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Ligusticum wallichi-induced vasorelaxation mediated by mitogen-activated protein kinase in rat aortic smooth muscle
Journal of Ethnopharmacology 90 (2004) 397–401
Ligusticum wallichi-induced vasorelaxation mediated by mitogen-activated protein kinase in rat aortic smooth muscle Bokyung Kim a,∗ , Junghwan Kim a , Aeran Kim a , Yoon-Sun Kim a , Youn Ri Lee a , Young Min Bae a , SungIl Cho a , Mee-Ra Rhyu b a
Department of Physiology, College of Medicine, Konkuk University, Danwol-dong 322, Chungju, Choong-Buk 380-701, South Korea b Food Chemistry & Biotechnology Division, Korea Food Research Institute, Kyonggi-Do 463-420, South Korea Received 16 May 2003; received in revised form 30 October 2003; accepted 3 November 2003
Abstract Traditional herbal medicines have been widely used for the treatment of cardiovascular disorders in oriental countries. To determine the effects of Ch1LW, a chloroform extract of Ligusticum wallichi, on the vascular system, we studied changes in rat aortic smooth muscle in terms of magnitude of contraction and the activity of mitogen-activated protein kinases (MAPKs). Ch1LW inhibited the muscle contraction induced by norepinephrine (NE) in aortic strips. Ch1LW also abolished Ca2+ -independent contraction evoked by 12-deoxyphorbol 13-isobutyrate in Ca2+ -free medium containing 1 mM EGTA. Furthermore, western blotting analysis using phosphorylated MAPK antibodies showed that NE increased the activity of both extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK, which were inhibited by PD98059 and SB203580, blockers of ERK1/2 and p38 MAPK, respectively. Furthermore, treatment with Ch1LW significantly abolished NE-mediated activation of ERK1/2, whereas the activity of p38 MAPK was not affected by the extract. These results suggest that Ch1LW induces vasorelaxation in rat aortic smooth muscle, which may be mediated by the inhibition of ERK1/2 pathway, but not p38 MAPK. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Aorta; Ligusticum wallichi; Mitogen-activated protein kinases; Vasorelaxation
1. Introduction Ligusticum wallichi, a popular Chinese herbal medicine, has been used orally with other herbs for “heart disease” for thousands of years. Current research in nutrition science provides a better understanding of the possible link between the plant and heart disease. A component of Ligusticum wallichi increases myocardial contractility and coronary circulation (Chiou et al., 1991; Hwang, 1993). In contrast, this plant can inhibit the muscle contractions induced by vasoconstrictors and low systemic blood pressure (Hwang, 1993). However, little is known about the effects of Ligusticum wallichi on the contraction of smooth muscle and the mechanisms underlying the contractile system. It is well established that smooth muscle contraction is regulated by intracellular Ca2+ ([Ca2+ ]i ) and the phosphorylation of myosin light chain (MLC) (Karaki, 1989; Somlyo and Himpens, 1989). However, various kinds of vasoconstrictors induce a further contraction at a given [Ca2+ ]i , and elicit a sustained contraction under ∗ Corresponding
author. Tel.: +82-43-8403726; fax: +82-43-8519392. E-mail address: [email protected] (B. Kim).
Ca2+ -depleted conditions, referred to as “Ca2+ -independent contraction,” in intact and membrane-permeabilized smooth muscle (Hori et al., 1992; Kim et al., 2003a). In previous reports, several molecules, including protein kinase C (PKC) and MAPKs, have been suggested as candidate regulators of Ca2+ -independent contraction (Nixon et al., 1995; Lee et al., 1999; Kim et al., 2003b). MAPKs constitute a family of serine/threonine-specific protein kinases which play a central role in intracellular signal transduction initiated by extracellular stimuli, including growth factors, neurotransmitters, and hormones (Kosako et al., 1994). The three MAPK isoforms, extracellular signal-regulated kinase (ERK1/2), p38 MAPK and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), are central elements in transducing the messages in mammalian cells (Miyata and Nishida, 1999). In vascular smooth muscle, MAPKs are activated by receptor agonists, including angiotensin II, phenylephrine, and endothelin-1 (Dessy et al., 1998; Touyz et al., 1999). There is accumulating evidence that the MAPK pathway is closely linked with the increase in smooth muscle contraction under Ca2+ -dependent and -independent conditions (Khalil et al., 1995; Dessy et al., 1998; Kwon et al., 2003). Further-
0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2003.11.003
398
B. Kim et al. / Journal of Ethnopharmacology 90 (2004) 397–401
more, recent studies have shown that MAPKs contribute to the intracellular signal transduction initiated by herbal medicines (Cheung et al., 2000; Kim et al., 2000), and it is assumed that MAPKs can be involved in the regulation of contractions mediated by Ligusticum wallichi. However, it has not been demonstrated that MAPKs contribute to the vascular activity mediated by Ligusticum wallichi. In this study, we investigated the effects of Ligusticum wallichi on vascular reactivity and the involvement of the MAPK pathway in Ligusticum wallichi-induced vasoactivity in rat aortic smooth muscle contraction, using a pharmacological approach, with parallel experiments based on the isolated-tissue bath and western blotting analyses. 2. Materials and methods 2.1. Preparation of extract Dried Ligusticum wallichi, obtained from the local market, was cut into small pieces and ground with a commercial mixer. The powder was homogenized with 10 volumes of chloroform and the homogenates were centrifuged at 10,000 × g for 20 min, and then filtered. The filtrates were evaporated and lyophilized at 37 ◦ C and the solids were dissolved in 100% ethanol at concentration of 2 g/ml to produce a stock for bioassay. A voucher specimen (No. LW-04) has been deposited in the Korea Food Research Institute (KFRI), Kyonggi-Do, Korea. 2.2. Animals and measurement of isometric contraction Male Sprague–Dawley rats (200–250 g) were stunned and bled. The thoracic aorta was isolated and cut into strips (2–3 mm wide and 7–8 mm in length). The endothelium was removed by gently rubbing the inner surface of the vessel with cotton thread moistened with physiological salt solution (PSS). Each strip was attached to a holder under a resting contraction of 10 mN. After equilibration for 20 min in a 5 ml muscle bath, each strip was repeatedly exposed to a 70 mM K+ solution until responses became stable. PSS contained (mM): NaCl 136.9, KCl 5.4, CaCl2 1.5, MgCl2 1.0, NaHCO3 23.8, glucose 5.5, and ethylenediaminetetraacetic acid (EDTA) 0.01. The high-K+ solution was prepared by replacing NaCl with equimolar K+ . These solutions were saturated with a 95% O2 and 5% CO2 mixture at 37 ◦ C and pH 7.4. Muscle contraction was recorded isometrically with a force-displacement transducer (FT03, Grass, RI, USA) connected to a polygraph system (7WC, Grass).
EGTA, 20 mM -glycerophosphate, 1 mM NaF, 2 mM Na3 VO4 , 5 g/ml aprotinin, 5 M leupeptin, 1% triton-X 100, 0.3 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol, 10% glycerol, and 150 mM NaCl. The homogenate was centrifuged at 14,000 × g for 10 min at 4 ◦ C, and the supernatant was collected. Protein concentrations were determined using a Bio-Rad protein assay kit (CA, USA), which is a colorimetric assay for protein based on the Bradford dye-binding procedure. Protein homogenates were diluted 1:1 (vol:vol) with sodium dodecyl sulphate (SDS) sample buffer containing 40 mM Tris–HCl (pH 6.8), 8 mM EGTA, 4% 2-mercaptoethanol, 40% glycerol, 0.01% bromophenol blue, and 4% SDS, and then boiled for 5 min. Equal amounts (30–50 g per lane) of proteins were separated in each lane of a 10% SDS–polyacrylamide gel. Electrophoretically separated proteins were transferred to a nitrocellulose membrane (Amersham Pharmacia, USA). Membranes were incubated with phosphate-buffered saline, 0.1% Tween 20 (PBST) containing 5% non-fat dried milk for 30 min, and then incubated with individual polyclonal anti-phosphorylated MAPK antibodies diluted 1:1000–5000 for 5 h at room temperature or overnight at 4 ◦ C. Following incubation with horseradish peroxide-conjugated anti-rabbit IgG (1:1000) for 60 min, the blots were developed using the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia, USA). Quantitative analysis of antibody-specific bands was performed with an image analyser (Bio-Profil, VL, France). 2.4. Materials Polyclonal anti-phosphorylated ERK1/2 antibody was purchased from Promega (Amersham Pharmacia, USA). Polyclonal anti-phosphorylated p38 MAPK antibody was purchased from Upstate Biotech (NY, USA). Norepinephrine, -glycerophosphate, NaF, Na3 VO4 , aprotinin, leupeptin, and phenylmethylsulfonyl fluoride were purchased from Sigma (MO, USA). 12-Deoxyphorbol 13-isobutyrate was purchased from Funakoshi (Japan). Triton-X 100 and dithiothreitol were purchased from Amersham Pharmacia (NJ, USA). PD98059 and SB203580 were purchased from Tocris Cookson (Bristol, UK). 2.5. Statistical analysis The results of experiments are expressed as means ± S.E.M. Unpaired Student’s t-test was used to compare the data, and P < 0.05 was considered to be significantly different.
2.3. Measurement of MAPK activity
3. Results
Aortic strips were isolated in the way described for contraction measurement experiments, and snap-frozen in liquid N2 after treatment with various stimulants and at different times. Samples were then homogenized in sample buffer containing 50 mM Tris–HCl (pH 7.4), 5 mM
3.1. Effects of Ch1LW on norepinephrine-induced contraction Isolated rat thoracic aorta was contracted with norepinephrine (NE) and then exposed to Ch1LW, a chloro-
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Fig. 1. Effects of Ch1LW on the vasoconstrictor-induced contraction in rat aortic smooth muscle. After the responses to 10 M norepinephrine (NE) was established, 1 and 10 mg/ml Ch1LW was added cumulatively (A). In Panel B, strips were pre-incubated with Ca2+ -free medium containing 1 mM EGTA for 30 min to remove external Ca2+ , and then 1 M DPB was added to the muscle strips. After establishing DPB-induced sustained contraction, 1 and 10 mg/ml Ch1LW was added sequentially.
form extract of Ligusticum wallichi. Ch1LW (1 mg/ml) significantly inhibited contraction induced by 10 M NE (52 ± 9.4% of NE-response), and the contraction was completely abolished with 10 mg/ml Ch1LW (Fig. 1A). To determine the effects of Ch1LW on Ca2+ -independent contraction, the extract was tested on phorbol ester-induced contractions in Ca2+ -free medium. After incubation of muscle strips in Ca2+ -free medium containing 1 mM EGTA for 30 min to remove external Ca2+ , 1 M 12-deoxyphorbol 13-isobutyrate (DPB) was added to the medium. When the DPB-elevated contraction reached a steady-state level, Ch1LW (1 mg/ml) was added, resulting in an inhibition of the contraction in Ca2+ -free medium (31 ± 7.1% of DPB-response) (Fig. 1B). Complete inhibition of DPB-induced contraction to the resting level was achieved with 10 mg/ml Ch1LW. In the quiescent preparation, Ch1LW (1–10 mg/ml) did not evoke any changes of contraction. Together, these results show the existence of additional mechanism involved in Ch1LW-induced vasorelaxation that inhibits both Ca2+ -dependent and -independent pathways. Neither NE- nor DPB-mediated contractions were affected by the equivalent concentration of the vehicle ethanol (data not shown). 3.2. Effects of Ch1LW on the activity of MAPK To determine whether MAPKs influence Ligusticum wallichi-induced vasorelaxation, the activity of MAPKs was measured using phosphorylated MAPK antibodies in rat aortic smooth muscle. To determine whether the activity of MAPKs measured with the antibodies reflected the real
Fig. 2. The activities of MAPKs during NE-stimulation in rat aortic smooth muscle. The time course of changes in phosphorylated ERK1/2 (A) and p38 MAPK (B) induced by NE. Strips were stimulated with 10 M NE for 0, 5, 15 and 30 min, respectively, and then western blotting analysis was carried out as described in Section 2. Results are presented as percent of phosphorylation relative to resting state. Values are means ± S.E.M. from three independent experiments. The insets indicate a representative result of western blotting with MAPK antibodies.
activation of the kinases, the selective inhibitors, PD98059 and SB203580, were used (Dessy et al., 1998). Fig. 2 illustrates the effects of NE on the activities of ERK1/2 and p38 MAPK in rat aortic smooth muscle strips. Treatment with NE (10 M) elicited a sustained increase in the activity of ERK1/2 in a time-dependent manner (Fig. 2A). The maximal response to NE was observed at 15 min. In addition, NE (10 M) also increased the activity of p38 MAPK with a pattern similar to that observed for ERK1/2 (Fig. 2B). PD98059 (10 M), an inhibitor of ERK1/2, was added to the medium at the beginning of incubation for 30 min, and inhibited NE-stimulated ERK1/2 activity (data not shown). SB203580 (10 M), an inhibitor of p38 MAPK, significantly diminished the activity of the kinase elevated by NE (10 M) (data not shown). The effects of Ch1LW on the activity of MAPKs induced by 10 M NE are illustrated in Figs. 3 and 4. Application of Ch1LW (10 mg/ml) to the resting muscle did not cause any change in the activity of
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ERK1/2 in rat aortic smooth muscle. However, the same concentration of Ch1LW significantly abolished the activity of ERK1/2 elevated by 10 M NE (Fig. 3). Furthermore, 10 mg/ml Ch1LW alone slightly increased p38 MAPK activity in resting muscle. However, in NE-stimulated muscle, Ch1LW did not reduce the activity of p38 MAPK, but caused it to increase further (Fig. 4).
4. Discussion
Fig. 3. Effects of Ch1LW on the increase in the activity of ERK1/2 evoked by NE in rat aortic smooth muscle. After aortic strips pre-treated with 10 M NE or saline for 15 min, Ch1LW (10 mg/ml) was treated for 15 min sequentially. Western blotting analysis was carried out as described in Section 2. Panel B shows the statistical results for changes of ERK1/2 activity, which are presented as percent of phosphorylation relative to non-stimulating resting state. Values are means ± S.E.M. from three independent experiments. ∗∗ P < 0.01 vs. control.
Fig. 4. Effects of Ch1LW on the increase in the activity of p38 MAPK evoked by NE in rat aortic smooth muscle. After aortic strips pre-treated with 10 M NE or saline for 15 min, 10 mg/ml Ch1LW was treated for 15 min. Panel B shows the statistical results for the changes of p38 MAPK activity, which are presented as percent of phosphorylation relative to non-stimulating resting state. Values are means ± S.E.M. from three independent experiments.
In the present study, we have demonstrated that a chloroform extract of Ligusticum wallichi inhibited the contraction induced by a vasoconstrictor in rat aortic smooth muscle. This result is consistent with an earlier study in which this plant inhibited the contractions induced by a receptor agonist and by membrane depolarization (Wu et al., 1989). It has been reported that tetramethylpyrazine (TMP), a component of Ligusticum wallichi, directly affects Ca2+ -influx through the cellular membrane and Ca2+ -release from the sarcoplasmic reticulum, which results in a decrease in [Ca2+ ]i in the vascular system (Dai and Bache, 1985; Pang et al., 1996). These results suggest that Ligusticum wallichi has a potent vasorelaxation effect and this effect may afford protection to the cardiovascular system. Furthermore, in the present study, an extract of this plant inhibited phorbol ester-induced Ca2+ -independent contractions in medium where external Ca2+ was chelated by EGTA. These results imply that Ligusticum wallichi causes vasorelaxation mediated by the inhibition of both Ca2+ -dependent and -independent pathways. Furthermore, these results suggest that TMP is not an essential component in the Ligusticum wallichi-induced inhibition of Ca2+ -independent contraction, and there may exist an unidentified substance(s) that inhibits phorbol ester-induced Ca2+ -independent contraction. MAPKs constitute a family of kinases believed to play important roles in the stimulus-induced contraction of smooth muscle (Kosako et al., 1994; Dessy et al., 1998; Kwon et al., 2003). In vascular smooth muscle, MAPKs have been implicated as components of the signal transduction that results in the activation of contractile proteins (Gerthoffer et al., 1997). Adam et al. (1995) established conclusively that MAPKs are modulators for Ca2+ -independent contraction in vascular smooth muscle. It has been suggested that activation of MAPKs initiates phosphorylation of caldesmon, an actin-regulating protein, and this might contribute to the sensitization of contractile proteins to intracellular Ca2+ (Gerthoffer et al., 1997). NE evokes activation of MAPKs in vascular smooth muscle (Yu et al., 1996). In the present study, to determine whether MAPKs influence Ligusticum wallichi-induced inhibition, the activity of MAPKs was measured using specific antibodies and inhibited with selective inhibitors in rat aortic smooth muscle (Dessy et al., 1998; Kwon et al., 2003). Although both kinases, ERK1/2 and p38 MAPK, were activated by NE with similar time courses, they displayed different patterns of inhibition by Ligusticum wal-
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lichi. The extract of Ligusticum wallichi completely blocked the activity of ERK1/2 elevated by the agonist. The activity of ERK1/2 induced by NE was also blocked by a selective inhibitor of the kinase. However, the extract did not inhibit the activity of p38 MAPK, which was reduced by a selective blocker of the kinase. In the present study, Ligusticum wallichi extract increased the activity level of p38 MAPK in both resting and NE-stimulated muscle. These results indicate that inhibition of ERK1/2, but not p38 MAPK, contributes to Ch1LW-induced vasorelaxation in rat aortic smooth muscle. In conclusion, Ch1LW, an extract of Ligusticum wallichi, strongly inhibited NE- and phorbol ester-mediated contractions, and the extract significantly attenuated the NE-induced activity of ERK1/2, but not p38 MAPK. These results suggest that Ligusticum wallichi extract-induced vasorelaxation is mediated by inhibition of ERK1/2 in rat aortic smooth muscle. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from Ministry of Agriculture and Forestry, Korea. References Adam, L.P., Franklin, M.T., Raff, G.J., Hathaway, D.R., 1995. Activation of mitogen-activated protein kinase in porcine carotid arteries. Circulation Research 76, 183–190. Cheung, W.M., Hui, W.S., Chu, P.W., Chiu, S.W., Ip, N.Y., 2000. Ganoderma extract activates MAP kinases and induces the neuronal differentiation of rat pheochromocytoma PC12 cells. FEBS Letters 486, 291–296. Chiou, G.C.Y., Yan, H.Y., Lei, H.Y., Li, B.H.P., Shen, Z.F., 1991. Ocular and cardiovascular pharmacology of tetramethylpyrazine isolated from Ligusticum wallichii Franch. Acta Pharmacologica Sinica 12, 99–104. Dai, X.Z., Bache, R.J., 1985. Coronary and systemic hemodynamic effects of tetramethylpyrazine in the dog. Journal of Cardiovascular Pharmacology 7, 841–849. Dessy, C., Kim, I., Sougnez, C.L., Laporte, R., Morgan, K.G., 1998. A role for MAP kinase in differentiated smooth muscle contraction evoked by alpha-adrenoceptor stimulation. American Journal of Physiology 275, C1081–C1086. Gerthoffer, W.T., Yamboliev, I.A., Pohl, J., Haynes, R., Dang, S., McHugh, J., 1997. Activation of MAP kinases in airway smooth muscle. American Journal of Physiology 272, 244–252. Hori, M., Sato, K., Sakata, K., Ozaki, H., Takano-Ohmuro, H., Tsuchiya, T., Sugi, H., Kato, I., Karaki, H., 1992. Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent
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contractions in vascular smooth muscle. Journal of Pharmacological and Experimental Therapeutics 261, 506–512. Hwang, K.C., 1993. The Pharmacology of Chinese Herb. CRC Press, Florida, p. 84. Karaki, H., 1989. Ca2+ localization and sensitivity in vascular smooth muscle. Trends in Pharmacological Science 10, 320–325. Khalil, R.A., Menice, C.B., Wang, C.L., Morgan, K.G., 1995. Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells. Circulation Research 76, 1101–1108. Kim, M.S., So, H.S., Park, J.S., Lee, K.M., Moon, B.S., Lee, H.S., Kim, T.Y., Moon, S.K., Park, R., 2000. Hwansodan protects PC12 cells against serum-deprivation-induced apoptosis via a mechanism involving Ras and mitogen-activated protein (MAP) kinase pathway. General Pharmacology 34, 227–235. Kim, Y.S., Kim, B., Karaki, H., Hori, M., Ozaki, H., 2003a. Up-regulation of Rnd1 during pregnancy serves as a negative-feedback control for Ca2+ sensitivity of contractile elements in rat myometrium. Biochemical and Biophysical Research Communications 311, 972–978. Kim, B., Kim, Y.S., Ahn, J., Kim, J., Cho, S., Won, K.J., Ozaki, H., Karaki, H., Lee, S.M., 2003b. Conventional-type protein kinase C contributes to phorbol ester-induced inhibition of rat myometrial tension. British Journal of Pharmacology 139, 408–414. Kosako, H., Gotoh, Y., Nishida, E., 1994. Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation. EMBO Journal 13, 2131–2138. Kwon, S., Lee, W.J., Fang, L.H., Kim, B., Ahn, H.Y., 2003. Mitogenactivated protein kinases partially regulate endothelin-1-induced contractions through a myosin light chain phosphorylation-independent pathway. Journal of Veterinary Medical Sciences 65, 225–230. Lee, Y.H., Kim, I., Laporte, R., Walsh, M.P., Morgan, K.G., 1999. Isozyme-specific inhibitors of protein kinase C translocation: effects on contractility of single permeabilized vascular muscle cells of the ferret. Journal of Physiology 517, 709–720. Miyata, Y., Nishida, E., 1999. Distantly related cousins of MAP kinase: biochemical properties and possible physiological functions. Biochemical and Biophysical Research Communications 266, 291–295. Nixon, G.F., Iizuka, K., Haystead, C.M., Haystead, T.A., Somlyo, A.P., Somlyo, A.V., 1995. Phosphorylation of caldesmon by mitogen-activated protein kinase with no effect on Ca2+ sensitivity in rabbit smooth muscle. Journal of Physiology 487, 283–289. Pang, P.K., Shan, J.J., Chiu, K.W., 1996. Tetramethylpyrazine, a calcium antagonist. Planta Medica 62, 431–435. Somlyo, A.P., Himpens, B., 1989. Cell calcium and its regulation in smooth muscle. FASEB Journal 3, 2266–2276. Touyz, R.M., El Mabrouk, M., He, G., Wu, X.H., Schiffrin, E.L., 1999. Mitogen-activated protein/extracellular signal-regulated kinase inhibition attenuates angiotensin II-mediated signaling and contraction in spontaneously hypertensive rat vascular smooth muscle cells. Circulation Research 84, 505–515. Wu, C.C., Chiou, W.F., Yen, M.H., 1989. A possible mechanism of action of tetramethylpyrazine on vascular smooth muscle in rat aorta. European Journal of Pharmacology 169, 189–195. Yu, S.M., Tsai, S.Y., Guh, J.H., Ko, F.N., Teng, C.M., Ou, J.T., 1996. Mechanism of catecholamine-induced proliferation of vascular smooth muscle cells. Circulation 94, 547–554.
Ligusticum wallichi-induced vasorelaxation mediated by mitogen-activated protein kinase in rat aortic smooth muscle Bokyung Kim a,∗ , Junghwan Kim a , Aeran Kim a , Yoon-Sun Kim a , Youn Ri Lee a , Young Min Bae a , SungIl Cho a , Mee-Ra Rhyu b a
Department of Physiology, College of Medicine, Konkuk University, Danwol-dong 322, Chungju, Choong-Buk 380-701, South Korea b Food Chemistry & Biotechnology Division, Korea Food Research Institute, Kyonggi-Do 463-420, South Korea Received 16 May 2003; received in revised form 30 October 2003; accepted 3 November 2003
Abstract Traditional herbal medicines have been widely used for the treatment of cardiovascular disorders in oriental countries. To determine the effects of Ch1LW, a chloroform extract of Ligusticum wallichi, on the vascular system, we studied changes in rat aortic smooth muscle in terms of magnitude of contraction and the activity of mitogen-activated protein kinases (MAPKs). Ch1LW inhibited the muscle contraction induced by norepinephrine (NE) in aortic strips. Ch1LW also abolished Ca2+ -independent contraction evoked by 12-deoxyphorbol 13-isobutyrate in Ca2+ -free medium containing 1 mM EGTA. Furthermore, western blotting analysis using phosphorylated MAPK antibodies showed that NE increased the activity of both extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK, which were inhibited by PD98059 and SB203580, blockers of ERK1/2 and p38 MAPK, respectively. Furthermore, treatment with Ch1LW significantly abolished NE-mediated activation of ERK1/2, whereas the activity of p38 MAPK was not affected by the extract. These results suggest that Ch1LW induces vasorelaxation in rat aortic smooth muscle, which may be mediated by the inhibition of ERK1/2 pathway, but not p38 MAPK. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Aorta; Ligusticum wallichi; Mitogen-activated protein kinases; Vasorelaxation
1. Introduction Ligusticum wallichi, a popular Chinese herbal medicine, has been used orally with other herbs for “heart disease” for thousands of years. Current research in nutrition science provides a better understanding of the possible link between the plant and heart disease. A component of Ligusticum wallichi increases myocardial contractility and coronary circulation (Chiou et al., 1991; Hwang, 1993). In contrast, this plant can inhibit the muscle contractions induced by vasoconstrictors and low systemic blood pressure (Hwang, 1993). However, little is known about the effects of Ligusticum wallichi on the contraction of smooth muscle and the mechanisms underlying the contractile system. It is well established that smooth muscle contraction is regulated by intracellular Ca2+ ([Ca2+ ]i ) and the phosphorylation of myosin light chain (MLC) (Karaki, 1989; Somlyo and Himpens, 1989). However, various kinds of vasoconstrictors induce a further contraction at a given [Ca2+ ]i , and elicit a sustained contraction under ∗ Corresponding
author. Tel.: +82-43-8403726; fax: +82-43-8519392. E-mail address: [email protected] (B. Kim).
Ca2+ -depleted conditions, referred to as “Ca2+ -independent contraction,” in intact and membrane-permeabilized smooth muscle (Hori et al., 1992; Kim et al., 2003a). In previous reports, several molecules, including protein kinase C (PKC) and MAPKs, have been suggested as candidate regulators of Ca2+ -independent contraction (Nixon et al., 1995; Lee et al., 1999; Kim et al., 2003b). MAPKs constitute a family of serine/threonine-specific protein kinases which play a central role in intracellular signal transduction initiated by extracellular stimuli, including growth factors, neurotransmitters, and hormones (Kosako et al., 1994). The three MAPK isoforms, extracellular signal-regulated kinase (ERK1/2), p38 MAPK and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), are central elements in transducing the messages in mammalian cells (Miyata and Nishida, 1999). In vascular smooth muscle, MAPKs are activated by receptor agonists, including angiotensin II, phenylephrine, and endothelin-1 (Dessy et al., 1998; Touyz et al., 1999). There is accumulating evidence that the MAPK pathway is closely linked with the increase in smooth muscle contraction under Ca2+ -dependent and -independent conditions (Khalil et al., 1995; Dessy et al., 1998; Kwon et al., 2003). Further-
0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2003.11.003
398
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more, recent studies have shown that MAPKs contribute to the intracellular signal transduction initiated by herbal medicines (Cheung et al., 2000; Kim et al., 2000), and it is assumed that MAPKs can be involved in the regulation of contractions mediated by Ligusticum wallichi. However, it has not been demonstrated that MAPKs contribute to the vascular activity mediated by Ligusticum wallichi. In this study, we investigated the effects of Ligusticum wallichi on vascular reactivity and the involvement of the MAPK pathway in Ligusticum wallichi-induced vasoactivity in rat aortic smooth muscle contraction, using a pharmacological approach, with parallel experiments based on the isolated-tissue bath and western blotting analyses. 2. Materials and methods 2.1. Preparation of extract Dried Ligusticum wallichi, obtained from the local market, was cut into small pieces and ground with a commercial mixer. The powder was homogenized with 10 volumes of chloroform and the homogenates were centrifuged at 10,000 × g for 20 min, and then filtered. The filtrates were evaporated and lyophilized at 37 ◦ C and the solids were dissolved in 100% ethanol at concentration of 2 g/ml to produce a stock for bioassay. A voucher specimen (No. LW-04) has been deposited in the Korea Food Research Institute (KFRI), Kyonggi-Do, Korea. 2.2. Animals and measurement of isometric contraction Male Sprague–Dawley rats (200–250 g) were stunned and bled. The thoracic aorta was isolated and cut into strips (2–3 mm wide and 7–8 mm in length). The endothelium was removed by gently rubbing the inner surface of the vessel with cotton thread moistened with physiological salt solution (PSS). Each strip was attached to a holder under a resting contraction of 10 mN. After equilibration for 20 min in a 5 ml muscle bath, each strip was repeatedly exposed to a 70 mM K+ solution until responses became stable. PSS contained (mM): NaCl 136.9, KCl 5.4, CaCl2 1.5, MgCl2 1.0, NaHCO3 23.8, glucose 5.5, and ethylenediaminetetraacetic acid (EDTA) 0.01. The high-K+ solution was prepared by replacing NaCl with equimolar K+ . These solutions were saturated with a 95% O2 and 5% CO2 mixture at 37 ◦ C and pH 7.4. Muscle contraction was recorded isometrically with a force-displacement transducer (FT03, Grass, RI, USA) connected to a polygraph system (7WC, Grass).
EGTA, 20 mM -glycerophosphate, 1 mM NaF, 2 mM Na3 VO4 , 5 g/ml aprotinin, 5 M leupeptin, 1% triton-X 100, 0.3 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol, 10% glycerol, and 150 mM NaCl. The homogenate was centrifuged at 14,000 × g for 10 min at 4 ◦ C, and the supernatant was collected. Protein concentrations were determined using a Bio-Rad protein assay kit (CA, USA), which is a colorimetric assay for protein based on the Bradford dye-binding procedure. Protein homogenates were diluted 1:1 (vol:vol) with sodium dodecyl sulphate (SDS) sample buffer containing 40 mM Tris–HCl (pH 6.8), 8 mM EGTA, 4% 2-mercaptoethanol, 40% glycerol, 0.01% bromophenol blue, and 4% SDS, and then boiled for 5 min. Equal amounts (30–50 g per lane) of proteins were separated in each lane of a 10% SDS–polyacrylamide gel. Electrophoretically separated proteins were transferred to a nitrocellulose membrane (Amersham Pharmacia, USA). Membranes were incubated with phosphate-buffered saline, 0.1% Tween 20 (PBST) containing 5% non-fat dried milk for 30 min, and then incubated with individual polyclonal anti-phosphorylated MAPK antibodies diluted 1:1000–5000 for 5 h at room temperature or overnight at 4 ◦ C. Following incubation with horseradish peroxide-conjugated anti-rabbit IgG (1:1000) for 60 min, the blots were developed using the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia, USA). Quantitative analysis of antibody-specific bands was performed with an image analyser (Bio-Profil, VL, France). 2.4. Materials Polyclonal anti-phosphorylated ERK1/2 antibody was purchased from Promega (Amersham Pharmacia, USA). Polyclonal anti-phosphorylated p38 MAPK antibody was purchased from Upstate Biotech (NY, USA). Norepinephrine, -glycerophosphate, NaF, Na3 VO4 , aprotinin, leupeptin, and phenylmethylsulfonyl fluoride were purchased from Sigma (MO, USA). 12-Deoxyphorbol 13-isobutyrate was purchased from Funakoshi (Japan). Triton-X 100 and dithiothreitol were purchased from Amersham Pharmacia (NJ, USA). PD98059 and SB203580 were purchased from Tocris Cookson (Bristol, UK). 2.5. Statistical analysis The results of experiments are expressed as means ± S.E.M. Unpaired Student’s t-test was used to compare the data, and P < 0.05 was considered to be significantly different.
2.3. Measurement of MAPK activity
3. Results
Aortic strips were isolated in the way described for contraction measurement experiments, and snap-frozen in liquid N2 after treatment with various stimulants and at different times. Samples were then homogenized in sample buffer containing 50 mM Tris–HCl (pH 7.4), 5 mM
3.1. Effects of Ch1LW on norepinephrine-induced contraction Isolated rat thoracic aorta was contracted with norepinephrine (NE) and then exposed to Ch1LW, a chloro-
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Fig. 1. Effects of Ch1LW on the vasoconstrictor-induced contraction in rat aortic smooth muscle. After the responses to 10 M norepinephrine (NE) was established, 1 and 10 mg/ml Ch1LW was added cumulatively (A). In Panel B, strips were pre-incubated with Ca2+ -free medium containing 1 mM EGTA for 30 min to remove external Ca2+ , and then 1 M DPB was added to the muscle strips. After establishing DPB-induced sustained contraction, 1 and 10 mg/ml Ch1LW was added sequentially.
form extract of Ligusticum wallichi. Ch1LW (1 mg/ml) significantly inhibited contraction induced by 10 M NE (52 ± 9.4% of NE-response), and the contraction was completely abolished with 10 mg/ml Ch1LW (Fig. 1A). To determine the effects of Ch1LW on Ca2+ -independent contraction, the extract was tested on phorbol ester-induced contractions in Ca2+ -free medium. After incubation of muscle strips in Ca2+ -free medium containing 1 mM EGTA for 30 min to remove external Ca2+ , 1 M 12-deoxyphorbol 13-isobutyrate (DPB) was added to the medium. When the DPB-elevated contraction reached a steady-state level, Ch1LW (1 mg/ml) was added, resulting in an inhibition of the contraction in Ca2+ -free medium (31 ± 7.1% of DPB-response) (Fig. 1B). Complete inhibition of DPB-induced contraction to the resting level was achieved with 10 mg/ml Ch1LW. In the quiescent preparation, Ch1LW (1–10 mg/ml) did not evoke any changes of contraction. Together, these results show the existence of additional mechanism involved in Ch1LW-induced vasorelaxation that inhibits both Ca2+ -dependent and -independent pathways. Neither NE- nor DPB-mediated contractions were affected by the equivalent concentration of the vehicle ethanol (data not shown). 3.2. Effects of Ch1LW on the activity of MAPK To determine whether MAPKs influence Ligusticum wallichi-induced vasorelaxation, the activity of MAPKs was measured using phosphorylated MAPK antibodies in rat aortic smooth muscle. To determine whether the activity of MAPKs measured with the antibodies reflected the real
Fig. 2. The activities of MAPKs during NE-stimulation in rat aortic smooth muscle. The time course of changes in phosphorylated ERK1/2 (A) and p38 MAPK (B) induced by NE. Strips were stimulated with 10 M NE for 0, 5, 15 and 30 min, respectively, and then western blotting analysis was carried out as described in Section 2. Results are presented as percent of phosphorylation relative to resting state. Values are means ± S.E.M. from three independent experiments. The insets indicate a representative result of western blotting with MAPK antibodies.
activation of the kinases, the selective inhibitors, PD98059 and SB203580, were used (Dessy et al., 1998). Fig. 2 illustrates the effects of NE on the activities of ERK1/2 and p38 MAPK in rat aortic smooth muscle strips. Treatment with NE (10 M) elicited a sustained increase in the activity of ERK1/2 in a time-dependent manner (Fig. 2A). The maximal response to NE was observed at 15 min. In addition, NE (10 M) also increased the activity of p38 MAPK with a pattern similar to that observed for ERK1/2 (Fig. 2B). PD98059 (10 M), an inhibitor of ERK1/2, was added to the medium at the beginning of incubation for 30 min, and inhibited NE-stimulated ERK1/2 activity (data not shown). SB203580 (10 M), an inhibitor of p38 MAPK, significantly diminished the activity of the kinase elevated by NE (10 M) (data not shown). The effects of Ch1LW on the activity of MAPKs induced by 10 M NE are illustrated in Figs. 3 and 4. Application of Ch1LW (10 mg/ml) to the resting muscle did not cause any change in the activity of
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ERK1/2 in rat aortic smooth muscle. However, the same concentration of Ch1LW significantly abolished the activity of ERK1/2 elevated by 10 M NE (Fig. 3). Furthermore, 10 mg/ml Ch1LW alone slightly increased p38 MAPK activity in resting muscle. However, in NE-stimulated muscle, Ch1LW did not reduce the activity of p38 MAPK, but caused it to increase further (Fig. 4).
4. Discussion
Fig. 3. Effects of Ch1LW on the increase in the activity of ERK1/2 evoked by NE in rat aortic smooth muscle. After aortic strips pre-treated with 10 M NE or saline for 15 min, Ch1LW (10 mg/ml) was treated for 15 min sequentially. Western blotting analysis was carried out as described in Section 2. Panel B shows the statistical results for changes of ERK1/2 activity, which are presented as percent of phosphorylation relative to non-stimulating resting state. Values are means ± S.E.M. from three independent experiments. ∗∗ P < 0.01 vs. control.
Fig. 4. Effects of Ch1LW on the increase in the activity of p38 MAPK evoked by NE in rat aortic smooth muscle. After aortic strips pre-treated with 10 M NE or saline for 15 min, 10 mg/ml Ch1LW was treated for 15 min. Panel B shows the statistical results for the changes of p38 MAPK activity, which are presented as percent of phosphorylation relative to non-stimulating resting state. Values are means ± S.E.M. from three independent experiments.
In the present study, we have demonstrated that a chloroform extract of Ligusticum wallichi inhibited the contraction induced by a vasoconstrictor in rat aortic smooth muscle. This result is consistent with an earlier study in which this plant inhibited the contractions induced by a receptor agonist and by membrane depolarization (Wu et al., 1989). It has been reported that tetramethylpyrazine (TMP), a component of Ligusticum wallichi, directly affects Ca2+ -influx through the cellular membrane and Ca2+ -release from the sarcoplasmic reticulum, which results in a decrease in [Ca2+ ]i in the vascular system (Dai and Bache, 1985; Pang et al., 1996). These results suggest that Ligusticum wallichi has a potent vasorelaxation effect and this effect may afford protection to the cardiovascular system. Furthermore, in the present study, an extract of this plant inhibited phorbol ester-induced Ca2+ -independent contractions in medium where external Ca2+ was chelated by EGTA. These results imply that Ligusticum wallichi causes vasorelaxation mediated by the inhibition of both Ca2+ -dependent and -independent pathways. Furthermore, these results suggest that TMP is not an essential component in the Ligusticum wallichi-induced inhibition of Ca2+ -independent contraction, and there may exist an unidentified substance(s) that inhibits phorbol ester-induced Ca2+ -independent contraction. MAPKs constitute a family of kinases believed to play important roles in the stimulus-induced contraction of smooth muscle (Kosako et al., 1994; Dessy et al., 1998; Kwon et al., 2003). In vascular smooth muscle, MAPKs have been implicated as components of the signal transduction that results in the activation of contractile proteins (Gerthoffer et al., 1997). Adam et al. (1995) established conclusively that MAPKs are modulators for Ca2+ -independent contraction in vascular smooth muscle. It has been suggested that activation of MAPKs initiates phosphorylation of caldesmon, an actin-regulating protein, and this might contribute to the sensitization of contractile proteins to intracellular Ca2+ (Gerthoffer et al., 1997). NE evokes activation of MAPKs in vascular smooth muscle (Yu et al., 1996). In the present study, to determine whether MAPKs influence Ligusticum wallichi-induced inhibition, the activity of MAPKs was measured using specific antibodies and inhibited with selective inhibitors in rat aortic smooth muscle (Dessy et al., 1998; Kwon et al., 2003). Although both kinases, ERK1/2 and p38 MAPK, were activated by NE with similar time courses, they displayed different patterns of inhibition by Ligusticum wal-
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lichi. The extract of Ligusticum wallichi completely blocked the activity of ERK1/2 elevated by the agonist. The activity of ERK1/2 induced by NE was also blocked by a selective inhibitor of the kinase. However, the extract did not inhibit the activity of p38 MAPK, which was reduced by a selective blocker of the kinase. In the present study, Ligusticum wallichi extract increased the activity level of p38 MAPK in both resting and NE-stimulated muscle. These results indicate that inhibition of ERK1/2, but not p38 MAPK, contributes to Ch1LW-induced vasorelaxation in rat aortic smooth muscle. In conclusion, Ch1LW, an extract of Ligusticum wallichi, strongly inhibited NE- and phorbol ester-mediated contractions, and the extract significantly attenuated the NE-induced activity of ERK1/2, but not p38 MAPK. These results suggest that Ligusticum wallichi extract-induced vasorelaxation is mediated by inhibition of ERK1/2 in rat aortic smooth muscle. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from Ministry of Agriculture and Forestry, Korea. References Adam, L.P., Franklin, M.T., Raff, G.J., Hathaway, D.R., 1995. Activation of mitogen-activated protein kinase in porcine carotid arteries. Circulation Research 76, 183–190. Cheung, W.M., Hui, W.S., Chu, P.W., Chiu, S.W., Ip, N.Y., 2000. Ganoderma extract activates MAP kinases and induces the neuronal differentiation of rat pheochromocytoma PC12 cells. FEBS Letters 486, 291–296. Chiou, G.C.Y., Yan, H.Y., Lei, H.Y., Li, B.H.P., Shen, Z.F., 1991. Ocular and cardiovascular pharmacology of tetramethylpyrazine isolated from Ligusticum wallichii Franch. Acta Pharmacologica Sinica 12, 99–104. Dai, X.Z., Bache, R.J., 1985. Coronary and systemic hemodynamic effects of tetramethylpyrazine in the dog. Journal of Cardiovascular Pharmacology 7, 841–849. Dessy, C., Kim, I., Sougnez, C.L., Laporte, R., Morgan, K.G., 1998. A role for MAP kinase in differentiated smooth muscle contraction evoked by alpha-adrenoceptor stimulation. American Journal of Physiology 275, C1081–C1086. Gerthoffer, W.T., Yamboliev, I.A., Pohl, J., Haynes, R., Dang, S., McHugh, J., 1997. Activation of MAP kinases in airway smooth muscle. American Journal of Physiology 272, 244–252. Hori, M., Sato, K., Sakata, K., Ozaki, H., Takano-Ohmuro, H., Tsuchiya, T., Sugi, H., Kato, I., Karaki, H., 1992. Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent
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