-stimulated with KCl. The average effects of DHE (10, 30, and 90 µM) and nifedipine (10 µM) on the " Fig. 3 A. On the DHE- and nicontractile tension are plotted in l fedipine-naïve jejunums, KCl (100 mM) triggered tonic contractions. Two min after the addition of KCl, either DHE or nifedipine was added. It was found that KCl-induced contractions were attenuated by nifedipine and DHE. Nifedipine caused an instant decline in contractile tension, while DHE was characterized by a gradual decrease. Quantification of contractile tension 5 min after nifedipine or DHE addition showed significant differences " Fig. 3 B: F [4, 25] = 18.16; among the treatment groups (l p < 0.001). Post-hoc tests revealed that nifedipine and DHE (30 and 90 µM) significantly inhibited KCl-induced jejunum contractions (p < 0.05). " Fig. 4) was designed to test the effects of DHE Experiment 3 (l and nifedipine on the contraction of jejunums stimulated by (±)-Bay-K8644, an L-type Ca2+ channel activator. The representative effects of (±)-Bay-K8644 (5 µM) on DHE- and nifedipine" Fig. 4 A. Addition of (±)-Baynaïve jejunums are plotted in l K8644 to the bath induced sustained and strong rhythmic contractile responses, a phenomenon consistent with previously reported results [8]. After confirmation of (±)-Bay-K8644-induced contraction (between 1 and 5 min), either nifedipine (10 µM) or DHE (30 µM) was added to the bath. The representative effects " Fig. 4 B and C, respectively. of nifedipine and DHE are plotted in l It was found that nifedipine and DHE reduced the frequency and average amplitude of the rhythmic contractions. Quantification of contractile tension 5 min after DHE or nifedipine addition showed significant differences among treatment groups
Fig. 2 Effects of dehydroeffusol and nifedipine on spontaneous and KClinduced contractions of rat jejunum. The average tension (A), tension quantification (B) at B time (2 min after DHE addition), and tension quantification (C) at C time (10 s after KCl addition) for each group are graphed. Nif: nifedipine at 10 µM; DE_10, DE_30, and DE_90: DHE at 10, 30, and 90 µM, respectively. Data are expressed as mean ± SEM, n = 6 for each group. * P < 0.05; *** p < 0.001 vs. vehicle control.
" Fig. 4 D; F [2, 15] = 11.04; p = 0.001). The contractile tensions (l of jejunum muscles after nifedipine and DHE treatment were significantly lower than that in the group treated only with (±)-BayK8644 (p < 0.001). These effects were not due to the potential toxicity of DHE or nifedipine, because the addition of KCl (100 mM) induced characteristic contractions at the end of the test. " Fig. 5) was designed to test the effects of preExperiment 4 (l treatment with DHE or nifedipine on jejunum contraction in-
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Fig. 1 Chemical structure of dehydroeffusol and its related phenanthrenes (1–5) with spasmolytic activity.
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Fig. 3 Effects of dehydroeffusol and nifedipine on KCl-induced contractions of the rat jejunum. The average tension (A) and the tension quantification (B) at B time (5 min after DHE addition) for each group are graphed. Nif: nifedipine at 10 µM; DE_10, DE_30, and DE_90: DHE at 10, 30, and 90 µM, respectively. Data are expressed as mean ± SEM, n = 6 for each group. * P < 0.05; *** p < 0.001 vs. vehicle control.
duced by pilocarpine, a non-selective muscarinic acetylcholine receptor agonist. In the absence of DHE or nifedipine pretreatment, the addition of pilocarpine (90 µM) to the bath induced an instant contraction characterized by high amplitude and rhythm, " Fig. 5 A. When the tissue bath as representatively plotted in l was pretreated with nifedipine (10 µM) or DHE (30 µM) for 10 min, the addition of pilocarpine to the bath did not produce the same tonic and rhythmic contractions, as representatively " Fig. 5 B and C, respectively. Quantification of contracplotted in l tile tension 10 s after pilocarpine addition (the peak effect of pilocarpine) showed significant differences among treatment " Fig. 5 D; F [2, 15] = 77.65; p < 0.001). The contractile groups (l tension in groups treated with nifedipine and DHE was significantly lower than that in the groups only treated with pilocarpine (p < 0.001). " Fig. 6) was designed to test the effects of preExperiment 5 (l treatment with DHE or nifedipine on jejunum contraction induced by histamine. In the absence of DHE or nifedipine pretreatment, the addition of histamine (100 µM) to the bath induced a transient contractile response, as representatively plotted in " Fig. 6 A. When pretreated with nifedipine (10 µM) or DHE l (30 µM) for 10 min, histamine did not produce any contractions, " Fig. 6 B and C, respectively. Quanas representatively plotted in l Di F et al. Effects of Dehydroeffusol …
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Fig. 4 Effects of dehydroeffusol and nifedipine on (±)-Bay K8644-induced contractions of the rat jejunum. The representative tensions in vehicle (A), nifedipine (B), and DHE (C) groups are plotted, and the tension quantifications for each group (D) at D time (5 min after DHE addition) are graphed. Nif: nifedipine at 10 µM; DE_30: DHE at 30 µM. Data are expressed as mean ± SEM, n = 6 for each group; *** p < 0.001 vs. vehicle control.
tification of contractile tension 25 s after histamine addition (the peak effect of histamine) showed significant differences among " Fig. 6 D; F [2, 15] = 15.47; p < 0.001). the treatment groups (l Post-hoc tests revealed that nifedipine and DHE inhibited histamine-induced contractions (p < 0.001).
Discussion !
DHE is a phenanthrene [1]. Previous publications have reported " Fig. 1) the spasmolytic effects of five phenanthrenes (1–5, in l with similar molecular structures to DHE [4–6]. In this study, we characterized the spasmolytic effects of DHE on the contraction of isolated rat jejunums induced by KCl, (±)-Bay-K8644, pilocarpine, and histamine. DHE displayed a dose-dependent inhibitory effect on the KCl-induced contractile response when administered either before or after KCl stimulation. Furthermore, DHE (30 µM) showed inhibitory effects on the contractile responses induced by (±)-Bay-K8644, pilocarpine, and histamine. Unex-
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Fig. 5 Effects of dehydroeffusol and nifedipine on pilocarpine-induced contraction of rat jejunum. The representative contractile tensions in vehicle (A), nifedipine (B), and DHE (C) groups are plotted, and the tension quantifications (D) for each group at D time (10 s after pilocarpine addition) are graphed. Nif: nifedipine at 10 µM; DE_30: DHE at 30 µM. Data are expressed as mean ± SEM, n = 6 for each group; *** p < 0.001 vs. vehicle control.
Fig. 6 Effects of dehydroeffusol and nifedipine on histamine-induced contraction of the rat jejunum. The representative contractile tensions in vehicle (A), nifedipine (B), and DHE (C) groups are plotted, and the tension quantifications (D) for each group at D time (25 s after histamine addition) are graphed. Nif: nifedipine at 10 µM; DE_30: DHE at 30 µM. Data are expressed as mean ± SEM, n = 6 for each group; *** p < 0.001 vs. vehicle control.
pectedly, in the absence of spasmogens, a high dose of DHE (90 µM) produced a transient, slight but significant increase in contraction. Since DHE together with phenanthrenes 1–5 represent a new kind of spasmolytic compounds, their mechanisms of action require addressing. Phenanthrene 1 antagonized contractions of rat ileum induced by histamine and BaCl2 [6]. Phenanthrene 2 inhibited spontaneous contractions of the guinea pig ileum [5]. Phenanthrenes 3, 4, and 5 inhibited contractions of the rat ileum induced by histamine, BaCl2, and L-NAME [4]. To date, the effects of six spasmolytic phenanthrenes have been tested on six spasmogens: KCl, (±)-Bay-K8644, BaCl2, pilocarpine, L-NAME, and histamine. The contraction of gastrointestinal smooth muscle is largely facilitated via a Ca2+-dependent pathway, which includes some key intermediaries such as extracellular Ca2+ entry or intercellular Ca2+ release into the cytoplasm, bonding of Ca2+ to calmodulin, activation of myosin light chain kinase, phosphorylation of myosin light chain, and hydrolysis of adenosine triphosphate
[9, 10]. The molecular signaling of many diverse spasmogens coincides with the rise of Ca2+ in muscle cells. KCl [11], Ba2+ (a K+ channel inhibitor) [12], pilocarpine (an agonist of muscarinic acetylcholine receptor) [13], and L-NAME (an inhibitor of nitric oxide synthase) [14] induce membrane depolarization, voltage-sensitive Ca2+ channel opening, and Ca2+ influx, whilst (±)-Bay-K8644 raises the opening probability of Ca2+ channels [15]. Histamine acts on H1-receptors to facilitate Ca2+ release from endoplasmic reticulum [16]. With common sense, it is unreasonable to expect that phenanthrenes act concurrently as enhancers specific for K+ channel opening, muscarinic, histamine, and nitric oxide signaling, although the involvement of neuronal nitric oxide release is suggested as a mechanism [6, 17]. It is acceptable to conclude, therefore, that phenanthrenes, including DHE, block Ca2+ channels and reduce intracellular Ca2+. However, there are still no supporting data on intracellular free Ca2+ in phenanthrene-treated muscle cells. Therefore, the possibility that phenanthrenes inhibit calmodulin, myosin light chain kinase, or other downstream
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molecules cannot be excluded. In the case of DHE, the pharmacological mechanism is complicated, in that DHE induced a slight but obvious contractile response in the spasmogen-naïve rat jejunum. We would like to propose a hypothesis that DHE may have a weak agonistic activity on Ca2+ channels at high concentrations, working as an antagonist with partial agonist properties [18]. In conclusion, the results of the present investigation provide pharmacological evidence that DHE can modulate the contraction of intestinal muscle, attenuating the tonic contraction induced by various nonspecific and specific spasmogens. These findings suggest that DHE may be used as therapeutics for intestinal spasms. The spasmolytic effects of DHE appear to act through a mechanism of decreasing the intracellular Ca2+ rise or inhibiting contractile mechanisms downstream from the Ca2+ signaling cascade. However, DHE is lightly spasmogenic at high doses. More studies should be done before clinical use of DHE as an anti-spasm medicine.
Materials and Methods !
Animals and drugs Male Sprague–Dawley rats (250–350 g) were used. They were housed under routine environmental conditions (temperature: 23–25 °C; humidity: 40–55%; 12-h light/dark cycle). The rats were provided unrestricted access to food and water. However, the day before the surgical procedures, the animals were fasted overnight but provided free access to water. Animal experiments were conducted in accordance with animal welfare regulations and guidelines, and the protocol was approved (Feb 24, 2012; No. LA2012–34) by the Biomedical Ethics Committee at Peking University Health Science Center, China. DHE (purity > 98%) was obtained from J. effusus as previously described [2]. Nifedipine (purity ≥ 98 %), (±)-Bay-K8644, histamine, and pilocarpine were obtained from Sigma-Aldrich Co. LLC. All other chemical reagents used in the study were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. DHE, (±)-Bay-K8644, and nifedipine were dissolved in DMSO, while other drugs were dissolved in Tyrodeʼs solution and added to the bath at appropriate volumes to obtain the predetermined concentrations. DMSO (0.3 % v/v) was used as the vehicle for DHE and was confirmed not to affect the contractile responses of the isolated tissues at its final concentration [19]. Nifedipine (end concentration of 10 µM) was used as the positive control.
Tissue preparation and recording of contractile tension The rats were killed by cervical dislocation. The abdomen was opened with a midline incision, and the jejunum was carefully removed and placed into Tyrodeʼs solution [20]. The composition of Tyrodeʼs solution was as follows: NaCl (136.9 mM), KCl (2.7 mM), CaCl2 (1.8 mM), MgCl2 (1.1 mM), NaHCO3 (11.9 mM), NaH2PO4 (0.4 mM), and glucose (5.5 mM). The pH of the solution was 7.4. The isolated jejunum was intraluminally washed, and remnants of the surrounding tissues were removed. Tubular segments (length, 8–10 mm) were cut from the jejunum and mounted horizontally in an organ bath containing 10 mL of Tyrodeʼs solution, with the help of two L-shaped holders. The organ bath was maintained at 37 °C and was constantly bubbled with air. The upper side of the tissue preparation was connected to a JH-2 force-displacement transducer [21]. The tissue was allowed to equilibrate for 30 min, and the bathing solution was changed every 15 min [22]. After equilibrium was attained, the tissue was given an ini-
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tial load of 0.5–0.7 g, and tissue responses were recorded for at least 10 min to obtain a control graph. Isometric forces of transverse muscle contractions were monitored using a Muscular Biosystem Software (developed by the National Institute on Drug Dependence, Peking University). Five experiments were performed with four spasmogenic reagents: KCl, (±)-Bay-K8644, pilocarpine, and histamine. Each experiment was performed using jejunum segments obtained from six different rats, and the order of segment sampling was altered among treatment groups to avoid any effects that may be related to segment localization along the intestine. A modified method, according to previous studies on KCl and nifedipine, was employed in experiments 1 and 2 [23]. In experiment 1, 30 µL of DHE was added to attain varying end concentrations (10, 30, and 90 µM). Ten minutes later, 100 mM KCl was added to induce jejunum contractions. Muscle tension was quantified 2 min after DHE addition and 10 s after KCl addition. In experiment 2, 100 mM KCl was first added to induce tetanic contractions. After 2.5 min, DHE was administered to discrete tissue baths to achieve varying end concentrations (10, 30, and 90 µM). Muscle tension was quantified 5 min after DHE addition. In experiment 3, the usage of (±)-Bay-K8644 was derived from a previous study [24]. The effect of DHE was explored with (±)-BayK8644, a 1,4-dihydropyridine Ca2+ channel activator. The tissue preparation was suspended in the bath solution containing 5 µM (±)-Bay-K8644. After confirming strong rhythmic contractions for at least 1 min, 30 µM DHE was added to the bath. Muscle tension was quantified 3 min after DHE addition. Following a period of 10 min, KCl (100 mM) was added to the bath to confirm the viability of the tissue. In experiments 4 and 5, the doses of pilocarpine [25] and histamine [26] were adopted from previous publications. The effects of DHE on pilocarpine-induced and histamine-induced contractions were studied. At 10 min after DHE (30 µM) addition, pilocarpine (90 µM) or histamine (100 µM) was added. Muscle tension was quantified 10 s after pilocarpine addition in experiment 4 and 25 s after histamine addition in experiment 5.
Statistical analysis The absolute tension force data were normalized with that of the average for the vehicle control in each experiment. The average of each treatment group (in experiments 1 and 2) or the representative tension progress (in experiments 3, 4, and 5) was used to determine the tension curves. Data in column figures are expressed as mean ± SEM of six separate samples from different rats. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc comparison tests. The differences were considered significant at p values of < 0.5. The statistical software used was OriginPro 9 from OriginLab Corporation.
Acknowledgements !
This project was sponsored in part by the National Science Foundation of China (No. 81173541, 81271473) and National Innovative Experiment Program for Undergraduate Students (MOE) of China (No. 101002625).
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Conflict of Interest !
The authors declare no conflict of interest.
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