Participation of cholinergic pathways in [alpha]-hederin-induced contraction of rat isolated stomach strips.
Received 19 October 2011
Received in revised form 15 December 2011
Accepted 18 February 2012
Isolated stomach strips
The dry extract of Hedra helix leaves and its main active compounds, predominantly [alpha]-hederin and hederacoside C, has been traditionally believed to act spasmolytic. However, it has been recently proved that both, the extract of ivy and triterpenoid saponins, exhibit strong contractile effect on rat isolated stomach smooth muscle strips. It turned out that the most potent contractile agent isolated from the extract of ivy leaves is [alpha]-hederin. Thus, it seems reasonable to estimate the mechanism of the contractile effect of this saponin.
The presented study was aimed at verifying the participation of cholinergic pathways (muscarinic and nicotine receptors) in [alpha]-hederin-induced contraction. The experiments were carried out on rat isolated stomach corpus and fundus strips under isotonic conditions. The preparations were preincubated with either atropine or hexamethonium and then exposed to [alpha]-hederin. All results are expressed as the percentage of the response to acetylcholine--a reference contractile agent.
The obtained results revealed that the pretreatment of isolated stomach strips (corpus and fundus) with atropine neither prevented nor remarkably reduced the reaction of the preparations to [alpha]-hederin. Similarly, if the application of saponin was preceded by the administration of hexamethonium, the strength of the contraction of stomach fundus strips induced by [alpha]-hederin was not modified.
Concluding, it can be assumed that the cholinergic pathways do not participate in [alpha]-hederin-evoked contraction of rat isolated stomach preparations.
[c] 2012 Elsevier GmbH. All rights reserved.
The traditional and contemporary use of the dry extract of Hedera helix leaves is based on the believe that it acts miorelaxant on the smooth muscle (Rippe and Medejsky, 2006). There is also experimental evidence that phytochemicals isolated from ivy plant leaves' extract (predominantly saponins) exhibit secretolytic and antispasmodic effects in in vitro conditions (Trute et al., 1997; Haberlein, 2008; Sieben et al., 2009). On the other hand, the clinical signs observed in the course of ivy poisoning (diarrhea, dyspnea) (Roth et al., 1994; Gfeller and Messonnier, 2004) indicate clearly that the extract of Hedera helix enhances contractile effect on the smooth muscle.Those findings were confirmed in experiments performed on rat isolated stomach corpus and fundus strips. It turned out that as well triterpenoid saponins ([alpha]-hederin and hederacoside C) as the whole dry extract of ivy leaves contracted stomach smooth muscle (Mendel et al., 2011). However, the mechanism of action of the active compounds remains unclear. Evaluation of the mode of action would allow to understand the signs of poisoning caused by Hedera helix (Gfeller and Messonnier, 2004) and adverse reactions noticed in patients treated with ivy-based drugs (Holzinger and Chenot, 2011). Besides, the results of in vitro studies could become an indication for the specific therapy in intoxications resulting from the digestion of the plant.
According to some preliminary study of Haberlein hederaco-side C undergoes in vivo biotransformation processes catalyzed by esterases; one sugar chain at C-28 is hydrolyzed and thus, [alpha]-hederin is created. The further hydrolysis does not happen as hederagenin was absent in blood plasma, in the performed study (Runkel et al., 2005). This finding explains why it seems reasonable to study the possible mechanism of action of [alpha]-hederin on gut smooth muscle. Thus, the aim of the study was to verify the participation of cholinergic pathways (muscarinic and nicotinic receptors) in signal transduction in [alpha]-hederin-induced contraction of isolated stomach corpus and fundus strips.
Materials and methods
Chemicals and incubation medium
Acetylcholine chloride, atropine sulfate, dimethylsulfondioxide (DMSO), hexamethonium bromide, isoproterenol hemisulfate, nicotine, (Sigma Chemicals Co, St. Louis, USA), [alpha]-hederin (ChromaDex, Irvine, Canada), [CaCl.sub.2] (Merck, Darmstadt, Germany), Na[H.sub.2][PO.sub.4] (Fluka Chemie, AG, Buchs, Switzerland), all other salts needed for the preparation of the incubation medium: NaCI, KCI, Mg[SO.sub.4], [NaHCO.sub.3]; glucose (POCh, Gliwice, Poland) were used for preparing experiments. Modified Krebs-Henseleit solution (M K-HS) was employed as an incubation medium and it maintained a pH value of 7.35-7.45 throughout long-term experiments while heated up to 37 [degrees] C and bubbled with carbogen (95% [O.sub.2] + 5% [CO.sub.2]). [alpha]-hederin was dissolved in DMSO (0.5%). The concentration of [alpha]-hederin, 100 [micro]M, was chosen on the basis of the previous studies (Mendel et al., 2011). Acetylcholine chloride, atropine sulfate, isoproterenol hemisulfate and hexamethonium were dissolved in modified Krebs-Henseleit solution.
All experiments were conducted on the tissues isolated from male Wistar rats. The animals were kept under standardized laboratory conditions for at least seven days prior the experiment. They had free access to pallet of food (Sniff Special Diaten, GmbH, Germany) and drinking water. The use of animals was approved by the local bioethics committee (approval number 16/2006).
Isolation and preparation of stomach strips
The animals were euthanized in chambers filled with carbon dioxide ([CO.sub.2]) (Everitt and Gross, 2006). After that, two strips, one of corpus and one of fundus, were isolated from rat's stomach as described before (Mendel et al., 2011). The prepared preparations were placed in incubation chambers (5 ml) (Schuler Organ Bath, Hugo Sachs Elektronic, Harvard Apparatus, Germany) filled with warmed (37 [degrees] C) M K-HS.
Registration of motoric activity of rat isolated stomach strips
Stomach corpus and fundus strips were attached with one end to a steel hook mounted at the end of a silk thread that was connected to an isotonic transducer (B 40, type 373, Hugo Sachs Elektronic, Germany). All experiments were carried out under a load of 0.5 g. Isotonic transducers were linked up to the analogue-digital registration set (PowerLab, ADInstruments, Australia) by a bridge amplifier (DBA, type 666, Hugo Sachs Elektronic, Germany). Recordings of isolated stomach strips' motoric activity were registered by Chart v5.0 program. All the calculations and analysis were completed by LabChart Reader 7 program and Excel (MS Office XP Professional).
The course of experiments
Investigations aimed at studying the effect of atropine (1 [micro]M) on the contraction caused by [alpha]-hederin (100 [micro]M) dissolved in DMSO (0.5%) started with 1 h equilibration. The preincubation was followed by the application of acetylcholine in a concentration of 10 [micro]M and 1 [micro]M for stomach corpus and fundus preparations, respectively. As soon as the spontaneous motoric activity of stomach strips stabilized, the application of atropine (1 [micro]M) was performed. Atropine was maintained in the incubation chambers for 5 min. and then the preparations were treated with acetylcholine in order to confirm the effectiveness of the blockade of muscarinic receptors. Acetylcholine remained in the incubation chambers for another 5 min and washed out after that time. Subsequently, the treatment of the stomach strips with atropine (1 [micro]M) was repeated and 5 minutes later followed by the application of [alpha]-hederin (100 [micro]M) dissolved in DMSO (0.5%). The preparations were exposed to saponin for at least 10 min.
Experiments designed to study the effect of hexamethonium (1 [micro]M) on [alpha]-hederin-induced contraction, started with a 1 h equilibration. The preincubation was followed by the application of acetylcholine in a concentration of 1 [micro]M. As soon as the spontaneous motoric activity of stomach strips stabilized, the application of hexamethonium (1 [micro]M) was performed. Hexamethonium was maintained in the incubation chambers for 5 min and then the preparations were treated with nicotine (1000 [micro]M). Nicotine was uphold in the incubation chambers for another 10 min and washed out. Subsequently, the treatment of the stomach fundus strips with hexamethonium (1 [micro]M) was repeated and 5 min later followed by the application of [alpha]-hederin (100 [micro]M) dissolved in DMSO (0.5%). The preparations were exposed to saponin for at least 10 min.
Expression of the obtained results and statistical analysis The results are expressed as the percent of the reaction evoked by acetylcholine applied in the optimal dose. All data were analyzed using Statistica PL for Windows (version 6.1). Results were expressed as mean values [+ or -] standard deviation (SD) of the average. Values of p [less than or equal to] 0.05 were considered to be significant. There were three tests employed in the statistical analysis: a one-way analysis of variance (ANOVA), t-Student test and LSD Fisher test. The change of the spontaneous motoric activity was considered as significant, if its strength of the reaction differed statistically from the force of the response to DMSO in a concentration of 0.5% (control).
Results and discussion
The administration of atropine (1 [micro]M) to the incubation chamber significantly inhibited the reaction of both types of isolated stomach strips (stomach corpus and fundus) to acetylcholine (Fig. 1). In the presence of atropine (1 [micro]M) in the incubation chamber, the response of stomach corpus and fundus preparations to acetylcholine (10 [micro]M) was reduced up to 8.97 [+ or -] 5.83% and 4.87 [+ or -] 4.91% of the preliminary response to acetylcholine without prior treatment with atropine, respectively (Fig. 2I and II). Furthermore. the pretreatment of isolated stomach strips (corpus and fundus) with atropine (1 [micro]M) did not remarkably change the reaction of the preparation to [alpha]-hederin applied in a concentration of 100 [micro]M (Fig. 2I and II). For isolated stomach corpus strips the reaction to [alpha]-hederin (100 [micro]M) without prior treatment with atropine amounted to 94.79 [+ or -] 75.91% of contraction caused by acetylcholine (10 [micro]M) and the strength of strips' reaction to the same dose of [alpha]-hederin but pretreated with atropine was similar and amounted to 96.02 [+ or -] 23.06% of the effect produced by acetylcholine (10 [micro]M). In the case of isolated stomach fundus strips the force of the response to [alpha]-hederin (100 [micro]M) was for 101.57 [+ or -] 27.75% and 102.73 [+ or -] 11.01% of the contraction caused by acetylcholine (1 [micro]M) for the preparations untreated and pretreated with atropine (1 [micro]M), respectively.
The administration of hexamethonium (1 [micro]M) to the incubation chamber significantly inhibited the reaction of isolated stomach fundus strips to nicotine (1000 [micro]M) (Fig. 3). The pretreatment of isolated stomach fundus strips with hexamethonium (1 [micro]M) did not change remarkably the reaction of the preparation to [alpha]-hederin applied in a concentration of 100 [micro]M. If the administration of saponin was preceded by a treatment with hexamethonium the strength of stomach fundus strips' contraction was 106.68 [+ or -] 11.90% of the reaction to acetylcholine (1 [micro]M) and the contraction was comparable with the one caused by [alpha]-hederin (100 [micro]M) without prior hexamethonium-treatment (Fig. 4).
An important role of parasympathetic cholinergic neurons and postsynaptic muscarinic acetylcholine receptors in the contraction of gastric smooth muscle has been provided in functional studies (Makhlouf and Murthy, 2006). The actions of acetylcholine in the periphery are the result of activation of either inotropic nicotinic receptor or the metabotropic muscarinic receptor (Caufield and Birdsall, 1998). The stomach is supplied with cholinergic nerves that produce contractions via muscarinic receptors, and these cholinergic nerves play an important role in the regulation of gastrointestinal motility (Makhlouf and Murthy, 2006). These observations highlight the central role of acetylcholine receptors in the control of gastrointestinal motor function.
On the other hand, the ganglion fibres emerging from ganglia network that contains Meissner's plexus and Auerbach's plexus, innervate the smooth muscle and secretory cells. The receptors on the ganglion cells are generally thought to be nicotinic, whereas those on the effector organs seem to be muscarinic (Wood, 2002). Thus, it is of the utmost importance to examine muscarinic and then also nicotinic receptors' participation in the mode of action of tested xenobiotics on smooth muscle activity.
Since atropine is a non-selective muscarinic antagonists, the application of atropine blocks completely the effect produced by acetylcholine (Perry, 1971). Thus, acetylcholine was used as a control of muscarinic receptors' blockage effectiveness in experiments designed to study the participation of cholinergic signal transduction in [alpha]-hederin-induced contraction. Due to the variety of muscarinic receptors taking part in signal transduction and activation of stomach smooth muscles it seemed reasonable to examine firstly general participation of M-type receptors in [alpha]-hederin-induced contraction of isolated stomach strips followed by the contribution of particular M-type receptors. The obtained results indicated clearly [alpha]-hederin induced stomach strips' contraction without activating any of muscarinic-type receptors (Fig. 2). Since, there was no notable reduction in the contractile response to [alpha]-hederin after pretreatment with atropine (non-selective muscarinic antagonist), any specific muscarinic antagonist was employed in further study of [alpha]-hederin mode of action. Moreover, the results indicate clearly that in the case of ivy poisoning in human and animal atropine should not be recommended neither for symptomatic nor for specific therapy although it is advised so in the available literature (Roth et al., 1994).
The results and conclusions referring to the effect of [alpha]-hederin on muscarinic receptors are in accordance with the remarks made by other authors regarding the impact of saponins. The investigations performed by Fu et al. (2005) in mice indicated that the effect of escin is not abolished by anisodamine hydrobromide, which means that escin, similarly like [alpha]-hederin does not exert its effect on cholinergic receptors. The idea that saponins do not interfere with muscarinic receptors can be also supported by the observation of Matsuda and coworkers (1999). Namely, they proved that contractile impact of saponins isolated from Aesculus hippocastanum (escins Ia, lb, IIa and IIb) was not inhibited in atropine-pretreated mice. There is also evidence that saponins can act as a stimulant on smooth muscle of other than gut tissues without involving muscarinic receptors' activation. The study performed by Uchendu and Leek (1999) revealed that saponins extracted from Dalbergia saxatilis displayed contractile effect on rat isolated uterine preparations which insensitive to atropine treatment. It insinuates that tissues' responses to those saponins were mediated by mechanism other than activation of myometrical cell membrane cholinoreceptors.
Nevertheless, it must be admitted that many literature examples show the possibility of saponins to interact with muscarinic receptors. For example: spasmogenic effect of crude saponin fraction isolated form Panax ginseng was completely inhibited by atropine (Takagi et al., 1972); radish extract, rich in saponins, stimulated GI motility in vitro and in vivo and its pharmacological activity was, at least partly, mediated by activation of muscarinic acetylcholinergic mechanism (Jung et al. 2000); the contractile activity of crude extracts of Fumaria indica and Hibiscus rosasinesis (both containing saponins) was abolished by pretreatment with atropine suggesting that the extract contains components with cholinomimetic activity (Gilani et al., 2005a,b).
Eventually, there are numerous saponins which act as relaxants on gut smooth muscle and are defined as muscarinic receptors' antagonist, e.g., ginsenosides: Rb1 and Rg3 (Tachikawa et al. 1999), Terminalia bellerica extract containing saponins (Gilani et al., 2008).
Considering two facts namely that: firstly, membrane proteins are thought to be localized selectively in cholesterol-rich domains (e.g. acetylcholine receptor) and secondly that saponins exhibit high affinity to cholesterol, it could be suspected that saponins selectively influence transmembrane protein function and mimic specificity at the effector level (Francis et al., 2002). This hypothesis could explain the participation of muscarinic receptors in many saponin-induced muscle contractions. However, as it had been shown in this paper, it is not the case of [alpha]-hederin.
The second step in determining the mode of chemical agents which induce contractions of isolated GI smooth muscle preparations is usually the verification of the participation of nicotinic receptors located in ganglions of enteric nervous system. The results disclosed that the pretreatment of isolated stomach fundus strips with hexamethonium (1 [micro]M) did not change remarkably the reaction of preparations to [alpha]-hederin applied in concentration of 100 [micro]M (Fig. 4). It suggests that nAChRs do not contribute to signal transduction in [alpha]-hederin-evoked reaction in the presented model.
Confirmation of the obtained results can be found in many studies aimed at investigating the mode of action of other phytochemicals and herbal preparations. Hexamethonium did not succeed in preventing or at least reducing the stimulant effect of saponin-rich ginger extract on rat and mouse stomach fundus strips (Ghayur and Gilani, 2005). On the other hand, Murata et al. (2001) and Shibata et al. (1999) tested the impact of Dai-Kenchu-Tou (DKT--herbal medicine containing as well P. ginseng root as dried ginger rhizome) on gut motility of conscious dogs and revealed that in the employed in vivo model the contractile activity of DKT can be inhibited by hexamethonium. It can indicate that the study of nAChRs participation in observed contraction of GI smooth muscle is strongly limited in models based on isolated gut strips and results observed in in vitro studies should not be extrapolated into in vivo conditions as they can be in opposition. The discrepancies between results gained in in vivo and in vitro experiments can result either from fast loss of vital activity of ENS in ex vivo models or from the inability of xenobiotics to reach neurogenal component in the studied model.
The study of Jeong et al. (2005) is the only example of an investigation which indicated the participation of nicotinic receptors' activation in contraction of rat small bowel segments evoked by radish root extract. Indeed, preparations' pretreatment with hexamethonium blocked, partially, the contraction produced by radish root extract. The results are, however, questionable. This is clue to the fact that the authors declared that hexamethonium blocked also in part ACh-induced contraction which is just impossible since acetylcholine evokes contractions by activating muscarinic receptors located directly on smooth muscle cells in this model (Bolton, 1979).
Summing up, it can be concluded without any doubts that nAChRs are not involved in [alpha]-hederin-induced contraction of rat isolated stomach corpus and fundus strips. Finally, it can be assumed that the cholinergic pathways (neither muscarinic nor nicotinic receptors) do not participate in [alpha]-hederin-evoked contraction of rat isolated stomach preparations.
The study was supported by a research grant from the Ministry of Science and Higher Education No 2496/B/P01/2009/36.
Bolton, T.B., 1979. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol. Pev. 59, 606-718.
Caufield, M.P., Birdsall, N.J.M., 1998. International union of pharmacology. XVII. Classification of muscarinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 50, 279-290.
Everitt, J.I., Gross, E.A., 2006. Euthanasia and necropsy. In: Suckow, M.A., Weisbroth, S.H., Franklin, C.L. (Eds.), The Laboratory Rat. Elsevier Academic Press, London, pp. 665-678.
Francis, G., Kerem, Z., Makkar, H.P.S., Becker, K., 2002. The biological action of saponins in animal system: a review. Br. J. Nutr. 88, 587-605.
Fu, F., Hou, Y., Jiang, W., Wang, R., Liu, K., 2005. Escin: inhibiting inflammation and promoting gastrointestinal transit to attenuate formation of postoperative adhesion. World J. Surg. 29, 1614-1620.
Gfeller, R.W., Messonnier, S.P., 2004. Handbook of Small Animal Toxicology and Poisonings. Mosby, Inc., St. Louis, p. 377.
Ghayur, M.N., Gilani, A.H., 2005. Pharmacological basis for the medical use of ginger in gastrointestinal disorders. Dif. Dis. Sci. 50, 1889-1897.
Gilani, A.H., Bashir, S., Janbaz, K.H., Khan, A., 2005a. Pharmacological basis for the use of Fumaria indica in constipation and diarrhea. J. Ethnopharmacol. 96, 585-589.
Gilani, A.H., Bashir, S., Janbaz, K.H., Shah, A.J., 2005b. Presence of cholinergic and calcium channel blocking activities explains the traditional use of Hibiscus rosasinesis in constipation and diarrhea. J. Ethnopharmacol. 102, 289-294.
Gilani, A.H., Khan, A., Ali, T., Ajmal, S., 2008. Mechanisms underlying the antispasmodic and bronchodilatory properties of Terminalia bellerica fruit. J. Ethnophamracol. 116, 528-538.
Haberlein, H., 2008. Hedera helix-mode of action evidenced by cell biologicals and biophysical investigation. Przew Lek. 1,255-256.
Holzinger, F., Chenot, J-F., 2011. Systematic review of clinical trials assessing the effectiveness of ivy leaf (Hedera Helix) for acute upper respiratory tract infections. Evid.-Based Complement. Alternat. Med. 2011, 382789.
Jeong,.S.1., Lee, S., Kim, KJ., Keum, K.S., Choo, Y.K., Choi, B.K., Jung, K.Y., 2005. Methylisogermabul Ione isolated from radish root stimulates small bowel motility via activation of acetylcholinergic receptors. J. Pharm. Pharmacol. 57, 1653-1659.
Jung, K.Y., Choo, Y.K., Kim, H.M., Choi, B.K., 2000. Radish extract stimulates motility of the intestine via the muscarinic receptors. J. Pharm. Pharmacol. 52, 1031-1036.
Makhlouf, G.M., Murthy, K.S., 2006. Cellular physiology of gastrointestinal smooth muscle. In: Johnson, L.R. (Ed.), Physiology of the Gastrointestinal Tract. Elsevier Academic Press, London, pp. 499-522.
Matsuda, H., Li, Y., Yokishawa, M., 1999. Effects of escins Ia, Ib, IIa and IIb from horse chestnuts on gastrointestinal transit and lieus in mice. Bioorg. Med. Chem. 7,1737-1741.
Mendel, M., Chlopecka, M., Dziekan, N., Wiechetek, M., 2011. The effect of the whole extract of common ivy (Hedera helix) leaves and selected active substances on the motoric activity of rat isolated stomach strips. J. Ethnopharmacol. 134, 796-802.
Murata, P., Hayakawa, T., Satoh, K., Kase, Y., Ishige, A., Sasaki, H., 2001. Effects of Dai-kenchu-to, a herbal medicine, on uterine and intestinal motility. Phytother. Res. 15,302-306.
Perry, W.L.M., 1971. Pharmacological Experiments on Isolated Preparations. E & S Churchill Livingstone, London.
Rippe. 0., Medejsky, M., 2006. Tartarus und Entgiftung: Stoffwechselkrankheiten und Ausleitungstherapien. In: Rippe, O., Medejsky, M. (Eds.), Die Krauterkunde des Paracelcus. Therapie mit Heilpflanzen nach abendlandischer Tradition. AT Verlag, Baden, pp. 256-257, 367.
Roth, L., Daunderer, M., Kormann, K., 1994. Giftpflanzen Pflanzgiften-Vorkommen, Wirkung, Therapie, Allergische and phototoxische Reaktionen. Nikol Verlagsgesellschaft mbH & Co, KG, Hamburg, pp. 390-392, 828.
Runkel, F., Prenner, L, Haberlein, H., 2005. Ein Beitrag zum Wirkmechanismus von Efeu. Pharm. Ztg. 150, 269-274.
Shibata, Ch., Saski, I., Naito, H., Ueno, T., Matsuno, S., 1999. The herbal medicine Dai-Kenchu-Tou stimulates upper gut motility through cholinergic and 5-hydroxytryptamine 3 receptors in conscious dogs. Surgery 126, 918-924.
Sieben, A., Prenner, L, Sorkalla, T., Wolf, A., Jakobs, D., Runkel, F., Haberlein, H., 2009. [alpha]-Hederin, but not hederacoside C and hederagenin from Hedera helix, affects the binding behavior, dynamics, and regulation of[32-adrenergic receptors. Biochemistry 48, 3477-3482.
Tachikawa, E., Kudo, K., Harada, K., Kashimoto, T., Miyate, Y., Kakizaki, A., Takahashi, E., 1999. Effects of ginseng saponins on responses induced by various receptor stimuli. Eur. J. Pharmacol. 396, 23-32.
Takagi, K, Saito, H., Tsuchiya, M., 1972. Pharmacological studies of Panax ginseng root: pharmacological properties of a crude saponin fraction. Jpn. J. Pharmacol. 22, 339-346.
Trute, A., Gross, J., Mutschler, E., Nahrstadt, A., 1997. In vitro antispasmodic compounds of the dry extract obtained from Hedera helix. Planta Med. 63,125-129.
Uchendu, C.N., Leek, B.F., 1999. Uterine muscle contractant from the root of Dalbergia saxatilis. Fitoterapia 70, 50-53.
Wood, J.D., 2002. Neural and humoral regulation of gastrointestinal motility. In: Schuster, M.M., Crowell, M.D., Koch, K.L. (Eds.), Hamilton Schuster atlas of gastrointestinal motility in health and disease. BC Decker Inc, London, pp. 19-42.
M. Mendel *, M. Chlopecka, N. Dziekan, W. Karlik, M. Wiechetek (1)
Division of Pharmacology and Toxicology, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, 8, Ciszewskiego St., 02-786 Warsaw, Poland
* Corresponding author. Tel.: +48 225936037; fax: +48 225936065.
E-mail address: email@example.com (M. Mendel).
0944-7113/$--see front matter [C] 2012 Elsevier GmbH. All rights reserved.