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Analysis of the Mechanisms Underlyingthe Vasorelaxant Action of Coptisine
in Rat Aortic Rings
Li-Li Gong,*,† Lian-Hua Fang,*
Hai-Lin Qin,* Yang Lv* and Guan-Hua Du*
*Beijing Key Laboratory of Drug Targets Identification and Drug Screening
Institute of Materia Medica
Chinese Academy of Medical Science and Peking Union Medical College
Beijing 100050, China†Beijing Chao-Yang Hospital affiliated with Beijing Capital Medical University
Beijing, China
Abstract: The aim of the present study was to evaluate the vasorelaxant effects of coptisineand its possible mechanisms in isolated rat aortic rings. Coptisine was evaluated on isolatedrat aortic rings precontracted with norepinephrine (NE) and KCl. The mechanisms wereevaluated in the presence or absence of specific pharmacological inhibitors. Coptisine(1 � 200�M) relaxed NE (1�M) or KCl (60mM) induced sustained contraction with pEC50
values of 4:49� 0:48 and 4:85� 0:57 in a concentration dependent manner. Pretreatmentwith coptisine (10, 50 or 100�M) also inhibited concentration-response curves to NE andKCl. The vasorelaxant effect of coptisine was attenuated significantly by endotheliumremoval, and incubation with N!-nitro-L-arginine methyl ester (L-NAME, 100�M),methylene blue (10�M) and indomethacin (5�M) partially reduced the vasorelaxant effectof coptisine. In endothelium-denuded rings, the vasorelaxant effect of coptisine was reducedsignificantly by 4-aminopyridine (4-AP, 100�M), but not glibenclamide (10�M) orte-traethylammonium (TEA, 5mM). Coptisine also reduced NE-induced transient contractionin Ca2þ-free solution, and inhibited contraction induced by increasing external calcium inCa2þ-free medium plus 60mM KCl. It was concluded that coptisine induced both endo-thelium-dependent and -independent relaxation in rat aortic rings. The NO-cGMP mediatedpathway may be involved in the endothelium-dependent relaxation and in the activation ofvoltage-dependent Kþ channels, contributing in part to the endothelium-independentrelaxation bycoptisine. Coptisine also blocks extracellular Ca2þ influx by interacting withboth voltage- and receptor-operated Ca2þ channels.
Keywords: Coptisine; Vasorelaxation; Aorta; Endothelium; Nitric Oxide.
Correspondence to: Dr. Lian-Hua Fang and Guan-Hua Du, Institute of Materia Medica, Chinese Academy of
Medical Science and Peking Union Medical College, 1 Xiannongtan Street, Beijing 100050, China. Tel: (þ86)10-6316-5184, Fax: (þ86) 10-6301-7757, E-mail: [email protected] (L. H. Fang); [email protected] (G. H. Du).
The American Journal of Chinese Medicine, Vol. 40, No. 2, 309–320© 2012 World Scientific Publishing Company
Institute for Advanced Research in Asian Science and MedicineDOI: 10.1142/S0192415X12500243
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Introduction
Coptis chinensis Franch, of the Ranunculaceae family, is a perennial, stemless herb thatgrows throughout China. Coptidis Rhizoma, the rhizome of C. chinensis, has been widelyused in traditional Chinese medicine for treating diarrhea and gastrointestinal disorders.Coptidis Rhizoma is known to harbor a diversity of alkaloids, including berberine, pal-matine, jateorrhizine, epiberberine and coptisine, which are considered to be its activeconstituents (Jung et al., 2008; Liu et al., 2011). Recently, several biological actions ofcoptisine have been reported, including anti-Alzheimer’s and antioxidant (Jung et al.,2009), anti-fungal (Kong et al., 2009), antidiabetic (Jung et al., 2008), antivirus (Li et al.,2008), antimicrobial (Yan et al., 2008), antihepatoma and antileukaemia (Lin et al., 2004)activities, inhibition of type A monamine oxidase (Ro et al., 2001) and inhibition ofacetylcholinesterase (Xiao et al., 2011). Previous studies showed that coptisine selectivelyinhibits vascular smooth muscle cell (VSMC) proliferation (Tanabe et al., 2006) and causesa double blockade of cell cycle progression in VSMCs (Tanabe et al., 2005). However, fewstudies have been carried out regarding the effect of coptisine on vascular smooth musclecontraction. Thus, the purpose of the present study was to evaluate the relaxant effects ofcoptisine and its possible mechanisms in the rat thoracic aorta.
Materials and Methods
Chemicals and Drugs
Norepinephrine (NE), acetylcholine (Ach), indomethacin, N!-nitro-L-arginine methylester (L-NAME), methylene blue, glibenclamide, tetraethylammonium (TEA) and4-aminopyridine (4-AP) were purchased from Sigma (St. Louis, MO, USA). All otherreagents were analytical purity.
Coptisine (Fig. 1) was extracted by the Department of Medicinal Chemistry of ourinstitute, and its structure was confirmed by the analysis of physical and chemical prop-erties and spectral evidence (Qin et al., 2004). Coptisine, glibenclamide and indomethacinwere dissolved in DMSO, while the other drugs were dissolved in distilled water, andfurther dilutions were made with distilled water. Preliminary experiments showed thatDMSO kept at concentrations less than 0.2% (v/v) had no effect on tension development ofisolated aorta.
N
O
O
O
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Figure 1. Chemical structure of coptisine.
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Preparation of Rat Aortic Rings
All studies were approved by the Laboratories Institutional Animal Care and Use Committeeof the Chinese Academy of Medical Sciences and Peking Union Medical College. MaleSprague-Dawley rats weighing 250–300 g were anesthetized with pentobarbitone sodium(60mg/kg, i.p.). Subsequently, a midline abdominal incision was performed to expose theaorta. The thoracic aorta was immediately excised and immersed in ice-cold Krebs-Henseleit(K-H) solution with the following composition (mM): NaCl 120, KCl 4.8, KH2PO4 1.2,NaHCO3 25, glucose 11, CaCl2 2.5, MgCl2 1.4 and ethylenediaminetetraacetic acid (EDTA)0.01. After removal of adhering fat and connective tissue, the aorta was cut into rings ofabout 2 � 3mm in length. For endothelium-denuded aorta, the endothelium wasmechanically removed by gently rubbing the lumen with a wet cotton ball, and the absenceof acetylcholine-induced relaxation was used as a denuding indicator (Brayden, 1990).
Measurement of Isometric Vascular Tone
The tension of aortic rings was recorded isometrically via a force displacement transducerconnected to a BIO-PAC polygraph (MP100A). The aortic rings were mounted betweentwo stainless steel wires in organ baths containing 10mL K-H solution, which wasmaintained at 37�C and gassed continuously with a 95% O2 and 5% CO2 mixture (Fanget al., 2006). After an initial 60min equilibration at a resting tension of 1.2 g, the aorticrings were given two successive stimulations with high Kþ (60mM) solution, which wasprepared by replacing NaCl with equimolar KCl in K-H solution. During the equilibrationperiod, the K-H solution was changed every 20min. The endothelial integrity was con-firmed by eliciting a relaxation with Ach (10�M) after contraction induced by NE (1�M).Only endothelium-intact rings exhibiting more than 60% relaxation in response to Achwere used for the experiments (Zhu et al., 2007). With the endothelium denuded rings, therelaxation in response to Ach was less than 5%.
Effects of Coptisine on the Contractions of Aortic Rings Induced by NE or KCl
In order to evaluate the effects of coptisine on the contraction induced by NE and KCl, twodifferent protocols were used. In the first study, the aortic rings were pre-contracted witheither NE (1�M) or high KCl (60mM). Once the plateau was attained, coptisine was addedcumulatively (1–200�M) to obtain the concentration-response curves. In the second study,coptisine (10, 50 or 100�M) was added to the bath, and after 20min incubation, theconcentration response curve for KCl or NE was generated by adding KCl (10–60mM)and NE (10�9–10�6 M) to the bath cumulatively, respectively.
Role of Endothelium in Coptisine-Induced Aorta Relaxation
To elucidate the role of endothelium in coptisine-mediated vasorelaxation, concentration-response to coptisine was studied in endothelium-intact and endothelium-denuded ringspre-contracted by NE (1�M). To determine which endothelial mediator(s) was/were
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related to the vasodilator effect of coptisine, the NO synthase (NOS) inhibitor L-NAME(100�M), the guanylate cyclase inhibitor methylene blue (10�M), and the cyclooxygenaseinhibitor indomethacin (5�M) were used. The endothelium-intact aortic rings were pre-incubated with these inhibitors for 20min before NE (1�M) was added to the bath, andthen coptisine (1–200�M) was added cumulatively.
Role of Kþ Channels in Coptisine-Induced Relaxation
To demonstrate the role of Kþ channels in coptisine-induced relaxation, endothelium-denuded aortic rings were pre-incubated with Kþ channel blockers, including TEA (5mM),glibenclamide (10�M) and 4-AP (100�M) for 20min before NE (1�M) was added. Whenthe contractile plateau was attained, coptisine (1–200�M) was added cumulatively.
Effect of Coptisine on Extracellular Ca2þ-Induced Contractionand Intracellular Ca2þ Release
To determine whether the inhibition of extracellular Ca2þ influx is involved in coptisine-induced relaxation, the experiments were carried out in Ca2þ-free K-H solution (Tirapelliet al., 2004). Endothelium-denuded aortic rings were washed with Ca2þ-free solution(approximately 12min) containing EGTA (1mM) and then rinsed with Ca2þ-free solution(without EGTA) containing KCl (60mM). The cumulative concentration-response curvesfor CaCl2 (0.1, 0.5, 1, 1.5, 2, and 2.5mM) were obtained in the absence of coptisine(vehicle group) or after a 20min incubation with coptisine (10, 50 or 100�M). With themaximal tension induced by 2.5mM Ca2þ in the vehicle group being considered as 100%,concentration-response curves for the added Ca2þ were constructed.
To clarify whether the relaxation induced by coptisine was related to the inhibition ofintracellular Ca2þ release, the experiments were carried out in Ca2þ-free K-H solutioncontaining 50�M EGTA (Jiang et al., 2005). The rings were first washed with Ca2þ-freesolution three times. After a 20min incubation with or without coptisine (10, 50 or100�M), NE (1�M) was added to stimulate the release of intracellular Ca2þ, and thecontraction was recorded.
Statistical Analysis
All data were expressed as the means � standard error of the mean (SEM). Statisticalanalysis was performed using the one-way ANOVA or Student’s t-test. A p value less than0.05 was regarded as significantly different.
Results
Relaxant Effects of Coptisine on Aortic Rings Contracted by NE and KCl
Coptisine relaxed the NE (1�M) pre-contracted aortic rings slowly in a dose-dependentmanner (pEC50 value 4:49� 0:48, n ¼ 6), with the maximal relaxation (Emax) of
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85:0� 3:21% reached at the concentration of 200�M (Fig. 2). Coptisine also relaxedaortic rings pre-contracted with KCl (60mM) in a similar way (pEC50 value 4:85� 0:57,Emax 87:6� 1:5%, n ¼ 6) (Fig. 2).
Effects of Coptisine on the Concentration-Response Curves of NE and KCl
Pre-incubation with various concentrations of coptisine (10, 50 and 100�M) inhibited theconcentration-response contraction of NE in a nonparallel fashion, and depressed themaximal responses to 108:8� 2:4%, 81:3� 3:1% and 53:6� 2:9%, respectively (vs.vehicle group 111:2� 2:0%, n ¼ 6) in endothelium-intact aortic rings (Fig. 3A). We alsoobserved that 10, 50 and 100�M coptisine inhibited the contractile response to KCl, anddepressed the maximal responses to 89:2� 1:7%, 55:1� 2:0% and 33:9� 1:5%,respectively (vs. vehicle group 93:4� 1:9%, n ¼ 6) in endothelium-intactaortic rings(Fig. 3B). These results, combined with the above results (Fig. 2), indicated that therelaxant effect of coptisine is more potent during pre-treatment than during post-treatment.
Role of Endothelium in Coptisine-Induced Relaxation of Aortic Rings
The relaxation effect of coptisine in endothelium-intact aorta pre-contracted by NE (1�M)was significantly stronger than that in endothelium-denuded aorta. In endothelium-denuded(E-) rings, coptisine produced a partial relaxation with a maximal effect of 73:2� 3:9%(vs. 85:0� 3:2% in the endothelium-intact group, n ¼ 6) (Fig. 4A). Since coptisineinduced both endothelium-dependent and -independent relaxation in rat isolated aorticrings, an attempt was made to investigate what endothelium-derived vasoactive factorscontributed to the coptisine-induced relaxation. Pre-incubation of endothelium-intact rings
Figure 2. Vasorelaxant effects of coptisine on endothelium-intact thoracic aorta rings pre-contracted with NE(1�M) or KCl (60mM). Results are presented as mean � SEM, n ¼ 6.
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with L-NAME (100�M), methylene blue (5�M) and indomethacin (5�M) significantlyreduced the coptisine-induced relaxation, with a maximal relaxant effect of 70:3� 2:8%,69:1� 1:9% and 58:7� 2:5%, respectively (vs. 85:0� 3:2% in the control group, n ¼ 6,Fig. 4B).
Role of Kþ Channels in Coptisine-Induced Relaxation
We used three Kþ channel blockers: the ATP-sensitive Kþ channel (KATP) blocker glib-enclamide, Ca2þ-activated Kþ channel (KCa) blocker TEA, and voltage-dependent Kþ
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Figure 3. Inhibitory effects of coptisine (10, 50, 100M) on the contraction induced by NE (10�9 � 10�6 M) (A)and KCl (10 � 60mM) (B) in endothelium-intact aortic rings. Results are presented as mean � SEM, n ¼ 6,*p < 0:05, **p < 0:01 compared with vehicle.
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channel (Kv) blocker 4-AP. Pretreatment with 4-AP (100�M) attenuated the coptisine-induced relaxation in endothelium-denuded rings pre-contracted by NE (1�M). However,glibenclamide (10�M) and TEA (5mM) did not significantly affect the coptisine-inducedrelaxation (Fig. 5). These results suggested that Kv channel opening may be involved incoptisine-induced relaxation at higher concentrations.
(A)
(B)
Figure 4. Vasorelaxant effects of coptisine (10, 50, 100�M) on the contraction induced by NE (1�M) in the aorticrings with (þEndo) or without (�Endo) endothelium (A). Effects of pre-incubation with L-NAME (100�M),
methylene blue (10�M) and indomethacin (5�M) on coptisine induced relaxation in endothelium-intact aorta (B).Results are presented as mean � SEM, n ¼ 6. **p < 0:01 compared with endothelium-intact aorta (A) or*p < 0:05, **p < 0:01 compared with control (B).
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Effect of Coptisine on Extracellular Ca2þ-Induced Contractionand Intracellular Ca2þ Release in Aortic Rings
The aortic contraction induced by high Kþ solution was mainly due to the depolarization ofVSMCs and the influx of extracellular Ca2þ through voltage-operated Ca2þ channels. Inthe Ca2þ-free solution plus 60mM KCl, cumulative addition of CaCl2 (0:1 � 2:5mM)induced a stepwise tension increase in aortic rings. Pretreatment with coptisine (50�M and100�M) for 20min noticeably attenuated CaCl2-induced contraction (Emax 41:3� 2:4%and 32:6� 4:1%, respectively, n ¼ 6); however, 10�M coptisine had no significant effect(Fig. 6A) at low concentrations of Ca2þ, suggesting that Ca2þ influx was reduced bycoptisine.
In the Ca2þ-free solution, NE (1�M) induced a transient contraction due to the releaseof intracellular Ca2þ. As shown in Fig. 6B, pretreatment with coptisine (10, 50 and100�M) for 20min significantly reduced the contraction induced by NE (1�M), and themaximal contraction was decreased to 0:28� 0:06 g, 0:12� 0:09 g and 0:06� 0:03,respectively (vs. 0:5� 0:07 g in the vehicle group, n ¼ 6). This suggests that coptisineattenuated calcium release from the sarcoplasmic reticulum.
Discussion
Coptisine is a natural compound which displays a broad range of pharmacological actions.The present study, which represents the first attempt to describe the vasodilatory propertiesof coptisine and its possible mechanisms, demonstrates that coptisine induces relaxation inrat aortic rings through both an endothelium-dependent and-independent mechanism.
Figure 5. Effects of pre-incubation with glibenclamide (10�M), TEA (5mM) and 4-AP (100�M) on coptisineinduced relaxation in thoracic aorta rings precontracted by NE (1�M). Results are presented as mean � SEM,n ¼ 6, **p < 0:01 compared with control.
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Vascular endothelium occupies the location between circulating blood and vascularsmooth muscle, and is considered to be important in the regulation of vascular tone.Key discoveries in the past decade revealed that the endothelium can modulate the toneof underlying vascular smooth muscle by the synthesis/release of potent vasorelaxant(endothelium-derived relaxing factors (EDRF) such as NO (Tolins et al., 1991) and pros-tacyclin) and vasoconstrictor substances (endothelium-derived contracting factors (EDCF)such as PGH2/thromboxane A2 (Maruyama et al., 1999), endothelin and ansiotensin II)
(A)
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Figure 6. Inhibitory effect of coptisine on the cumulative-contraction curve dependent on extracellular Ca2þ
influx induced by KCl (60mM) in Ca2þ-free solution (A), and on the NE (1�M) induced contraction in Ca2þ-freesolution (B). Results are presented as mean � SEM, n ¼ 6, **p < 0:01 compared with vehicle.
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(Rubanyi, 1993). Removal of functional endothelium inhibited the relaxant responseto coptisine, suggesting that the vasorelaxation caused by coptisine was endothelium-dependent.We found that L-NAME, methylene blue and indomethacin significantly reducedthe coptisine-induced vasorelaxation. These results indicate that the NO-cGMP pathwaymay be involved in the relaxation due to coptisine in endothelium-intact aorta.
Kþ channels play an important role in the regulation of muscle contractility andvascular tone (Nelson and Quayle, 1995). Direct activation of Kþ channels on VSMCsnormally hyperpolarizes the cell membrane and thus inhibits Ca2þ influx through voltage-sensitive Ca2þ channels. There are several types of Kþ conductance present in vascularsmooth muscle, and they are subject to modulation by various factors (Nelson and Quayle,1995). The observation that the vasorelaxant effect of coptisine was still present in theendothelium-denuded aortic rings and in those treated with NOS inhibitor, suggests thatcoptisine has a direct effect on VSMCs. In the results obtained in endothelium-denudedaortic preparations, the relaxant effect of coptisine was effectively attenuated by theputative Kv channel blocker 4-AP. Glibenclamide and TEA did not significantly inhibitcoptisine-induced relaxation, suggesting that KATP and KCa channels are not involved inthe coptisine-induced relaxation effect. It is probable that coptisine activates Kv channels inrat endothelium-denuded arteries. These results indicate that the vasorelaxant effect ofcoptisine is partially mediated by the opening of the Kþ channels in VSMCs.
The influx of external Ca2þ through specific Ca2þ channels, or Ca2þ release frominternal stores, plays an important role in the excitation-contraction coupling of smoothmuscle. As we know, there are two kinds of Ca2þ channels in VSMCs: voltage-dependentCa2þ channels, and receptor-operated Ca2þ channels (Horowitz et al., 1996). By acting onspecific membrane receptors, NE induces Ca2þ influx through receptor-operated channels,causing tonic contraction (Paoletti and Govoni, 1987). NE also stimulates the formation ofinositol 1,4,5-triphosphate (IP3), which binds to and opens specific IP3-receptor operatedchannels in the sarcoplasmic reticulum membrane and induces Ca2þ release, causingphasic contraction (Broekaert and Godfraind, 1979). On the other hand, the high Kþ-induced contraction of smooth muscle is the result of an increase in Ca2þ influx throughvoltage-dependent Ca2þ channels (Van Breemen et al., 1972) and the activation ofpotential-operated calcium channels is also involved in NE-induced contraction. In thepresent experiments, coptisine (10, 50 and 100�M) was able to inhibit contractile effectsinduced by NE and KCl in endothelium-intact aortic rings in a nonparallel fashion, indi-cating that coptisine might interfere with both voltage-dependent and receptor-operatedCa2þ channels. We found that coptisine (50 and 100�M) significantly inhibited CaCl2-induced contraction in endothelium-denuded rings in Ca2þ-free medium containing 60mMKCl, supporting that coptisine exhibits Ca2þ entry blocking activity. Coptisine (10, 50 and100�M) also inhibited the contraction triggered by NE in endothelium-denuded rings inCa2þ-free medium, suggesting that coptisine may also inhibit Ca2þ mobilization fromintracellular stores. Taken together, these results indicate that coptisine may be acting as aCa2þ antagonist.
In conclusion, the results suggest that coptisine exerts its vasodilatory effects by actingon multiple sites (Fig. 7). Coptisine induces relaxation in rat aortic rings through
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endothelium-dependent and -independent pathways. The NO-cGMP mediated pathwaymay be involved in the endothelium-dependent relaxation, and the activation of Kvchannels contributes in part to the endothelium-independent relaxation due to coptisine.Coptisine also blocks extracellular Ca2þ influx by interacting with both voltage- andreceptor-operated Ca2þ channels.
Acknowledgments
This study was supported by the National Natural Science Foundation of China(No. 30572182) and the special foundation on scientific and technological basic work thatis provided by China Ministry of Science and Technology (No. 2007FY130100).
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