Clinical and Experimental Hypertension

ISSN: 1064-1963 (Print) 1525-6006 (Online) Journal homepage: http://www.tandfonline.com/loi/iceh20

Comparison of aldosterone synthesis in adrenal cells, effect of various AT1 receptor blockers with or without atrial natriuretic peptide Shin-ichiro Miura, Asuka Nakayama, Sayo Tomita, Yoshino Matsuo, Yasunori Suematsu & Keijiro Saku To cite this article: Shin-ichiro Miura, Asuka Nakayama, Sayo Tomita, Yoshino Matsuo, Yasunori Suematsu & Keijiro Saku (2015) Comparison of aldosterone synthesis in adrenal cells, effect of various AT1 receptor blockers with or without atrial natriuretic peptide, Clinical and Experimental Hypertension, 37:5, 353-357, DOI: 10.3109/10641963.2014.987391 To link to this article: http://dx.doi.org/10.3109/10641963.2014.987391

Published online: 12 Dec 2014.

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Date: 06 November 2015, At: 16:38

http://informahealthcare.com/ceh ISSN: 1064-1963 (print), 1525-6006 (electronic) Clin Exp Hypertens, 2015; 37(5): 353–357 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/10641963.2014.987391

Comparison of aldosterone synthesis in adrenal cells, effect of various AT1 receptor blockers with or without atrial natriuretic peptide Shin-ichiro Miura1,2,3, Asuka Nakayama1, Sayo Tomita1, Yoshino Matsuo1, Yasunori Suematsu1, and Keijiro Saku1,2 Department of Cardiology and 2Department of Molecular Cardiovascular Therapeutics, Fukuoka University School of Medicine, Fukuoka, Japan, and 3Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic Foundation, OH, USA

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Abstract

Keywords

Bifunctional angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) that can block the activation of not only AT1 receptor, but also neprilysin, which metabolizes vasoactive peptides including atrial natriuretic peptide (ANP), are currently being developed. However, the usefulness of the inactivation of ANP in addition to the AT1 receptor with regard to aldosterone (Ald) synthesis is not yet clear. We evaluated the inhibitory effects of various ARBs combined with or without ANP on Ang II-induced adrenal Ald synthesis using a human adrenocortical cell line (NCI-H295R). Ang II increased Ald synthesis in a dose- and time-dependent manner. Ald synthesis induced by Ang II was completely blocked by azilsartan, but not PD123319 (AT2 receptor antagonist). CGP42112 AT2 receptor agonist did not affect Ald synthesis. While most ARBs block Ang II-induced Ald synthesis to different extents, azilsartan and olmesartan have similar blocking effects on Ald synthesis. The different effects of ARBs were particularly observed at 107 and 108 M. ANP attenuated Ang II-induced Ald synthesis, and ANP-mediated attenuation of Ang II-induced Ald synthesis were blocked by inhibitors of G-protein signaling subtype 4 and protein kinase G. ANP (108 and 107 M) without ARBs inhibited Ald synthesis, and the combination of ANP (107 M) and ARB (108 M) had an additive effect with respect to the inhibition of Ald synthesis. In conclusions, ARBs had differential effects on Ang II-induced Ald synthesis, and ANP may help to block Ald synthesis when the dose of ARB is not sufficient to block its secretion.

Adrenocortical cell, aldosterone, atrial natriuretic peptide, bifunctional angiotensin II type 1 receptor blocker, molecule-specific effects

Introduction Eight different angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) have been approved worldwide for the treatment of hypertension (HTN) and cardiovascular diseases. Although ARBs have been shown to have molecule-specific (differential) effects in addition to class (common) effects in basic experimental studies (1), it is still controversial whether ARBs have molecule-specific (differential) effects in a clinical setting (2). To resolve this issue, bifunctional ARBs that not only block AT1 receptor activation, but also inhibit neprilysin activity, by blocking endothelin-1 receptor or the release of nitric oxide, are currently being developed (3–5). Among these, a single molecule that can inhibit both AT1 receptor and neprilysin is being developed for the treatment of resistant HTN and heart failure (HF) (6–8). Neprilysin is a membrane-bound zinc metalloprotease that metabolizes vasoactive peptides including atrial natriuretic peptide (ANP) and bradykinin, which are involved in controlling BP. ANP has exactly the opposite function of aldosterone (Ald). ANP inhibits Ald synthesis in the

Correspondence: Shin-ichiro Miura, Department of Cardiology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. Tel: +81 92 801 1011. Fax: +81 91 865 2692. E-mail: [email protected]

History Received 31 July 2014 Revised 31 October 2014 Accepted 4 November 2014 Published online 11 December 2014

biosynthetic pathway from cholesterol to pregnenolone and from corticosterone to Ald (9,10). Ald antagonists, including spironolactone and eplerenone, have been shown to reduce the risk of cardiovascular death in patients with HF (11). It would be very interesting to determine whether AT1 receptor blockade and treatment with ANP have an additive or synergistic inhibitory effect on Ald synthesis. Therefore, in the present study, we confirmed whether ARBs have molecule-specific effects in addition to class effects with regard to Ald synthesis in adrenal cells. Moreover, we examined whether combined treatment with ARB and ANP is more effective than the respective monotherapies.

Materials and methods Materials The following reagents were purchased: azilsartan, Exp3174, irbesartan, olmesartan and telmisartan (Toronto Research Chemicals Inc., Toronto, Canada); Ang II and CGP42112 (AT2 receptor agonist) (Pepitide Institute Inc., Osaka, Japan); PD123319 (AT2 receptor antagonist) and ANP (SigmaAldrich, St. Louis, MO); CCG63802 [inhibitor of G-protein signaling subtype 4 (RGS4)] (Santa Cruz Biotechnology, Inc., Dallas, TX); and KT5823 [inhibitor of protein kinase G (PKG)] (Cayman Chemical Company, Ann Arbor, MI).

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Cell cultures Human adrenocortical cells [NCI-H295R, American Type Culture Collection (ATCC) CRL-10296] (ATCC, Manassas, VA) were cultured and maintained in Nu-Serum (BD Biosciences, Baltimore, MD) and penicillin- and streptomycin-supplemented Dulbecco’s modified Eagle’s essential medium-F12 (Wako Chemicals, Osaka, Japan) in 5% CO2 at 37  C.

For the experiments, cells were grown for 24 h under serumfree conditions at 37  C in 5% CO2, and then incubated with or without various treatments for additional indicated times under serum-free conditions. The levels of Ald in the cell culture medium (Ald synthesis) after various treatments for 6–48 h were measured in duplicate by an enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). Statistical analysis The results are expressed as the mean ± standard deviation of three or more independent determinations. The significance of differences in measured values was evaluated with an analysis of variance using Fisher’s t-test and unpaired or paired Student’s t-test, as appropriate. Statistical significance was set at 50.05.

Results Ang II increased Ald synthesis in a dose- and time-dependent manner We examined whether Ang II significantly increased Ald synthesis for up to 48 h in NCI-H295R (Figure 1A). Ang II (107 M) significantly increased Ald synthesis in a timedependent manner (6, 24 and 48 h). Ang II (108 to 106 M) also significantly increased Ald synthesis in a dose-dependent manner (Figure 1B).

Figure 1. Ang II increased Ald synthesis in a dose- and time-dependent manner. Ald synthesis was analyzed by EIA at the indicated incubation times (A; 6, 24 and 48 h) and the indicated concentrations of Ang II (B, 106 to 109 M) for 48 h. *p50.05 versus Ang II (–) at 6 h. #p50.05 versus Ang II (–) for each incubation time.

Ang II-induced Ald synthesis was completely blocked by 106 M azilsartan, but not 106 M PD123319 (AT2 receptor antagonist) (Figure 2A). In addition, CGP42112 (108 to 106 M) did not affect Ald synthesis (Figure 2B). Each ARB has differential blocking effects on Ang II-induced Ald synthesis Next, we analyzed the blocking effects of ARBs (106 to 1010 M) on Ang II (107 M)-induced Ald synthesis (Figure 3). Although all the ARBs significantly blocked Ang II-induced Ald synthesis at 106 M, ARBs had different blocking effects on Ang II-induced Ald synthesis at 107 and 108 M. The blocking effects of azilsartan (107 and 108 M) on Ald synthesis were significantly stronger than those of irbesartan (107 and 108 M) and telmisartan and Exp3174 (108 M). Azilsartan and olmesartan had similar blocking effects on Ang II-induced Ald synthesis. ANP-mediated attenuation of Ang II-induced Ald synthesis and its mechanisms We performed to examine the effects of 106 M ANPmediated attenuation of 106 to 108 M Ang II-induced Ald synthesis (Figure 4). The effects of ANP were significant, but relatively small even if we use 106 to 108 M Ang II. To find the mechanisms responsible for ANP-mediated attenuation of Ang II-induced Ald synthesis, we used the inhibitors of RGS4 (CCG63802) and PKG (KT5823). ANP-mediated attenuation of Ang II-induced aldosterone synthesis was blocked by CCG63802 and KT5823. ANP with or without ARBs inhibited Ald synthesis ANP (107 and 108 M) without ARBs inhibited Ald synthesis, and the combination of ANP (107 M) and ARB (108 M) had an additive blocking effect on Ald synthesis (Figure 5).

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treatment with ARB and ANP is more effective than the respective monotherapies when the ARB does not completely block Ang II-induced Ald synthesis. We have proposed that small differences in the molecular structures of ARBs lead to differences in their abilities to influence the AT1 receptor (1,12,13). In this study, azilsartan

Discussion In the present study, we confirmed that ARBs have moleculespecific effects with regard to blocking effect on Ang IIinduced Ald synthesis in adrenal cells. Moreover, combined

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Figure 2. Ang II-induced Ald synthesis was mediated by AT1 receptor. (A) Ald synthesis was analyzed by EIA with or without Ang II (107 M), azilsartan (106 M) and PD123319 (106 M) for 48 h. (B) Ald synthesis was analyzed by EIA using the indicated concentrations of CPG42112 (106 to 108 M) for 48 h. % Max aldosterone secretion indicates the aldosterone secretion (440 pg/ml) (100%) induced by Ang II (107 M) after adjusting for the basal aldosterone secretion (281 pg/ml) without treatment (0%). *p50.05 versus Ang II ().

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Figure 3. ARBs have differential blocking effects on Ang II-induced Ald synthesis. Ald synthesis was analyzed by EIA with Ang II (107 M) and the indicated concentrations of ARBs (106 to 1010 M) for 48 h. % Max aldosterone secretion indicates the aldosterone secretion (100%) induced by Ang II (107 M) after adjusting for the basal aldosterone secretion without treatment (0%). *p50.05 versus Ang II () + ARBs (). #p50.05 versus Ang II (+) + azilsartan.

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Figure 4. ANP-mediated attenuation of Ang II-induced Ald synthesis and its mechanisms. Ald synthesis was analyzed by EIA with Ang II (106 to 108 M) in the presence and absence of 106 M ANP, 107 M KT6823 and 106 M CCG63802 for 48 h. % Max aldosterone secretion indicates the aldosterone secretion (100 %) induced by Ang II (107 M) after adjusting for the basal aldosterone secretion without treatment (0%). *p50.05 versus ANP () in each concentration of Ang II. #p50.05 versus Ang II (+) alone.

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Figure 5. ANP with or without ARBs inhibited Ald synthesis. Ald synthesis was analyzed by EIA with or without Ang II (107 M), the indicated concentration of ANP (106 and 107 M) and ARBs (106 M) for 48 h. % Max aldosterone secretion indicates the aldosterone secretion (100%) induced by Ang II (107 M) after adjusting for the basal aldosterone secretion without treatment (0%). *p50.05 versus Ang II (). yp50.05 versus Ang II (+). #p50.05 versus Ang II (+) + ARB (+).

and olmesartan were superior to other ARBs with respect to their blocking effects on Ang II-induced Ald synthesis. The superiority of these two ARBs may be due to their unique interactions with the AT1 receptor. The ability of azilsartan may be associated with its unique moiety, a 5-oxo-1,2,4oxadiazole, in place of a tetrazole ring (14), and azilsartan has been shown to bind tightly to and dissociate slowly from AT1 receptors in comparison to other ARBs (15). We also previously reported that the interactions between the AT1

receptor and the hydroxyl and carboxyl groups of olmesartan play an important role in its tight binding to the receptor (16). We also found that combined treatment with an ARB and ANP may be more effective than treatment with either alone. There are two possible explanations for how ANP can block Ald synthesis. First, we found that ANP-mediated attenuation of Ang II-induced aldosterone synthesis was blocked by CCG63802 and KT5823. A guanylyl cyclase-A, a natriuretic peptide receptor, activates PKG-RGS4 pathway, which

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DOI: 10.3109/10641963.2014.987391

attenuates a Galpha(q)-dependent pathway (17). In this study, Ang II-induced Gq-dependent Ald synthesis was blocked by ANP-induced activation of PKG-RGS4 pathway. Second, ANP may block Ald synthesis in the biosynthetic pathway from cholesterol to pregnenolone and from corticosterone to Ald (9,10). Interestingly, irbesartan at 107 to 108 M and telmisartan at 108 M partially blocked Ang II-induced Ald synthesis, although all of the ARBs completely blocked Ald synthesis at a high concentration (106 M). For example, a 100-mg dose of irbesartan results in a peak irbesartan concentration of around 1 mM in human plasma (18), and 1 mM of irbesartan could completely block Ang II-induced Ald synthesis. Generally, human plasma concentrations of ARBs decrease to one-tenth of their initial level after 12 h, and return to the basal level after 24 h. In this situation, the combination of ARB with ANP may be useful for blocking Ald synthesis. In addition, plasma Ald levels are elevated in a subset of patients despite ARB therapy. This phenomenon is known as ‘‘aldosterone escape’’ or ‘‘aldosterone breakthrough’’ (19). The combination of ARB with ANP may also be useful against such ‘‘aldosterone escape’’. In conclusion, this study demonstrated that ARBs have differential effects on Ang II-induced Ald synthesis, and ANP may help to block Ald synthesis when the dose of ARB is not sufficient to block its secretion. Although our finding is based on basic research, it may lead to exciting insights into the blockade of Ald by ARBs with ANP in a clinical setting.

Declaration of interest KS and SM have received grants and lecture honoraria from Daiichi-Sankyo Co. Ltd., MSD, Dainippon Sumitomo Pharma Co. Ltd., Takeda Pharma Co. Ltd., Boehringer Ingelheim and Astellas Pharma Inc. KS is a Chief Director and SM is a Director of NPO Clinical and Applied Science, Fukuoka, Japan. KS has an Endowed Department of ‘‘Department of Molecular Cardiovascular Therapeutics’’ supported by MSD, Co. Ltd. SM belongs to the Department of Molecular Cardiovascular Therapeutics supported by MSD, Co. Ltd.

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3. Kurtz TW, Klein U. Next generation multifunctional angiotensin receptor blockers. Hypertens Res 2009;32:826–34. 4. Mohanan A, Gupta R, Dubey A, et al. TRC120038, a novel dual AT(1)/ET(A) receptor blocker for control of hypertension, diabetic nephropathy, and cardiomyopathy in ob-ZSF1 rats. Int J Hypertens 2011:751513. 5. Breschi MC, Calderone V, Digiacomo M, et al. New NO-releasing pharmacodynamic hybrids of losartan and its active metabolite: design, synthesis, and biopharmacological properties. J Med Chem 2006;49:2628–39. 6. Monge M, Lorthioir A, Bobrie G, Azizi M. New drug therapies interfering with the renin–angiotensin–aldosterone system for resistant hypertension. J Renin Angiotensin Aldosterone Syst 2013;14:285–9. 7. Kario K, Sun N, Chiang FT, et al. Efficacy and safety of LCZ696, a first-in-class angiotensin receptor neprilysin inhibitor, in Asian patients with hypertension: a randomized, double-blind, placebocontrolled study. Hypertension 2014;63:698–705. 8. Jhund PS, Claggett B, Packer M, et al. Independence of the blood pressure lowering effect and efficacy of the angiotensin receptor neprilysin inhibitor, LCZ696, in patients with heart failure with preserved ejection fraction: an analysis of the PARAMOUNT trial. Eur J Heart Fail 2014;16:671–7. 9. Campbell WB, Currie MG, Needleman P. Inhibition of aldosterone biosynthesis by atriopeptins in rat adrenal cells. Circ Res 1985;57: 113–18. 10. Olson LJ, Ho BY, Cashdollar LW, Drewett JG. Functionally active catalytic domain is essential for guanylyl cyclase-linked receptor mediated inhibition of human aldosterone synthesis. Mol Pharmacol 1998;54:761–9. 11. Chatterjee S, Moeller C, Shah N, et al. Eplerenone is not superior to older and less expensive aldosterone antagonists. Am J Med 2012; 125:817–25. 12. Fujino M, Miura S, Kiya Y, et al. A small difference in the molecular structure of angiotensin II receptor blockers induces AT1 receptor-dependent and -independent beneficial effects. Hypertens Res 2010;33:1044–52. 13. Miura S, Nakao N, Hanzawa H, et al. Reassessment of the unique mode of binding between angiotensin II type 1 receptor and their blockers. PLoS One 2013;8:e79914. 14. Miura S, Okabe A, Matsuo Y, et al. Unique binding behavior of the recently approved angiotensin II receptor blocker azilsartan compared with that of candesartan. Hypertens Res 2013;36:134–9. 15. Ojima M, Igata H, Tanaka M, et al. In vitro antagonistic properties of a new angiotensin type 1 receptor blocker, azilsartan, in receptor binding and function studies. J Pharmacol Exp Ther 2011;336: 801–8. 16. Miura S, Fujino M, Hanzawa H, et al. Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor. J Biol Chem 2006;281:19288–95. 17. Tokudome T, Kishimoto I, Horio T, et al. Regulator of G-protein signaling subtype 4 mediates antihypertrophic effect of locally secreted natriuretic peptides in the heart. Circulation 2008;117: 2329–39. 18. Pool JL, Guthrie RM, Littlejohn III TW, et al. Dose-related antihypertensive effects of irbesartan in patients with mild-tomoderate hypertension. Am J Hypertens 1998;11:462–70. 19. Bomback AS, Klemmer PJ. The incidence and implications of aldosterone breakthrough. Nat Clin Pract Nephrol 2007;3: 486–92.

Comparison of aldosterone synthesis in adrenal cells, effect of various AT1 receptor blockers with or without atrial natriuretic peptide.

Bifunctional angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) that can block the activation of not only AT1 receptor, but also neprilysin...
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