Chapter 5

AT1R–AT2R Cross Talk

Carmine Savoia, Massimo Volpe
Clinical and Molecular Medicine Department, Sapienza University of Rome, Rome, Italy

CROSS REGULATION OF ANGIOTENSIN II TYPE 1 RECEPTOR AND THE ANGIOTENSIN II

TYPE 2 RECEPTOR
The renin–angiotensin system (RAS) is an enzymatic and hormonal cascade involved in blood pressure regulation and
cardiovascular and renal homeostasis. Angiotensin II (Ang II) is the main effector of the RAS and exerts its biological
­action by binding with high affinity to two distinct subtype receptors, the Ang II type 1 receptor (AT1R) and the Ang II
type 2 receptor (AT2R).1–5 Both AT1R and AT2R belong to the family of seven-transmembrane G protein-coupled receptors
(GPCRs).1–3
The functional significance of AT1R and AT2R in cardiovascular and renal pathophysiology has been investigated in vivo
and in vitro.1–5 It has been recognized that AT1R plays a critical role in the Ang II-mediated actions in the cardiovascular sys-
tem and the kidney, by inducing vasoconstriction, blood pressure increase, water and sodium retention, growth promotion,
fibrosis, and inflammation in several pathophysiological conditions.3–5 Conversely, the role of AT2R is much less character-
ized, particularly in humans.1–3 One of the limitations for the characterization of this subtype receptor is that, in adult life,
AT2R is often expressed at the detection limit in few organs and tissues including the coronary arteries, cardiomyocytes,
ventricular myocardium,1,2,6 peripheral vasculature,7,8 and kidney.9 Despite its low expression level, the distribution and den-
sity of AT2R in adult tissues may be related to the involvement of this subtype receptor in cardiovascular and renal function.
In vitro and in vivo studies have shown that AT1R stimulation may regulate the expression of AT2R, suggesting the
existence of a complex cross-regulatory mechanism between the two subtype receptors.2,3,7–11 For instance, Ang II-induced
AT2R expression was enhanced in vascular smooth muscle cells (VSMCs) by AT1R blockade, suggesting that AT1R may
control AT2R expression.10 In endothelial cells transfected with the AT2R promoter, Ang II-induced expression of AT2R
mRNA was blunted by AT1R stimulation.11 Low levels of AT2R mRNA and protein have been detected in the vasculature
of normotensive and hypertensive rats.7,10 The expression of the receptor was increased only in hypertensive animals, in
which it contributed to vasodilation after in vivo chronic AT1R blockade,7,10 suggesting that the cross-regulatory mechanism
between the Ang II subtype receptors works mostly in pathological conditions at transcription level in the vasculature of
rodents.
Few studies have investigated the expression and the function of AT2R in humans, particularly in the cardiovascular sys-
tem. AT2R has been detected in vascular endothelial cells, fibroblasts, and macrophages of the human lung.12 In the human
peripheral arteries, AT2R has been detected in the coronary circulation,13 particularly in the coronary microcirculation.14 We
have reported15 that AT2R is upregulated in the vascular wall of peripheral resistance arteries from hypertensive diabetic
patients only in the presence of selective AT1R blockade with the angiotensin receptor blocker (ARB) valsartan, suggest-
ing that the cross talk regulatory mechanism between AT1R and AT2R may occur also in humans, particularly in high-risk
cardiovascular patients.15
In the vasculature, the negative cross talk between AT2R and AT1R may be characterized also by a negative modulation
of AT1R expression and AT1R-mediated signaling in the presence of AT2R stimulation and/or overexpression.16,17 Several
lines of evidence support this hypothesis. It was reported that AT1R expression was significantly higher in AT2R knockout
mice than in control animals.16 Overexpression and activation of AT2R downregulated AT1aR expression in rat VSMC in an
Ang II-independent manner that involved the activation of the bradykinin (BK)/nitric oxide pathway.18 Moreover, transfec-
tion of the AT2R gene in rat VSMC inhibited AT1R-mediated tyrosine phosphorylation of signal transducers and activators
of transcription (STAT) 1a/b, STAT2, and STAT3.17 The counterregulatory activity of AT2R on AT1R may be relevant also
in the renal pathophysiology involving increased activation of RAS. In the kidney, AT2R regulated AT1R expression at tran-
scriptional and functional level via the nitric oxide/cyclic guanosine monophosphate (cGMP)/Sp1 pathway.19 In particular,
The Protective Arm of the Renin–Angiotensin System (RAS). http://dx.doi.org/10.1016/B978-0-12-801364-9.00005-5
© 2015 Elsevier Inc. All rights reserved. 35
36  The Protective Arm of the Renin–Angiotensin System (RAS)

AT2R attenuated the ability of Ang II to increase Na+/K+/ATPase activity in rat immortalized renal proximal tubule (RPT)
cells, effects that were supported also in studies in RPT cells from AT2R mice.19 These evidences may further explain the
mechanisms of modulation of sodium excretion and blood pressure control.
Apart from the regulation of AT1R transcription and function via nitric oxide/cGMP pathway, AT2R may also regulate
AT1R expression via a direct protein–protein interaction. In this regard, it has been shown that GPCRs are able to both
homo­dimerize and heterodimerize.20–22 For instance, following agonist stimulation, beta-2 adrenergic, muscarinic, dopa-
mine D2, and opioid receptors undergo homodimerization,21,22 suggesting that it may be possible to modulate receptor
function through intermolecular interactions. Homodimers of AT1R or AT2R are important for inducing cell signaling. The
elevated levels of AT1R dimers on monocytes promoted atherogenesis in ApoE-deficient mice. In addition, constitutively
active homodimerization of AT2R was localized in the cell membrane without Ang II stimulation and induced apoptosis
without changes in receptor conformation.23 AT1R may heterodimerize with BK type B2 receptor,24 dopamine receptor,25
endothelin type B receptor,26 Mas receptor,27 and AT2R.28 The functional role of homo- or heterodimerization of Ang II
receptors, however, still remains controversial.
There is evidence that heterodimerization may explain the antagonistic functions of AT1R and AT2R. The occurrence
of a detected AT1R and AT2R heterodimerization that leads to AT1R signal inhibition may be independent of the bind-
ing of Ang II to AT2 receptor. The overexpression of AT2R induced apoptosis of fibroblasts in the absence of Ang II.29
Furthermore, the overexpression of the AT2R after transfection into cultured rat VSMC may induce downregulation of
AT1aR expression in a ligand-independent manner that is possibly mediated by the BK/nitric oxide pathway. This, in turn,
can reduce the basal DNA synthesis and proliferation of VSMCs and abolish the response of DNA synthesis to Ang II
in VSMCs.18 It is therefore possible that increased production of nitric oxide by overexpression of AT2R could suppress
AT1aR expression by the inhibition of its transcription. These data underline that AT2R has constitutive activity,23,29 which
might induce heterodimerization independent of Ang II stimulation.
Conversely, it has been shown that AT2R formed preferably homodimers, rather than heterodimers with AT1R, on the
cell surface, and subsequently induced cell signaling including the antagonism of phospholipase C-b3 phosphorylation.30
Interestingly, the expression levels of homodimerized AT1R or AT2R on the cell surface did not change after treatment with
Ang II or with the selective ARBs and PD123319, respectively.30 Hence, that evidence suggests that Ang II-induced AT1R
signaling may be mainly blocked by AT2R signaling through their negative cross talk in the cytoplasm rather than by the
heterodimerization of both receptors on the cell surface. In this regard, the balance of the expression levels of AT1R and
AT2R receptors may be critical for the mutual antagonistic actions.

FUNCTIONAL SIGNIFICANCE OF THE AT1R–AT2R CROSS TALK

In the vasculature, AT2R induces vasorelaxation in vivo in both resistance and capacitance vessels.7,10,15,31 In vitro experi-
ments in cultured cells and in isolated vessels as well as in vivo experiments in mice and rats have shown a link between
AT2R stimulation and the nitric oxide/cGMP-dependent pathway.31 This occurs either directly32 or indirectly trough en-
hanced BK formation7 or increased eNOS activity/expression.33
A large body of evidence suggests that AT2R may countervail many of the AT1R-mediated actions in the vasculature
under physiological and pathological circumstances. This may occur through mechanisms independent of direct AT2R
stimulation or by a cross-interaction with AT1R functional expression and signaling,9 suggesting that the regulatory cross
talk between AT1R and AT2R is important at a functional level.
Aortic AT1R expression and vascular response to Ang II are greater in AT2R knockout than wild-type mice,16 sug-
gesting that AT2R may counter the AT1R-induced effect on blood pressure. Chronic Ang II administration induced vaso-
dilation and hypotension in rats after AT1R blockade, and this response was abolished by coadministration of the AT2R
antagonist PD123319.7 Recently, it has been shown that direct AT2R agonist compound 21 (C21) promoted vasorelax-
ation in vitro, which in turn was associated with vasodepressor responses only in conscious spontaneously hypertensive
rats (SHR) previously treated with an ARB.34 Data from Ang II receptor knockout mice also confirm the existence of
a physiological cross talk between AT1R and AT2R at the level of the modulation of vascular tone.9,35 Several reports
indicate that the vascular functions of AT2R are unmasked when AT1R is inhibited7,10,33,36 using ARBs. Taken together,
those data further underline the occurrence of a functional cross talk between AT1R and AT2R in blood vessel function.
AT2R is highly expressed in the fetal kidney. Its expression gradually decreases after birth, but AT2R continues to be
expressed in the adult kidney, though at low levels.37 AT2R is expressed throughout the kidney in both vascular and tubular
elements mainly in RPT cells.34 AT2R stimulation inhibits the activity of Na+/K+/ATPase in the proximal tubules isolated
from adult rats, mice, and rabbits,38,39 inducing in turn natriuresis, which may also be modulated by the interactions of AT2R
with dopamine D1 receptors.40
 AT1R–AT2R Cross Talk Chapter | 5  37

Selective intrarenal AT1R blockade in rats induced a highly significant natriuretic response that was abolished with
intrarenal coinfusion of the AT2R antagonist PD 123319.39 These results further indicate that, similar to vasodilation, the
beneficial natriuretic response to ARB administration is related to AT2R activation. In the 5/6 nephrectomy model, which
developed a time-dependent increase in AT2R expression at 7, 15, and 30 days after renal ablation, pretreatment with the
ARB losartan showed a further increase in AT2R expression. Treatment with the AT2R antagonist PD123319 was asso­ciated
with downregulation of AT2R, increased renal damage, and increased blood pressure, suggesting that the AT2R represents
a beneficial counterregulatory mechanism to protect the kidney from ischemic injury.41 Recent evidence showed that the
AT2R-mediated renal vasodilator effects after stimulation with C21 are unmasked by ACE inhibition in SHR but not in
normotensive rats. Thus, the upregulation of renal vascular AT2R, particularly in hypertension, is associated with a coun-
tervailing function opposing the increased AT1R-mediated tonic renal vasoconstriction and underlines the relevance of the
occurrence of a functional cross talk between AT1R and AT2R also in the kidney.42
A functional counterregulatory mechanism at the expression and signaling level among Ang II subtype receptors and
growth-promoting receptors may also occur and contribute to the pathophysiology of the Ang II-induced damage in the
cardiovascular system.
It has been well established that Ang II exerts growth-promoting effects via activation of multiple signaling cascades
including the activation of ERK and PI 3-K/Akt pathways and increased expression of growth factors including transform-
ing growth factor-β I (TGF-β type I),43,44 in the cardiovascular system. It has been shown that the Ang II-induced activation
of ERK and PI 3-K/Akt cascades required AT1R-operated transactivation of EGFR in VSMC45 and fibroblasts.46 These
actions were counteracted by AT2R activation in VSMC47 and in Ang II-stimulated fibroblasts of human hypertrophic
scars.46
In mouse fibroblasts, both Ang II receptors were able to phosphorylate ERK1/2. However, in the cells expressing only
AT1R, the EGF-induced MAPK pathway was enhanced in the presence of Ang II in a synergistic fashion. In contrast, a
reduction of EGF-induced MAPK activation was observed in the cells expressing only AT2R. In fibroblasts expressing both
Ang II subtype receptors, Ang II promoted an enhancement of EGF-induced MAPK activation. However, in the presence of
an ARB, the effect of the EGF receptor was reduced, suggesting the existence of an opposite cross talk of AT1R and AT2R
with EGF receptors. This contributes to explain a complex functional interaction between these pathways in the regulation
of cellular growth processes.48 Furthermore, there is evidence that overexpression of AT2R downregulated the AT1aR in
VSMCs from WKY but not in cells from SHR. Similarly, overexpression of AT2R abolished DNA synthesis in response
to Ang II in VSMCs from WKY, whereas DNA synthesis in response to Ang II was not altered in SHR. These phenomena
may be a consequence of the downregulation of the AT1aR only in VSMCs from WKY, and not from SHR. The lack of
downregulation of the AT1aR in SHR may contribute, in part, to the exaggerated VSMC growth in SHR.49

CONCLUSION
In conclusion, the occurrence of a functional counterregulatory mechanism between AT1R and AT2R, as well as a functional
cross talk among Ang II receptors and growth-promoting receptors, may further explain the mechanisms of the pathophysi-
ology of cardiovascular and renal damage linked to RAS activation. These mechanisms are also relevant to understand the
pathophysiology and pharmacology of Ang II receptors, which are important to find specific and selective therapies for
cardiovascular protection.

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 AT1R–AT2R Cross Talk Chapter | 5  39

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