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ETB receptor-mediated pathway in beating rabbit atria
Life Sciences 90 (2012) 793–798
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Ouabain stimulates atrial natriuretic peptide secretion via the endothelin-1/ETB receptor-mediated pathway in beating rabbit atria Li-ping Liu a, Lan Hong a, Li Yu a, Hai-yan Li a, Da-zhi Ding c, Shan-ji Jin c, Xun Cui a, b,⁎ a b c
Department of Physiology, School of Basic Medical sciences, Yanbian University, Yanji 133-002, China Key Laboratory of Organism Functional Factors of the Changbai Mountain, Ministry of Education, Yanbian University, Yanji, 133002, China Institute of Clinical Medicine, Yanbian University, Yanji 133-000, China
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Article history: Received 24 December 2011 Accepted 3 April 2012 Keywords: Atrial natriuretic peptide Endothelin-1 Endothelin-1 receptor L-type Ca2+ channel Na+–Ca2+ exchanger
a b s t r a c t Aims: Ouabain has been reported to increase the secretion of atrial natriuretic peptide (ANP) in vitro. However, the mechanism by which ouabain increases ANP secretion is not well known. Therefore, the purpose of the present study was to investigate the underlying mechanism of ouabain-stimulated ANP secretion. Main methods: A perfused beating rabbit atrial model was used. The ANP and ET-1 levels in the atrial perfusates were measured by radioimmunoassays. Key findings: Ouabain (1.0, 3.0 and 6.0 μmol/L) significantly increased atrial ANP secretion in a dose-dependent manner, while the endothelin (ET)-1 levels were increased by the higher doses (3.0 and 6.0 μmol/L) of ouabain. Ouabain-increased atrial ET-1 release was blocked by PD98059 (30.0 μmol/L), an inhibitor of mitogen-activated protein kinase (MAPK). Nifedipine (1.0 μmol/L), an inhibitor of L-type Ca2+ channels, completely abolished ouabain-increased ANP secretion without changing the ouabain-induced atrial dynamics. KB-R7943 (3.0 μmol/L), an inhibitor of Na+–Ca2+ exchangers, completely blocked the effects of ouabain-increased atrial dynamics, but did not modulate ouabain-increased ANP secretion. ET-1 significantly stimulated atrial ANP release in a dosedependent manner. The effects of ET-1 and ouabain on ANP secretion were completely blocked by BQ788 (0.3 μmol/L), an inhibitor of ET-1 type B (ETB) receptors, but not by BQ123 (0.3 μM), an inhibitor of ET-1 type A receptors. Ouabain-increased atrial ANP secretion was blocked by PD98059 and indomethacin (30.0 μmol/L), an inhibitor of cyclooxygenase. Significance: Ouabain significantly stimulated atrial ANP secretion via an ET-1-ETB receptor-mediated pathway involving MAPK signaling pathway activation and prostaglandin formation. © 2012 Elsevier Inc. All rights reserved.
Introduction The cardiac atrium synthesizes and secretes atrial natriuretic peptide (ANP) in myocytes (De Bold, 1985). As a cardiac hormone, ANP plays important roles in modulating blood pressure and cardiovascular homeostasis. Many factors, such as mechanical stretching of the atrial wall, hypoxia and endothelin (ET)-1, are closely related to the regulation of atrial ANP secretion. Among these factors, mechanical stretching of the atrial wall is considered to be the predominant stimulus for ANP secretion. Ouabain, as an inhibitor of Na +–K +/ATPase, increases ANP secretion (Bloch et al., 1988; Morise et al., 1991), and calcium was reported to be an important second messenger in the regulation of ANP secretion (Schiebinger and Cragoe, 1993). However, there have been conflicting reports regarding the role of intracellular Ca 2+ in the regulation of ANP secretion (Peters et al., 2006; Wen et al., 2000; Zhang et al., ⁎ Corresponding author at: Department of Physiology, School of Basic Medical Sciences, Yanbian University, Gongyuan Road 977, Yanji Jilin 133-002, China. Tel.: +86 433 243 5132; fax: +86 433 243 5104. E-mail address: [email protected] (X. Cui). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2012.04.008
2008), and the mechanism underlying ouabain-stimulated ANP secretion therefore remains to be defined. One of the most potent stimuli for ANP secretion is the endothelial cell-derived peptide ET-1. ET-1 is the predominant ET isopeptide in the heart (Plumpton et al., 1996), and both ET-1 type A (ETA) and type B (ETB) receptor mRNAs are found in the atrial and ventricular myocardium, the conducting system and endocardial cells as well as in coronary arteries (Awane-Igata et al., 1997; Molenaar et al., 1993). Furthermore, Lew and Baertschi (1992) demonstrated that ET-1 enhances ANP secretion in cocultured cardiac myocytes and endothelial cells. Fukuda et al. (1989) showed that ET-1 increases ANP secretion and upregulates ANP mRNA in isolated rat cardiac myocytes. Skvorak et al. (1995) demonstrated that ET-1 augments the ANP secretory response to mechanical stretching in isolated perfused rat atria. Owing to the effects of ouabain as well as ET-1 on ANP secretion and the potential paracrine nature of ET-1, the hypothesis of the present study was that the effect of ouabain-stimulated ANP secretion is correlated with atrial ET-1 and its regulatory response for ANP secretion. Therefore, the present study was designed to investigate the effects and mechanisms of ouabain on the atrial ET-1 levels and ANP secretion. In addition, the regulatory effects of ET-1
L. Liu et al. / Life Sciences 90 (2012) 793–798
Materials and methods Preparation of perfused beating rabbit atria New Zealand white rabbits of either sex were used and the mean wet weight of the atria was 2.50 ± 0.08 g. Isolated perfused beating left atria were prepared using previously described methods (Cho et al., 1993). Soon after setting up each perfused atrium, transmural electrical field stimulation with a luminal electrode was started at 1.5 Hz (0.3 ms, 30–40 V) and the atrium was perfused with HEPES buffer solution using a peristaltic pump (1 ml/min) that allowed atrial pacing for measurements of the changes in atrial volume (stroke volume), pulse pressure, ANP and ET-1 secretion. The HEPES buffer contained (in mmol/L) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 10.0 glucose and 10.0 HEPES (pH 7.4 with NaOH) as well as 0.1% bovine serum albumin. Experimental protocols Each atrium was perfused for 60 min to stabilize the parameters of ANP secretion and atrial dynamics. The perfusates were collected at 2-min intervals at 4 °C for measurements of the ANP and ET-1 levels. The control cycle (12 min as an experimental cycle) was followed by infusion of 1.0, 3.0 or 6.0 μmol/L ouabain for one cycle and recovery for two cycles by infusion of HEPES buffer, and the levels of ANP and ET-1 in the perfusates were determined by radioimmunoassays. In other experiments, the control cycle was followed by infusion of different doses of ET-1 (0.003, 0.03 or 0.3 μmol/L) to observe the effects of ET-1 on atrial ANP secretion (n = 6). The effects were evaluated by comparing values from a control period with those from the second cycle of the experimental period. To investigate the mechanisms of ouabain-promoted ET-1 production and ANP secretion, another series of experiments was performed (Table 1). After one control period, one treatment agent cycle was followed by one cycle of infusion of the treatment agents plus ouabain (3.0 μmol/L) and then continued for two cycles of recovery in the presence of the prior agents. The treatment agents used in the present study were as follows: 1) KB-R7943 (3.0 μmol/L), an Na +–Ca 2+ exchanger inhibitor; 2) nifedipine (3.0 μmol/L; n = 6), an L-type Ca 2+ channel inhibitor; 3) BQ123 (0.3 μmol/L), an ETA receptor inhibitor; 4) BQ788 (0.3 μmol/L), an ETB receptor inhibitor; 5) PD98059 (30.0 μmol/L), a mitogen-activated protein kinase (MAPK) inhibitor; and 6) indomethacin (30.0 μmol/L), a cyclooxygenase antagonist.
Biological Technology, Beijing, China) after an extraction procedure on Sep-Pak C18 cartridges (Waters, Milford, MA). Intra-assay and interassay coefficients of variation were less than 10 and 15% respectively. The sensitivity of the assay is 0.5 pg/tube; cross-reactivity with ET-2 and ET-3 were less than 1%. All samples were assayed in the same run and at least in duplicate. The amount of ET-1 in perfusate was expressed as pg/min/g of wet atrial tissue. Statistical analysis The significance of differences between values was determined by one-way ANOVA followed by Dunnett's multiple comparison test (Figs. 1B and C, 2, 3, 6–9). An unpaired t-test was also applied to data (Figs. 1A, 4, 5). Statistical significance was defined as values of P b 0.05. All of the data were represented as means ± SEM. Results Effects of KB-R7943 and nifedipine on ouabain-increased ANP secretion In the present study, ANP secretion were increased in a dosedependent and long-lasting manner during the period of recovery
A Changes of ANP secretion (fold)
receptor antagonists on the ouabain-stimulated atrial ANP secretion were also determined.
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Table 1 Scheme of the experimental protocol. Each rectangle indicates one 12-min cycle during the experimental period. After one control cycle, one treatment agent cycle is followed by one cycle of infusion of the treatment agents plus ouabain (3.0 μmol/L) and then continued for two cycles of recovery in the presence of the prior agents. Control
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The significance of shaded and checkered is to distinguish the control period with or without ouabain treatment underlying treated agent.
C Changes of stroke volume (fold)
The levels of immunoreactive ANP and ET-1 in the perfusates were measured by specific radioimmunoassays as described previously (Cho et al., 1993). The amounts of secreted immunoreactive ANP were expressed as ng/minute/g of wet atrial tissue. Most of the secreted ANP was processed ANP (Cho et al., 1993). The ET-1 levels of atrial perfusates were determined by competitive RIA (North Institute Of
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Fraction number Fig. 1. Effects of different doses of ouabain (1.0, 3.0 and 6.0 μmol/L) on ANP secretion (A, B) and atrial stroke volume (C). Data are means ± SEM (n = 6). **P b 0.01, ***P b 0.001 vs. control period.
L. Liu et al. / Life Sciences 90 (2012) 793–798
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Fraction number Fig. 2. Effects of the Na+–Ca2+ exchanger inhibitor KB-R7943 (3.0 μmol/L) on ouabaininduced ANP secretion (A) and atrial stroke volume (B). Data are means ± SEM (n = 6). **P b 0.01 vs. control period.
after infusion of different doses of ouabain (n = 6 for each groups; at 1.0 μmol/L of ouabain P b 0.01, at 3.0 and 6.0 μmol/L of ouabain P b 0.001 vs. control period respectively; Fig. 1A, B). An increase in the atrial stroke volume occurred at an early point during treatment with ouabain (n = 6; P b 0.001 vs. control; Fig. 1C) and continued for the recovery period (P b 0.01 vs. control; Fig. 1C) in the perfused
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To define the relationship between ET-1 and ouabain-induced ANP release, another series of experiments was performed in rabbit beating atria. As shown in Fig. 4, high doses of ouabain (3.0 and 6.0 μmol/L) significantly increased atrial ET-1 secretion (n= 6; P b 0.05 and P b 0.01 vs. control, respectively). Moreover, low concentrations of ET-1 significantly increased ANP secretion in a dose-dependent manner (n=6; Pb 0.001 vs. control, respectively; Fig. 5A) concomitantly with increase in atrial stroke volume (n =6; P b 0.05, Pb 0.01, Pb 0.001 vs control, respectively; Fig. 5B). In the presence of ET-1 receptor antagonists, ET-1-increased atrial ANP secretion was slightly attenuated by the ETA inhibitor BQ123 (n= 6; P > 0.05 vs. ET-1; Fig. 6A), but completely blocked by the ETB inhibitor BQ788 (n=6; P >0.05 vs. control and P b 0.001 vs. ET-1 period;
Changes of ANP secretion (fold)
Changes of stroke volume (fold)
beating rabbit atria. The Na +–Ca 2+ exchanger inhibitor KB-R7943 completely blocked the effect of ouabain-increased atrial dynamics during the recovery period (n= 6; P > 0.05 vs. control; Fig. 2B), but did not modulate ouabain-increased ANP secretion (n= 6; P b 0.01 vs. control; Fig. 2A). In contrast, the L-type Ca 2+ channel inhibitor nifedipine completely inhibited ouabain-increased ANP secretion (n = 6; P > 0.05 vs. control; Fig. 3A), but did not change the ouabain-increased atrial dynamics (n=6; Pb 0.001 vs. control; Fig. 3B).
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Fig. 4. Effects of different doses of ouabain (1.0, 3.0 and 6.0 μmol/L) on atrial ET-1 production in isolated perfused beating rabbit atria. Data are means ± SEM (n = 6). *P b 0.05, **P b 0.01 vs. control period.
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Fig. 5. Effects of ET-1 (3, 30, and 300 nmol/L) on atrial ANP secretion (A) and stroke volume (B). Data are means ± SEM (n = 6). *P b 0.05, **P b 0.01, and ***P b 0.001 vs. control period.
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Fraction number Fig. 6. Effects of the ETA receptor inhibitor BQ123 (0.3 μmol/L) and ETB receptor inhibitor BQ788 (0.3 μmol/L) on ET-1-induced ANP secretion. Data are means± SEM (n= 6). **P b 0.01, △△△P b 0.001 vs. control period.
Fig. 6B). In addition, ouabain-stimulated ANP secretion was completely inhibited in the presence of BQ788 alone (n=6; Pb 0.001 vs. ouabain; Fig. 7B) or together with BQ123 (n =6; Pb 0.001 vs. ouabain; Fig. 7C), but not in the presence of BQ123 alone (n=6; P>0.05 vs. ouabain; Fig. 7A). These data suggest that ouabain-increased atrial ANP secretion is promoted by ET-1, and that this mainly occurs via ETB receptors in the isolated perfused beating rabbit atria. Role of the MAPK pathway in ouabain-increased atrial ET-1 and ANP secretion To investigate the mechanism by which ouabain increased atrial ET-1 production and ANP secretion, another series of experiments was performed in isolated perfused beating rabbit atria with PD98059. On its own, PD98059 (30.0 μmol/L) did not modulate the atrial ET-1 production, ANP secretion or atrial stroke volume (n= 6; P > 0.05 vs. control cycle, respectively; Fig. 8A–C). In the presence of PD98059, ouabain-promoted ET-1 production as well as ANP secretion was completely abolished (n= 6; P > 0.05 vs. control cycle, respectively; Fig. 8A, B) and ouabain-increased atrial stroke volume was also blocked (n= 6; P > 0.05 vs. control cycle; Fig. 8C). These findings suggest that the MAPK signaling pathway is involved in the ouabain-induced atrial ET-1 production and ANP secretion. Effect of indomethacin on ouabain-increased ANP secretion To determine the role of prostaglandins in ouabain-increased ANP secretion, the effects of indomethacin on the ouabain-induced ANP secretion and atrial stroke volume were investigated. Indomethacin (30.0 μmol/L) alone slightly decreased atrial ANP secretion, but the difference did not reach statistical significance compared with the
Changes ofANP serection (fold)
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control cycle (n= 6; P > 0.05; Fig. 9A). In the presence of indomethacin plus ouabain (3.0 μmol/L), ANP secretion was slightly lower than that in the presence of indomethacin alone with no significant differences compared with the control period and indomethacin alone period (n= 6; P > 0.05, respectively; Fig. 9A). On the other hand, ouabainincreased ANP secretion in the recovery period was completely abolished by indomethacin (n= 6; P > 0.05 vs. control, indomethacin and/or plus ouabain cycle; Fig. 9A). The ouabain-increased atrial stroke volume was also blocked by indomethacin (n= 6; P > 0.05 vs. control, indomethacin and/or plus ouabain cycle; Fig. 9B). These findings indicate that prostaglandins are involved in the ouabain-induced atrial ANP secretion. Discussion The present study has demonstrated the long-lasting and potent effect of ouabain on the atrial ANP secretion. Ouabain-stimulated ANP secretion was correlated with the ET-1-regulated ANP secretion via ETB receptors. Na +–K +/ATPase is known to play important roles in the regulation of intracellular sodium and calcium, since binding of ouabain to Na +– K +/ATPase increased intracellular Ca2+ through Na+–Ca2+ exchangers
L. Liu et al. / Life Sciences 90 (2012) 793–798
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by altering the intracellular Na+ concentration in cardiac myocytes, and thereby regulating myocardial contractility (Lee, 1985; Levi et al., 1994). In contrast, early studies suggested that ouabain might activate L-type Ca2+ channels and stimulate Ca2+ release from the sarcoplasmic reticulum and/or endoplasmic reticulum (McGarry and Williams, 1993). The present study showed that ouabain-increased atrial dynamics was blocked by the Na+–Ca 2+ exchanger inhibitor KB-R7943, but not by the L-type Ca 2+ channel inhibitor nifedipine. Consequently, these findings indicate that ouabain increased the atrial dynamics via Ca 2+ influx through Na +–Ca 2+ exchangers, but not L-type Ca 2+ channels, consistent with previous studies (Schwartz et al., 1988; Eisner and Smith, 1992; Barry et al., 1985). Furthermore, the data showed that ouabain has a long-lasting effect on the atrial ANP secretion in period of the recovery after treatment with ouabain in a dose-dependent manner. The regulatory effect of ouabain on atrial ANP secretion was blocked by nifedipine concomitantly with an increase in stroke volume, and the KB-R7943 blocked the effect of ouabain-increased atrial stroke volume only but failed in the modulation of ouabain-increased ANP secretion. These results not only demonstrated that there was no significant correlation between ouabain-induced ANP secretion and the atrial hemodyamics because there was an increase in stroke volume even though there was no significant secretion of ANP by ouabain plus nifedipine, but also indicated
that ouabain stimulated ANP secretion via Ca2+ influx through L-type Ca2+ channels, but not Na+–Ca2+ exchangers. However, in our previous study the atrial ANP secretion was inhibited by Ca 2+ influx through L-type Ca 2+ channels (Wen et al., 2000; Zhang et al., 2008), suggesting that there was an unknown mechanism involved in ouabain-stimulated ANP secretion. Accordingly, on the basis of the paracrine nature of ET-1 and its effect on atrial ANP secretion, the present study investigated the relationship between ET-1 and ouabain-stimulated ANP secretion in perfused beating rabbit atria. In the present study, ouabain significantly increased the levels of atrial ET-1 and that exogenous ET-1 potently stimulated atrial ANP secretion in a dose-dependent manner. ET-1 is best known for its ability to induce ANP secretion, and the present findings are consistent with an earlier study (Lew and Baertschi, 1992). In addition, the ouabain-increased ET-1 levels and ANP secretion were completely blocked by the MAPK inhibitor PD98059, and the ouabainincreased atrial stroke volume was also inhibited by PD98059. These results suggest that ouabain enhanced atrial ET-1 release via activation of MAPK signaling pathway, and thus ouabain-induced ANP secretion was augmented by ET-1. Previous reports showed that ET-1 elicits the negative inotropic myocardial effect from ETB receptor activation (Konrad et al., 2005), and that this requires an intact endocardial endothelium and is mediated by nitric oxide and prostaglandins (Leite-Moreira and Brás-Silva, 2004). The present study observed that ET-1-activated atrial ANP secretion was completely blocked by BQ788, an ETB receptor inhibitor, while the ETA receptor inhibitor BQ123 only slightly attenuated the effect of ET-1-activated ANP secretion. The results of the present study differed with previous reports that ET-1 stimulated ANP secretion was mainly via ETA receptor activation (Irons et al., 1993; Thibault et al., 1994), but similarly to the report (Zhang et al., 2004) that non-selective ET receptors inhibitor bosentan, but not BQ-123, significantly attenuated hypoxia-induced ANP release which was partially modulated via interaction with endogenous ET. The reason of these controversial observations with ET receptor-mediated regulation of ANP secretion was not well known, perhaps correlated with different experimental model conditions, methods, and some others. Furthermore, ouabainstimulated ANP secretion was also inhibited by BQ788 as well as
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indomethacin, a cyclooxygenase inhibitor, but not by BQ123. However, the ouabain-increased atrial stroke volume was also inhibited by indomethacin, although the reason for this observation remains to be defined. The present data have therefore demonstrated that ET-1 augmented stretch-activated atrial ANP secretion by ETB receptor activation and that this effect was observed in the ouabain-stimulated ANP secretion processes. In conclusion, results of the present study indicate that activation of MAPK signaling pathway and ET-1 release are main mechanisms by which ouabain-increased ANP secretion in isolated beating rabbit atria and suggest that a substantial part of the ouabain-stimulated ANP secretion occurs via increased ET-1 production leading to the formation of prostaglandin. Conclusion Ouabain significantly stimulated atrial ANP secretion via an ET-1-ETB receptor-mediated pathway involving MAPK signaling pathway activation and prostaglandin formation. Conflict of interest statement None.
Acknowledgments The authors thank Su Xiang Li for expert technical assistance. This work was supported by the National Natural Science Foundation of China (No. 30860091). References Awane-Igata Y, Ikeda S, Watanabe T. Inhibitory effects of TAK-044 on endothelin induced vasoconstriction in various canine arteries and porcine coronary arteries: a comparison with selective ETA and ETB receptor antagonists. Br J Pharmacol 1997;120:516–22. Barry WH, Hasin Y, Smith TW. Sodium pump inhibition, enhanced calcium influx via sodium–calcium exchange, and positive inotropic response in cultured heart cells. Circ Res 1985;56:231–41. Bloch KD, Zamir N, Lichtstein D, Seidman CE, Seidman JG. Ouabain induces secretion of proatrial natriuretic factor by rat atrial cardiocytes. Am J Physiol Endocrinol Metab 1988;255:E383–7. Cho KW, Kim SH, Hwang YH, Seul KH. Extracellular fluid translocation in perfused rabbit atria: implication in control of atrial natriuretic peptide secretion. J Physiol (Lond) 1993;468:591–607.
De Bold AJ. Atrial natriuretic factor: a hormone produced by the heart. Science 1985;230: 767–70. Eisner DA, Smith TW. The Heart and Cardiovascular System. 2nd ed. Morgan Raven Press; 1992. p. 863–902. Fukuda Y, Hirata Y, Taketani S, Kojima T, Oikawa S, Nakazato H, et al. Endothelin stimulates accumulations of cellular atrial natriuretic peptide and its messenger RNA in rat cardiocytes. Biochem Biophys Res Commun 1989;164:1431–6. Irons CE, Murray SF, Glembotski CC. Identification of the receptor subtype responsible for endothelin-mediated protein kinase C activation and atrial natriuretic factor secretion from atrial myocytes. J Biol Chem 1993;268(31):23417–21. Konrad D, Oldner A, Wanecek M, Rudehill A, Weitzberg E, Biber B, et al. Positive inotropic and negative lusitropic effects of endothelin receptor agonism in vivo. Am J Physiol Heart Circ Physiol 2005;289:H1702–9. Lee CO. 200 years of digitalis: the emerging central role of the sodium ion in the control of cardiac force. Am J Physiol Cell Physiol 1985;249:C367–78. Leite-Moreira AF, Brás-Silva C. Inotropic effects of ETB receptor stimulation and their modulation by endocardial endothelium, NO, and prostaglandins. Am J Physiol Heart Circ Physiol 2004;287:H1194–9. Levi AJ, Boyett MR, Lee CO. The cellular actions of digitalis glycosides on the heart. Prog Biophys Mol Biol 1994;62:1-54. Lew RA, Baertschi AJ. Endothelium-dependent ANF secretion in vitro. Am J Physiol Heart Circ Physiol 1992;263:H1071–7. McGarry SJ, Williams AJ. Digoxin activates sarcoplasmic reticulum Ca2+-release channels: a possible role in cardiac inotropy. Br J Pharmacol 1993;108:1043–50. Molenaar P, O'Reilly G, Sharkey A, Kuc RE, Harding DP, Plumpton C, et al. Characterization and localization of endothelin receptor subtypes in the human atrioventricular conducting system and myocardium. Circ Res 1993;72:526–38. Morise T, Takeuchi Y, Okamoto S, Tadeda R. Stimulation of atrial natriuretic peptide secretion and syntheses by Na–K-ATPase inhibitors. Biochem Biophys Res Commun 1991;176:875–81. Peters CG, Miller DF, Giovannucci DR. Identification, localization and interaction of SNARE proteins in atrial cardiac myocytes. J Mol Cell Cardiol 2006;40:361–74. Plumpton C, Ferro CJ, Haynes WG, Webb DJ, Davenport AP. The increase in human plasma immunoreactive endothelin but not big endothelin-1 or its C-terminal fragment induced by systemic administration of the endothelin antagonist TAK-044. Br J Pharmacol 1996;119:311–4. Schiebinger RJ, Cragoe Jr EJ. Ouabain. A stimulator of atrial natriuretic peptide secretion and its mechanism. Circ Res 1993;72:1035–43. Schwartz A, Grupp G, Wallick E, Grupp IL, Ball Jr WJ. Role of the Na+, K+-ATPase in the cardiotonic action of cardiac glycosides. Prog Clin Biol Res 1988;268B:321–38. Skvorak JP, Nazian SJ, Dietz JR. Endothelin acts as a paracrine regulator of stretchinduced atrial natriuretic peptide release. Am J Physiol Regul Integr Comp Physiol 1995;269:R1093–8. Thibault G, Doubell AF, Garcia R, Larivière R, Schiffrin EL. Endothelin-stimulated secretion of natriuretic peptides by rat atrial myocytes is mediated by endothelin A receptors. Circ Res 1994;74(3):460–70. Wen JF, Cui X, Ahn JS, Kim SH, Seul KH, Kim SZ, et al. Distinct roles for L- and T-type Ca2+ channels in regulation of atrial ANP release. Am J Physiol Heart Circ Physiol 2000;279: H2879–88. Zhang Y, Oliver JR, Horowitz JD. The role of endothelin in mediating ischemia/hypoxiainduced atrial natriuretic peptide release. J Cardiovasc Pharmacol 2004;43(2): 227–33. Zhang Y, Liu L, Liang ZL, Li XL, Jin YZ, Cui X. cAMP produced by pituitary adenylate cyclase activating polypeptide 27 inhibits atrial natriuretic peptide secretion in rabbit beating atria. Clin Exp Pharmacol Physiol 2008;35:1233–7.
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Ouabain stimulates atrial natriuretic peptide secretion via the endothelin-1/ETB receptor-mediated pathway in beating rabbit atria Li-ping Liu a, Lan Hong a, Li Yu a, Hai-yan Li a, Da-zhi Ding c, Shan-ji Jin c, Xun Cui a, b,⁎ a b c
Department of Physiology, School of Basic Medical sciences, Yanbian University, Yanji 133-002, China Key Laboratory of Organism Functional Factors of the Changbai Mountain, Ministry of Education, Yanbian University, Yanji, 133002, China Institute of Clinical Medicine, Yanbian University, Yanji 133-000, China
a r t i c l e
i n f o
Article history: Received 24 December 2011 Accepted 3 April 2012 Keywords: Atrial natriuretic peptide Endothelin-1 Endothelin-1 receptor L-type Ca2+ channel Na+–Ca2+ exchanger
a b s t r a c t Aims: Ouabain has been reported to increase the secretion of atrial natriuretic peptide (ANP) in vitro. However, the mechanism by which ouabain increases ANP secretion is not well known. Therefore, the purpose of the present study was to investigate the underlying mechanism of ouabain-stimulated ANP secretion. Main methods: A perfused beating rabbit atrial model was used. The ANP and ET-1 levels in the atrial perfusates were measured by radioimmunoassays. Key findings: Ouabain (1.0, 3.0 and 6.0 μmol/L) significantly increased atrial ANP secretion in a dose-dependent manner, while the endothelin (ET)-1 levels were increased by the higher doses (3.0 and 6.0 μmol/L) of ouabain. Ouabain-increased atrial ET-1 release was blocked by PD98059 (30.0 μmol/L), an inhibitor of mitogen-activated protein kinase (MAPK). Nifedipine (1.0 μmol/L), an inhibitor of L-type Ca2+ channels, completely abolished ouabain-increased ANP secretion without changing the ouabain-induced atrial dynamics. KB-R7943 (3.0 μmol/L), an inhibitor of Na+–Ca2+ exchangers, completely blocked the effects of ouabain-increased atrial dynamics, but did not modulate ouabain-increased ANP secretion. ET-1 significantly stimulated atrial ANP release in a dosedependent manner. The effects of ET-1 and ouabain on ANP secretion were completely blocked by BQ788 (0.3 μmol/L), an inhibitor of ET-1 type B (ETB) receptors, but not by BQ123 (0.3 μM), an inhibitor of ET-1 type A receptors. Ouabain-increased atrial ANP secretion was blocked by PD98059 and indomethacin (30.0 μmol/L), an inhibitor of cyclooxygenase. Significance: Ouabain significantly stimulated atrial ANP secretion via an ET-1-ETB receptor-mediated pathway involving MAPK signaling pathway activation and prostaglandin formation. © 2012 Elsevier Inc. All rights reserved.
Introduction The cardiac atrium synthesizes and secretes atrial natriuretic peptide (ANP) in myocytes (De Bold, 1985). As a cardiac hormone, ANP plays important roles in modulating blood pressure and cardiovascular homeostasis. Many factors, such as mechanical stretching of the atrial wall, hypoxia and endothelin (ET)-1, are closely related to the regulation of atrial ANP secretion. Among these factors, mechanical stretching of the atrial wall is considered to be the predominant stimulus for ANP secretion. Ouabain, as an inhibitor of Na +–K +/ATPase, increases ANP secretion (Bloch et al., 1988; Morise et al., 1991), and calcium was reported to be an important second messenger in the regulation of ANP secretion (Schiebinger and Cragoe, 1993). However, there have been conflicting reports regarding the role of intracellular Ca 2+ in the regulation of ANP secretion (Peters et al., 2006; Wen et al., 2000; Zhang et al., ⁎ Corresponding author at: Department of Physiology, School of Basic Medical Sciences, Yanbian University, Gongyuan Road 977, Yanji Jilin 133-002, China. Tel.: +86 433 243 5132; fax: +86 433 243 5104. E-mail address: [email protected] (X. Cui). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2012.04.008
2008), and the mechanism underlying ouabain-stimulated ANP secretion therefore remains to be defined. One of the most potent stimuli for ANP secretion is the endothelial cell-derived peptide ET-1. ET-1 is the predominant ET isopeptide in the heart (Plumpton et al., 1996), and both ET-1 type A (ETA) and type B (ETB) receptor mRNAs are found in the atrial and ventricular myocardium, the conducting system and endocardial cells as well as in coronary arteries (Awane-Igata et al., 1997; Molenaar et al., 1993). Furthermore, Lew and Baertschi (1992) demonstrated that ET-1 enhances ANP secretion in cocultured cardiac myocytes and endothelial cells. Fukuda et al. (1989) showed that ET-1 increases ANP secretion and upregulates ANP mRNA in isolated rat cardiac myocytes. Skvorak et al. (1995) demonstrated that ET-1 augments the ANP secretory response to mechanical stretching in isolated perfused rat atria. Owing to the effects of ouabain as well as ET-1 on ANP secretion and the potential paracrine nature of ET-1, the hypothesis of the present study was that the effect of ouabain-stimulated ANP secretion is correlated with atrial ET-1 and its regulatory response for ANP secretion. Therefore, the present study was designed to investigate the effects and mechanisms of ouabain on the atrial ET-1 levels and ANP secretion. In addition, the regulatory effects of ET-1
L. Liu et al. / Life Sciences 90 (2012) 793–798
Materials and methods Preparation of perfused beating rabbit atria New Zealand white rabbits of either sex were used and the mean wet weight of the atria was 2.50 ± 0.08 g. Isolated perfused beating left atria were prepared using previously described methods (Cho et al., 1993). Soon after setting up each perfused atrium, transmural electrical field stimulation with a luminal electrode was started at 1.5 Hz (0.3 ms, 30–40 V) and the atrium was perfused with HEPES buffer solution using a peristaltic pump (1 ml/min) that allowed atrial pacing for measurements of the changes in atrial volume (stroke volume), pulse pressure, ANP and ET-1 secretion. The HEPES buffer contained (in mmol/L) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 10.0 glucose and 10.0 HEPES (pH 7.4 with NaOH) as well as 0.1% bovine serum albumin. Experimental protocols Each atrium was perfused for 60 min to stabilize the parameters of ANP secretion and atrial dynamics. The perfusates were collected at 2-min intervals at 4 °C for measurements of the ANP and ET-1 levels. The control cycle (12 min as an experimental cycle) was followed by infusion of 1.0, 3.0 or 6.0 μmol/L ouabain for one cycle and recovery for two cycles by infusion of HEPES buffer, and the levels of ANP and ET-1 in the perfusates were determined by radioimmunoassays. In other experiments, the control cycle was followed by infusion of different doses of ET-1 (0.003, 0.03 or 0.3 μmol/L) to observe the effects of ET-1 on atrial ANP secretion (n = 6). The effects were evaluated by comparing values from a control period with those from the second cycle of the experimental period. To investigate the mechanisms of ouabain-promoted ET-1 production and ANP secretion, another series of experiments was performed (Table 1). After one control period, one treatment agent cycle was followed by one cycle of infusion of the treatment agents plus ouabain (3.0 μmol/L) and then continued for two cycles of recovery in the presence of the prior agents. The treatment agents used in the present study were as follows: 1) KB-R7943 (3.0 μmol/L), an Na +–Ca 2+ exchanger inhibitor; 2) nifedipine (3.0 μmol/L; n = 6), an L-type Ca 2+ channel inhibitor; 3) BQ123 (0.3 μmol/L), an ETA receptor inhibitor; 4) BQ788 (0.3 μmol/L), an ETB receptor inhibitor; 5) PD98059 (30.0 μmol/L), a mitogen-activated protein kinase (MAPK) inhibitor; and 6) indomethacin (30.0 μmol/L), a cyclooxygenase antagonist.
Biological Technology, Beijing, China) after an extraction procedure on Sep-Pak C18 cartridges (Waters, Milford, MA). Intra-assay and interassay coefficients of variation were less than 10 and 15% respectively. The sensitivity of the assay is 0.5 pg/tube; cross-reactivity with ET-2 and ET-3 were less than 1%. All samples were assayed in the same run and at least in duplicate. The amount of ET-1 in perfusate was expressed as pg/min/g of wet atrial tissue. Statistical analysis The significance of differences between values was determined by one-way ANOVA followed by Dunnett's multiple comparison test (Figs. 1B and C, 2, 3, 6–9). An unpaired t-test was also applied to data (Figs. 1A, 4, 5). Statistical significance was defined as values of P b 0.05. All of the data were represented as means ± SEM. Results Effects of KB-R7943 and nifedipine on ouabain-increased ANP secretion In the present study, ANP secretion were increased in a dosedependent and long-lasting manner during the period of recovery
A Changes of ANP secretion (fold)
receptor antagonists on the ouabain-stimulated atrial ANP secretion were also determined.
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Table 1 Scheme of the experimental protocol. Each rectangle indicates one 12-min cycle during the experimental period. After one control cycle, one treatment agent cycle is followed by one cycle of infusion of the treatment agents plus ouabain (3.0 μmol/L) and then continued for two cycles of recovery in the presence of the prior agents. Control
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The levels of immunoreactive ANP and ET-1 in the perfusates were measured by specific radioimmunoassays as described previously (Cho et al., 1993). The amounts of secreted immunoreactive ANP were expressed as ng/minute/g of wet atrial tissue. Most of the secreted ANP was processed ANP (Cho et al., 1993). The ET-1 levels of atrial perfusates were determined by competitive RIA (North Institute Of
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after infusion of different doses of ouabain (n = 6 for each groups; at 1.0 μmol/L of ouabain P b 0.01, at 3.0 and 6.0 μmol/L of ouabain P b 0.001 vs. control period respectively; Fig. 1A, B). An increase in the atrial stroke volume occurred at an early point during treatment with ouabain (n = 6; P b 0.001 vs. control; Fig. 1C) and continued for the recovery period (P b 0.01 vs. control; Fig. 1C) in the perfused
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To define the relationship between ET-1 and ouabain-induced ANP release, another series of experiments was performed in rabbit beating atria. As shown in Fig. 4, high doses of ouabain (3.0 and 6.0 μmol/L) significantly increased atrial ET-1 secretion (n= 6; P b 0.05 and P b 0.01 vs. control, respectively). Moreover, low concentrations of ET-1 significantly increased ANP secretion in a dose-dependent manner (n=6; Pb 0.001 vs. control, respectively; Fig. 5A) concomitantly with increase in atrial stroke volume (n =6; P b 0.05, Pb 0.01, Pb 0.001 vs control, respectively; Fig. 5B). In the presence of ET-1 receptor antagonists, ET-1-increased atrial ANP secretion was slightly attenuated by the ETA inhibitor BQ123 (n= 6; P > 0.05 vs. ET-1; Fig. 6A), but completely blocked by the ETB inhibitor BQ788 (n=6; P >0.05 vs. control and P b 0.001 vs. ET-1 period;
Changes of ANP secretion (fold)
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beating rabbit atria. The Na +–Ca 2+ exchanger inhibitor KB-R7943 completely blocked the effect of ouabain-increased atrial dynamics during the recovery period (n= 6; P > 0.05 vs. control; Fig. 2B), but did not modulate ouabain-increased ANP secretion (n= 6; P b 0.01 vs. control; Fig. 2A). In contrast, the L-type Ca 2+ channel inhibitor nifedipine completely inhibited ouabain-increased ANP secretion (n = 6; P > 0.05 vs. control; Fig. 3A), but did not change the ouabain-increased atrial dynamics (n=6; Pb 0.001 vs. control; Fig. 3B).
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Fig. 4. Effects of different doses of ouabain (1.0, 3.0 and 6.0 μmol/L) on atrial ET-1 production in isolated perfused beating rabbit atria. Data are means ± SEM (n = 6). *P b 0.05, **P b 0.01 vs. control period.
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Fig. 5. Effects of ET-1 (3, 30, and 300 nmol/L) on atrial ANP secretion (A) and stroke volume (B). Data are means ± SEM (n = 6). *P b 0.05, **P b 0.01, and ***P b 0.001 vs. control period.
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Fig. 6B). In addition, ouabain-stimulated ANP secretion was completely inhibited in the presence of BQ788 alone (n=6; Pb 0.001 vs. ouabain; Fig. 7B) or together with BQ123 (n =6; Pb 0.001 vs. ouabain; Fig. 7C), but not in the presence of BQ123 alone (n=6; P>0.05 vs. ouabain; Fig. 7A). These data suggest that ouabain-increased atrial ANP secretion is promoted by ET-1, and that this mainly occurs via ETB receptors in the isolated perfused beating rabbit atria. Role of the MAPK pathway in ouabain-increased atrial ET-1 and ANP secretion To investigate the mechanism by which ouabain increased atrial ET-1 production and ANP secretion, another series of experiments was performed in isolated perfused beating rabbit atria with PD98059. On its own, PD98059 (30.0 μmol/L) did not modulate the atrial ET-1 production, ANP secretion or atrial stroke volume (n= 6; P > 0.05 vs. control cycle, respectively; Fig. 8A–C). In the presence of PD98059, ouabain-promoted ET-1 production as well as ANP secretion was completely abolished (n= 6; P > 0.05 vs. control cycle, respectively; Fig. 8A, B) and ouabain-increased atrial stroke volume was also blocked (n= 6; P > 0.05 vs. control cycle; Fig. 8C). These findings suggest that the MAPK signaling pathway is involved in the ouabain-induced atrial ET-1 production and ANP secretion. Effect of indomethacin on ouabain-increased ANP secretion To determine the role of prostaglandins in ouabain-increased ANP secretion, the effects of indomethacin on the ouabain-induced ANP secretion and atrial stroke volume were investigated. Indomethacin (30.0 μmol/L) alone slightly decreased atrial ANP secretion, but the difference did not reach statistical significance compared with the
Changes ofANP serection (fold)
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control cycle (n= 6; P > 0.05; Fig. 9A). In the presence of indomethacin plus ouabain (3.0 μmol/L), ANP secretion was slightly lower than that in the presence of indomethacin alone with no significant differences compared with the control period and indomethacin alone period (n= 6; P > 0.05, respectively; Fig. 9A). On the other hand, ouabainincreased ANP secretion in the recovery period was completely abolished by indomethacin (n= 6; P > 0.05 vs. control, indomethacin and/or plus ouabain cycle; Fig. 9A). The ouabain-increased atrial stroke volume was also blocked by indomethacin (n= 6; P > 0.05 vs. control, indomethacin and/or plus ouabain cycle; Fig. 9B). These findings indicate that prostaglandins are involved in the ouabain-induced atrial ANP secretion. Discussion The present study has demonstrated the long-lasting and potent effect of ouabain on the atrial ANP secretion. Ouabain-stimulated ANP secretion was correlated with the ET-1-regulated ANP secretion via ETB receptors. Na +–K +/ATPase is known to play important roles in the regulation of intracellular sodium and calcium, since binding of ouabain to Na +– K +/ATPase increased intracellular Ca2+ through Na+–Ca2+ exchangers
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Fraction number Fig. 8. Effects of the MAPK inhibitor PD98059 (30.0 μmol/L) on ouabain-induced atrial ET-1 production (A), ANP secretion (B) and stroke volume (C). Data are means ± SEM (n = 6).
by altering the intracellular Na+ concentration in cardiac myocytes, and thereby regulating myocardial contractility (Lee, 1985; Levi et al., 1994). In contrast, early studies suggested that ouabain might activate L-type Ca2+ channels and stimulate Ca2+ release from the sarcoplasmic reticulum and/or endoplasmic reticulum (McGarry and Williams, 1993). The present study showed that ouabain-increased atrial dynamics was blocked by the Na+–Ca 2+ exchanger inhibitor KB-R7943, but not by the L-type Ca 2+ channel inhibitor nifedipine. Consequently, these findings indicate that ouabain increased the atrial dynamics via Ca 2+ influx through Na +–Ca 2+ exchangers, but not L-type Ca 2+ channels, consistent with previous studies (Schwartz et al., 1988; Eisner and Smith, 1992; Barry et al., 1985). Furthermore, the data showed that ouabain has a long-lasting effect on the atrial ANP secretion in period of the recovery after treatment with ouabain in a dose-dependent manner. The regulatory effect of ouabain on atrial ANP secretion was blocked by nifedipine concomitantly with an increase in stroke volume, and the KB-R7943 blocked the effect of ouabain-increased atrial stroke volume only but failed in the modulation of ouabain-increased ANP secretion. These results not only demonstrated that there was no significant correlation between ouabain-induced ANP secretion and the atrial hemodyamics because there was an increase in stroke volume even though there was no significant secretion of ANP by ouabain plus nifedipine, but also indicated
that ouabain stimulated ANP secretion via Ca2+ influx through L-type Ca2+ channels, but not Na+–Ca2+ exchangers. However, in our previous study the atrial ANP secretion was inhibited by Ca 2+ influx through L-type Ca 2+ channels (Wen et al., 2000; Zhang et al., 2008), suggesting that there was an unknown mechanism involved in ouabain-stimulated ANP secretion. Accordingly, on the basis of the paracrine nature of ET-1 and its effect on atrial ANP secretion, the present study investigated the relationship between ET-1 and ouabain-stimulated ANP secretion in perfused beating rabbit atria. In the present study, ouabain significantly increased the levels of atrial ET-1 and that exogenous ET-1 potently stimulated atrial ANP secretion in a dose-dependent manner. ET-1 is best known for its ability to induce ANP secretion, and the present findings are consistent with an earlier study (Lew and Baertschi, 1992). In addition, the ouabain-increased ET-1 levels and ANP secretion were completely blocked by the MAPK inhibitor PD98059, and the ouabainincreased atrial stroke volume was also inhibited by PD98059. These results suggest that ouabain enhanced atrial ET-1 release via activation of MAPK signaling pathway, and thus ouabain-induced ANP secretion was augmented by ET-1. Previous reports showed that ET-1 elicits the negative inotropic myocardial effect from ETB receptor activation (Konrad et al., 2005), and that this requires an intact endocardial endothelium and is mediated by nitric oxide and prostaglandins (Leite-Moreira and Brás-Silva, 2004). The present study observed that ET-1-activated atrial ANP secretion was completely blocked by BQ788, an ETB receptor inhibitor, while the ETA receptor inhibitor BQ123 only slightly attenuated the effect of ET-1-activated ANP secretion. The results of the present study differed with previous reports that ET-1 stimulated ANP secretion was mainly via ETA receptor activation (Irons et al., 1993; Thibault et al., 1994), but similarly to the report (Zhang et al., 2004) that non-selective ET receptors inhibitor bosentan, but not BQ-123, significantly attenuated hypoxia-induced ANP release which was partially modulated via interaction with endogenous ET. The reason of these controversial observations with ET receptor-mediated regulation of ANP secretion was not well known, perhaps correlated with different experimental model conditions, methods, and some others. Furthermore, ouabainstimulated ANP secretion was also inhibited by BQ788 as well as
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indomethacin, a cyclooxygenase inhibitor, but not by BQ123. However, the ouabain-increased atrial stroke volume was also inhibited by indomethacin, although the reason for this observation remains to be defined. The present data have therefore demonstrated that ET-1 augmented stretch-activated atrial ANP secretion by ETB receptor activation and that this effect was observed in the ouabain-stimulated ANP secretion processes. In conclusion, results of the present study indicate that activation of MAPK signaling pathway and ET-1 release are main mechanisms by which ouabain-increased ANP secretion in isolated beating rabbit atria and suggest that a substantial part of the ouabain-stimulated ANP secretion occurs via increased ET-1 production leading to the formation of prostaglandin. Conclusion Ouabain significantly stimulated atrial ANP secretion via an ET-1-ETB receptor-mediated pathway involving MAPK signaling pathway activation and prostaglandin formation. Conflict of interest statement None.
Acknowledgments The authors thank Su Xiang Li for expert technical assistance. This work was supported by the National Natural Science Foundation of China (No. 30860091). References Awane-Igata Y, Ikeda S, Watanabe T. Inhibitory effects of TAK-044 on endothelin induced vasoconstriction in various canine arteries and porcine coronary arteries: a comparison with selective ETA and ETB receptor antagonists. Br J Pharmacol 1997;120:516–22. Barry WH, Hasin Y, Smith TW. Sodium pump inhibition, enhanced calcium influx via sodium–calcium exchange, and positive inotropic response in cultured heart cells. Circ Res 1985;56:231–41. Bloch KD, Zamir N, Lichtstein D, Seidman CE, Seidman JG. Ouabain induces secretion of proatrial natriuretic factor by rat atrial cardiocytes. Am J Physiol Endocrinol Metab 1988;255:E383–7. Cho KW, Kim SH, Hwang YH, Seul KH. Extracellular fluid translocation in perfused rabbit atria: implication in control of atrial natriuretic peptide secretion. J Physiol (Lond) 1993;468:591–607.
De Bold AJ. Atrial natriuretic factor: a hormone produced by the heart. Science 1985;230: 767–70. Eisner DA, Smith TW. The Heart and Cardiovascular System. 2nd ed. Morgan Raven Press; 1992. p. 863–902. Fukuda Y, Hirata Y, Taketani S, Kojima T, Oikawa S, Nakazato H, et al. Endothelin stimulates accumulations of cellular atrial natriuretic peptide and its messenger RNA in rat cardiocytes. Biochem Biophys Res Commun 1989;164:1431–6. Irons CE, Murray SF, Glembotski CC. Identification of the receptor subtype responsible for endothelin-mediated protein kinase C activation and atrial natriuretic factor secretion from atrial myocytes. J Biol Chem 1993;268(31):23417–21. Konrad D, Oldner A, Wanecek M, Rudehill A, Weitzberg E, Biber B, et al. Positive inotropic and negative lusitropic effects of endothelin receptor agonism in vivo. Am J Physiol Heart Circ Physiol 2005;289:H1702–9. Lee CO. 200 years of digitalis: the emerging central role of the sodium ion in the control of cardiac force. Am J Physiol Cell Physiol 1985;249:C367–78. Leite-Moreira AF, Brás-Silva C. Inotropic effects of ETB receptor stimulation and their modulation by endocardial endothelium, NO, and prostaglandins. Am J Physiol Heart Circ Physiol 2004;287:H1194–9. Levi AJ, Boyett MR, Lee CO. The cellular actions of digitalis glycosides on the heart. Prog Biophys Mol Biol 1994;62:1-54. Lew RA, Baertschi AJ. Endothelium-dependent ANF secretion in vitro. Am J Physiol Heart Circ Physiol 1992;263:H1071–7. McGarry SJ, Williams AJ. Digoxin activates sarcoplasmic reticulum Ca2+-release channels: a possible role in cardiac inotropy. Br J Pharmacol 1993;108:1043–50. Molenaar P, O'Reilly G, Sharkey A, Kuc RE, Harding DP, Plumpton C, et al. Characterization and localization of endothelin receptor subtypes in the human atrioventricular conducting system and myocardium. Circ Res 1993;72:526–38. Morise T, Takeuchi Y, Okamoto S, Tadeda R. Stimulation of atrial natriuretic peptide secretion and syntheses by Na–K-ATPase inhibitors. Biochem Biophys Res Commun 1991;176:875–81. Peters CG, Miller DF, Giovannucci DR. Identification, localization and interaction of SNARE proteins in atrial cardiac myocytes. J Mol Cell Cardiol 2006;40:361–74. Plumpton C, Ferro CJ, Haynes WG, Webb DJ, Davenport AP. The increase in human plasma immunoreactive endothelin but not big endothelin-1 or its C-terminal fragment induced by systemic administration of the endothelin antagonist TAK-044. Br J Pharmacol 1996;119:311–4. Schiebinger RJ, Cragoe Jr EJ. Ouabain. A stimulator of atrial natriuretic peptide secretion and its mechanism. Circ Res 1993;72:1035–43. Schwartz A, Grupp G, Wallick E, Grupp IL, Ball Jr WJ. Role of the Na+, K+-ATPase in the cardiotonic action of cardiac glycosides. Prog Clin Biol Res 1988;268B:321–38. Skvorak JP, Nazian SJ, Dietz JR. Endothelin acts as a paracrine regulator of stretchinduced atrial natriuretic peptide release. Am J Physiol Regul Integr Comp Physiol 1995;269:R1093–8. Thibault G, Doubell AF, Garcia R, Larivière R, Schiffrin EL. Endothelin-stimulated secretion of natriuretic peptides by rat atrial myocytes is mediated by endothelin A receptors. Circ Res 1994;74(3):460–70. Wen JF, Cui X, Ahn JS, Kim SH, Seul KH, Kim SZ, et al. Distinct roles for L- and T-type Ca2+ channels in regulation of atrial ANP release. Am J Physiol Heart Circ Physiol 2000;279: H2879–88. Zhang Y, Oliver JR, Horowitz JD. The role of endothelin in mediating ischemia/hypoxiainduced atrial natriuretic peptide release. J Cardiovasc Pharmacol 2004;43(2): 227–33. Zhang Y, Liu L, Liang ZL, Li XL, Jin YZ, Cui X. cAMP produced by pituitary adenylate cyclase activating polypeptide 27 inhibits atrial natriuretic peptide secretion in rabbit beating atria. Clin Exp Pharmacol Physiol 2008;35:1233–7.