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Ursolic acid increases the secretion of atrial natriuretic peptide in isolated perfused beating rabbit atria
European Journal of Pharmacology 653 (2011) 63–69
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Ursolic acid increases the secretion of atrial natriuretic peptide in isolated perfused beating rabbit atria Hao Zhen Cui a,b, Jin Fu Wen c, Hye Ran Choi a, Xiang Li a,b, Kyung Woo Cho a, Dae Gill Kang a,b,⁎, Ho Sub Lee a,b,⁎ a b c
Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, 570-749, Republic of Korea Hanbang Body-fluid Research Center(HBRC), Wonkwang University, Iksan, Jeonbuk, 570-749, Republic of Korea Department of Physiology, Institute of Atherosclerosis, Taishan Medical University, 2 East Yingsheng Road, Taian, Shandong 271000, China
a r t i c l e
i n f o
Article history: Received 14 July 2010 Received in revised form 19 October 2010 Accepted 31 October 2010 Available online 29 November 2010 Keywords: Atrial natriuretic peptide Atrium K+ATP channel Na+–K+-ATPase Ursolic acid
a b s t r a c t Ursolic acid is reported to have beneficial effects on the regulation of cardiovascular homeostasis. However, the effects of ursolic acid on cardiac hormone secretion are yet to be defined. The present study was designed to test the effects of ursolic acid on the secretory and contractile functions of the atria. Experiments were conducted in isolated perfused beating rabbit atria. We measured the changes in atrial dynamics, pulse pressure, stroke volume, cAMP efflux, as well as the secretion of atrial natriuretic peptide (ANP). Ursolic acid increased ANP secretion and mechanical dynamics in a concentration-dependent manner. The inhibition of Ltype Ca2+ channels with nifedipine attenuated the ursolic acid-induced increase in ANP secretion but not mechanical dynamics. The inhibition of K+ATP channels with glibenclamide attenuated the ursolic acidinduced increase in ANP secretion—but not atrial dynamics—in a concentration-dependent manner. The selective Na+–K+-ATPase inhibitor ouabain blocked the ursolic acid-induced increase in atrial dynamics but not ANP secretion. These findings show that ursolic acid increases ANP secretion via its activation of K+ATP channels and subsequent inhibition of Ca2+ entry through L-type Ca2+ channels in rabbit atria. These data also suggest that ursolic acid increases atrial dynamics via its inhibition of Na+–K+-ATPase activity. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ursolic acid and its isomer, oleanolic acid, are triterpenoid compounds that are found in various plants, edible vegetables, and many traditional medicinal herbs (Liu, 1995). Ursolic acid and oleanolic acid are known to have anti-inflammatory, anti-proliferatory, antianalgesic, anti-hyperlipidemic, and hepatoprotective effects (Liu, 1995; Dzubak et al., 2006). Ursolic acid is also known to have beneficial effects in the regulation of cardiovascular homeostasis. Ursolic acid also elicits cardioprotection in ischemia-induced cardiac dysfunction (Senthil et al., 2007). Furthermore, ursolic acid decreases arterial blood pressure and increases cardiac contractility (Somova et al., 2004). Olive oil, which contains oleanolic acid and ursolic acid, is known to have beneficial effects on the cardiovascular system (Psaltopoulou et al., 2004). However, the mechanisms by which ursolic acid elicits cardioprotection are not completely understood. The cardiac atrium is an endocrine organ that secretes cardiac hormones. These hormones are members of a family of natriuretic peptides that includes atrial natriuretic peptide (ANP), and brain and C⁎ Corresponding authors. Department of Physiology, College of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea. Lee is to be contacted at Tel.: + 82 63 850 6841. Kang, Tel.: + 82 63 850 6933. E-mail addresses: [email protected] (D.G. Kang), [email protected] (H.S. Lee). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.10.098
type natriuretic peptide (De Bold, 1985; Forssmann et al., 1998). ANP is involved in the regulation of blood pressure and the homeostasis of fluid volume. In addition to possessing cardiovascular effects, ANP is involved in lipid homeostasis via hormone-sensitive lipase in human adipocytes (Sengenes et al., 2003). The plasma levels of ANP are controlled by the balance between its secretion from cardiac atria and its metabolism, cellular uptake and degradation. 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). The role of changes in the intracellular levels of Ca2+, cAMP, and cGMP for the regulation of ANP secretion is a subject of interest. Some authors report a decrease in ANP secretion by increasing Ca2+ entry via L-type Ca2+ channels (De Bold and De Bold, 1989; Ruskoaho et al., 1990; Wen et al., 2000), whereas others report an increase (Schiebinger et al., 1994). K+ATP channels also modulate ANP secretion from atria (Xu et al., 1996; Kim et al., 1997). The effects of ursolic acid on ANP secretion have yet to be clarified. Oleanolic acid (an isomer of ursolic acid) activates K+ATP channels (Maia et al., 2006). K+ATP channels are well expressed in cardiac atria (Flagg et al., 2008) and are involved in the regulation of ANP secretion (Xu et al., 1996; Kim et al., 1997). Therefore, we hypothesized that ursolic acid activates ANP secretion. The purpose of the present study was to clarify the effects of ursolic acid on ANP secretion and its related mechanisms in isolated perfused
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beating atria from rabbits. Our study is the first to show that ursolic acid increases ANP secretion via its activation of K+ATP channels.
The pericardial space of the organ chamber was open to the air to avoid the restriction of atrial dynamics. The atrium was immediately perfused with a HEPES buffer solution using a peristaltic pump (1 ml/ min). Changes in atrial stroke volume were 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.
2. Methods and materials The investigation was carried out with the approval of the Institutional Animal Care and Utilization Committee for Medical Science of Wonkwang University. 2.1. Preparations of isolated perfused beating atria
2.2. Experimental protocols
An isolated atrial preparation was set up using a previously described method (Cho et al., 1995). To maintain a homeostatic balance between the variable ion channels and the excitability of the atrial myocytes, the setup was electrically paced (1.3 Hz). In brief, rabbit hearts were rapidly removed and placed in warm oxygenated physiological (0.9%) saline. The left atrium was then dissected. A calibrated transparent atrial cannula containing 2 small catheters within it was inserted into the left atrium through the atrioventricular orifice. The outer tip of the atrial cannula was left open to allow outflow from the atrium. One of the 2 catheters located in the atrium was for inflow and the other was used to record pressure changes within the atrium itself. The cannulated atrium was then transferred to an organ chamber containing HEPES buffer at 36.5 °C.
Beating atria were perfused for 60 min to stabilize ANP secretion. The perfusate was collected for analyses at 2-min intervals at 4 °C. Experiments were carried out with a total of 16 groups of atria in order to answer the questions detailed below.
A
2.2.1. Does ursolic acid increase ANP secretion and mechanical dynamics? An initial control period of 12 min was followed by ursolic acid infusion (3 μM, group 1, n = 5; 10 μM, group 2, n = 5; 30 μM, group 3, n = 5; vehicle, group 4, n = 6) for 72 min. To evaluate the effects of ursolic acid, values (mean of 2 fractions) obtained before and 36 min after the addition were compared. Data are expressed as percentage differences from the control (before the addition of ursolic acid).
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Fig. 1. Effects of ursolic acid on secretory and contractile functions in isolated perfused beating rabbit atria. A, Time-matched controls for ANP secretion (a), pulse pressure (b), stroke volume (c), and cAMP efflux (d). B, Effects of ursolic acid (UA, 30 μM) on ANP secretion (a), pulse pressure (b), stroke volume (c), and cAMP efflux (d). C, Summary data showing the effects of ursolic acid on ANP secretion (a), pulse pressure (b), stroke volume (c), and cAMP efflux (d). n = 5–6 experiments per treatment. The mean values of 2 fractions before (fraction numbers 5 and 6) and after 36 min of infusion of ursolic acid (fraction numbers 23 and 24) were compared. A control (Cont) period of 12 min was followed by infusion with ursolic acid or vehicle. *P b 0.05, **P b 0.01, ***P b 0.001 vs. control; #P b 0.05, ##P b 0.001 vs. ursolic cid (10 μM).
H.Z. Cui et al. / European Journal of Pharmacology 653 (2011) 63–69
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Fig. 2. Effects of the inhibition of L-type Ca2+ channels on the ursolic acid-induced increases in secretory and contractile functions in perfused beating atria. A, Effects of ursolic acid (UA, 30 μM) on ANP secretion (a), pulse pressure (b), and stroke volume (c). B, Effects of nifedipine (1 μM) on the ursolic acid-induced increases in ANP secretion (a), pulse pressure (b), and stroke volume (c). n = 7–8 experiments per treatment. The mean values of two fractions before (fraction numbers 23 and 24) and after infusion of ursolic acid were compared. A control (Cont) period of 12 min was followed by infusion with vehicle or nifedipine and then ursolic acid in the presence of vehicle or nifedipine. *P b 0.01, **P b 0.001 vs. control; #P b 0.05, ##P b 0.001 vs. values before ursolic acid infusion.
2.2.2. Are the effects of ursolic acid related to the activity of L-type Ca2+ channels? Nifedipine (1 μM) was applied to identify the role of Ca2+ entry via Ltype Ca2+ channels in ursolic acid-induced changes in secretory and contractile function. Thirty-six minutes of infusion with nifedipine or vehicle was followed by 36 min of ursolic acid (30 μM) in the continuous presence of nifedipine or vehicle (vehicle + ursolic acid, group 5, n = 8; nifedipine+ ursolic acid, group 6, n = 7; nifedipine+ vehicle, group 7, n = 8). Values obtained before and after ursolic acid infusion were compared. Data are expressed as percentage differences from the control (before the addition of ursolic acid). 2.2.3. Are the effects of ursolic acid related to K+ATP channel activity? The effects of glibenclamide—a K+ATP channel inhibitor—were tested to identify the role of K+ATP channels in ursolic acid-induced changes in secretory and contractile functions. Thirty-six minutes of infusion with glibenclamide was followed by 36 min of infusion with ursolic acid (30 μM) or vehicle in the continuous presence of the previous agent (glibenclamide 3 μM + ursolic acid, group 8, n = 6; glibenclamide 10 μM + ursolic acid, group 9, n = 7; glibenclamide 10 μM + vehicle, group 10, n = 3; glibenclamide 30 μM + ursolic acid, group 11, n = 6; glibenclamide 30 μM + vehicle, group 12, n = 3).
ouabain + vehicle, group 14, n = 6). In another series of experiments, the effects of β1- and β2-adrenoceptor antagonists on ursolic acidinduced changes were tested (CGP 0.3 μM + ursolic acid, group 15, n = 5–6; ICI 0.1 μM + ursolic acid, group 16, n = 5). 2.3. Radioimmunoassay of ANP The levels of immunoreactive ANP in the perfusates were measured with a specific radioimmunoassay as described previously (Cho et al., 1995; Cui et al., 2009). The amount of ANP secreted is expressed as nanograms of ANP per minute per gram of atrial tissue. 2.4. Radioimmunoassay of cAMP cAMP was measured using an equilibrated radioimmunoassay (Cui et al., 2002; Wen et al., 2004). The level of cAMP efflux was expressed as pmol of cAMP per minute per gram of atrial tissue. The level of cAMP efflux is linearly correlated with the atrial tissue content of cAMP (Wen et al., 2004). Non-specific binding was b2.0%. The intraand inter-assay coefficients of variation were 5.0% (n = 10) and 9.6% (n = 10) respectively. 2.5. Drugs
2.2.4. Are the effects of ursolic acid related to the activity of Na+–K+-ATPase? The effects of ouabain—a selective Na+–K+-ATPase inhibitor— were tested to identify the role of Na+–K+-ATPase in ursolic acidinduced changes in secretory and contractile functions. Thirty-six minutes of infusion with ouabain was followed by 36 min of infusion with ursolic acid (30 μM) or vehicle in the continuous presence of the previous agent (ouabain 0.3 μM + ursolic acid, group 13, n = 8;
Ursolic acid, nifedipine and glibenclamide (Sigma-Aldrich, Yongin, Korea) were first dissolved in dimethyl sulfoxide (DMSO): the final concentration of DMSO was b0.1%. CGP 20712A, ICI 118551, and ouabain (Sigma-Aldrich) were dissolved in distilled water. The concentrations of inhibitors for receptors, Ca2+ channels, K+ATP channels, and Na+–K+ATPase were within previously used ranges (Xiao and Lakatta, 1993; Kim et al., 1997; Cui et al., 2002; Dostanic et al., 2003).
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Δ% ANP scretion
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3.2. Blockage of L-type Ca2+ channels with nifedipine attenuated ursolic acid-induced increases in ANP secretion
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L-type Ca2+ channels were inhibited by nifedipine in order to identify the mechanisms by which ursolic acid increases ANP secretion and atrial dynamics. The baseline levels of ANP secretion and atrial dynamics were stable. Ursolic acid increased ANP secretion and pulse pressure (Fig. 2A). Nifedipine (1 μM) increased ANP secretion and concomitantly decreased atrial dynamics (Fig. 2B). The nifedipine-induced increase in ANP secretion and decrease in atrial dynamics were maintained during the presence of the agent. Nifedipine attenuated the ursolic acid-induced increase in ANP secretion (Figs 2Ba and 3A). However, nifedipine had no significant effect on the ursolic acid-induced increase in atrial dynamics (Figs. 2Bb, Bc, 3B, and C). These findings indicate that intact Ca2+ entry via L-type Ca2+ channels is involved in the ursolic acid-induced increase in ANP secretion but not atrial dynamics.
Δ% stroke volume
60 40
3.3. Blockage of K+ATP channels attenuates ursolic acid-induced increases in ANP secretion
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Fig. 3. Summary data showing the effects of nifedipine and ouabain on the ursolic acidinduced increases in secretory and contractile functions in perfused beating atria. Effects of nifedipine (1 μM) and ouabain (0.3 μM) on the ursolic acid (UA, 30 μM)induced increases in ANP secretion (A), pulse pressure (B), and stroke volume (C). n = 6–8 experiments per treatment. Control, data from Fig. 1A; ursolic acid, data from Fig. 2A; nifedipine + ursolic acid, data from Fig. 2B; ouabain + ursolic acid, data from Fig. 6B. The mean values of two fractions before (fraction numbers 23 and 24) and after 36 min of infusion of ursolic acid (fraction numbers 41 and 42) were compared. *P b 0.001 vs. control; +P b 0.001 vs. ursolic acid.
Blockage of L-type Ca2+ channels modulates ursolic acid-induced increases in ANP secretion. Because ursolic acid is thought to activate K+ATP channels (Maia et al. 2006), this defines the role of K+ATP channels as endogenous modulators of L-type Ca2+ channels. In the next series of experiments, the effects of glibenclamide were tested. Glibenclamide (30 μM) had no significant effect on the baseline levels of ANP secretion (Fig. 4Ba). However, glibenclamide increased atrial dynamics slightly but significantly up to ~36 min, and the effect was maintained during the presence of the agent (Fig. 4, Bb and Bc). Glibenclamide attenuated the ursolic acid-induced increase in ANP secretion in a concentration-dependent manner (Figs 4Aa, Ba and 5A; data for Fig. 4A from Fig. 2A). The greatest concentration of glibenclamide (30 μM) completely blocked the ursolic acid-induced increase in ANP secretion. However, the treatment had no effect on the ursolic acidinduced increase in atrial dynamics (Figs 4Ab, Ac, Bb, Bc, 5B, and C). These findings indicate that K+ATP channels are involved in the ursolic acid-induced increase in ANP secretion but not in atrial dynamics.
3.4. Inhibition of Na+–K+-ATPase with ouabain attenuates ursolic acid-induced increase in atrial dynamics 2.6. Statistical analyses Statistical significance was determined using repeated measures ANOVA followed by Bonferroni's multiple comparison test (Figs. 1A, B, 2, 4, and 6). Student's t-test for unpaired data (see Figs. 1C, 3, and 5) was also performed. P values less than 0.05 were considered significant. Data are expressed as mean ± S.E.M. 3. Results 3.1. Ursolic acid increases ANP secretion and mechanical dynamics The baseline levels of ANP secretion, atrial dynamics, pulse pressure, stroke volume, and cAMP efflux were stable during the experimental period in perfused beating atria (Fig. 1A). Ursolic acid (30 μM) increased ANP secretion, atrial pulse pressure, and stroke volume (Fig. 1B). The ursolic acid-induced increase in ANP secretion and atrial dynamics were maintained continuously up to 36 min and declined slightly thereafter during the latter part of the experiment. Ursolic acid had no significant effect on cAMP efflux. The effects of ursolic acid on the secretory and contractile functions of the cardiac atrium were concentration-dependent (Fig. 1C).
To identify the mechanisms involved in the ursolic acid-induced increase in atrial dynamics, the role of Na+–K+-ATPase was clarified. Ouabain increased atrial dynamics continuously during the presence of the agent (Figs 3B, 6Bb, and Bc) but had no effect on the baseline levels of ANP secretion (Fig. 6Ba). Ouabain attenuated the ursolic acidinduced increase in atrial dynamics (Figs 3B, C, 6Ab, Ac, Bb, and Bc) but had no effect on the ursolic acid-induced increase in ANP secretion (Figs 3A, 6Aa, and Ba). These findings indicate that Na+–K+-ATPase is involved in the ursolic acid-induced increase in atrial dynamics but not in ANP secretion. The effects of β-adrenoceptor antagonists were tested to identify their involvement in ursolic acid-induced changes in secretory and contractile function. β1-adrenoceptor inhibition with CGP 20712A (0.3 μM) had no effect on the ursolic acid-induced increase in ANP secretion (168.80 ± 12.69 vs. 170.35 ± 16.85) and pulse pressure (19.6 ± 1.2 vs. 25.0 ± 1.8) (n = 5–8 experiments per treatment). Similarly, β2-adrenoceptor inhibition with ICI 118551 (0.1 μM) had no significant effects on ANP secretion (168.36±7.76 vs. 170.35 ± 16.85) or pulse pressure (24.8 ±4.2 vs. 25.0 ±1.8) (n = 5–8 experiments per treatment). These findings indicate that β-adrenoceptors are not involved in the ursolic acid-induced increases in ANP secretion and atrial dynamics.
H.Z. Cui et al. / European Journal of Pharmacology 653 (2011) 63–69
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Fig. 4. Effects of the inhibition of K+ATP channels on the ursolic acid-induced increases in secretory and contractile functions in perfused beating atria. A, Effects of ursolic acid (UA, 30 μM) on ANP secretion (a), pulse pressure (b), and stroke volume (c). Data from Fig. 2A. B, Effects of glibenclamide (Glib, 30 μM) on the ursolic acid-induced increases in ANP secretion (a), pulse pressure (b), and stroke volume (c). n = 6–8 experiments per treatment. **P b 0.001 vs. control; #P b 0.05, ##P b 0.01, ###P b 0.001 vs. values before ursolic acid infusion.
4. Discussion
Fig. 5. Summary data showing the effects of glibenclamide on the ursolic acid-induced increases in secretory and contractile functions in isolated perfused beating rabbit atria. Effects of glibenclamide (G) on the ursolic acid (UA)-induced increase in ANP secretion (A), pulse pressure (B), and stroke volume (C). n=6–8 experiments per treatment. C, control, data from Fig. 1A; G0, 30 μM ursolic acid, data from Fig. 2A; G30 (30 μM glibenclamide)+ursolic acid, data from Fig. 4B; G30 +vehicle, data from G10 + vehicle, n=3, and G30 +vehicle, n=3, were pooled. *Pb 0.001 vs. Control; +Pb 0.01, ++Pb 0.001 vs. ursolic acid.
The present study shows for the first time that ursolic acid increases ANP secretion in isolated perfused beating rabbit atria. It also shows that ursolic acid increases atrial dynamics, pulse pressure, and stroke volume. The ursolic acid-induced increase in ANP secretion (but not mechanical dynamics) was attenuated by K+ATP channel blockage with glibenclamide. These findings suggest that ursolic acid increases ANP secretion via its activation of K+ATP channels. Activation of K+ATP channels increases ANP secretion (Kim et al., 1997). In addition, oleanolic acid (an isomer of ursolic acid) reportedly activates K+ATP channels (Maia et al., 2006). Maia et al. observed that glibenclamide blocks the antinociception produced by oleanolic acid (Maia et al., 2006). An inhibitor of sarcolemmal K+ATP channels attenuates the ursolic acid-induced increase in ANP secretion. Furthermore, the ursolic acid-induced increase in ANP secretion was blocked by the inhibition of L-type Ca2+ channels. The present study and previous reports showed that Ca2+ entry through L-type Ca2+ channels tonically inhibits ANP secretion (Fig. 7; Wen et al., 2000). Wen et al. showed that the activation of L-type Ca2+ channels decreases ANP secretion, whereas the inhibition of these channels increases secretion. In the present study, the blockage of the ursolic acid-induced increase in ANP secretion by nifedipine suggests that intact Ca2+ entry through L-type Ca2+ channels is a prerequisite for the ursolic acid-induced increase in ANP secretion. Ursolic acid may activate sarcolemmal K+ATP channels; this causes hyperpolarization that inhibits Ca2+ entry through L-type Ca2+ channels. This relieves the Ca2+ inhibition of ANP secretion, thereby leading to an increase in ANP secretion (Fig. 7). The activation of K+ATP channels shortens the duration of the action potential and decreases Ca2+ entry through L-type Ca2+ channels (Tamargo et al., 2004). If ursolic acid is a secretagogue for ANP as shown in the present study, the result showing a decrease in arterial blood pressure by ursolic acid (Somova et al., 2004) is explainable. Consequently, an increase in the plasma levels of ANP is expected to decrease blood pressure.
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Fig. 6. Effects of the inhibition of Na+–K+-ATPase on the ursolic acid-induced increases in secretory and contractile functions in isolated perfused beating rabbit atria. A, Effects of ursolic acid (UA, 30 μM) on ANP secretion (a), pulse pressure (b), and stroke volume (c). Data from Fig. 2A. B, Effects of ouabain (0.3 μM) on the ursolic acid-induced increases in ANP secretion (a), pulse pressure (b), and stroke volume (c). n = 8 experiments per treatment. *P b 0.001 vs. control; ##P b 0.001 vs. values before ursolic acid infusion.
The present study shows that ursolic acid has a positive inotropic effect. The ursolic acid-induced increase in atrial dynamics was blocked by a low concentration of ouabain but not by glibenclamide or nifedipine. Ouabain is a well-known selective inhibitor of Na+–K+ATPase and positive inotropic agent. Na+–K+-ATPase inhibition increases subsarcolemmal Na+ concentration and activates the reverse mode of Na+–Ca2+ exchangers, which is involved in the positive inotropic effect of ouabain (Leblanc and Hume, 1990). Therefore, this finding suggests that Na+–K+-ATPase is involved in the ursolic acid-induced positive inotropic effect. Both ursolic acid and oleanolic acid inhibit Na+–K+-ATPase (Yan et al., 2010; Chen et al., 2009); the present study corroborates this. Selective β1- or β2-adrenoceptor inhibitors did not affect the ursolic acid-induced increase in atrial dynamics. These findings suggest that β1- and β2-adrenoceptors are not involved in the ursolic acid-induced positive inotropic effect. Similarly, ursolic acid had no
effect on atrial cAMP efflux levels. Ursolic acid has a cardiotonic effect in anesthetized rats (Somova et al., 2004); the present study is in accordance with this report. Ursolic acid is known to have cardioprotective effects. Ursolic acid protects the rat myocardium against ischemic insult because of its antioxidant properties (Senthil et al., 2007). ANP is known to protect against ischemia/reperfusion injury and oxidative stress via cGMP and other signaling pathways in the cardiovascular system (Sangawa et al., 2004; De Vito et al., 2010). Further, ANP elicits anti-hypertrophic and anti-fibrotic effects via natriuretic peptide receptor-A-cGMP signaling in the heart (Nishikimi et al., 2006). ANP may possess intracardiac antirenin–angiotensin–aldosterone pathway properties; ANP inhibits aldosterone synthase gene expression in cultured neonatal rat cardiocytes (Ito et al., 2003). The ursolic acid-induced accentuation of ANP secretion may have beneficial effects on the regulation of the cardiovascular homeostasis. 5. Conclusions The present study shows that ursolic acid increases ANP secretion via its activation of K+ATP channels and subsequent inhibition of Ca2+ entry through L-type Ca2+ channels. It also suggests that ursolic acid increases atrial mechanical dynamics via Na+–K+-ATPase inhibition. It is, therefore, proposed that ursolic acid obtained by diets such as olive oil may have beneficial effects on the regulation of cardiovascular homeostasis through activation of cardiac hormone ANP secretion. Acknowledgments
Fig. 7. Schematic mechanism for the accentuation of ANP secretion by ursolic acid (UA) in isolated perfused beating rabbit atria. ursolic acid may activate sarcolemmal K+ATP channels, and the resulting hyperpolarization inhibits the entry of Ca2+ through L-type Ca2+ channels. This relieves the Ca2+ inhibition of ANP secretion thereby leading to an increase in ANP secretion. Glib, glibenclamide; Nife, nifedipine; LTCC, L-type Ca2+ channel; , activation; ⊢, inhibition; ⊖, inhibition.
This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea and funded by the Ministry of Education, Science, and Technology (MEST) (No. 2010-0029467) and grants from the National Natural Science Foundation of China (No. 30971080).
H.Z. Cui et al. / European Journal of Pharmacology 653 (2011) 63–69
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Nishikimi, T., Maeda, N., Matsuoka, H., 2006. The role of natriuretic peptides in cardioprotection. Cardiovasc. Res. 69, 318–328. Psaltopoulou, T., Naska, A., Orfanos, P., Trichopoulos, D., Mountokalakis, T., Trichopoulou, A., 2004. Olive oil, the Mediterranean diet, and arterial blood pressure: the Greek European perspective investigation into cancer and nutrition (EPIC) study. Am. J. Clin. Nutr. 80, 1012–1018. Ruskoaho, H., 1992. Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacol. Rev. 44, 479–601. 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. Schiebinger, R.J., Li, Y., Cragoe Jr., E.J., 1994. Calcium dependency of frequencystimulated atrial natriuretic peptide secretion. Hypertension 23, 710–716. Sengenes, C., Bouloumie, A., Hauner, H., Berlan, M., Busse, R., Lafontan, M., 2003. Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes. J. Biol. Chem. 278, 48617–48626. Senthil, S., Chandramohan, G., Pugalendi, K.V., 2007. Isomers (oleanolic and ursolic acids) differ in their protective effect against isoproterenol-induced myocardial ischemia in rats. Int. J. Cardiol. 119, 131–133. Somova, L.I., Shode, F.O., Mipando, M., 2004. Cardiotonic and antidysrhythmic effects of oleanolic and ursolic acids, methyl maslinate and uvaol. Phytomedicine 11, 121–129. Tamargo, J., Caballero, R., Gomez, R., Valenzuela, C., Delpon, E., 2004. Pharmacology of cardiac potassium channels. Cardiovasc. Res. 62, 9–33. Wen, J.F., Cui, X., Ahn, J.S., Kim, S.H., Seul, K.H., Kim, S.Z., Park, Y.K., Lee, H.S., Cho, K.W., 2000. Distinct roles for L- and T-type Ca2+ channels in regulation of atrial ANP release. Am. J. Physiol. 279, H2879–H2888. Wen, J.F., Cui, X., Jin, J.Y., Kim, S.M., Kim, S.Z., Kim, S.H., 2004. High and low gain switches for regulation of cAMP efflux concentration. Distinct roles for particulate GC- and soluble GC-cGMP-PDE3 signaling in rabbit atria. Circ. Res. 94, 936–943. Xiao, R.P., Lakatta, E.G., 1993. β1-Adrenoceptor stimulation and β2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca2+, and Ca2+ current in single rat ventricular cells. Circ. Res. 73, 286–300. Xu, T., Jiao, J.H., Pence, R.A., Baertschi, A.J., 1996. ATP-sensitive potassium channels regulate stimulated ANF secretion in isolated rat heart. Am. J. Physiol. 271, H2339–H2345. Xu, D.Y., Wen, J.F., Quan, H.X., Zhou, G.H., Kim, S.Y., Park, S.H., Kim, S.Z., Lee, H.S., Cho, K.W., 2008. K+ACh channel activation with carbachol increases atrial ANP secretion. Life Sci. 82, 1083–1091. Yan, S.-L., Huang, C.-Y., Wu, S.-T., Yin, M.-C., 2010. Oleanolic acid and ursolic acid induce apoptosis in four human liver cancer cell lines. Toxicol. In Vitro 24, 842–848.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Ursolic acid increases the secretion of atrial natriuretic peptide in isolated perfused beating rabbit atria Hao Zhen Cui a,b, Jin Fu Wen c, Hye Ran Choi a, Xiang Li a,b, Kyung Woo Cho a, Dae Gill Kang a,b,⁎, Ho Sub Lee a,b,⁎ a b c
Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, 570-749, Republic of Korea Hanbang Body-fluid Research Center(HBRC), Wonkwang University, Iksan, Jeonbuk, 570-749, Republic of Korea Department of Physiology, Institute of Atherosclerosis, Taishan Medical University, 2 East Yingsheng Road, Taian, Shandong 271000, China
a r t i c l e
i n f o
Article history: Received 14 July 2010 Received in revised form 19 October 2010 Accepted 31 October 2010 Available online 29 November 2010 Keywords: Atrial natriuretic peptide Atrium K+ATP channel Na+–K+-ATPase Ursolic acid
a b s t r a c t Ursolic acid is reported to have beneficial effects on the regulation of cardiovascular homeostasis. However, the effects of ursolic acid on cardiac hormone secretion are yet to be defined. The present study was designed to test the effects of ursolic acid on the secretory and contractile functions of the atria. Experiments were conducted in isolated perfused beating rabbit atria. We measured the changes in atrial dynamics, pulse pressure, stroke volume, cAMP efflux, as well as the secretion of atrial natriuretic peptide (ANP). Ursolic acid increased ANP secretion and mechanical dynamics in a concentration-dependent manner. The inhibition of Ltype Ca2+ channels with nifedipine attenuated the ursolic acid-induced increase in ANP secretion but not mechanical dynamics. The inhibition of K+ATP channels with glibenclamide attenuated the ursolic acidinduced increase in ANP secretion—but not atrial dynamics—in a concentration-dependent manner. The selective Na+–K+-ATPase inhibitor ouabain blocked the ursolic acid-induced increase in atrial dynamics but not ANP secretion. These findings show that ursolic acid increases ANP secretion via its activation of K+ATP channels and subsequent inhibition of Ca2+ entry through L-type Ca2+ channels in rabbit atria. These data also suggest that ursolic acid increases atrial dynamics via its inhibition of Na+–K+-ATPase activity. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ursolic acid and its isomer, oleanolic acid, are triterpenoid compounds that are found in various plants, edible vegetables, and many traditional medicinal herbs (Liu, 1995). Ursolic acid and oleanolic acid are known to have anti-inflammatory, anti-proliferatory, antianalgesic, anti-hyperlipidemic, and hepatoprotective effects (Liu, 1995; Dzubak et al., 2006). Ursolic acid is also known to have beneficial effects in the regulation of cardiovascular homeostasis. Ursolic acid also elicits cardioprotection in ischemia-induced cardiac dysfunction (Senthil et al., 2007). Furthermore, ursolic acid decreases arterial blood pressure and increases cardiac contractility (Somova et al., 2004). Olive oil, which contains oleanolic acid and ursolic acid, is known to have beneficial effects on the cardiovascular system (Psaltopoulou et al., 2004). However, the mechanisms by which ursolic acid elicits cardioprotection are not completely understood. The cardiac atrium is an endocrine organ that secretes cardiac hormones. These hormones are members of a family of natriuretic peptides that includes atrial natriuretic peptide (ANP), and brain and C⁎ Corresponding authors. Department of Physiology, College of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea. Lee is to be contacted at Tel.: + 82 63 850 6841. Kang, Tel.: + 82 63 850 6933. E-mail addresses: [email protected] (D.G. Kang), [email protected] (H.S. Lee). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.10.098
type natriuretic peptide (De Bold, 1985; Forssmann et al., 1998). ANP is involved in the regulation of blood pressure and the homeostasis of fluid volume. In addition to possessing cardiovascular effects, ANP is involved in lipid homeostasis via hormone-sensitive lipase in human adipocytes (Sengenes et al., 2003). The plasma levels of ANP are controlled by the balance between its secretion from cardiac atria and its metabolism, cellular uptake and degradation. 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). The role of changes in the intracellular levels of Ca2+, cAMP, and cGMP for the regulation of ANP secretion is a subject of interest. Some authors report a decrease in ANP secretion by increasing Ca2+ entry via L-type Ca2+ channels (De Bold and De Bold, 1989; Ruskoaho et al., 1990; Wen et al., 2000), whereas others report an increase (Schiebinger et al., 1994). K+ATP channels also modulate ANP secretion from atria (Xu et al., 1996; Kim et al., 1997). The effects of ursolic acid on ANP secretion have yet to be clarified. Oleanolic acid (an isomer of ursolic acid) activates K+ATP channels (Maia et al., 2006). K+ATP channels are well expressed in cardiac atria (Flagg et al., 2008) and are involved in the regulation of ANP secretion (Xu et al., 1996; Kim et al., 1997). Therefore, we hypothesized that ursolic acid activates ANP secretion. The purpose of the present study was to clarify the effects of ursolic acid on ANP secretion and its related mechanisms in isolated perfused
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beating atria from rabbits. Our study is the first to show that ursolic acid increases ANP secretion via its activation of K+ATP channels.
The pericardial space of the organ chamber was open to the air to avoid the restriction of atrial dynamics. The atrium was immediately perfused with a HEPES buffer solution using a peristaltic pump (1 ml/ min). Changes in atrial stroke volume were 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.
2. Methods and materials The investigation was carried out with the approval of the Institutional Animal Care and Utilization Committee for Medical Science of Wonkwang University. 2.1. Preparations of isolated perfused beating atria
2.2. Experimental protocols
An isolated atrial preparation was set up using a previously described method (Cho et al., 1995). To maintain a homeostatic balance between the variable ion channels and the excitability of the atrial myocytes, the setup was electrically paced (1.3 Hz). In brief, rabbit hearts were rapidly removed and placed in warm oxygenated physiological (0.9%) saline. The left atrium was then dissected. A calibrated transparent atrial cannula containing 2 small catheters within it was inserted into the left atrium through the atrioventricular orifice. The outer tip of the atrial cannula was left open to allow outflow from the atrium. One of the 2 catheters located in the atrium was for inflow and the other was used to record pressure changes within the atrium itself. The cannulated atrium was then transferred to an organ chamber containing HEPES buffer at 36.5 °C.
Beating atria were perfused for 60 min to stabilize ANP secretion. The perfusate was collected for analyses at 2-min intervals at 4 °C. Experiments were carried out with a total of 16 groups of atria in order to answer the questions detailed below.
A
2.2.1. Does ursolic acid increase ANP secretion and mechanical dynamics? An initial control period of 12 min was followed by ursolic acid infusion (3 μM, group 1, n = 5; 10 μM, group 2, n = 5; 30 μM, group 3, n = 5; vehicle, group 4, n = 6) for 72 min. To evaluate the effects of ursolic acid, values (mean of 2 fractions) obtained before and 36 min after the addition were compared. Data are expressed as percentage differences from the control (before the addition of ursolic acid).
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Fig. 1. Effects of ursolic acid on secretory and contractile functions in isolated perfused beating rabbit atria. A, Time-matched controls for ANP secretion (a), pulse pressure (b), stroke volume (c), and cAMP efflux (d). B, Effects of ursolic acid (UA, 30 μM) on ANP secretion (a), pulse pressure (b), stroke volume (c), and cAMP efflux (d). C, Summary data showing the effects of ursolic acid on ANP secretion (a), pulse pressure (b), stroke volume (c), and cAMP efflux (d). n = 5–6 experiments per treatment. The mean values of 2 fractions before (fraction numbers 5 and 6) and after 36 min of infusion of ursolic acid (fraction numbers 23 and 24) were compared. A control (Cont) period of 12 min was followed by infusion with ursolic acid or vehicle. *P b 0.05, **P b 0.01, ***P b 0.001 vs. control; #P b 0.05, ##P b 0.001 vs. ursolic cid (10 μM).
H.Z. Cui et al. / European Journal of Pharmacology 653 (2011) 63–69
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Fig. 2. Effects of the inhibition of L-type Ca2+ channels on the ursolic acid-induced increases in secretory and contractile functions in perfused beating atria. A, Effects of ursolic acid (UA, 30 μM) on ANP secretion (a), pulse pressure (b), and stroke volume (c). B, Effects of nifedipine (1 μM) on the ursolic acid-induced increases in ANP secretion (a), pulse pressure (b), and stroke volume (c). n = 7–8 experiments per treatment. The mean values of two fractions before (fraction numbers 23 and 24) and after infusion of ursolic acid were compared. A control (Cont) period of 12 min was followed by infusion with vehicle or nifedipine and then ursolic acid in the presence of vehicle or nifedipine. *P b 0.01, **P b 0.001 vs. control; #P b 0.05, ##P b 0.001 vs. values before ursolic acid infusion.
2.2.2. Are the effects of ursolic acid related to the activity of L-type Ca2+ channels? Nifedipine (1 μM) was applied to identify the role of Ca2+ entry via Ltype Ca2+ channels in ursolic acid-induced changes in secretory and contractile function. Thirty-six minutes of infusion with nifedipine or vehicle was followed by 36 min of ursolic acid (30 μM) in the continuous presence of nifedipine or vehicle (vehicle + ursolic acid, group 5, n = 8; nifedipine+ ursolic acid, group 6, n = 7; nifedipine+ vehicle, group 7, n = 8). Values obtained before and after ursolic acid infusion were compared. Data are expressed as percentage differences from the control (before the addition of ursolic acid). 2.2.3. Are the effects of ursolic acid related to K+ATP channel activity? The effects of glibenclamide—a K+ATP channel inhibitor—were tested to identify the role of K+ATP channels in ursolic acid-induced changes in secretory and contractile functions. Thirty-six minutes of infusion with glibenclamide was followed by 36 min of infusion with ursolic acid (30 μM) or vehicle in the continuous presence of the previous agent (glibenclamide 3 μM + ursolic acid, group 8, n = 6; glibenclamide 10 μM + ursolic acid, group 9, n = 7; glibenclamide 10 μM + vehicle, group 10, n = 3; glibenclamide 30 μM + ursolic acid, group 11, n = 6; glibenclamide 30 μM + vehicle, group 12, n = 3).
ouabain + vehicle, group 14, n = 6). In another series of experiments, the effects of β1- and β2-adrenoceptor antagonists on ursolic acidinduced changes were tested (CGP 0.3 μM + ursolic acid, group 15, n = 5–6; ICI 0.1 μM + ursolic acid, group 16, n = 5). 2.3. Radioimmunoassay of ANP The levels of immunoreactive ANP in the perfusates were measured with a specific radioimmunoassay as described previously (Cho et al., 1995; Cui et al., 2009). The amount of ANP secreted is expressed as nanograms of ANP per minute per gram of atrial tissue. 2.4. Radioimmunoassay of cAMP cAMP was measured using an equilibrated radioimmunoassay (Cui et al., 2002; Wen et al., 2004). The level of cAMP efflux was expressed as pmol of cAMP per minute per gram of atrial tissue. The level of cAMP efflux is linearly correlated with the atrial tissue content of cAMP (Wen et al., 2004). Non-specific binding was b2.0%. The intraand inter-assay coefficients of variation were 5.0% (n = 10) and 9.6% (n = 10) respectively. 2.5. Drugs
2.2.4. Are the effects of ursolic acid related to the activity of Na+–K+-ATPase? The effects of ouabain—a selective Na+–K+-ATPase inhibitor— were tested to identify the role of Na+–K+-ATPase in ursolic acidinduced changes in secretory and contractile functions. Thirty-six minutes of infusion with ouabain was followed by 36 min of infusion with ursolic acid (30 μM) or vehicle in the continuous presence of the previous agent (ouabain 0.3 μM + ursolic acid, group 13, n = 8;
Ursolic acid, nifedipine and glibenclamide (Sigma-Aldrich, Yongin, Korea) were first dissolved in dimethyl sulfoxide (DMSO): the final concentration of DMSO was b0.1%. CGP 20712A, ICI 118551, and ouabain (Sigma-Aldrich) were dissolved in distilled water. The concentrations of inhibitors for receptors, Ca2+ channels, K+ATP channels, and Na+–K+ATPase were within previously used ranges (Xiao and Lakatta, 1993; Kim et al., 1997; Cui et al., 2002; Dostanic et al., 2003).
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Δ% ANP scretion
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L-type Ca2+ channels were inhibited by nifedipine in order to identify the mechanisms by which ursolic acid increases ANP secretion and atrial dynamics. The baseline levels of ANP secretion and atrial dynamics were stable. Ursolic acid increased ANP secretion and pulse pressure (Fig. 2A). Nifedipine (1 μM) increased ANP secretion and concomitantly decreased atrial dynamics (Fig. 2B). The nifedipine-induced increase in ANP secretion and decrease in atrial dynamics were maintained during the presence of the agent. Nifedipine attenuated the ursolic acid-induced increase in ANP secretion (Figs 2Ba and 3A). However, nifedipine had no significant effect on the ursolic acid-induced increase in atrial dynamics (Figs. 2Bb, Bc, 3B, and C). These findings indicate that intact Ca2+ entry via L-type Ca2+ channels is involved in the ursolic acid-induced increase in ANP secretion but not atrial dynamics.
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3.3. Blockage of K+ATP channels attenuates ursolic acid-induced increases in ANP secretion
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Fig. 3. Summary data showing the effects of nifedipine and ouabain on the ursolic acidinduced increases in secretory and contractile functions in perfused beating atria. Effects of nifedipine (1 μM) and ouabain (0.3 μM) on the ursolic acid (UA, 30 μM)induced increases in ANP secretion (A), pulse pressure (B), and stroke volume (C). n = 6–8 experiments per treatment. Control, data from Fig. 1A; ursolic acid, data from Fig. 2A; nifedipine + ursolic acid, data from Fig. 2B; ouabain + ursolic acid, data from Fig. 6B. The mean values of two fractions before (fraction numbers 23 and 24) and after 36 min of infusion of ursolic acid (fraction numbers 41 and 42) were compared. *P b 0.001 vs. control; +P b 0.001 vs. ursolic acid.
Blockage of L-type Ca2+ channels modulates ursolic acid-induced increases in ANP secretion. Because ursolic acid is thought to activate K+ATP channels (Maia et al. 2006), this defines the role of K+ATP channels as endogenous modulators of L-type Ca2+ channels. In the next series of experiments, the effects of glibenclamide were tested. Glibenclamide (30 μM) had no significant effect on the baseline levels of ANP secretion (Fig. 4Ba). However, glibenclamide increased atrial dynamics slightly but significantly up to ~36 min, and the effect was maintained during the presence of the agent (Fig. 4, Bb and Bc). Glibenclamide attenuated the ursolic acid-induced increase in ANP secretion in a concentration-dependent manner (Figs 4Aa, Ba and 5A; data for Fig. 4A from Fig. 2A). The greatest concentration of glibenclamide (30 μM) completely blocked the ursolic acid-induced increase in ANP secretion. However, the treatment had no effect on the ursolic acidinduced increase in atrial dynamics (Figs 4Ab, Ac, Bb, Bc, 5B, and C). These findings indicate that K+ATP channels are involved in the ursolic acid-induced increase in ANP secretion but not in atrial dynamics.
3.4. Inhibition of Na+–K+-ATPase with ouabain attenuates ursolic acid-induced increase in atrial dynamics 2.6. Statistical analyses Statistical significance was determined using repeated measures ANOVA followed by Bonferroni's multiple comparison test (Figs. 1A, B, 2, 4, and 6). Student's t-test for unpaired data (see Figs. 1C, 3, and 5) was also performed. P values less than 0.05 were considered significant. Data are expressed as mean ± S.E.M. 3. Results 3.1. Ursolic acid increases ANP secretion and mechanical dynamics The baseline levels of ANP secretion, atrial dynamics, pulse pressure, stroke volume, and cAMP efflux were stable during the experimental period in perfused beating atria (Fig. 1A). Ursolic acid (30 μM) increased ANP secretion, atrial pulse pressure, and stroke volume (Fig. 1B). The ursolic acid-induced increase in ANP secretion and atrial dynamics were maintained continuously up to 36 min and declined slightly thereafter during the latter part of the experiment. Ursolic acid had no significant effect on cAMP efflux. The effects of ursolic acid on the secretory and contractile functions of the cardiac atrium were concentration-dependent (Fig. 1C).
To identify the mechanisms involved in the ursolic acid-induced increase in atrial dynamics, the role of Na+–K+-ATPase was clarified. Ouabain increased atrial dynamics continuously during the presence of the agent (Figs 3B, 6Bb, and Bc) but had no effect on the baseline levels of ANP secretion (Fig. 6Ba). Ouabain attenuated the ursolic acidinduced increase in atrial dynamics (Figs 3B, C, 6Ab, Ac, Bb, and Bc) but had no effect on the ursolic acid-induced increase in ANP secretion (Figs 3A, 6Aa, and Ba). These findings indicate that Na+–K+-ATPase is involved in the ursolic acid-induced increase in atrial dynamics but not in ANP secretion. The effects of β-adrenoceptor antagonists were tested to identify their involvement in ursolic acid-induced changes in secretory and contractile function. β1-adrenoceptor inhibition with CGP 20712A (0.3 μM) had no effect on the ursolic acid-induced increase in ANP secretion (168.80 ± 12.69 vs. 170.35 ± 16.85) and pulse pressure (19.6 ± 1.2 vs. 25.0 ± 1.8) (n = 5–8 experiments per treatment). Similarly, β2-adrenoceptor inhibition with ICI 118551 (0.1 μM) had no significant effects on ANP secretion (168.36±7.76 vs. 170.35 ± 16.85) or pulse pressure (24.8 ±4.2 vs. 25.0 ±1.8) (n = 5–8 experiments per treatment). These findings indicate that β-adrenoceptors are not involved in the ursolic acid-induced increases in ANP secretion and atrial dynamics.
H.Z. Cui et al. / European Journal of Pharmacology 653 (2011) 63–69
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Fig. 4. Effects of the inhibition of K+ATP channels on the ursolic acid-induced increases in secretory and contractile functions in perfused beating atria. A, Effects of ursolic acid (UA, 30 μM) on ANP secretion (a), pulse pressure (b), and stroke volume (c). Data from Fig. 2A. B, Effects of glibenclamide (Glib, 30 μM) on the ursolic acid-induced increases in ANP secretion (a), pulse pressure (b), and stroke volume (c). n = 6–8 experiments per treatment. **P b 0.001 vs. control; #P b 0.05, ##P b 0.01, ###P b 0.001 vs. values before ursolic acid infusion.
4. Discussion
Fig. 5. Summary data showing the effects of glibenclamide on the ursolic acid-induced increases in secretory and contractile functions in isolated perfused beating rabbit atria. Effects of glibenclamide (G) on the ursolic acid (UA)-induced increase in ANP secretion (A), pulse pressure (B), and stroke volume (C). n=6–8 experiments per treatment. C, control, data from Fig. 1A; G0, 30 μM ursolic acid, data from Fig. 2A; G30 (30 μM glibenclamide)+ursolic acid, data from Fig. 4B; G30 +vehicle, data from G10 + vehicle, n=3, and G30 +vehicle, n=3, were pooled. *Pb 0.001 vs. Control; +Pb 0.01, ++Pb 0.001 vs. ursolic acid.
The present study shows for the first time that ursolic acid increases ANP secretion in isolated perfused beating rabbit atria. It also shows that ursolic acid increases atrial dynamics, pulse pressure, and stroke volume. The ursolic acid-induced increase in ANP secretion (but not mechanical dynamics) was attenuated by K+ATP channel blockage with glibenclamide. These findings suggest that ursolic acid increases ANP secretion via its activation of K+ATP channels. Activation of K+ATP channels increases ANP secretion (Kim et al., 1997). In addition, oleanolic acid (an isomer of ursolic acid) reportedly activates K+ATP channels (Maia et al., 2006). Maia et al. observed that glibenclamide blocks the antinociception produced by oleanolic acid (Maia et al., 2006). An inhibitor of sarcolemmal K+ATP channels attenuates the ursolic acid-induced increase in ANP secretion. Furthermore, the ursolic acid-induced increase in ANP secretion was blocked by the inhibition of L-type Ca2+ channels. The present study and previous reports showed that Ca2+ entry through L-type Ca2+ channels tonically inhibits ANP secretion (Fig. 7; Wen et al., 2000). Wen et al. showed that the activation of L-type Ca2+ channels decreases ANP secretion, whereas the inhibition of these channels increases secretion. In the present study, the blockage of the ursolic acid-induced increase in ANP secretion by nifedipine suggests that intact Ca2+ entry through L-type Ca2+ channels is a prerequisite for the ursolic acid-induced increase in ANP secretion. Ursolic acid may activate sarcolemmal K+ATP channels; this causes hyperpolarization that inhibits Ca2+ entry through L-type Ca2+ channels. This relieves the Ca2+ inhibition of ANP secretion, thereby leading to an increase in ANP secretion (Fig. 7). The activation of K+ATP channels shortens the duration of the action potential and decreases Ca2+ entry through L-type Ca2+ channels (Tamargo et al., 2004). If ursolic acid is a secretagogue for ANP as shown in the present study, the result showing a decrease in arterial blood pressure by ursolic acid (Somova et al., 2004) is explainable. Consequently, an increase in the plasma levels of ANP is expected to decrease blood pressure.
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A
B
Ouabain (0.3 μM) Cont Ouabain + UA (30 μM)
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ANP secretion (ng/min/g)
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0 0
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36
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42
0
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42
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Fig. 6. Effects of the inhibition of Na+–K+-ATPase on the ursolic acid-induced increases in secretory and contractile functions in isolated perfused beating rabbit atria. A, Effects of ursolic acid (UA, 30 μM) on ANP secretion (a), pulse pressure (b), and stroke volume (c). Data from Fig. 2A. B, Effects of ouabain (0.3 μM) on the ursolic acid-induced increases in ANP secretion (a), pulse pressure (b), and stroke volume (c). n = 8 experiments per treatment. *P b 0.001 vs. control; ##P b 0.001 vs. values before ursolic acid infusion.
The present study shows that ursolic acid has a positive inotropic effect. The ursolic acid-induced increase in atrial dynamics was blocked by a low concentration of ouabain but not by glibenclamide or nifedipine. Ouabain is a well-known selective inhibitor of Na+–K+ATPase and positive inotropic agent. Na+–K+-ATPase inhibition increases subsarcolemmal Na+ concentration and activates the reverse mode of Na+–Ca2+ exchangers, which is involved in the positive inotropic effect of ouabain (Leblanc and Hume, 1990). Therefore, this finding suggests that Na+–K+-ATPase is involved in the ursolic acid-induced positive inotropic effect. Both ursolic acid and oleanolic acid inhibit Na+–K+-ATPase (Yan et al., 2010; Chen et al., 2009); the present study corroborates this. Selective β1- or β2-adrenoceptor inhibitors did not affect the ursolic acid-induced increase in atrial dynamics. These findings suggest that β1- and β2-adrenoceptors are not involved in the ursolic acid-induced positive inotropic effect. Similarly, ursolic acid had no
effect on atrial cAMP efflux levels. Ursolic acid has a cardiotonic effect in anesthetized rats (Somova et al., 2004); the present study is in accordance with this report. Ursolic acid is known to have cardioprotective effects. Ursolic acid protects the rat myocardium against ischemic insult because of its antioxidant properties (Senthil et al., 2007). ANP is known to protect against ischemia/reperfusion injury and oxidative stress via cGMP and other signaling pathways in the cardiovascular system (Sangawa et al., 2004; De Vito et al., 2010). Further, ANP elicits anti-hypertrophic and anti-fibrotic effects via natriuretic peptide receptor-A-cGMP signaling in the heart (Nishikimi et al., 2006). ANP may possess intracardiac antirenin–angiotensin–aldosterone pathway properties; ANP inhibits aldosterone synthase gene expression in cultured neonatal rat cardiocytes (Ito et al., 2003). The ursolic acid-induced accentuation of ANP secretion may have beneficial effects on the regulation of the cardiovascular homeostasis. 5. Conclusions The present study shows that ursolic acid increases ANP secretion via its activation of K+ATP channels and subsequent inhibition of Ca2+ entry through L-type Ca2+ channels. It also suggests that ursolic acid increases atrial mechanical dynamics via Na+–K+-ATPase inhibition. It is, therefore, proposed that ursolic acid obtained by diets such as olive oil may have beneficial effects on the regulation of cardiovascular homeostasis through activation of cardiac hormone ANP secretion. Acknowledgments
Fig. 7. Schematic mechanism for the accentuation of ANP secretion by ursolic acid (UA) in isolated perfused beating rabbit atria. ursolic acid may activate sarcolemmal K+ATP channels, and the resulting hyperpolarization inhibits the entry of Ca2+ through L-type Ca2+ channels. This relieves the Ca2+ inhibition of ANP secretion thereby leading to an increase in ANP secretion. Glib, glibenclamide; Nife, nifedipine; LTCC, L-type Ca2+ channel; , activation; ⊢, inhibition; ⊖, inhibition.
This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea and funded by the Ministry of Education, Science, and Technology (MEST) (No. 2010-0029467) and grants from the National Natural Science Foundation of China (No. 30971080).
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