European Journal of Pharmacology 735 (2014) 44–51
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Cardiovascular pharmacology
Emodin accentuates atrial natriuretic peptide secretion in cardiac atria Guang Hai Zhou a,1, Feng Zhang a,1, Xin Nong Wang a, Oh Jeong Kwon c, Dae Gill Kang c, Ho Sub Lee c, Song Nan Jin b,n, Kyung Woo Cho a, Jin Fu Wen a,nn a Institute of Atherosclerosis, Key Laboratory of Atherosclerosis in Universities of Shandong, Taishan Medical University, 2 East Yingsheng Road, Taian 271000, Shandong, China b Institute of Materia Medica, Taishan Medical University, Middle of Changcheng Road, Taian 271016, Shandong, China c Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan 570-749, Jeonbuk, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 25 January 2014 Received in revised form 4 April 2014 Accepted 9 April 2014 Available online 18 April 2014
Emodin, an active anthraquinone constituent isolated from the rhubarb, a traditional Chinese herbal medicine which is widely used in clinical treatment, has cardiovascular protective properties. However, it remains unclear whether the cardiovascular protective actions of emodin are related to an activation of cardiac natriuretic hormone secretion. The purpose of the present study was to explore the effect of emodin on the secretion of ANP, a member of the family of cardiac natriuretic hormones, and its mechanisms involved. Experiments were performed in isolated perfused beating rabbit atria allowing measurement of ANP secretion, atrial pulse pressure, and stroke volume. Emodin increased ANP secretion concomitantly with a decrease in atrial pulse pressure and stroke volume in a concentration-dependent manner. These effects were reversible. Inhibition of K þ channels with tetraethylammonium and glibenclamide attenuated the emodin-induced changes in ANP secretion and atrial dynamics. Furthermore, the emodin-induced changes in ANP secretion and atrial dynamics were attenuated by inhibition of L-type Ca2 þ channels with nifedipine. Atropine, methoctramine, tertiapin-Q, and pertussis toxin had no significant effect on the emodin-induced changes in ANP secretion and mechanical dynamics. The present study demonstrates that emodin increases þ channel in isolated ANP secretion via inhibition of L-type Ca2 þ channels through an activation of KATP beating rabbit atria. The results also provide a rationale for the use of emodin in the treatment of impairment of the regulation of the cardiovascular homeostasis. & 2014 Published by Elsevier B.V.
Keywords: Emodin Atrial natriuretic peptide secretion Cardiac natriuretic hormone þ KATP channel L-type Ca2 þ channel
1. Introduction Emodin (1,3,8-trihydroxy-6-methylanthraquinone), an active anthraquinone constituent isolated from the rhubarb (Wu et al., 2013), has cardiovascular protective actions, such as anti-hypertensive, anti-hyperlipidemic, anti-inflammatory, and vasorelaxant effects (Huang et al., 1991; Meng et al., 2010; Chen et al., 2012; Tzeng et al., 2012). Effects of emodin on cardiac endocrine function are unidentified. Atrial cardiomyocytes synthesize atrial natriuretic peptide (ANP) and secrete it into the bloodstream. ANP has various physiological effects: natriuretic, diuretic, vasorelaxant, anti-hypertensive, antiinflammatory, anti-proliferative, and anti-oxidative actions (Kiemer and Vollmar, 2001; Sangawa et al., 2004; Vesely, 2009; De Vito et al., 2010). Recently, it has also been shown that ANP is involved in lipid
n
Corresponding author. Tel.: þ 86 538 623 7282; fax: þ 86 538 622 2651. Corresponding author. Tel.: þ 86 538 622 3006; fax: þ86 538 622 2651. E-mail addresses: [email protected] (S.N. Jin), [email protected], [email protected] (J.F. Wen). 1 The two first authors contributed equally to this work. nn
http://dx.doi.org/10.1016/j.ejphar.2014.04.014 0014-2999/& 2014 Published by Elsevier B.V.
homeostasis via cGMP signaling (Moro and Lafontan, 2013). The ANP system is considered as an endogenous counterbalancing limb for the regulation of the renin–angiotensin–aldosterone system. These notions indicate that ANP exhibits potent protective actions on the cardiovascular system through its multiple targets. ANP secretion is primarily controlled by changes in atrial volume and cardiac workload and is also modulated by neurotransmitters and hormones (Ruskoaho, 1992; Xu et al., 2008; Kim et al., 2013). Intracellular second messengers, Ca2þ , cAMP, and cGMP, and K þ channels are involved in the regulation of ANP secretion (Ito et al., 1988; Xu et al., 1996; Wen et al., 2004). Recently, it was shown that endogenous acetylcholine (ACh) is positively involved in the regulation of ANP secretion (Kim et al., 2013). M2 muscarinic ACh receptor (mAChR) þ stimulation accentuates ANP secretion via activation of Gαi/o–KACh channel signaling (Xu et al., 2008; Kim et al., 2013). In addition, þ activation of KATP channel is also known to increase ANP secretion (Kim et al., 1997). In contrast, it has been shown that increase in intracellular Ca2þ concentration and Ca2 þ entry via L-type Ca2þ channel is an inhibitory modulator for ANP secretion (Ito et al., 1988; de Bold and de Bold, 1989; Ruskoaho et al., 1990; Wen et al., 2000). K þ þ þ channels, KACh channel and KATP channel in particular, are indirectly
G.H. Zhou et al. / European Journal of Pharmacology 735 (2014) 44–51
involved in the regulation of ANP secretion through inhibiting Ca2 þ entry via L-type Ca2þ channel (Kim et al., 1997; Xu et al., 2008). þ Shortening of the action potential caused by activation of KATP channel is an endogenous modulator for the regulation of intracellular Ca2 þ levels and L-type Ca2 þ channel activity (Noma, 1983). The effect of emodin on ANP secretion from the heart has not yet been identified. It is known that emodin affects ion channels (Liu et al., 2004; Li et al., 1998). Emodin is also known to have cholinergic properties (Ali et al., 2004; Lu et al., 2007; Lenta et al., 2008; Xu et al., 2012). Therefore, we hypothesized that emodin affects ANP secretion from the cardiac atria through modulation of ion channel activity and muscarinic signaling. The purpose of the present study was to investigate the effect of emodin on ANP secretion and its underlying mechanisms in isolated beating rabbit atria. From this study, we find that emodin accentuates ANP secretion via inhibition of L-type Ca2 þ channel þ activity through an activation of KATP channels.
2. Materials and methods All of the experimental protocols were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996) and were approved by the Institutional Animal Care and Utilization Committee for Medical Science of Taishan Medical University.
2.1. Experimental protocols Experiments were performed to identify effects of emodin on atrial ANP secretion and its mechanisms involved in isolated beating rabbit atria. The atrium was perfused for 60 min to stabilize ANP secretion and atrial dynamics. [3H]Inulin was introduced to the pericardial fluid 20 min before the start of sample collection (Wen et al., 2000). The perfusate was collected for ANP analysis at 2-min intervals at 4 1C. Experiments were carried out in order to solve the questions detailed below. 2.1.1. Does emodin increase ANP secretion and mechanical dynamics? An initial control period of 48 min was followed by emodin infusion (0, 10, 30, and 100 μM) for 36 min (vehicle, n¼6; emodin 10 μM, n¼6; emodin 30 μM, n¼ 12; emodin 100 μM, n¼8). To evaluate the effects of emodin on ANP secretion and atrial dynamics, stroke volume and pulse pressure, values (mean of 2 fractions) obtained before and 36 min after the addition of the agent were compared. Data are expressed as percent difference from the control (before the addition of emodin). 2.1.2. Are the effects of emodin related to K þ channel activity? þ þ K þ channels, KATP channel and KACh channel in particular, are known to be involved in the regulation of ANP secretion (Xu et al., 1996; Kim et al., 1997; Xu et al., 2008). To test an involvement of
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Fig. 1. Effects of emodin on secretory and contractile function in isolated beating rabbit atria. (A) Effects of vehicle on atrial natriuretic peptide (ANP) secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n¼6). (B) Effects of emodin (100 μM) on ANP secretion (a), ANP concentration (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n ¼8). Values are means 7S.E.M. nnnPo 0.001 vs. values before emodin.
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K þ channels, experiments were performed in the presence of selective and non-selective K þ channel inhibitors. Effets of tetraethylammonium (TEA), a non-selective inhibitor of K þ channels, þ glibenclamide (Glib), a selective KATP channel inhibitor, and þ tertiapin-Q (TPN), a selective KACh channel inhibitor, were tested. Thirty-six min of infusion with inhibitor was followed by 36 min of emodin (30 μM) or vehicle in the continuous presence of the previous agent (TEA 10 mMþemodin, n¼8; TEAþvehicle, n¼ 3; Glib 100 μMþemodin, n¼8; Glibþvehicle, n¼ 4; TPN 0.3 μMþemodin, n¼ 8; TPNþvehicle, n¼4).
2.1.3. Are the effects of emodin related to an activation of the muscarinic system? Emodin has been known to have cholinergic properties (Ali et al., 2004; Lu et al., 2007; Lenta et al., 2008; Xu et al., 2012) and also
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Fig. 2. Concentration-dependence of effects of emodin on ANP concentration (A), stroke volume (B), and pulse pressure (C). The effects are expressed as percent difference (Δ% changes) before and 36 min after emodin. Cont, vehicle control, data from Fig. 1A, n¼6; 10, emodin 10 μM, n¼ 6; 30, emodin 30 μM, n¼ 12; 100, emodin 100 μM, data from Fig. 1B, n¼ 8. nPo0.05, nnPo0.01, nnnPo0.001; NS, not significant.
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the cardiac muscarinic system is known to be involved in the regulation of ANP secretion (Xu et al., 2008; Kim et al., 2013). To define the role of M2 mAChR–Gαi/o signaling in the regulation of ANP secretion by emodin, experiments were performed. The control period (12 min) was followed by hemicholinium-3 (HC-3), an inhibitor of high-affinity choline transporter which is rate-limiting step of ACh synthesis, and atropine (Atro) and methoctramin (M), non-selective and selective M2 mAChR inhibitor, respectively, for 36 min and then emodin (30 mM) or vehicle for 36 min in the continuous presence of the inhibitor (HC-3 10 μMþ emodin, n ¼ 5; HC-3 þvehicle, n ¼3; Atro 3 μM þemodin, n¼ 6; Atroþvehicle, n¼ 3; M 1 μMþ emodin, n ¼6; M þvehicle, n¼ 4). In another series of experiments, the role of inhibitory G-protein αi/o in the effects of emodin was investigated. The effect of emodin was tested in the atria from rabbits treated with pertussis toxin (PTX) or vehicle (sham, vehicle-treated, emodin 30 μM and ACh 0.3 μM, n ¼ 8; PTX-treated, emodin 30 μM and ACh 0.3 μM, n ¼8). For the treatment of PTX, rabbits were intravenously injected with PTX (5 mg/kg) or vehicle (1 ml/rabbit) 3 days prior to experiment. The dose of PTX and duration of the treatment were in the range previously reported (Endoh et al., 1985; Xu et al., 2008).
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Time in min Fig. 3. Recovery from the emodin (30 μM)-induced changes in ANP secretion and atrial dynamics. Effects of emodin (30 μM) on ANP secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e), and its recovery by replacing buffer without the agent. Number of experiments, n ¼6. n Po 0.05, nnP o0.01 vs. values before emodin.
G.H. Zhou et al. / European Journal of Pharmacology 735 (2014) 44–51
2.1.4. Are the effects of emodin related to the activity of L-type Ca2 þ channels? Nifedipine (Nife) was applied to identify the role of Ca2 þ entry via L-type Ca2 þ channels in the emodin-induced changes in secretory and contractile function. Thirty-six min of infusion with Nife was followed by 36 min of emodin (30 μM) or vehicle in the continuous presence of Nife (1 μM) (Nife 1 μM þemodin, n ¼6; Nife þvehicle, n ¼3).
out the blood. The left atrium was dissected and inserted into cannula and then ligated by silk. The cannulated atrium was transferred to an organ chamber containing HEPES buffer at 36.5 1C. The atrium was immediately perfused with a HEPES buffer solution by a peristaltic pump (1 ml/min). The composition of the buffer was as follows (mM): 10 HEPES, 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 25 NaHCO3, 10 glucose, and 0.1% bovine serum albumin (BSA) (pH 7.4 with NaOH). The setup was electrically paced at 1.3 Hz (duration, 0.3 ms; voltage, 30–40 V) that allowed for measurement of the changes in atrial stroke volume, pulse pressure, transmural extracellular fluid (ECF) translocation, and ANP secretion. Atrial stroke volume was monitored by reading the lowest level of the water column in the calibrated atrial cannula at end diastole. Atrial pulse pressure was measured via a pressure transducer connected to the intra-atrial catheter and recorded on a physiograph (Model BL-420S, Chengdu TME Technology Co, Ltd, Chengdu, Sichuan, China).
2.1.5. Are the effects of emodin related to AMP-activated protein kinase activity? Emodin has been known to activate AMP-activated protein kinase (AMPK) signaling (Tzeng et al., 2012; Song et al., 2013). To identify an involvement of AMPK signaling in the emodin-induced changes in ANP secretion, experiments were performed in the presence of Compound C, an inhibitor of AMPK signaling (Compound C 20 μMþemodin 30 μM, n¼4; vehicleþemodin, n¼4).
2.3. Measurement of the ECF translocation and molar concentration of ANP
2.2. Preparation of isolated perfused beating atria Male New Zealand White rabbits (weighing about 2 kg) obtained from Shandong Lukang Pharmaceutical Co., Ltd. (Jining, Shandong, China) were used in this study. An isolated atrial preparation was set up using a previously described method (Wen et al., 2004). In brief, rabbit heart was rapidly removed and placed in warm oxygenated physiological saline and washed
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The amount of ANP was expressed in ANP secretion and ANP concentration in terms of ECF translocation. The amount of ECF translocated through the atrial wall was calculated as follows (Cho et al., 1995), ECF translocated (μl/min g atrial wet/weight) ¼{[total radioactivity of 3H-inulin in perfusate (cpm/min)]/[radioactivity of Glib (100 μM) Cont Glib+ Emodin (30 μM)
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Fig. 4. Effects of glibenclamide on the emodin-induced changes in ANP secretion and atrial dynamics. (A) Effects of emodin (30 μM) on ANP secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n¼ 12). (B) Effects of glibenclamide (Glib, 100 μM) on the emodin-induced changes in ANP secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n¼ 8). nPo 0.05, nnP o0.01 vs. values before emodin; #P o0.05, ## Po 0.01, ###Po 0.001 vs. control.
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3 H-inulin in pericardial reservoir (cpm/μl)]}/atrial wet weight (mg) 1000. The molar concentration of ANP (μM) ¼(ANP secreted/ECF translocated)/molecular mass of ANP (Cho et al., 1995). The molar concentration of ANP reflects the rate of atrial myocyte ANP release in the extracellular space.
2.4. Radioimmunoassay of ANP The levels of immunoreactive ANP in the perfusates were measured by a specific radioimmunoassay (RIA) as described previously (Cho et al., 1995; Wen et al., 2000). The level of ANP secreted was expressed as nanograms of ANP per minute per gram of atrial tissue. 2.5. Reagents Emodin (Xian Jiatian Biological Technology Co. Ltd., Xian, Shanxi, China), nifedipine, glibenclamide and Compound C (Sigma Chemical Co. St. Louis, MO, USA) were first dissolved in dimethyl sulfoxide (DMSO): the final concentration of DMSO was o 0.1%. TEA, HC-3, atropine, methoctramine and Tertiapin-Q (Sigma Chemical Co. St. Louis, MO, USA) were dissolved in distilled water. Pertussis toxin (Tocris, Ellisville, MO, USA) was dissolved in 0.9% saline. [3H]Inulin was purchased from PerkinElmer Inc (Waltham, MA, USA).
2.6. Statistical analyses Statistical significance was determined using repeated measures ANOVA followed by Bonferroni's multiple comparison test (Figs. 1, 3, 4, and 5). Student's t-test for unpaired data (Figs. 2, 6, 7, and 8) was also performed. P values less than 0.05 were considered significant. All data are expressed as means 7S.E.M.
3. Results 3.1. Emodin accentuates ANP secretion in isolated beating rabbit atria Baseline levels of ANP secretion and ANP concentration which reflects the rate of atrial myocyte release of ANP into the extracellular space (in Section 2) were stable during the period of experiment (Fig. 1Aa and Ab). Atrial mechanical dynamics, stroke volume and pulse pressure, and ECF translocation were also stable (Fig. 1Ac–Ae). Administration of emodin (100 μM) increased ANP secretion and ANP concentration concomitantly with a decrease in stroke volume, pulse pressure, and ECF translocation (Fig. 1Ba–Be). The changes of ANP secretion and ANP concentration in response to emodin showed similar pattern. The emodininduced changes in ANP concentration and atrial dynamics were concentration-dependent (Fig. 2). The effects were reversible.
Fig. 5. Effects of nifedipine on the emodin-induced changes in ANP secretion and atrial dynamics. (A) Effects of emodin (30 μM) on ANP secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n¼ 12). Data are the same as Fig. 4A. (B) Effects of nifedipine (Nife, 1 μM) on the emodin-induced changes in ANP secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n ¼6). nPo 0.05, nnP o0.01 vs. values before emodin; #Po 0.05, ##Po 0.01, ###Po 0.001 vs. control.
G.H. Zhou et al. / European Journal of Pharmacology 735 (2014) 44–51
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-50 Fig. 6. Summarized effects of inhibitors of ion channels on the emodin (30 μM)induced changes in ANP secretion and atrial dynamics. Effects of tetraethylammonium (TEA, 10 mM), glibenclamide (Glib, 100 μM) and nifedipine (Nife, 1 μM) on the emodin (30 μM)-induced increase in ANP concentration (concn) (A), and decrease in stroke volume (B) and pulse pressure (C). #Po 0.05, ##P o 0.01, ###P o0.001 vs. vehicle (V); nP o0.05, nnPo 0.01, nnnP o 0.001 vs. emodin (E) (Cont). Number of experiments; Cont-E, n¼ 12, data from Fig. 4A, Cont-V, n¼ 6, data from Fig. 1A; TEAE, n¼ 8, TEA-V, n¼ 3; Glib-E, n ¼8, data from Fig. 4B, Glib-V, n¼ 4; Nife-E, n¼ 6, data from Fig. 5B, Nife-V, n¼ 3.
Emodin-induced changes in ANP secretion and atrial dynamics returned back to the baseline levels by replacing buffer without the agent (Fig. 3). þ 3.2. Emodin accentuates ANP secretion via activation of KATP channel
To identify the mechanism by which emodin accentuates ANP secretion, modulators were used. Because an activation of K þ channel is involved in the accentuation of ANP secretion from the cardiac atria (Kim et al., 1997), effects of inhibition of K þ channels were tested. Administration of TEA (10 mM), a non-selective inhibitor of K þ channels, decreased ANP secretion and ANP concentration concomitantly with an increase in atrial dynamics. Pretreatment with TEA significantly attenuated the emodin (30 μM)-induced increase in ANP concentration (Fig. 6). TEA also attenuated the emodin-induced decrease in atrial dynamics. In the next step, to further define the involvement of K þ channels, effects þ of Glib, a selective inhibitor of KATP channel, were tested. As shown in Fig. 4A, emodin (30 μM) increased ANP secretion and ANP concentration concomitantly with a decrease in atrial dynamics. Glib (100 μM) decreased ANP secretion and ANP concentration and increased atrial dynamics and ECF translocation (Fig. 4B). Pretreatment with Glib significantly attenuated the emodininduced increase in ANP secretion and ANP concentration
Δ% Pulse pressure
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-60 Fig. 7. Summarized effects of modulators of the M2 muscarinic acetylcholine receptor signaling on the emodin-induced changes in ANP secretion and atrial dynamics. Effects of hemicholinium-3 (HC-3, 10 μM), atropine (Atro, 3 μM), methoctramine (M, 1 μM), and tertiapin (TPN, 0.3 μM) on the emodin (30 μM)induced increase in ANP concentration (concn) (A), and decrease in stroke volume (B) and pulse pressure (C). Number of experiments; Cont-Emodin (E), n¼ 12, data from Fig. 4A, Cont-vehicle (V), n¼6, data from Fig. 1A; HC-3þ E, n¼ 5, HC-3, n¼ 3; Atro þ E, n¼ 6, Atro, n ¼3; Mþ E, n¼ 6, M, n¼4; TPN þ E, n¼8, TPN, n ¼4.
(Fig. 4Ba and Bb, and Fig. 6). Glib also attenuated the emodininduced decrease in atrial dynamics (Fig. 4Bc and Bd, and Fig. 6). Because Ca2 þ entry via L-type Ca2 þ channel is a prerequisite for þ the accentuation of ANP secretion by activation of KATP channel þ and KACh channel (Kim et al., 1997; Xu et al., 2008), effects of inhibition of L-type Ca2 þ channel on the emodin accentuation of ANP secretion were tested. As shown in Fig. 5, inhibition of L-type Ca2 þ channel with Nife increased ANP secretion and ANP concentration concomitantly with a decrease in atrial dynamics and ECF translocation (Fig. 5B). Pretreatment with Nife attenuated the emodin-induced changes in ANP secretion and atrial dynamics (Fig. 5B and Fig. 6). Previously, it was shown that emodin has cholinergic properties (Ali et al., 2004; Lu et al., 2007; Lenta et al., 2008; Xu et al., 2012). To identify the role of the muscarinic system in the emodininduced accentuation of ANP secretion, effects of modulators of the M2 mAChR signaling were tested. Inhibition of ACh synthesis with HC-3, an inhibitor of high-affinity choline transporter which is rate limiting step of ACh synthesis (Okuda et al., 2000), had no significant effect on the emodin-induced changes in ANP concentration and atrial dynamics (Fig. 7). Furthermore, inhibitors of the þ M2 mAChR, Atro and M, and the downstream KACh channel, TPN, had no significant effects on the emodin-induced changes in ANP
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Fig. 9. Proposed mechanism for the accentuation of ANP secretion by emodin. Emodin accentuates atrial myocyte ANP release (Exo). Non-selective K þ channel þ inhibitor, tetraethylammonium, and selective KATP channel inhibitor, glibenclamide (Glib), attenuate the emodin-induced increase in ANP release which indicates a role þ for KATP channel opening. An inhibition of L-type Ca2 þ channel (LTCC) with nifedipine (Nife) also attenuates the emodin-induced increase in atrial myocyte ANP release. Ca2 þ entry through LTCC is inhibitory for ANP release in this experimental model. Also, K þ channel activation is a physiological regulator for Ca2 þ entry through LTCC. Therefore, it is possible to propose that emodin accentuates atrial myocyte ANP release via inhibition of Ca2 þ entry through LTCC (an inhibitory regulator for ANP release) by shortening of the action potential þ caused by KATP channel activation. M2 muscarinic acetylcholine receptor (mAChR)– þ KACh channel signaling is not involved in the emodin-induced accentuation of ANP release in cardiac atria. TPN, tertiapin-Q; SL, atrial myocyte sarcolemma;(-) inhibition.
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Fig. 8. Effects of emodin (30 μM) and acetylcholine (ACh, 0.3 μM) on ANP concentration (A), stroke volume (B), and pulse pressure (C) in the atria from Sham- and pertussis toxin (PTX)-treated rats. Values are means 7SE. nP o 0.05, nn P o0.01, nnnPo 0.001 vs. corresponding values of Sham (vehicle). Number of experiments; Sham group, n ¼8: PTX-treated group, n¼ 8.
concentration and atrial dynamics (Fig. 7). To further define the role of inhibitory G-protein αi/o, experiments were performed in the atria from rabbits treated with PTX. PTX treatment had no significant effect on the emodin-induced increase in ANP concentration (Fig. 8). Emodin-induced decrease in atrial dynamics was rather slightly accentuated in the atria treated with PTX. As expected, both the ACh-induced increase in ANP concentration and decrease in atrial dynamics were attenuated in atria from rabbits treated with PTX. Emodin is known to activate AMP-activated protein kinase (AMPK) signaling (Tzeng et al., 2012; Song et al., 2013). To identify an involvement of AMPK signaling in the emodin-induced accentuation of ANP secretion, experiments were performed in the presence of Compound C, an inhibitor of AMPK signaling. Pretreatment with Compound C (20 μM) had no significant effect on the emodin-induced accentuation of ANP secretion and inhibition of atrial dynamics (for ANP secretion in Δ% changes, 181.82 7 52.71%, n ¼4, vs. 97.14 718.76% in control, n¼ 4, not significant; for pulse pressure in Δ% changes, 41.25 72.39%, n¼ 4, vs. 41.04 7 7.65% in control, n¼ 4, not significant).
The present study shows for the first time that emodin accentuates ANP secretion in a concentration-dependent manner in isolated beating rabbit atria. The accentuation of the ANP secretion by emodin was accompanied by a decrease in atrial pulse pressure and stroke volume. The emodin-induced changes in ANP secretion and atrial dynamics returned back to baseline levels by replacing buffer solution without the agent. The emodininduced changes in ANP secretion and atrial dynamics were significantly attenuated by an inhibition of K þ channels with þ TEA and Glib. These findings indicate that an activation of KATP channel is involved in the emodin-induced increase in ANP þ secretion. KATP channel is well expressed in the cardiac myocytes þ (Noma, 1983). Previously, it was shown that KATP channel is involved in the regulation of ANP secretion (Xu et al., 1996; Kim et al., 1997; Ogawa et al., 2009). Kim et al. (1997) showed that þ activation of KATP channel is positively involved in the regulation of ANP secretion. The present finding is consistent with this report. Recently, Xu et al. (2008) showed that activation of M2 mAChR increased ANP secretion via activation of inhibitory G-protein αi/o– þ KACh channel signaling. Because emodin possesses cholinergic properties (Ali et al., 2004; Lu et al., 2007; Lenta et al., 2008; Xu et al., 2012) and M2 mAChR is involved in the regulation of ANP secretion (Xu et al., 2008; Kim et al., 2013), emodin was expected to accentuate ANP secretion via activation of cardiac muscarinic signaling pathway. But it was not the case. Not only the M2 mAChR þ but also inhibitory G-protein αi/o and KACh channels was not involved in the emodin-induced increase in ANP secretion. The þ present findings suggest that K þ channels, KATP channel in particular, are involved in the emodin-induced accentuation of ANP secretion. Ca2 þ entry via activation of L-type Ca2 þ channel is known to inhibit ANP secretion in the atria (Ito et al., 1988; de Bold and de Bold, 1989; Ruskoaho et al., 1990; Wen et al., 2000). Intracellular Ca2 þ tonically inhibits ANP secretion in beating rabbit atria (Wen et al., 2000). The present study shows that emodin-induced
G.H. Zhou et al. / European Journal of Pharmacology 735 (2014) 44–51
increase in ANP secretion was attenuated by inhibition of L-type Ca2 þ channel which suggests that Ca2 þ entry is involved. Previously, it was shown that Ca2 þ entry through L-type Ca2 þ þ þ channel is a prerequisite for the KACh channel activation- and KATP channel activation-induced accentuation of ANP secretion (Kim et al., 1997; Xu et al., 2008). Taken together, we propose that emodin accentuates ANP secretion via inhibition of Ca2 þ entry through L-type Ca2 þ channel by shortening of the action potential caused þ by KATP channel activation (Fig. 9). Alternatively, emodin may increase ANP secretion via inhibition of L-type Ca2 þ channel. It has been shown that emodin has two-way regulation on the intracellular Ca2 þ concentration and L-type Ca2 þ channel activity in cardiomyocytes: an increase at low concentration and inhibition at higher concentration (Liu et al., 2004). Emodin is known to induce an activation of AMPK signaling (Tzeng et al., 2012; Song et al., 2013). However, in the present study, it was shown that AMPK signaling is not involved in the emodin-induced accentuation of ANP secretion. In summary, emodin accentuates ANP secretion via activation þ of KATP channels in cardiac atria. Emodin in rhubarb, Rheum officinale Baill, that has long been used in traditional Chinese herbal medicine, is known to possess cardiovascular protective properties (Huang et al., 1991; Du and Ko, 2005; Meng et al., 2010; Chen et al., 2012; Tzeng et al., 2012). Considering the physiological and pathophysiological roles of ANP, it is expected that effects of emodin may closely be related with the accentuation of ANP secretion from the atria. In a series of preliminary experiments, emodin increased plasma levels of ANP concomitantly with an accentuation of atrial ANP gene expression.
Acknowledgements This work was supported by research grants from the National Natural Science Foundation of China (Nos. 30971080, 81270920), the Natural Science Foundation of Shandong Province (No. ZR2011HM022), and the National Research Foundation of Korea (NRF) funded by the Korea government (MSIP) (2008-0062484).
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Endoh, M., Maruyama, M., Iijima, T., 1985. Attenuation of muscarinic cholinergic inhibition by islet-activating protein in the heart. Am. J. Physiol. Heart Circ. Physiol 249, H309–H320. Huang, H.C., Lee, C.R., Chao, P.D., Chen, C.C., Chu, S.H., 1991. Vasorelaxant effect of emodin, an anthraquinone from a Chinese herb. Eur. J. Pharmacol. 205, 289–294. Ito, T., Toki, Y., Siegel, N., Gierse, J.K., Needleman, P., 1988. Manipulation of stretchinduced atriopeptin prohormone release and processing in the perfused rat heart. Proc. Nat. Acad. Sci. U.S.A. 85, 8365–8369. Kiemer, A., Vollmar, A., 2001. The atrial natriuretic peptide regulates the production of inflammatory mediators in macrophages. Ann. Rheum. Dis. 60 (Suppl. 3) (iii68-iii70). Kim, H.Y., Cho, K.W., Xu, D.Y., Kang, D.G., Lee, H.S., 2013. Endogenous ACh tonically stimulates ANP secretion in rat atria. Am. J. Physiol. Heart Circ. Physiol 305, H1050–H1056. Kim, S.H., Cho, K.W., Chang, S.H., Kim, S.Z., Chae, S.W., 1997. 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Identification and characterization of the high-affinity choline transporter. Nat. Neurosci. 3, 120–125. Ruskoaho, H., 1992. Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacol. Rev. 44, 479–602. Ruskoaho, H., Vuolteenaho, O., Leppaluoto, J., 1990. Phorbol esters enhance stretchinduced atrial natriuretic peptide secretion. Endocrinology 127, 2445–2455. Sangawa, K., Nakanishi, K., Ishino, K., Inoue, M., Kawada, M., Sano, S., 2004. Atrial natriuretic peptide protects against ischemia–reperfusion injury in the isolated rat heart. Ann. Thorac. Surg. 77, 233–237. Song, P., Kim, J.H., Ghim, J., Yoon, J.H., Lee, A., Kwon, Y., Hyun, H., Moon, H.Y., Choi, H. S., Berggren, P.O., Suh, P.G., Ryu, S.H., 2013. Emodin regulates glucose utilization by activating AMP-activated protein kinase. J. Biol. Chem. 288, 5732–5742. Tzeng, T.-F., Lu, H.-J., Liou, S.-S., Chang, C.J., Liu, I.-M., 2012. 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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Emodin accentuates atrial natriuretic peptide secretion in cardiac atria Guang Hai Zhou a,1, Feng Zhang a,1, Xin Nong Wang a, Oh Jeong Kwon c, Dae Gill Kang c, Ho Sub Lee c, Song Nan Jin b,n, Kyung Woo Cho a, Jin Fu Wen a,nn a Institute of Atherosclerosis, Key Laboratory of Atherosclerosis in Universities of Shandong, Taishan Medical University, 2 East Yingsheng Road, Taian 271000, Shandong, China b Institute of Materia Medica, Taishan Medical University, Middle of Changcheng Road, Taian 271016, Shandong, China c Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan 570-749, Jeonbuk, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 25 January 2014 Received in revised form 4 April 2014 Accepted 9 April 2014 Available online 18 April 2014
Emodin, an active anthraquinone constituent isolated from the rhubarb, a traditional Chinese herbal medicine which is widely used in clinical treatment, has cardiovascular protective properties. However, it remains unclear whether the cardiovascular protective actions of emodin are related to an activation of cardiac natriuretic hormone secretion. The purpose of the present study was to explore the effect of emodin on the secretion of ANP, a member of the family of cardiac natriuretic hormones, and its mechanisms involved. Experiments were performed in isolated perfused beating rabbit atria allowing measurement of ANP secretion, atrial pulse pressure, and stroke volume. Emodin increased ANP secretion concomitantly with a decrease in atrial pulse pressure and stroke volume in a concentration-dependent manner. These effects were reversible. Inhibition of K þ channels with tetraethylammonium and glibenclamide attenuated the emodin-induced changes in ANP secretion and atrial dynamics. Furthermore, the emodin-induced changes in ANP secretion and atrial dynamics were attenuated by inhibition of L-type Ca2 þ channels with nifedipine. Atropine, methoctramine, tertiapin-Q, and pertussis toxin had no significant effect on the emodin-induced changes in ANP secretion and mechanical dynamics. The present study demonstrates that emodin increases þ channel in isolated ANP secretion via inhibition of L-type Ca2 þ channels through an activation of KATP beating rabbit atria. The results also provide a rationale for the use of emodin in the treatment of impairment of the regulation of the cardiovascular homeostasis. & 2014 Published by Elsevier B.V.
Keywords: Emodin Atrial natriuretic peptide secretion Cardiac natriuretic hormone þ KATP channel L-type Ca2 þ channel
1. Introduction Emodin (1,3,8-trihydroxy-6-methylanthraquinone), an active anthraquinone constituent isolated from the rhubarb (Wu et al., 2013), has cardiovascular protective actions, such as anti-hypertensive, anti-hyperlipidemic, anti-inflammatory, and vasorelaxant effects (Huang et al., 1991; Meng et al., 2010; Chen et al., 2012; Tzeng et al., 2012). Effects of emodin on cardiac endocrine function are unidentified. Atrial cardiomyocytes synthesize atrial natriuretic peptide (ANP) and secrete it into the bloodstream. ANP has various physiological effects: natriuretic, diuretic, vasorelaxant, anti-hypertensive, antiinflammatory, anti-proliferative, and anti-oxidative actions (Kiemer and Vollmar, 2001; Sangawa et al., 2004; Vesely, 2009; De Vito et al., 2010). Recently, it has also been shown that ANP is involved in lipid
n
Corresponding author. Tel.: þ 86 538 623 7282; fax: þ 86 538 622 2651. Corresponding author. Tel.: þ 86 538 622 3006; fax: þ86 538 622 2651. E-mail addresses: [email protected] (S.N. Jin), [email protected], [email protected] (J.F. Wen). 1 The two first authors contributed equally to this work. nn
http://dx.doi.org/10.1016/j.ejphar.2014.04.014 0014-2999/& 2014 Published by Elsevier B.V.
homeostasis via cGMP signaling (Moro and Lafontan, 2013). The ANP system is considered as an endogenous counterbalancing limb for the regulation of the renin–angiotensin–aldosterone system. These notions indicate that ANP exhibits potent protective actions on the cardiovascular system through its multiple targets. ANP secretion is primarily controlled by changes in atrial volume and cardiac workload and is also modulated by neurotransmitters and hormones (Ruskoaho, 1992; Xu et al., 2008; Kim et al., 2013). Intracellular second messengers, Ca2þ , cAMP, and cGMP, and K þ channels are involved in the regulation of ANP secretion (Ito et al., 1988; Xu et al., 1996; Wen et al., 2004). Recently, it was shown that endogenous acetylcholine (ACh) is positively involved in the regulation of ANP secretion (Kim et al., 2013). M2 muscarinic ACh receptor (mAChR) þ stimulation accentuates ANP secretion via activation of Gαi/o–KACh channel signaling (Xu et al., 2008; Kim et al., 2013). In addition, þ activation of KATP channel is also known to increase ANP secretion (Kim et al., 1997). In contrast, it has been shown that increase in intracellular Ca2þ concentration and Ca2 þ entry via L-type Ca2þ channel is an inhibitory modulator for ANP secretion (Ito et al., 1988; de Bold and de Bold, 1989; Ruskoaho et al., 1990; Wen et al., 2000). K þ þ þ channels, KACh channel and KATP channel in particular, are indirectly
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involved in the regulation of ANP secretion through inhibiting Ca2 þ entry via L-type Ca2þ channel (Kim et al., 1997; Xu et al., 2008). þ Shortening of the action potential caused by activation of KATP channel is an endogenous modulator for the regulation of intracellular Ca2 þ levels and L-type Ca2 þ channel activity (Noma, 1983). The effect of emodin on ANP secretion from the heart has not yet been identified. It is known that emodin affects ion channels (Liu et al., 2004; Li et al., 1998). Emodin is also known to have cholinergic properties (Ali et al., 2004; Lu et al., 2007; Lenta et al., 2008; Xu et al., 2012). Therefore, we hypothesized that emodin affects ANP secretion from the cardiac atria through modulation of ion channel activity and muscarinic signaling. The purpose of the present study was to investigate the effect of emodin on ANP secretion and its underlying mechanisms in isolated beating rabbit atria. From this study, we find that emodin accentuates ANP secretion via inhibition of L-type Ca2 þ channel þ activity through an activation of KATP channels.
2. Materials and methods All of the experimental protocols were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996) and were approved by the Institutional Animal Care and Utilization Committee for Medical Science of Taishan Medical University.
2.1. Experimental protocols Experiments were performed to identify effects of emodin on atrial ANP secretion and its mechanisms involved in isolated beating rabbit atria. The atrium was perfused for 60 min to stabilize ANP secretion and atrial dynamics. [3H]Inulin was introduced to the pericardial fluid 20 min before the start of sample collection (Wen et al., 2000). The perfusate was collected for ANP analysis at 2-min intervals at 4 1C. Experiments were carried out in order to solve the questions detailed below. 2.1.1. Does emodin increase ANP secretion and mechanical dynamics? An initial control period of 48 min was followed by emodin infusion (0, 10, 30, and 100 μM) for 36 min (vehicle, n¼6; emodin 10 μM, n¼6; emodin 30 μM, n¼ 12; emodin 100 μM, n¼8). To evaluate the effects of emodin on ANP secretion and atrial dynamics, stroke volume and pulse pressure, values (mean of 2 fractions) obtained before and 36 min after the addition of the agent were compared. Data are expressed as percent difference from the control (before the addition of emodin). 2.1.2. Are the effects of emodin related to K þ channel activity? þ þ K þ channels, KATP channel and KACh channel in particular, are known to be involved in the regulation of ANP secretion (Xu et al., 1996; Kim et al., 1997; Xu et al., 2008). To test an involvement of
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Fig. 1. Effects of emodin on secretory and contractile function in isolated beating rabbit atria. (A) Effects of vehicle on atrial natriuretic peptide (ANP) secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n¼6). (B) Effects of emodin (100 μM) on ANP secretion (a), ANP concentration (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n ¼8). Values are means 7S.E.M. nnnPo 0.001 vs. values before emodin.
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K þ channels, experiments were performed in the presence of selective and non-selective K þ channel inhibitors. Effets of tetraethylammonium (TEA), a non-selective inhibitor of K þ channels, þ glibenclamide (Glib), a selective KATP channel inhibitor, and þ tertiapin-Q (TPN), a selective KACh channel inhibitor, were tested. Thirty-six min of infusion with inhibitor was followed by 36 min of emodin (30 μM) or vehicle in the continuous presence of the previous agent (TEA 10 mMþemodin, n¼8; TEAþvehicle, n¼ 3; Glib 100 μMþemodin, n¼8; Glibþvehicle, n¼ 4; TPN 0.3 μMþemodin, n¼ 8; TPNþvehicle, n¼4).
2.1.3. Are the effects of emodin related to an activation of the muscarinic system? Emodin has been known to have cholinergic properties (Ali et al., 2004; Lu et al., 2007; Lenta et al., 2008; Xu et al., 2012) and also
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Fig. 2. Concentration-dependence of effects of emodin on ANP concentration (A), stroke volume (B), and pulse pressure (C). The effects are expressed as percent difference (Δ% changes) before and 36 min after emodin. Cont, vehicle control, data from Fig. 1A, n¼6; 10, emodin 10 μM, n¼ 6; 30, emodin 30 μM, n¼ 12; 100, emodin 100 μM, data from Fig. 1B, n¼ 8. nPo0.05, nnPo0.01, nnnPo0.001; NS, not significant.
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the cardiac muscarinic system is known to be involved in the regulation of ANP secretion (Xu et al., 2008; Kim et al., 2013). To define the role of M2 mAChR–Gαi/o signaling in the regulation of ANP secretion by emodin, experiments were performed. The control period (12 min) was followed by hemicholinium-3 (HC-3), an inhibitor of high-affinity choline transporter which is rate-limiting step of ACh synthesis, and atropine (Atro) and methoctramin (M), non-selective and selective M2 mAChR inhibitor, respectively, for 36 min and then emodin (30 mM) or vehicle for 36 min in the continuous presence of the inhibitor (HC-3 10 μMþ emodin, n ¼ 5; HC-3 þvehicle, n ¼3; Atro 3 μM þemodin, n¼ 6; Atroþvehicle, n¼ 3; M 1 μMþ emodin, n ¼6; M þvehicle, n¼ 4). In another series of experiments, the role of inhibitory G-protein αi/o in the effects of emodin was investigated. The effect of emodin was tested in the atria from rabbits treated with pertussis toxin (PTX) or vehicle (sham, vehicle-treated, emodin 30 μM and ACh 0.3 μM, n ¼ 8; PTX-treated, emodin 30 μM and ACh 0.3 μM, n ¼8). For the treatment of PTX, rabbits were intravenously injected with PTX (5 mg/kg) or vehicle (1 ml/rabbit) 3 days prior to experiment. The dose of PTX and duration of the treatment were in the range previously reported (Endoh et al., 1985; Xu et al., 2008).
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Time in min Fig. 3. Recovery from the emodin (30 μM)-induced changes in ANP secretion and atrial dynamics. Effects of emodin (30 μM) on ANP secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e), and its recovery by replacing buffer without the agent. Number of experiments, n ¼6. n Po 0.05, nnP o0.01 vs. values before emodin.
G.H. Zhou et al. / European Journal of Pharmacology 735 (2014) 44–51
2.1.4. Are the effects of emodin related to the activity of L-type Ca2 þ channels? Nifedipine (Nife) was applied to identify the role of Ca2 þ entry via L-type Ca2 þ channels in the emodin-induced changes in secretory and contractile function. Thirty-six min of infusion with Nife was followed by 36 min of emodin (30 μM) or vehicle in the continuous presence of Nife (1 μM) (Nife 1 μM þemodin, n ¼6; Nife þvehicle, n ¼3).
out the blood. The left atrium was dissected and inserted into cannula and then ligated by silk. The cannulated atrium was transferred to an organ chamber containing HEPES buffer at 36.5 1C. The atrium was immediately perfused with a HEPES buffer solution by a peristaltic pump (1 ml/min). The composition of the buffer was as follows (mM): 10 HEPES, 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 25 NaHCO3, 10 glucose, and 0.1% bovine serum albumin (BSA) (pH 7.4 with NaOH). The setup was electrically paced at 1.3 Hz (duration, 0.3 ms; voltage, 30–40 V) that allowed for measurement of the changes in atrial stroke volume, pulse pressure, transmural extracellular fluid (ECF) translocation, and ANP secretion. Atrial stroke volume was monitored by reading the lowest level of the water column in the calibrated atrial cannula at end diastole. Atrial pulse pressure was measured via a pressure transducer connected to the intra-atrial catheter and recorded on a physiograph (Model BL-420S, Chengdu TME Technology Co, Ltd, Chengdu, Sichuan, China).
2.1.5. Are the effects of emodin related to AMP-activated protein kinase activity? Emodin has been known to activate AMP-activated protein kinase (AMPK) signaling (Tzeng et al., 2012; Song et al., 2013). To identify an involvement of AMPK signaling in the emodin-induced changes in ANP secretion, experiments were performed in the presence of Compound C, an inhibitor of AMPK signaling (Compound C 20 μMþemodin 30 μM, n¼4; vehicleþemodin, n¼4).
2.3. Measurement of the ECF translocation and molar concentration of ANP
2.2. Preparation of isolated perfused beating atria Male New Zealand White rabbits (weighing about 2 kg) obtained from Shandong Lukang Pharmaceutical Co., Ltd. (Jining, Shandong, China) were used in this study. An isolated atrial preparation was set up using a previously described method (Wen et al., 2004). In brief, rabbit heart was rapidly removed and placed in warm oxygenated physiological saline and washed
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(μl/min/g)
Pulse pressure
The amount of ANP was expressed in ANP secretion and ANP concentration in terms of ECF translocation. The amount of ECF translocated through the atrial wall was calculated as follows (Cho et al., 1995), ECF translocated (μl/min g atrial wet/weight) ¼{[total radioactivity of 3H-inulin in perfusate (cpm/min)]/[radioactivity of Glib (100 μM) Cont Glib+ Emodin (30 μM)
Emodin (30 μM)
Cont
ECF translocation
47
50
∗∗
∗
25
25
0
0
1.5
1.5
∗∗
1.0
##
∗
1.0
.5
.5
0.0
0.0
800
800
400
400
# ##
∗
∗ 0
0
12
12
6
6
###
∗
∗ 0
0
30
30
# 15
15
0
0 0
12
24
36
48
Time in min
60
72
84
0
12
24
36
48
60
72
84
Time in min
Fig. 4. Effects of glibenclamide on the emodin-induced changes in ANP secretion and atrial dynamics. (A) Effects of emodin (30 μM) on ANP secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n¼ 12). (B) Effects of glibenclamide (Glib, 100 μM) on the emodin-induced changes in ANP secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n¼ 8). nPo 0.05, nnP o0.01 vs. values before emodin; #P o0.05, ## Po 0.01, ###Po 0.001 vs. control.
48
G.H. Zhou et al. / European Journal of Pharmacology 735 (2014) 44–51
3 H-inulin in pericardial reservoir (cpm/μl)]}/atrial wet weight (mg) 1000. The molar concentration of ANP (μM) ¼(ANP secreted/ECF translocated)/molecular mass of ANP (Cho et al., 1995). The molar concentration of ANP reflects the rate of atrial myocyte ANP release in the extracellular space.
2.4. Radioimmunoassay of ANP The levels of immunoreactive ANP in the perfusates were measured by a specific radioimmunoassay (RIA) as described previously (Cho et al., 1995; Wen et al., 2000). The level of ANP secreted was expressed as nanograms of ANP per minute per gram of atrial tissue. 2.5. Reagents Emodin (Xian Jiatian Biological Technology Co. Ltd., Xian, Shanxi, China), nifedipine, glibenclamide and Compound C (Sigma Chemical Co. St. Louis, MO, USA) were first dissolved in dimethyl sulfoxide (DMSO): the final concentration of DMSO was o 0.1%. TEA, HC-3, atropine, methoctramine and Tertiapin-Q (Sigma Chemical Co. St. Louis, MO, USA) were dissolved in distilled water. Pertussis toxin (Tocris, Ellisville, MO, USA) was dissolved in 0.9% saline. [3H]Inulin was purchased from PerkinElmer Inc (Waltham, MA, USA).
2.6. Statistical analyses Statistical significance was determined using repeated measures ANOVA followed by Bonferroni's multiple comparison test (Figs. 1, 3, 4, and 5). Student's t-test for unpaired data (Figs. 2, 6, 7, and 8) was also performed. P values less than 0.05 were considered significant. All data are expressed as means 7S.E.M.
3. Results 3.1. Emodin accentuates ANP secretion in isolated beating rabbit atria Baseline levels of ANP secretion and ANP concentration which reflects the rate of atrial myocyte release of ANP into the extracellular space (in Section 2) were stable during the period of experiment (Fig. 1Aa and Ab). Atrial mechanical dynamics, stroke volume and pulse pressure, and ECF translocation were also stable (Fig. 1Ac–Ae). Administration of emodin (100 μM) increased ANP secretion and ANP concentration concomitantly with a decrease in stroke volume, pulse pressure, and ECF translocation (Fig. 1Ba–Be). The changes of ANP secretion and ANP concentration in response to emodin showed similar pattern. The emodininduced changes in ANP concentration and atrial dynamics were concentration-dependent (Fig. 2). The effects were reversible.
Fig. 5. Effects of nifedipine on the emodin-induced changes in ANP secretion and atrial dynamics. (A) Effects of emodin (30 μM) on ANP secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n¼ 12). Data are the same as Fig. 4A. (B) Effects of nifedipine (Nife, 1 μM) on the emodin-induced changes in ANP secretion (a), ANP concentration (concn) (b), stroke volume (c), pulse pressure (d), and ECF translocation (e) (n ¼6). nPo 0.05, nnP o0.01 vs. values before emodin; #Po 0.05, ##Po 0.01, ###Po 0.001 vs. control.
G.H. Zhou et al. / European Journal of Pharmacology 735 (2014) 44–51
TEA
Glib
Nife
Cont
HC-3
Atro
M
TPN
E
E V
E V
E V
E V
E V
E V
E V
E V
V
### ##
∗
##
∗∗
50
##
∗∗∗
0
0
∗∗ ##
-25
∗
∗
##
### -50
150
Δ% ANP concn
100
Cont
Δ% Stroke volume
Δ% Stroke volume
Δ% ANP concn
150
49
100 50 0
0
-30
0
∗∗
-25
##
###
∗
##
∗
-50 Fig. 6. Summarized effects of inhibitors of ion channels on the emodin (30 μM)induced changes in ANP secretion and atrial dynamics. Effects of tetraethylammonium (TEA, 10 mM), glibenclamide (Glib, 100 μM) and nifedipine (Nife, 1 μM) on the emodin (30 μM)-induced increase in ANP concentration (concn) (A), and decrease in stroke volume (B) and pulse pressure (C). #Po 0.05, ##P o 0.01, ###P o0.001 vs. vehicle (V); nP o0.05, nnPo 0.01, nnnP o 0.001 vs. emodin (E) (Cont). Number of experiments; Cont-E, n¼ 12, data from Fig. 4A, Cont-V, n¼ 6, data from Fig. 1A; TEAE, n¼ 8, TEA-V, n¼ 3; Glib-E, n ¼8, data from Fig. 4B, Glib-V, n¼ 4; Nife-E, n¼ 6, data from Fig. 5B, Nife-V, n¼ 3.
Emodin-induced changes in ANP secretion and atrial dynamics returned back to the baseline levels by replacing buffer without the agent (Fig. 3). þ 3.2. Emodin accentuates ANP secretion via activation of KATP channel
To identify the mechanism by which emodin accentuates ANP secretion, modulators were used. Because an activation of K þ channel is involved in the accentuation of ANP secretion from the cardiac atria (Kim et al., 1997), effects of inhibition of K þ channels were tested. Administration of TEA (10 mM), a non-selective inhibitor of K þ channels, decreased ANP secretion and ANP concentration concomitantly with an increase in atrial dynamics. Pretreatment with TEA significantly attenuated the emodin (30 μM)-induced increase in ANP concentration (Fig. 6). TEA also attenuated the emodin-induced decrease in atrial dynamics. In the next step, to further define the involvement of K þ channels, effects þ of Glib, a selective inhibitor of KATP channel, were tested. As shown in Fig. 4A, emodin (30 μM) increased ANP secretion and ANP concentration concomitantly with a decrease in atrial dynamics. Glib (100 μM) decreased ANP secretion and ANP concentration and increased atrial dynamics and ECF translocation (Fig. 4B). Pretreatment with Glib significantly attenuated the emodininduced increase in ANP secretion and ANP concentration
Δ% Pulse pressure
Δ% Pulse pressure
-60
0
-30
-60 Fig. 7. Summarized effects of modulators of the M2 muscarinic acetylcholine receptor signaling on the emodin-induced changes in ANP secretion and atrial dynamics. Effects of hemicholinium-3 (HC-3, 10 μM), atropine (Atro, 3 μM), methoctramine (M, 1 μM), and tertiapin (TPN, 0.3 μM) on the emodin (30 μM)induced increase in ANP concentration (concn) (A), and decrease in stroke volume (B) and pulse pressure (C). Number of experiments; Cont-Emodin (E), n¼ 12, data from Fig. 4A, Cont-vehicle (V), n¼6, data from Fig. 1A; HC-3þ E, n¼ 5, HC-3, n¼ 3; Atro þ E, n¼ 6, Atro, n ¼3; Mþ E, n¼ 6, M, n¼4; TPN þ E, n¼8, TPN, n ¼4.
(Fig. 4Ba and Bb, and Fig. 6). Glib also attenuated the emodininduced decrease in atrial dynamics (Fig. 4Bc and Bd, and Fig. 6). Because Ca2 þ entry via L-type Ca2 þ channel is a prerequisite for þ the accentuation of ANP secretion by activation of KATP channel þ and KACh channel (Kim et al., 1997; Xu et al., 2008), effects of inhibition of L-type Ca2 þ channel on the emodin accentuation of ANP secretion were tested. As shown in Fig. 5, inhibition of L-type Ca2 þ channel with Nife increased ANP secretion and ANP concentration concomitantly with a decrease in atrial dynamics and ECF translocation (Fig. 5B). Pretreatment with Nife attenuated the emodin-induced changes in ANP secretion and atrial dynamics (Fig. 5B and Fig. 6). Previously, it was shown that emodin has cholinergic properties (Ali et al., 2004; Lu et al., 2007; Lenta et al., 2008; Xu et al., 2012). To identify the role of the muscarinic system in the emodininduced accentuation of ANP secretion, effects of modulators of the M2 mAChR signaling were tested. Inhibition of ACh synthesis with HC-3, an inhibitor of high-affinity choline transporter which is rate limiting step of ACh synthesis (Okuda et al., 2000), had no significant effect on the emodin-induced changes in ANP concentration and atrial dynamics (Fig. 7). Furthermore, inhibitors of the þ M2 mAChR, Atro and M, and the downstream KACh channel, TPN, had no significant effects on the emodin-induced changes in ANP
50
G.H. Zhou et al. / European Journal of Pharmacology 735 (2014) 44–51
Sham E
ACh
PTX-treated E
ACh
Δ% changes in ANP concentration
150
100
50
** 0
Δ % changes in stroke volume
0
-20
*
***
-40
-60
4. Discussion
0
Δ % changes in pulse pressure
Fig. 9. Proposed mechanism for the accentuation of ANP secretion by emodin. Emodin accentuates atrial myocyte ANP release (Exo). Non-selective K þ channel þ inhibitor, tetraethylammonium, and selective KATP channel inhibitor, glibenclamide (Glib), attenuate the emodin-induced increase in ANP release which indicates a role þ for KATP channel opening. An inhibition of L-type Ca2 þ channel (LTCC) with nifedipine (Nife) also attenuates the emodin-induced increase in atrial myocyte ANP release. Ca2 þ entry through LTCC is inhibitory for ANP release in this experimental model. Also, K þ channel activation is a physiological regulator for Ca2 þ entry through LTCC. Therefore, it is possible to propose that emodin accentuates atrial myocyte ANP release via inhibition of Ca2 þ entry through LTCC (an inhibitory regulator for ANP release) by shortening of the action potential þ caused by KATP channel activation. M2 muscarinic acetylcholine receptor (mAChR)– þ KACh channel signaling is not involved in the emodin-induced accentuation of ANP release in cardiac atria. TPN, tertiapin-Q; SL, atrial myocyte sarcolemma;(-) inhibition.
***
-20
* -40
Fig. 8. Effects of emodin (30 μM) and acetylcholine (ACh, 0.3 μM) on ANP concentration (A), stroke volume (B), and pulse pressure (C) in the atria from Sham- and pertussis toxin (PTX)-treated rats. Values are means 7SE. nP o 0.05, nn P o0.01, nnnPo 0.001 vs. corresponding values of Sham (vehicle). Number of experiments; Sham group, n ¼8: PTX-treated group, n¼ 8.
concentration and atrial dynamics (Fig. 7). To further define the role of inhibitory G-protein αi/o, experiments were performed in the atria from rabbits treated with PTX. PTX treatment had no significant effect on the emodin-induced increase in ANP concentration (Fig. 8). Emodin-induced decrease in atrial dynamics was rather slightly accentuated in the atria treated with PTX. As expected, both the ACh-induced increase in ANP concentration and decrease in atrial dynamics were attenuated in atria from rabbits treated with PTX. Emodin is known to activate AMP-activated protein kinase (AMPK) signaling (Tzeng et al., 2012; Song et al., 2013). To identify an involvement of AMPK signaling in the emodin-induced accentuation of ANP secretion, experiments were performed in the presence of Compound C, an inhibitor of AMPK signaling. Pretreatment with Compound C (20 μM) had no significant effect on the emodin-induced accentuation of ANP secretion and inhibition of atrial dynamics (for ANP secretion in Δ% changes, 181.82 7 52.71%, n ¼4, vs. 97.14 718.76% in control, n¼ 4, not significant; for pulse pressure in Δ% changes, 41.25 72.39%, n¼ 4, vs. 41.04 7 7.65% in control, n¼ 4, not significant).
The present study shows for the first time that emodin accentuates ANP secretion in a concentration-dependent manner in isolated beating rabbit atria. The accentuation of the ANP secretion by emodin was accompanied by a decrease in atrial pulse pressure and stroke volume. The emodin-induced changes in ANP secretion and atrial dynamics returned back to baseline levels by replacing buffer solution without the agent. The emodininduced changes in ANP secretion and atrial dynamics were significantly attenuated by an inhibition of K þ channels with þ TEA and Glib. These findings indicate that an activation of KATP channel is involved in the emodin-induced increase in ANP þ secretion. KATP channel is well expressed in the cardiac myocytes þ (Noma, 1983). Previously, it was shown that KATP channel is involved in the regulation of ANP secretion (Xu et al., 1996; Kim et al., 1997; Ogawa et al., 2009). Kim et al. (1997) showed that þ activation of KATP channel is positively involved in the regulation of ANP secretion. The present finding is consistent with this report. Recently, Xu et al. (2008) showed that activation of M2 mAChR increased ANP secretion via activation of inhibitory G-protein αi/o– þ KACh channel signaling. Because emodin possesses cholinergic properties (Ali et al., 2004; Lu et al., 2007; Lenta et al., 2008; Xu et al., 2012) and M2 mAChR is involved in the regulation of ANP secretion (Xu et al., 2008; Kim et al., 2013), emodin was expected to accentuate ANP secretion via activation of cardiac muscarinic signaling pathway. But it was not the case. Not only the M2 mAChR þ but also inhibitory G-protein αi/o and KACh channels was not involved in the emodin-induced increase in ANP secretion. The þ present findings suggest that K þ channels, KATP channel in particular, are involved in the emodin-induced accentuation of ANP secretion. Ca2 þ entry via activation of L-type Ca2 þ channel is known to inhibit ANP secretion in the atria (Ito et al., 1988; de Bold and de Bold, 1989; Ruskoaho et al., 1990; Wen et al., 2000). Intracellular Ca2 þ tonically inhibits ANP secretion in beating rabbit atria (Wen et al., 2000). The present study shows that emodin-induced
G.H. Zhou et al. / European Journal of Pharmacology 735 (2014) 44–51
increase in ANP secretion was attenuated by inhibition of L-type Ca2 þ channel which suggests that Ca2 þ entry is involved. Previously, it was shown that Ca2 þ entry through L-type Ca2 þ þ þ channel is a prerequisite for the KACh channel activation- and KATP channel activation-induced accentuation of ANP secretion (Kim et al., 1997; Xu et al., 2008). Taken together, we propose that emodin accentuates ANP secretion via inhibition of Ca2 þ entry through L-type Ca2 þ channel by shortening of the action potential caused þ by KATP channel activation (Fig. 9). Alternatively, emodin may increase ANP secretion via inhibition of L-type Ca2 þ channel. It has been shown that emodin has two-way regulation on the intracellular Ca2 þ concentration and L-type Ca2 þ channel activity in cardiomyocytes: an increase at low concentration and inhibition at higher concentration (Liu et al., 2004). Emodin is known to induce an activation of AMPK signaling (Tzeng et al., 2012; Song et al., 2013). However, in the present study, it was shown that AMPK signaling is not involved in the emodin-induced accentuation of ANP secretion. In summary, emodin accentuates ANP secretion via activation þ of KATP channels in cardiac atria. Emodin in rhubarb, Rheum officinale Baill, that has long been used in traditional Chinese herbal medicine, is known to possess cardiovascular protective properties (Huang et al., 1991; Du and Ko, 2005; Meng et al., 2010; Chen et al., 2012; Tzeng et al., 2012). Considering the physiological and pathophysiological roles of ANP, it is expected that effects of emodin may closely be related with the accentuation of ANP secretion from the atria. In a series of preliminary experiments, emodin increased plasma levels of ANP concomitantly with an accentuation of atrial ANP gene expression.
Acknowledgements This work was supported by research grants from the National Natural Science Foundation of China (Nos. 30971080, 81270920), the Natural Science Foundation of Shandong Province (No. ZR2011HM022), and the National Research Foundation of Korea (NRF) funded by the Korea government (MSIP) (2008-0062484).
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