Nicklin, Stuart A.

English

Enlighten: Publications

 
 
 
 
Nicklin, S. A. (2016) A novel mechanism of action for angiotensin-(1-7) via 
the angiotensin type 1 receptor. Hypertension, 68(6), pp. 1342-1343. 
(doi:10.1161/HYPERTENSIONAHA.116.08215) 
 
This is the author’s final accepted version. 
 
There may be differences between this version and the published version. 
You are advised to consult the publisher’s version if you wish to cite from 
it. 
 
 
 
 
http://eprints.gla.ac.uk/128323/ 
     
 
 
 
 
 
 
Deposited on: 15 September 2016 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enlighten – Research publications by members of the University of Glasgow 
http://eprints.gla.ac.uk 
 A novel mechanism of action for angiotensin-(1-7) via the angiotensin type 1 receptor 
 
Running title: Ang-(1-7) is a biased AT1R agonist 
 
Stuart A Nicklin 
 
Corresponding author: 
Stuart A Nicklin, PhD 
Institute of Cardiovascular and Medical Sciences, University of Glasgow, 126 University 
Place, Glasgow, G12 8TA, UK. Tel: +44-141-3302521. Fax: +44-1413306997. Email: 
stuart.nicklin@glasgow.ac.uk 
  
1 
 
Angiotensin-(1-7) [Ang-(1-7)] was originally thought to be an inactive metabolite of 
angiotensin II (Ang II) in the renin angiotensin system (RAS). However, reported biological 
effects and seminal discoveries of an angiotensin converting enzyme (ACE) homologue, 
ACE2 which metabolises Ang II to Ang-(1-7),
1
 combined with identification of the G protein 
coupled receptor Mas as an endogenous Ang-(1-7) receptor
2
 defined a natural counter-
regulatory ACE2/Ang-(1-7)/Mas axis of the RAS. The counter-regulatory axis of the RAS is 
firmly established to inhibit detrimental effects mediated through the classical ACE/ Ang II/ 
angiotensin type 1 receptor (AT1R) axis. Moreover, it is simplistic to separate the RAS into 
these two axes as the systemic and local tissue-specific RAS includes a wide range of 
molecules with biological action, including the alternative Ang II receptor, the angiotensin 
type 2 receptor (AT2R), other peptide metabolites, e.g. Ang-(1-9), Ang III and Ang IV, and 
recent discoveries including an Ang-(1-7) metabolite, Alamandine, and its receptor Mas-
related G protein-coupled receptor D (MrgD).
3
 
Ang-(1-7) mediates varied physiological and therapeutic effects via Mas, including 
preventing adverse tissue remodelling, improving vascular and cardiac function and 
promoting wound healing. Importantly, effects of Ang-(1-7) are not all directly through 
antagonising the ACE/Ang II/AT1R axis. In fact Ang-(1-7), through Mas and Gs,
4
 activates 
distinct intracellular signalling leading to production of nitric oxide, modulation of ERK 
signalling and stimulation of cAMP release [reviewed in
5
]. Therefore, Ang-(1-7) is well 
established as an independent RAS component and is being explored clinically, e.g. for 
stimulating haematopoietic recovery in cancer patients following chemotherapy.
6
 
In this issue of Hypertension the complexity of RAS interactions, and particularly Ang-(1-7) 
function, is further expanded by Galandrin et al, via a series of elegant molecular 
pharmacology studies that decipher a novel role for Ang-(1-7) as a biased AT1R agonist
7
 and 
2 
 
therefore define a previously unrecognised mechanism of action. The concept of biased 
agonism at the AT1R is relatively new and stems from the recognition that biased AT1R 
agonists, e.g. the peptide [Sar
1
, Ile
4
, Ile
8
]-angiotensin II, stimulate -arrestin recruitment to 
the receptor leading to AT1R internalisation, -arrestin-dependent signalling and potential 
therapeutic outcomes which are distinct to the classical G protein-coupled responses.
8
 
Galandrin et al performed a systematic evaluation of angiotensin peptides was performed in 
AT1R-overexpressing HEK293T cells.
7
 Bioluminescence resonance energy transfer (BRET) 
was used to assess abilities of Ang II, Ang III, Ang IV and Ang-(1-7) to activate classical G 
signalling. While Ang III and AngIV mimicked Ang II in activating G signalling via Gi/o, 
Gq/11 and G13 families, Ang-(1-7) did not. Binding affinity studies revealed all angiotensin 
peptides displaced 
125
I-Ang II from the AT1R, with affinities from nM (Ang II and Ang III) to 
M (Ang IV). Ang-(1-7) was reported to have a Ki of 360 nM and act as a natural neutral 
antagonist at the AT1R by shifting the Ang II response curve to the right for activation of Gi3 
and, with lower potency, Gq. Importantly, Ang-(1-7) also acted as a biased AT1R agonist by 
stimulating -arrestin2 recruitment. The potency of Ang-(1-7) was lower than Ang II, but in 
line with its binding affinity and inhibited by the AT1R blocker candesartan. 
Biased AT1R agonism is being actively investigated for therapeutic applications. The 
classical actions of Ang II at the AT1R in hypertension and cardiovascular disease (CVD) are 
mainly mediated through activating Gq. Conversely, AT1R signalling also leads to non-G 
protein-mediated signalling through -arrestin2 which is therapeutic in CVD. Currently, the 
-arrestin2-biased AT1R molecule TRV1200027 is being explored in clinical trials for heart 
failure after demonstrating efficacy in reducing blood pressure and improving cardiac 
function in rodent models.
9
 The therapeutic development of biased AT1R agonists is 
important in the context of Ang-(1-7) as a biased AT1R agonist. Galendrin et al moved from 
3 
 
in vitro molecular pharmacology studies to a whole organ model in isolated aortas from wild 
type and AT1R knockout mice.
7
 Ang-(1-7) inhibited phenylephrine-mediated contraction in 
wild type, but not AT1R knockout aortas. Moreover, effects of Ang-(1-7) in wild type aorta 
were not inhibited by the mas antagonist A-779, nor the AT2R antagonist PD-123,319, but 
were by candesartan. Intriguingly, when Ang II was utilised as a vasoconstrictor in aortas 
from wild type mice, co-application of Ang-(1-7) potentiated the effect in a manner sensitive 
to the Mas antagonist A-779. The reason for this finding is not clear but highlights the 
complexity of RAS interactions. Previous studies have highlighted cross-talk between Ang-
(1-7) and other RAS receptors, with effects reported via the AT2R
10
 and MrgD.
4
 However, 
AT2R studies have predominantly utilised the AT2R antagonist PD-123,319 which is also 
reported to inhibit MrgD which may complicate interpretation.
3, 4
 Furthermore, Mas is also a 
physiological antagonist of the AT1R, again highlighting the complexity of cross-talk in the 
RAS.
11
 These new studies
7
 are the first demonstration that Ang-(1-7) can both interact with 
the AT1R and mediate distinct signalling effects and provide new knowledge regarding how 
RAS components interact via crosstalk.  
What remains to be dissected is how these individual peptide/ receptor interactions function 
in vivo. In a diseased tissue there are likely to be differing levels of RAS-related receptors 
and dynamically changing angiotensin peptide levels and it will be difficult to determine the 
relative contribution of individual peptides acting at different receptors. Nevertheless it will 
be important to research the contribution of biased agonism from Ang-(1-7) at the AT1R and 
integrate the knowledge with that known through Mas agonism.  
In summary, this study
7
 has defined novel interactions in the RAS and contributes to 
increased knowledge of how RAS peptides and receptors intersect to mediate biological 
outcomes. Further understanding of these aspects of the RAS may lead to the next generation 
4 
 
of RAS-targeting medicines capable of integrating differential signalling outcomes into 
synergistic action in CVD. 
 
Sources of funding 
Work on the renin angiotensin system in the author’s laboratory is supported by the Medical 
Research Council (MR/L019108/1). 
 
Disclosure statement 
None. 
  
5 
 
References 
1.  Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, 
Woolf B, Robison K, Jeyaseelan R, Breitbart RE, Acton S. A novel angiotensin-converting 
enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res. 
2000;87:E1-9. 
2.  Santos RA, Simoes e Silva AC, Maric C, et al. Angiotensin-(1-7) is an endogenous ligand 
for the G protein-coupled receptor Mas. Proc Natl Acad Sci U S A. 2003;100:8258-8263. 
3.  Lautner RQ, Villela DC, Fraga-Silva RA, et al. Discovery and characterization of 
alamandine: A novel component of the renin-angiotensin system. Circ Res. 2013;112:1104-
1111. 
4.  Tetzner A, Gebolys K, Meinert C, Klein S, Uhlich A, Trebicka J, Villacanas O, Walther T. 
G-protein-coupled receptor MrgD is a receptor for angiotensin-(1-7) involving adenylyl 
cyclase, cAMP, and phosphokinase A. Hypertension. 2016;68:185-194. 
5.  McKinney CA, Fattah C, Loughrey CM, Milligan G, Nicklin SA. Angiotensin-(1-7) and 
angiotensin-(1-9): Function in cardiac and vascular remodelling. Clin Sci (Lond). 
2014;126:815-827. 
6.  Rodgers KE, Oliver J, diZerega GS. Phase I/II dose escalation study of angiotensin 1-7 
[a(1-7)] administered before and after chemotherapy in patients with newly diagnosed breast 
cancer. Cancer Chemother Pharmacol. 2006;57:559-568. 
7.  Galandrin S, Denis C, Boularan C, Marie J, M'Kadmi C, Pilette C, Dubroca C, Nicasie Y, 
Seguelas M-H, N'Guyen D, Baneres J-L, Pathak A, Senard J-M, Gales CG. The 
cardioprotective angiotensin 1-7 peptide acts as a natural biased ligand at the angiotensin ii 
type 1 receptor. Hypertension. 2016;in press. 
8.  Rajagopal K, Whalen EJ, Violin JD, Stiber JA, Rosenberg PB, Premont RT, Coffman TM, 
Rockman HA, Lefkowitz RJ. Beta-arrestin2-mediated inotropic effects of the angiotensin II 
6 
 
type 1a receptor in isolated cardiac myocytes. Proc Natl Acad Sci U S A. 2006;103:16284-
16289. 
9.  Felker GM, Butler J, Collins SP, Cotter G, Davison BA, Ezekowitz JA, Filippatos G, 
Levy PD, Metra M, Ponikowski P, Soergel DG, Teerlink JR, Violin JD, Voors AA, Pang PS. 
Heart failure therapeutics on the basis of a biased ligand of the angiotensin-2 type 1 receptor. 
Rationale and design of the BLAST-AHF study (biased ligand of the angiotensin receptor 
study in acute heart failure). JACC Heart Fail. 2015;3:193-201. 
10.  Walters PE, Gaspari TA, Widdop RE. Angiotensin-(1-7) acts as a vasodepressor agent 
via angiotensin II type 2 receptors in conscious rats. Hypertension. 2005;45:960-966. 
11. Canals M, Jenkins L, Kellett E, Milligan G. Up-regulation of the angiotensin II type 1 
receptor by the mas proto-oncogene is due to constitutive activation of Gq/G11 by mas. J Biol 
Chem. 2006;281:16757-16767. 
 
  
7 
 
Figure Legend 
Figure 1. New model of angiotensin-(1-7) action. Angiotensin II (Ang II) acts at the 
angiotensin type 1 receptor (AT1R) and stimulates (patho)physiological G protein signalling 
in cardiovascular disease, mainly through the Gq/i family. Ang II also activates G protein-
independent signalling via recruitment of -Arrestin2 (Arr2) leading to therapeutic effects. 
The counter-regulatory renin angiotensin system axis peptide angiotensin-(1-7) [Ang-(1-7)] 
mainly acts at Mas to stimulate diverse therapeutic effects in different diseases via Gs 
activation. Ang-(1-7) also binds the AT1R leading to Arr2 recruitment and vasorelaxation in 
isolated aortas. 
 
 
 
 
 
 
 
 
 
 
 
8 
 
 
 
 


A novel mechanism of action for angiotensin-(1-7) via the angiotensin type 1 receptor

Enlighten

    Nicklin, S. A. (2016) A novel mechanism of action for angiotensin-(1-7) via the angiotensin type 1 receptor. Hypertension, 68(6), pp. 1342-1343. (doi:10.1161/HYPERTENSIONAHA.116.08215)  This is the author’s final accepted version.  There may be differences between this version and the published version. You are advised to consult the publisher’s version if you wish to cite from it.     http://eprints.gla.ac.uk/128323/            Deposited on: 15 September 2016              Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk  A novel mechanism of action for angiotensin-(1-7) via the angiotensin type 1 receptor  Running title: Ang-(1-7) is a biased AT1R agonist  Stuart A Nicklin  Corresponding author: Stuart A Nicklin, PhD Institute of Cardiovascular and Medical Sciences, University of Glasgow, 126 University Place, Glasgow, G12 8TA, UK. Tel: +44-141-3302521. Fax: +44-1413306997. Email: stuart.nicklin@glasgow.ac.uk   1  Angiotensin-(1-7) [Ang-(1-7)] was originally thought to be an inactive metabolite of angiotensin II (Ang II) in the renin angiotensin system (RAS). However, reported biological effects and seminal discoveries of an angiotensin converting enzyme (ACE) homologue, ACE2 which metabolises Ang II to Ang-(1-7),1 combined with identification of the G protein coupled receptor Mas as an endogenous Ang-(1-7) receptor2 defined a natural counter-regulatory ACE2/Ang-(1-7)/Mas axis of the RAS. The counter-regulatory axis of the RAS is firmly established to inhibit detrimental effects mediated through the classical ACE/ Ang II/ angiotensin type 1 receptor (AT1R) axis. Moreover, it is simplistic to separate the RAS into these two axes as the systemic and local tissue-specific RAS includes a wide range of molecules with biological action, including the alternative Ang II receptor, the angiotensin type 2 receptor (AT2R), other peptide metabolites, e.g. Ang-(1-9), Ang III and Ang IV, and recent discoveries including an Ang-(1-7) metabolite, Alamandine, and its receptor Mas-related G protein-coupled receptor D (MrgD).3 Ang-(1-7) mediates varied physiological and therapeutic effects via Mas, including preventing adverse tissue remodelling, improving vascular and cardiac function and promoting wound healing. Importantly, effects of Ang-(1-7) are not all directly through antagonising the ACE/Ang II/AT1R axis. In fact Ang-(1-7), through Mas and Gs,4 activates distinct intracellular signalling leading to production of nitric oxide, modulation of ERK signalling and stimulation of cAMP release [reviewed in5]. Therefore, Ang-(1-7) is well established as an independent RAS component and is being explored clinically, e.g. for stimulating haematopoietic recovery in cancer patients following chemotherapy.6 In this issue of Hypertension the complexity of RAS interactions, and particularly Ang-(1-7) function, is further expanded by Galandrin et al, via a series of elegant molecular pharmacology studies that decipher a novel role for Ang-(1-7) as a biased AT1R agonist7 and 2  therefore define a previously unrecognised mechanism of action. The concept of biased agonism at the AT1R is relatively new and stems from the recognition that biased AT1R agonists, e.g. the peptide [Sar1, Ile4, Ile8]-angiotensin II, stimulate -arrestin recruitment to the receptor leading to AT1R internalisation, -arrestin-dependent signalling and potential therapeutic outcomes which are distinct to the classical G protein-coupled responses.8 Galandrin et al performed a systematic evaluation of angiotensin peptides was performed in AT1R-overexpressing HEK293T cells.7 Bioluminescence resonance energy transfer (BRET) was used to assess abilities of Ang II, Ang III, Ang IV and Ang-(1-7) to activate classical G signalling. While Ang III and AngIV mimicked Ang II in activating G signalling via Gi/o, Gq/11 and G13 families, Ang-(1-7) did not. Binding affinity studies revealed all angiotensin peptides displaced 125I-Ang II from the AT1R, with affinities from nM (Ang II and Ang III) to M (Ang IV). Ang-(1-7) was reported to have a Ki of 360 nM and act as a natural neutral antagonist at the AT1R by shifting the Ang II response curve to the right for activation of Gi3 and, with lower potency, Gq. Importantly, Ang-(1-7) also acted as a biased AT1R agonist by stimulating -arrestin2 recruitment. The potency of Ang-(1-7) was lower than Ang II, but in line with its binding affinity and inhibited by the AT1R blocker candesartan. Biased AT1R agonism is being actively investigated for therapeutic applications. The classical actions of Ang II at the AT1R in hypertension and cardiovascular disease (CVD) are mainly mediated through activating Gq. Conversely, AT1R signalling also leads to non-G protein-mediated signalling through -arrestin2 which is therapeutic in CVD. Currently, the -arrestin2-biased AT1R molecule TRV1200027 is being explored in clinical trials for heart failure after demonstrating efficacy in reducing blood pressure and improving cardiac function in rodent models.9 The therapeutic development of biased AT1R agonists is important in the context of Ang-(1-7) as a biased AT1R agonist. Galendrin et al moved from 3  in vitro molecular pharmacology studies to a whole organ model in isolated aortas from wild type and AT1R knockout mice.7 Ang-(1-7) inhibited phenylephrine-mediated contraction in wild type, but not AT1R knockout aortas. Moreover, effects of Ang-(1-7) in wild type aorta were not inhibited by the mas antagonist A-779, nor the AT2R antagonist PD-123,319, but were by candesartan. Intriguingly, when Ang II was utilised as a vasoconstrictor in aortas from wild type mice, co-application of Ang-(1-7) potentiated the effect in a manner sensitive to the Mas antagonist A-779. The reason for this finding is not clear but highlights the complexity of RAS interactions. Previous studies have highlighted cross-talk between Ang-(1-7) and other RAS receptors, with effects reported via the AT2R10 and MrgD.4 However, AT2R studies have predominantly utilised the AT2R antagonist PD-123,319 which is also reported to inhibit MrgD which may complicate interpretation.3, 4 Furthermore, Mas is also a physiological antagonist of the AT1R, again highlighting the complexity of cross-talk in the RAS.11 These new studies7 are the first demonstration that Ang-(1-7) can both interact with the AT1R and mediate distinct signalling effects and provide new knowledge regarding how RAS components interact via crosstalk.  What remains to be dissected is how these individual peptide/ receptor interactions function in vivo. In a diseased tissue there are likely to be differing levels of RAS-related receptors and dynamically changing angiotensin peptide levels and it will be difficult to determine the relative contribution of individual peptides acting at different receptors. Nevertheless it will be important to research the contribution of biased agonism from Ang-(1-7) at the AT1R and integrate the knowledge with that known through Mas agonism.  In summary, this study7 has defined novel interactions in the RAS and contributes to increased knowledge of how RAS peptides and receptors intersect to mediate biological outcomes. Further understanding of these aspects of the RAS may lead to the next generation 4  of RAS-targeting medicines capable of integrating differential signalling outcomes into synergistic action in CVD.  Sources of funding Work on the renin angiotensin system in the author’s laboratory is supported by the Medical Research Council (MR/L019108/1).  Disclosure statement None.   5  References 1.  Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, Breitbart RE, Acton S. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res. 2000;87:E1-9. 2.  Santos RA, Simoes e Silva AC, Maric C, et al. Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas. Proc Natl Acad Sci U S A. 2003;100:8258-8263. 3.  Lautner RQ, Villela DC, Fraga-Silva RA, et al. Discovery and characterization of alamandine: A novel component of the renin-angiotensin system. Circ Res. 2013;112:1104-1111. 4.  Tetzner A, Gebolys K, Meinert C, Klein S, Uhlich A, Trebicka J, Villacanas O, Walther T. G-protein-coupled receptor MrgD is a receptor for angiotensin-(1-7) involving adenylyl cyclase, cAMP, and phosphokinase A. Hypertension. 2016;68:185-194. 5.  McKinney CA, Fattah C, Loughrey CM, Milligan G, Nicklin SA. Angiotensin-(1-7) and angiotensin-(1-9): Function in cardiac and vascular remodelling. Clin Sci (Lond). 2014;126:815-827. 6.  Rodgers KE, Oliver J, diZerega GS. Phase I/II dose escalation study of angiotensin 1-7 [a(1-7)] administered before and after chemotherapy in patients with newly diagnosed breast cancer. Cancer Chemother Pharmacol. 2006;57:559-568. 7.  Galandrin S, Denis C, Boularan C, Marie J, M'Kadmi C, Pilette C, Dubroca C, Nicasie Y, Seguelas M-H, N'Guyen D, Baneres J-L, Pathak A, Senard J-M, Gales CG. The cardioprotective angiotensin 1-7 peptide acts as a natural biased ligand at the angiotensin ii type 1 receptor. Hypertension. 2016;in press. 8.  Rajagopal K, Whalen EJ, Violin JD, Stiber JA, Rosenberg PB, Premont RT, Coffman TM, Rockman HA, Lefkowitz RJ. Beta-arrestin2-mediated inotropic effects of the angiotensin II 6  type 1a receptor in isolated cardiac myocytes. Proc Natl Acad Sci U S A. 2006;103:16284-16289. 9.  Felker GM, Butler J, Collins SP, Cotter G, Davison BA, Ezekowitz JA, Filippatos G, Levy PD, Metra M, Ponikowski P, Soergel DG, Teerlink JR, Violin JD, Voors AA, Pang PS. Heart failure therapeutics on the basis of a biased ligand of the angiotensin-2 type 1 receptor. Rationale and design of the BLAST-AHF study (biased ligand of the angiotensin receptor study in acute heart failure). JACC Heart Fail. 2015;3:193-201. 10.  Walters PE, Gaspari TA, Widdop RE. Angiotensin-(1-7) acts as a vasodepressor agent via angiotensin II type 2 receptors in conscious rats. Hypertension. 2005;45:960-966. 11. Canals M, Jenkins L, Kellett E, Milligan G. Up-regulation of the angiotensin II type 1 receptor by the mas proto-oncogene is due to constitutive activation of Gq/G11 by mas. J Biol Chem. 2006;281:16757-16767.    7  Figure Legend Figure 1. New model of angiotensin-(1-7) action. Angiotensin II (Ang II) acts at the angiotensin type 1 receptor (AT1R) and stimulates (patho)physiological G protein signalling in cardiovascular disease, mainly through the Gq/i family. Ang II also activates G protein-independent signalling via recruitment of -Arrestin2 (Arr2) leading to therapeutic effects. The counter-regulatory renin angiotensin system axis peptide angiotensin-(1-7) [Ang-(1-7)] mainly acts at Mas to stimulate diverse therapeutic effects in different diseases via Gs activation. Ang-(1-7) also binds the AT1R leading to Arr2 recruitment and vasorelaxation in isolated aortas.            8     

Stuart A. Nicklin

Crossref

Hypertension