C.B. Li, W.B. Tang, and C.Y. Liu Institute of Physiology, School of Medicine,

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1 Title: Arecoline excites the contraction of distal colonic smooth muscle strips in rats via M 3 receptors - extracellular Ca 2+ influx - Ca 2+ store release pathway Short Title: Arecoline and colon motility Authors: Chuan-Bao Li, Xiao Yang, Wen-Bo Tang, Chuan-Yong Liu, and Dong-Ping Xie C.B. Li, W.B. Tang, and C.Y. Liu Institute of Physiology, School of Medicine, Shandong University, 44 West Wenhua Rd. Jinan, Shandong , P. R. China X.Yang, and D.P. Xie. 1 Department of Physiology, School of Medicine, Tongji University, 50 Chifeng Rd. Shanghai , P. R. China 1 Corresponding author: ( xiedping@tongji.edu.cn). This work was supported by National Natural Science Foundation of China (No ) and the 985 Foundation of Tongji University in China. Accepted by Can. J. Physiol. Pharmacol. (SCI) 1

2 Abstract: Areca is a Chinese herbal medicine that is widely used for constipation. However the mechanisms of its action are not clear. We investigated the effects of arecoline, the most active component of areca, on the motility of rat distal colonic smooth muscle strips. In longitudinal muscle of distal colon (LMDC) and circular muscle of distal colon (CMDC), arecoline increased the contraction in a dose-dependent manner. Tetrodotoxin (TTX) did not inhibit the effects of arecoline. The contractile response to arecoline was completely antagonized by atropine. 4-diphenylacetoxy-N-methylpiperidine-methiodide (4- DAMP) severely depressed the response for arecoline, but neither gallamine nor methoctramine did. Nifedipine, 2-aminoethoxydiphenyl borate (2 APB) and Ca 2+ free Krebs solution with ethylene glycol-bis ( -aminoethyl ether)-n, N, N', N'-tetraacetic acid (EGTA) partly inhibited the effects of arecoline. Sum of Ca 2+ free Krebs solution, EGTA and 2 APB completely inhibited the effects of arecoline. Conclusion: The results show that arecoline stimulates distal colonic contraction in rats via M 3 receptors - extracellular Ca 2+ influx - Ca 2+ store release pathway. It is likely that the action of areca in relieving constipation is due to its stimulation of muscle contraction. Key words: M 2 and M 3 receptors, colon, smooth muscle, intestinal motility, arecoline, IP 3 receptor, extracellular Ca 2+ influx, Ca 2+ store release. 2

3 Introduction Areca (Areca catechu L., betel nuts; a Chinese herbal medicine named pinang or bing-lang) has been shown to relieve indigestion, and used clinically to treat abdominal distention and constipation caused by stagnation of the circulation (Jia 1994; Zhang et al. 2000; Eglen 2001). Our previous study indicated that areca stimulated the motility of colonic smooth muscle strips in rats (Xie et al. 2002). Arecoline is a major betel-nut alkaloid and one of the most important and effective extractions from areca (Arjungi 1976; Panigrahi and Rao 1982). The pharmaceutical importance of areca is probably due to the presence of arecoline (Yang et al. 2002). In our studies, we observed the effect of arecoline on the contraction of distal colonic smooth muscle strips in rats. Arecoline was considered as a nonselective muscarinic receptor agonist. Arecoline dose dependently increases the contraction of the guinea pig ileum, the gastrointestinal transit of the mouse and the colonic smooth muscle strips of rabbits (Williams et al. 1992; Xie et al. 2004). Muscarinic receptor consists of five subtypes (M 1 - M 5 ). All the subtypes of muscarinic receptor are present in circular smooth muscle cells of guinea pig gastric antrum (So et al. 2003). M 2 and M 3 receptors are expressed abundantly throughout the gastrointestinal tract to regulate the contraction of smooth muscle (Sawyer and Ehlert 1998; Lambrecht et al. 1999). Zhang et al. have demonstrated the coexistence of muscarinic M 3 (45%) and M 2 (55%) receptors in adult rat colonic smooth muscle cells (Zhang et al. 1996). In the present experiment, we investigate the involvement of 3

4 muscarinic receptor subtypes in arecoline induced contraction of colonic smooth muscle strips. After binding with the agonists, M 2 and M 3 receptors inhibit adenylyl cyclase (AC) and activate phospholipase C (PLC) (Caulfield 1993; Eglen et al. 1996; Caulfield and Birdsall 1998). Our previous study showed that arecoline increased the contraction of the rabbit colonic smooth muscle strips via M 3 receptor (Xie et al. 2004). Arecoline induces epithelial changes by activation of the M 3 receptor via the induction of calcium (Thangjam et al. 2009). Arecoline also stimulates testosterone production of rat Leydig cells by activating L-type calcium channels (Wang et al. 2008). In this study we further investigate the source of Ca 2+ in arecoline induced contraction of colonic smooth muscle strips. Materials and methods Animals Wistar rats, female, weighing g, were housed in a temperature (22 C)-controlled environment. The use and treatment of animals followed the guidelines of the International Animal Care and Use Committee of Shandong University. All animals were cared for in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, published by the National Science Council, China. Tissue culture 4

5 Animals were fasted overnight and killed. The distal colon (5 cm proximal to the anus) was removed (Xie et al. 2006). The segment of the colon was opened along the mesentery. Muscle strips (8 2 mm) parallel to either the circular or the longitudinal fibers were excised and named circular muscle of distal colon (CMDC) and longitudinal muscle of distal colon (LMDC), respectively. The mucosa on each strip was carefully removed. The muscle strip was suspended in a tissue chamber containing 5 ml Krebs solution (37 C) and bubbled continuously with 95% O 2 and 5% CO 2. One end of the strip was fixed on a hook at the bottom of the chamber. Another end was connected to an external isometric force transducer (JH-2B, Peijing, China). Spontaneous contractile activity of the colonic strips (under an initial tension of 1 g) was recorded simultaneously in an MFlab system by an SMUP-PC amplifier (Fudan University, Shanghai, China). The Krebs solution was composed of the following reagents (mmol/l): NaCl 120.6, KCl 5.9, CaCl 2 2.5, KH 2 PO 4 1.2, MgCl 2 1.2, NaHCO and glucose Experimental protocols Dose response effect of arecoline on distal colonic motility The distal colonic strips were stabilized for 60 min. Preliminary experiments showed that arecoline produced maximal effect within 3 minutes. Dose-dependent curves of arecoline (1 nmol/l, 10 nmol/l, 30 nmol/l, 100 nmol/l, 1 μmol/l, 10 μmol/l) were recorded by successively applying arecoline at 5 minutes intervals. 5

6 Effect of tetrodotoxin (TTX) on arecoline induced response TTX is a selective Na + channel blocker. To investigate whether arecoline acts directly on smooth muscle or on nerve fiber, the effect of TTX (10 µmol/l) on arecoline induced response was examined. TTX was added separately to tissue chamber 30 min before addition of arecoline (1 nmol/l - 10 μmol/l). Antagonism by muscarinic receptor antagonists Antagonism by muscarinic receptor antagonists was tested using the nonselective muscarinic receptor antagonist atropine (10 μmol/l), the M 2 - selective antagonist gallamine (1 μmol/l), methoctramine (3 nmol/l), or the M 3 - selective antagonist 4 - DAMP (0.4 μmol/l). The muscle strips were separately pretreated with these agents for 20 min before the arecoline (1 nmol/l - 10 μmol/l) was administered. Ca 2+ store release and Ca 2+ influx Activation of M 3 /PLC/IP 3 system induces the release of stored intracellular Ca 2+. Therefore IP 3 receptor antagonists were used to investigate whether the arecoline-induced contractions depended on Ca 2+ store release. Muscle strips were pretreated for 20 min using the selective IP 3 receptor antagonist 2 - APB (30 μmol/l) or 6

7 L- type Ca 2+ channel blocker nifedipine (0.1 μmol/l). Arecoline (1 nmol/l - 10 μmol/l) was then administered. Drugs The following drugs were used: arecoline, atropine sulfate, gallamine, methoctramine, 4-Diphenylacetoxy-N-methylpiperidine-methiodide (4 - DAMP), 2-Aminoethoxydiphenyl borate (2 - APB), nifedipine, ethylene glycol-bis ( -aminoethyl ether)-n,n, N',N'-tetraacetic acid (EGTA), tetrodotoxin (TTX). All the chemicals were purchased from Sigma. Statistical analysis The area under contraction curve of LMDC and CMDC were measured by using MFlab system (Fudan University, Shanghai, China). Data were presented as means ± standard error of the mean, with n indicating the number of rats, and statistically analyzed by one-way Analysis of Variance. Significant differences were determined by Dunnett, s method. A probability level of P < 0.05 was considered to be significant for statistical analysis. Results Dose response effect of arecoline on distal colonic motility 7

8 The lowest concentration of arecoline (1 nmol/l) failed to elicit a contractile response. Arecoline(10 nmol/l - 10 μmol/l) dose dependently increased the contractions of LMDC and CMDC (Fig. 1A). The dose response curves of the arecoline are shown in Fig. 1B, in which the contractile activities were expressed as area under the contraction curve. Effect of tetrodotoxin (TTX) on arecoline induced response TTX (10 µmol/l) had no significant effect on the area under contraction curve of LMDC or CMDC. As shown in Fig. 2, the dose response curves of the arecoline were almost not affected after a 30-min treatment with the Na + channel blocker TTX (P > 0.05 compared with the data administration of TTX). Antagonism by muscarinic receptor antagonists The nonselective muscarinic receptor antagonist atropine (10 μmol/l) had no significant effect on distal colonic smooth muscle strips. The dose response curves of the arecoline were completely eliminated after treatment with atropine (Fig. 3A, E, F. P < 0.05 compared with the data administration of atropine, n = 10). The M 2 selective antagonist gallamine (1 μmol/l) and methoctramine (3 nmol/l) did not affect the area under contractions of LMDC and CMDC (P > 0.05 respectively). 8

9 Pretreatment with gallamine or methoctramine did not produce shift of arecoline-induced curves of LMDC and CMDC (Fig. 3B, E, F). As shown in Fig. 3E and F, pretreatment with M 3 selective antagonist 4 - DAMP (0.4 μmol/l) produced a rightward shift of arecoline-induced curves of LMDC and CMDC. Actually, the dose response curves of the arecoline were almost completely eliminated after treatment with 4 DAMP except that the concentration of arecoline was increased to 10 μmol/l (Fig. 3C). Pretreatment with 4 - DAMP (0.4 μmol/l) and gallamine (1 μmol/l) simultaneously behaved similarly (Fig. 3D, E, F). Ca 2+ store release and Ca 2+ influx The selective IP 3 receptor antagonist 2 - APB (30 μmol/l) shifted the arecoline-induced curves of LMDC and CMDC to the right (Fig. 4A). The contractile response (area under the contraction curve) to cumulative applications of arecoline from 10 nmol/l to 100 nmol/l, but not applications of arecoline from 1 μmol/l to 10 μmol/l, were decreased by administration of 2 APB (Fig. 4B, C). At the dose of 30 nmol/l arecoline, area under the contraction curve of LMDC and CMDC with 2 APB decreased from 66.5 ± 9.6 gs to 39.1 ± 8.6 gs, from ± 48.1 gs to 55.6 ± 14.0 gs respectively (P < 0.05 compared with the data administration of 2 - APB, n = 10). But at the highest dose of 10 μmol/l arecoline, area under the contraction curve of LMDC and CMDC decreased from ± 11.6 gs to 99.7 ± 6.8 gs, from ± 9

10 30.6 gs to ± 14.8 gs respectively (P > 0.05 compared with the data administration of 2 - APB, n = 10). The L- type Ca 2+ channel blocker nifedipine (0.1 μmol/l) produced a parallel rightward shift of arecoline-induced curves of LMDC and CMDC in a contraction dependent manner (Fig. 5A, C, D). Ca 2+ free Krebs solution plus EGTA also produced a parallel rightward shift of arecoline-induced curves in a contraction dependent manner (Fig. 5B, C, D). At the highest dose of 10 μmol/l arecoline, area under the contraction curve of LMDC and CMDC with nifedipine decreased from ± 9.8 gs to 64.2 ± 4.9 gs, from ± 42.1 gs to ± 21.1 gs respectively (P < 0.05 compared with the data administration of nifedipine, n = 10); pretreated with Ca 2+ free solution and EGTA, area under the contraction curve of LMDC and CMDC decreased from ± 13.8 gs to 36.7 ± 3.0 gs, from ± 36.1 gs to 38.4 ± 3.6 gs respectively (P < 0.05 compared with the data administration of both Ca 2+ free solution and EGTA, n = 10). As shown in Fig. 6A, B and C, pretreatment with Ca 2+ free Krebs solution, EGTA (10 μmol/l) and 2 - APB (30 μmol/l) simultaneously completely blocked the arecoline-induced curves of LMDC and CMDC (P < 0.05 compared with the data administration of both 2 - APB and EGTA in Ca 2+ free Krebs solution, n = 10). Discussion 10

11 Our results showed that arecoline dose dependently stimulated the contraction of colonic smooth muscle in rats. Since arecoline is one of the most important and effective extractions from areca, it is likely that the effect of areca on rat colonic smooth muscle is mainly due to arecoline. The nonselective M receptor antagonist atropine completely blocked the arecoline-induced contractions suggesting that arecoline acts on rat colonic smooth muscle via M receptors. A variety of neurons in gastrointestinal smooth muscle layers express presynaptic muscarinic receptors, which serve to modulate the rate of neuronal firing and the release of neurotransmitters. Muscarinic agonists cause a TTX sensitive increase in spontaneous acetylcholine release from myenteric neurons (Kilbinger and Stein 1988). In our studies, TTX did not block the arecoline-induced contractions, which suggests that the action of arecoline is TTX insensitive and that arecoline acts directly on the colonic smooth muscle. Coexistence of M 3 and M 2 receptors in rat colonic smooth muscle cell has been previously reported (Zhang 1996; Gomez et al. 1992). A lot of previous evidence suggests that an interaction between M 2 and M 3 receptors plays a crucial role in mediating the gastrointestinal contraction response (Singer et al. 2002; Unno et al. 2003). Iwanaga et al found that carbachol induces Ca 2+ -dependent contraction via muscarinic M 2 and M 3 receptors in rat colonic subepithelial myofibroblasts (Iwanaga et al. 2009). Acetylcholine (Ach) activates both M 2 and M 3 signalling pathways in rat duodenum myocytes (Fritz et al. 2008). Although both M 2 and M 3 receptors mediate 11

12 cholinergic contraction in human esophageal smooth muscle, the M 3 receptor is predominant (Braverman et al. 2009). The M 3 receptor also mediates magnolol induced contractions of colonic circular muscles in rats (Jeong et al. 2008). In our previous study in rabbits (Xie et al. 2004), M 2 receptor antagonist gallamine (0.4 μmol/l) had no effect on arecoline (80 nm) induced colonic smooth muscle contraction. In this study, we increased the concentration of gallamine from 0.4 μmol/l to 1 μmol/l. We further observed the effect of another M 2 receptor antagonist methoctramine on arecoline-induced dose response curve. Neither gallamine nor methoctramine had effect on arecoline-induced contraction. It was the M 3 receptor antagonist (4 - DAMP) that inhibited the arecoline-induced contraction, which suggested that arecoline stimulated the contraction of distal colonic smooth muscle in rat via M 3 receptor but not M 2 receptors. The M 3 receptor causes the contraction via Gq type of GTP-binding protein - phospholipase C - IP 3 - Ca 2+ stores release pathway (Okamoto et al. 2002). In our studies, the IP 3 receptor antagonist 2 - APB partly inhibited the arecoline-induced contraction (see Figure 4B: for LMDC, suppressed about 22%; for CMDC, about 13.9%), which suggested that although arecoline-induced contraction is mediated by IP 3 - Ca 2+ store release, Ca 2+ influx mechanisms might also involved. We further studied the involvement of extracellular Ca 2+ influx in the arecoline-induced contraction. We found that the L type Ca 2+ channel blocker nifedipine significantly reduced the arecoline-induced contraction (see Fig. 5C, D: for LMDC, 12

13 suppressed about 50%; for CMDC, about 55%). When the normal Krebs solution is substituted with the Ca 2+ free Krebs solution, the arecoline-induced contraction decreased severely (also see Fig. 5C, D: for LMDC, suppressed about 71.4%; for CMDC, about 88.9%). The results showed that the arecoline-induced colonic contraction mainly depends on influx of extracellelar Ca 2+. Arecoline-induced contraction disappeared pretreated with both Ca 2+ free Krebs solution and 2-APB, which suggested that both Ca 2+ influx and Ca 2+ store release contribute to the arecoline-induced contraction of the colonic smooth muscle. When both M 2 and M 3 receptors mediate the gastrointestinal contraction response, cationic channel opening is mediated by M 2 receptors (Zholos and Bolton 1997; Komori et al. 1998) and potentiated by M 3 receptors through Ca 2+ store release (Pacaud and Bolton 1991; Zholos et al. 1994) or direct interaction with M 2 receptors (Zholos and Bolton 1997; Okamoto et al. 2002). Sakamoto et al (Sakamoto et al. 2006) found that muscarinic receptor-operated cationic current was inhibited competitively by an M 2 -selective antagonist but non-competitively by an M 3 -selective one. The muscarinic agonist oxotremorine methiodide (OxoM) elicited transient repeatable increases in the intracellular calcium concentration. M 2 machr antagonist AF-DX 116 reduced the responses to OxoM. Activation of M 3 but not M 2 receptors increased intracellular calcium via activation the release of calcium from the intracellular stores (Kullmann et al. 2008). But in our studies, the arecoline-evoked colonic contraction is mediated by only M 3 receptors, the L-type Ca 2+ channels are involved in this pathway (Fig. 3C, E, F and Fig. 5A, C, D). Sakamoto et al (Sakamoto et al. 2007) found that 15% of 13

14 M 2 -knockout (M 3 receptors pathways) mouse gut smooth muscle cells had significant channel activity when exposed to carbachol. And the ongoing channel activity was terminated upon addition of atropine in the bath solution containing carbachol. Our studies in rats suggested that arecoline functioning independently stimulates the contraction of colonic smooth muscle strips via M 3 receptor - cationic channel activation pathway but not interaction with M 2 receptors - cationic channel activation pathway. The primary role of the muscarinic cationic channel is to depolarize the membrane, so admitting Ca 2+ into the cell via voltage-gated Ca 2+ channels (Bolton 1979; Unno et al. 2003). The M 3 -mediated depolarization has been suggested to be due to the activity of both 70-pS and 120-pS channels (Sakamoto et al. 2007). It is possible that arecoline activates the M 3 pathway, via the M 3 Gq PLCβ system, leads to activation of the 70-pS and 120-pS cationic channels in brief opening mode, the depolarization of the membrane activates the L-type voltage-gated Ca 2+ channels, so admitting Ca 2+ into the cell, and concurrently to IP 3 -induced Ca 2+ release, then eventually causes the contraction of colonic smooth muscle strips. However, further work is needed to test this possibility. In summary, arecoline functioning independently stimulates the contraction of isolated distal colonic smooth muscle strips in rats via M 3 receptor, and the M 3 receptors mediate the arecoline-induced colonic contraction via both extracellular Ca 2+ influx and Ca 2+ store release. It is likely that the action of areca in relieving constipation is due to the stimulation of arecoline on smooth muscle via M 3 receptor directly. The findings 14

15 suggested the new highlight of areca to relieve constipation as the rectal suppository clinically. Acknowledgements We thank David C. Randall and Peter Grigg for their critical reading of the manuscript. This work was supported by National Natural Science Foundation of China (No ) and the 985 Foundation of Tongji University in China. 15

16 References Argunji, K.N Areca nut: a review. Arzneimittelforschung, 26 (5): PMID: Bolton, T.B Mechanisms of action of transmitters and other substances on smoothmuscle. Physiol. Rev. 59 (3): PMID: Braverman, A.S., Miller, L.S., Vegesna, A.K., Tiwana, M.I., Tallarida, R.J., and Ruggieri, M.R.Sr Quantitation of the contractile response mediated by two receptors: M 2 and M 3 muscarinic receptor-mediated contractions of human gastroesophageal smooth muscle. J. Pharmacol. Exp. Ther. 329(1): PMID: Caulfield, M.P Muscarinic receptors characterization, coupling and function. Pharmacol. Ther. 58 (3): PMID: Caulfield, M.P., and Birdsall, N.J International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev. 50 (2): PMID: Eglen, R.M Muscarinic receptors and gastrointestinal tract smooth muscle function. Life Sci. 68 (22-23): PMID: Eglen, R.M., Hegde, S.S., and Watson, N Muscarinic receptor subtypes and smooth muscle function. Pharmacol. Rev. 48 (4): PMID: Fritz, N., Dabertrand, F., Mironneau, J., Macrez, N., and Morel, J.L

17 Acetylcholine evokes an InsP3R1-dependent transient Ca 2+ signal in rat duodenum myocytes. Can. J. Physiol. Pharmacol. 86(9): PMID: Gomez, A., Martos F., Bellido, I., Marquez, E., Garcia, A.J., Pavia, J., et al Muscarinic receptor subtypes in human and rat colon smooth muscle. Biochem. Pharmacol. 43 (11): PMID: Iwanaga, K., Murata, T., Okada, M., Hori, M., and Ozaki, H Carbachol induces Ca(2+)-dependent contraction via muscarinic M 2 and M 3 receptors in rat intestinal subepithelial myofibroblasts. J. Pharmacol. Sci. 110(3): PMID: Jeong, S.I., Kim, Y.S., Lee, M.Y., Kang, J.K., Lee, S., Choi, B.K., et al Regulation of contractile activity by magnolol in the rat isolated gastrointestinal tracts. Pharmacol. Res. 59(3): PMID: Jia, M Clinical importance of areca. Liaoning J. Traditional Chinese Medicine, 21(1): Kilbinger, H., and Stein, A Dicyclomine discriminates between M 1 - and M 2 -muscarinic receptors in the guinea-pig ileum. Br. J. Pharmacol. 94 (4): PMID: Komori, S., Unno, T., Nakayama, T., and Ohashi, H M 2 and M 3 muscarinic receptors couple, respectively, with activation of nonselective cationic channels and potassium channels in intestinal smooth muscle cells. Jpn. J. Pharmacol. 76 (2): PMID: Kullmann, F.A., Artim, D., Beckel, J., Barrick, S., de Groat, W.C., and Birder, L.A Heterogeneity of muscarinic receptor-mediated Ca 2+ responses in cultured 17

18 urothelial cells from rat. Am. J. Physiol. Renal. Physiol. 294(4): F971- F981. PMID: Lambrecht, G., Gross, J., and Mutschler, E Neuronal soma-dendritic and prejunctional M 1 -M 4 receptors in gastrointestinal and genitourinary smooth muscle. Life Sci. 64 (6-7): PMID: Okamoto, H., Prestwich, S.A., Asai, S., Unno, T., Bolton, T.B., and Komori, S Muscarinic agonist potencies at three different effector systems linked to the M 2 or M 3 receptor in longitudinal smooth muscle of guinea-pig small intestine. Br. J. Pharmacol. 135 (7): PMID: Pacaud, P., and Bolton, T.B Relation between muscarinic receptor cationic current and internal calcium in guinea-pig jejunal smooth muscle cells. J. Physiol. 441: PMID: Panigrahi, G.., and Rao, A Chromosome-breaking ability of arecoline, a major betel-nut alkaloid, in mouse bone-marrow cells in vivo. Mutat. Res. 103 (2): PMID: Sakamoto, T., Unno, T., Kitazawa, T., Taneike, T., Yamada, M., Wess, J., et al Three distinct muscarinic signalling pathways for cationic channel activation in mouse gut smooth muscle cells. J. Physiol. 582 (Pt 1): PMID: Sakamoto, T., Unno, T., Matsuyama, H., Uchiyama, M., Hattori, M., Nishimura, M., et al Characterization of muscarinic receptor-mediated cationic currents in longitudinal smooth muscle cells of mouse small intestine. J. Pharmacol. Sci. 100 (3): PMID:

19 Sawyer, G.W., and Ehlert, F.J Contractile roles of the M 2 and M 3 muscarinic receptors in the guinea pig colon. J. Pharmacol. Exp. Ther. 284 (1): PMID: Singer, C.A., Vang, S., and Gerthoffer, W.T Coupling of M 2 muscarinic receptors to Src activation in cultured canine colonic smooth muscle cells. Am. J. Physiol. Gastrointest. Liver Physiol. 282 (1): G61-G68. PMID: So, I., Yang, D.K., Kim, H.J, Min, K.W., Kang, T.M, Kim, S.J, et al Five subtypes of muscarinic receptors are expressed in gastric smooth muscles of guinea pig. Exp. Mol. Med. 35 (1): PMID: Thangjam, G.S., Agarwal, P., Balapure, A.K., Girish, Rao S., and Kondaiah, P Regulation of extracellular matrix genes by arecoline in primary gingival fibroblasts requires epithelial factors. J. Periodontal. Res. 44(6): PMID: Unno, T., Kwon, S.C., Okamoto, H., Kato, Y., and Matsuyama, H Receptor signaling mechanisms underlying muscarinic agonist-evoked contraction in guinea-pig ileal longitudinal smooth muscle. Br. J. Pharmacol. 139 (2): PMID: Wang, S.W., Hwang, G.S., Chen, T.J., and Wang, P.S Effects of arecoline on testosterone release in rats. Am. J. Physiol. Endocrinol. Metab. 295 (2): E PMID: Williams, P.D., Colbert, W.E., Shetler, T.J., and Turk, J.A Comparative pharmacological profile of muscarinic agonists in the isolated ileum, the pithed rat, and the mouse charcoal meal transit test. Gen. Pharmacol. 23 (2): PMID: 19

20 Xie, D., Chen, L., Liu, C., and Liu, K The inhibitory effects of oxytocin on distal colonic contractile activity in rabbits are enhanced by ovarian steroids. Acta Physiol. (Oxf) 186 (2): PMID: Xie, D.P., Chen, L.B., Liu, C.Y., Zhang C.L., Liu, K.J., and Wang, S. P Arecoline excites the colonic smooth muscle motility via M 3 receptor in rabbits. Chin. J. Physiolo. 47 (2): PMID: Xie, D.P., Li, W., Qu, S.Y., Zheng, T.Z., Yang, Y.L., Ding, Y.H., et al Effect of areca on contraction of colonic muscle strips in rats. World J. Gastroenterol. 8 (2): PMID: Yang, Y.L., Cheng, F., Wang, H., and Si, K.Y Effects of Areca catechu L. on gastrointestinal motility in animals. Journal of Northwest Normal University, 138(1): 1-3. Zhang, L Muscarinic receptors in developing rat colon. Eur. J. Pharmacol. 304 (1-3): PMID: Zhang., Ren, P., Huang, X., and Li, Y Regulation of Chinese herbal on gastrointestinal hormones and gastrointestinal motility. Shijie Huaren Xiaohua Zazhi, 8(12): Zholos, A.V., and Bolton, T.B Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle. Br. J. Pharmacol. 122 (5): PMID:

21 Zholos, A.V., Komori, S., Ohashi, H., and Bolton, T.B Ca 2+ inhibition of inositol trisphosphate-induced Ca 2+ release in single smooth muscle cells of guinea-pig small intestine. J. Physiol. 481 ( Pt 1): PMID:

22 Figure Legends Fig. 1. The contractile effects of arecoline on longitudinal muscle of distal colon (LMDC) and circular muscle of distal colon (CMDC) in rats. (A) The contractile responses and area under the contraction curve produced by the application of ascending concentrations of arecoline (1 nmol/l - 10 μmol/l) on colonic LMDC and CMDC. Arecoline was applied at stepwise increasing concentrations at points marked by the arrow down. (B) Averaged concentration response curves induced by arecoline on the contraction of colonic LMDC and CMDC. Each point represents the mean SE of area under the contraction curve. Control means the data prior to arecoline treatment. P < 0.05 compared with the control (n = 10). Fig. 2. (A) Contractions of LMDC and CMDC produced by arecoline (1 nmol/l - 10 μmol/l) in the absence and presence of Na + channel blocker TTX (10 µmol/l). Arrows down means the administration of arecoline. TTX was applied at points marked by the open triangles down. Averaged concentration response curves for contraction of LMDC (B) and CMDC (C) induced by arecoline in the absence and presence of Na + channel blocker TTX. LMDC, longitudinal muscle of distal colon; CMDC, circular muscle of distal colon; TTX, tetrodotoxin. TTX + arecoline, both TTX and arecoline are used.control means the data prior to arecoline administration. Each point indicates the mean SE of area under the contraction curve. 22

23 Fig. 3. Contractions of LMDC and CMDC produced by arecoline (1 nmol/l - 10 μmol/l) in the absence and presence of nonselective muscarinic antagonist atropine (10 μmol/l, fig. 3A), selective M 2 receptor antagonist gallamine (1 μmol/l) and methoctramine (3 nmol/l, fig. 3B), selective M 3 receptor antagonist 4-DAMP (0.4 μmol/l, fig. 3C), or gallamine as well as 4-DAMP (fig. 3D). Arrows down means the administration of arecoline. Antagonist was applied at points marked by the open triangles down. Averaged concentration response curves for contraction of LMDC (E) and CMDC (F) induced by arecoline in the absence and presence of nonselective or selective muscarinic antagonist. LMDC, longitudinal muscle of distal colon; CMDC, circular muscle of distal colon; 4-DAMP, 4-Diphenylacetoxy-Nmethylpiperidine-methiodide. Control means the data prior to arecoline administration. Each point indicates the mean SE of area under the contraction curve. P < 0.05 compared with the data of arecoline in the absence of antagonist. Fig. 4. Contractions of LMDC and CMDC produced by arecoline (1 nmol/l - 10 μmol/l) in the absence and presence of 2-APB (30 μmol/l, Fig. 4A). Arrows down means the administration of arecoline. 2-APB was applied at points marked by the open triangles down. Averaged concentration response curves for contraction of LMDC (B) and CMDC (C) induced by arecoline in the absence and presence of 2-APB. LMDC, longitudinal muscle of distal colon; CMDC, circular muscle of distal colon; 2-APB, 2-aminoethoxydiphenyl borate. 2-APB + arecoline, both 2-APB and arecoline are added.control means the data prior to arecoline administration. Each 23

24 point indicates the mean SE of area under the contraction curve. P < 0.05 compared with the data of arecoline in the absence of 2-APB (n = 10). Fig. 5. Effect of nifedipine (0.1 μmol/l) and Ca 2+ free Krebs solution (with EGTA, 10 μmol/l) on arecoline (1 nmol/l - 10 μmol/l) induced contraction of longitudinal muscle of distal colon (LMDC) and circular muscle of distal colon (CMDC) in rats. (A) Representative of the recording of the colonic motility. Arrows down means the administration of arecoline. Nifedipine or Ca 2+ free Krebs solution was applied at points marked by the open triangles down. (B) Satatistic analysis of the effect of nifedipine and Ca 2+ free Krebs solution on arecoline induced contraction of LMDC in rats. (C) Satatistic analysis of the effect of nifedipine and Ca 2+ free Krebs solution on arecoline induced contraction of CMDC in rats. EGTA, ethylene glycol-bis ( -aminoethyl ether)-n, N, N', N'-tetraacetic acid. nifedipine + arecoline, both nifedipine and arecoline are added. Ca 2+ free Krebs solution + EGTA, the strips are incubated in the arecoline of Ca 2+ free Krebs solution with EGTA.Control means the data prior to arecoline administration. Each point indicates the mean SE of area under the contraction curve. P < 0.05 compared with the data of arecoline in the absence of nifedipine or Ca 2+ free Krebs solution (n = 10). Fig. 6. Effect of Ca 2+ free Krebs solution and 2 - APB simultaneously on arecoline (1 nmol/l - 10 μmol/l) induced contraction of longitudinal muscle of distal colon (LMDC) and circular muscle of distal colon (CMDC) in rats. (A) Representative of the recording of the colonic motility. Arrows down means the administration of 24

25 arecoline. Ca 2+ free Krebs solution and 2 - APB were applied at points marked by the open triangles down. (B) Satatistic analysis of the effect of Ca 2+ free Krebs solution and 2 - APB on arecoline induced contraction of LMDC in rats. (C) Satatistic analysis of the effect of Ca 2+ free Krebs solution and 2 - APB on arecoline induced contraction of CMDC in rats. 2-APB, 2-aminoethoxydiphenyl borate. EGTA, ethylene glycol-bis ( -aminoethyl ether)-n, N, N', N'-tetraacetic acid. Ca 2+ free Krebs solution + EGTA + 2-APB + arecoline, the strips are incubated in Ca 2+ free Krebs solution with EGTA, then both 2-APB and arecoline are added.control means the data prior to arecoline administration. Each point indicates the mean SE of area under the contraction curve. P < 0.05 compared with the data of arecoline pretreatment with Ca 2+ free Krebs solution and 2 - APB (n = 10). 25

26 A B 600 Area under the contraction curve (gs) arecoline LMDC (n = 10) arecoline CMDC (n = 10) control log molar concentration of arecoline Fig. 1. (Li et al) 26

27 Contraction (g) A TTX (10 μm) LMDC LMDC TTX (10 μm) CMDC CMDC Arecoline (nm) B 200 Area under the contraction curve (gs) LMDC arecoline (n = 10) TTX + arecoline (n = 10) control log molar concentration of arecoline 27

28 C 600 CMDC Area under the contraction curve (gs) arecoline (n = 12) TTX + arecoline (n = 12) control log molar concentration of arecoline Fig. 2. (Li et al) A 28

29 B C 29

30 D E Area under the contraction curve (gs) LMDC arecoline (n = 10) atropine + arecoline (n = 10) gallamine + arecoline (n = 10) methoctramine + arecoline (n = 10) 4-DAMP + arecoline (n = 10) gallamine + 4-DAMP + arecoline (n = 10) control log molar concentration of arecoline 30

31 F Area under the contraction curve (gs) 600 arecoline (n = 15) atropine + arecoline (n = 10) 500 gallamine + arecoline (n = 10) methoctramine + arecoline (n = 10) 4-DAMP + arecoline (n = 10) 400 gallamine + 4-DAMP + arecolline (n = 10) CMDC control log molar concentration of arecoline A Fig. 3. (Li et al) 31

32 Area under the contraction curve (gs) B LMDC arecoline (n = 10) 2-APB + arecoline (n = 10) control log molar concentration of arecoline C Area under the contraction curve (gs) CMDC arecoline (n = 10) 2-APB + arecoline (n = 10) control log molar concentration of arecoline Fig. 4. (Li et al) 32

33 A B 33

34 c C 200 Area under the contraction curve (gs) LMDC arecoline (n = 10) nifedipine + arecoline (n = 10) Ca 2+ free Krebs solution + EGTA (n = 10) control log molar concentration of arecoline D Area under the contraction curve (gs) arecoline (n = 10) nifedipine + arecoline (n = 10) Ca 2+ free Krebs solution + EGTA (n = 10) CMDC control log molar concentration of arecoline Fig. 5. (Li et al) 34

35 A B Area under the contraction curve (gs) LMDC arecoline (n = 10) Ca 2+ free Krebs solution + EGTA + 2-APB + arecoline (n = 10) control log molar concentration of arecoline 35

36 C Area under the contraction curve (gs) CMDC arecoline (n = 10) Ca 2+ free Krebs solution + EGTA + 2-APB + arecoline (n = 10) control log molar concentration of arecoline Fig. 6. (Li et al) 36