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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2018 Apr 17;175(15):3063–3079. doi: 10.1111/bph.14172

GPCRs in pulmonary arterial hypertension: tipping the balance

Jean Iyinikkel 1, Fiona Murray 1,✉
PMCID: PMC6031878  PMID: 29468655

Abstract

Pulmonary arterial hypertension (PAH) is a progressive, fatal disease characterised by increased pulmonary vascular resistance and excessive proliferation of pulmonary artery smooth muscle cells (PASMC). GPCRs, which are attractive pharmacological targets, are important regulators of pulmonary vascular tone and PASMC phenotype. PAH is associated with the altered expression and function of a number of GPCRs in the pulmonary circulation, which leads to the vasoconstriction and proliferation of PASMC and thereby contributes to the imbalance of pulmonary vascular tone associated with PAH; drugs targeting GPCRs are currently used clinically to treat PAH and extensive preclinical work supports the utility of a number of additional GPCRs. Here we review how GPCR expression and function changes with PAH and discuss why GPCRs continue to be relevant drug targets for the disease.

Abbreviations

BMPR2

bone morphogenetic protein receptor 2

CH

chronic hypoxic

IPAH

idiopathic pulmonary arterial hypertension

MCT

monocrotaline

NAM

negative allosteric modulator

PAEC

pulmonary artery endothelial cells

PAH

pulmonary arterial hypertension

PAM

positive allosteric modulator

PASMC

pulmonary artery smooth muscle cells

Introduction

The normal pulmonary circulation is a low resistance and high flow circulation, which is maintained by locally produced or circulating vasomodulators. An imbalance in the activity of vasoconstrictor/proliferative and vasodilator/anti‐proliferative mediators in the pulmonary circulation leads to remodelling of the pulmonary artery. Structural changes in the pulmonary artery, a key feature of which is the proliferation of pulmonary artery smooth muscle cells (PASMC) leads to increased pulmonary vascular tone that can manifest as pulmonary arterial hypertension (PAH). PAH is a progressive disease that is characterized by pulmonary artery pressure greater than 25 mmHg (Lau et al., 2017); right ventricular function and hypertrophy are major determinants in the prognosis of PAH (Maarman et al., 2017). PAH includes patients with similar pathophysiological, histological and prognostic features; PAH can be idiopathic, heritable [70% of which are associated with mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene], or secondary to drug/toxin exposure or to other conditions, such as connective tissue disease (Lau et al., 2017). Restoring the imbalance in pulmonary vascular tone is a key endpoint of drugs used clinically to treat PAH.

Many of the vasoactive mediators in the pulmonary circulation, such as http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=989, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2504 (ANG‐II), http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1915 and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1152, act via GPCRs expressed on the vasculature, in particular on PASMC and pulmonary artery endothelial cells (PAEC) (Barnes and Liu, 1995; Morrell et al., 2009; Murray et al., 2011). GPCRs are the largest receptor family in the human genome and successful therapeutic targets due to their tissue and cell specific distribution and accessibility on the plasma membrane (Insel et al., 2012). Altered expression and function of a number of GPCRs and circulating levels of their endogenous ligand are associated with the progression of PAH, which when taken together, contributes to the increased pulmonary vascular tone by tipping the balance of homeostatic signalling in PASMC to favour vasoconstriction and proliferation. We will provide an overview of GPCRs, in particular those whose expression are altered in PASMC with PAH, and discuss how both ‘old’ and ‘new’ GPCRs are relevant targets to restore the imbalance in pulmonary vascular tone.

GPCRs in the pulmonary circulation

GPCRs are guanine nucleotide exchange factors for heterotrimeric G proteins, whose α and βγ subunits dissociate upon ligand binding leading to the activation/inactivation of signalling pathways that control the production of second messengers, the activity of intracellular proteins and the expression of various genes (Rajagopal et al., 2010; Murray et al., 2011; Insel et al., 2012). Although GPCRs can couple to more than one G protein, they are usually classified based on the G protein they preferentially activate. G proteins are divided into four main classes according to their α subunit: Gαs, Gαi, Gαq/11 and Gα12/13, although Gβγ can also act as a single entity to initiate signalling. G protein‐independent signalling also occurs on GPCR activation via β‐arrestin recruitment that contributes to GPCR internalization and downstream signalling such as ERK activation, gene transcription and growth factor receptor transactivation (Figure 1). Both G protein and β‐arrestin mediated signalling are key components in a complex signalling network that controls the pulmonary circulation; however, for many GPCRs, the relative contribution of each of these pathways to the overall physiological response of ligands in the pulmonary artery remains to be fully explored (Murray et al., 2011).

Figure 1.

Figure 1

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GPCR‐mediated signalling in pulmonary artery smooth muscle cells. Gα and βγ subunits dissociate upon receptor activation and initiate signalling. Additionally, recruitment of β‐arrestins can also initiate G‐protein independent signalling and trafficking. Gαs stimulates the production of cAMP via AC, leading to the activation of PKA and Epac, thus vasodilating PASMCs and decreasing proliferation. Gαi activation inhibits AC activity thereby reducing cAMP, which in turn leads to PASMC vasoconstriction and proliferation. Gαq activation promotes the hydrolysis of phosphatidylinositol 4,5‐bisphosphate (PIP2) generating intracellular messengers 1,2‐DAG and inositol 1,4,5‐trisphosphate (IP3). DAG activates PKC while IP3 stimulates intracellular release of Ca2+ which then form a complex with a Ca2+ binding protein‐calmodulin. Gα12/13 activation increases RhoGEF and Rho kinase (ROCK) further promoting vasoconstriction. AR: adrenoceptor.

In general, activating Gαi‐, Gαq/11‐ and Gα12/13‐dependent signalling leads to vasoconstriction and proliferation of PASMC, whereas Gαs‐dependent signalling leads to vasodilatation and decreased proliferation (Figure 1). Gαs‐coupled GPCRs increase [cAMP]i, by activating http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=257 (ACs), which increases the activity of downstream mediators such as PKA and exchange protein directly activated by cAMP (http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=259). PKA also phosphorylates targets such as myosin light chain (MLC) kinase to decrease its activity, whereas Epac increase Rap‐1, both resulting in vasodilatation and decreased proliferation of PASMC. The ability of cAMP to also regulate gene transcription, for example, via the cAMP response element binding protein, means its physiological effects can persist long after GPCR activation. In contrast, activation of Gαi, Gαq/11 and Gα12/13 tend to produce overlapping biological responses in PASMC, which leads to increased Ca2+ sensitization and the phosphorylation of MLC, promoting actin–myosin cross‐bridging and PASMC contraction. Activation of Gαi‐coupled GPCRs oppose the effects of Gαs‐coupled GPCRs by decreasing [cAMP]i by inhibiting ACs. Gαq/11‐coupled GPCRs activate PLC leading to increased inositol‐1,4,5‐triphosphate (IP3) and [Ca2+]i and the phosphorylation of target proteins, such as Ca2+‐calmodulin dependent PK that activates MLC kinase, leading to vasoconstriction. In parallel, DAG promotes the association of PKC to the membrane, which phosphorylates a number of contractile proteins. Increased Ca2+ sensitization and sustained PASMC vasoconstriction can also result in stimulation of Gα12/13‐coupled GPCRS, which activate Rho GEFs (a low MW monomeric G protein) and Rho kinase (ROCK) that phosphorylate the MLC phosphatase and inhibit its activity; Gαq/11 also increases ROCK. The expression and activity of GPCRs is an important determinant in the amplitude of second messengers and downstream signalling in PASMC.

An updated list of GPCRs that are known regulators of the pulmonary vascular circulation is provided in Table 1. In PASMC, at least 33 GPCRs have been characterized, some with multiple coupling; Gαs (11 GPCRs), Gαi (eight GPCRs), Gαq/11 (16 GPCRs) and Gα12/13 (four GPCRs). In parallel, in PAEC at least 18 GPCRs have been characterized, again with multiple coupling; Gαs (two GPCRs), Gαi (five GPCRs), Gαq/11 (14 GPCRs) and Gα12/13 (11 GPCRs). Taken together, these data show that the low tone of the pulmonary circulation is, at least in part, the consequence of the relative high abundance of Gαq/Gα12/13‐coupled GPCRs in PAEC and Gαs‐coupled GPCRs in PASMC. GPCR ligands can have differing effects in the pulmonary circulation depending on the expression of the predominant receptor subtype, the function of the endothelium, the species being investigated and the initial tone of the pulmonary circulation; low basal tone in the pulmonary artery means vasodilators have little effect (Barnes and Liu, 1995; Murray et al., 2011). ET‐1 or 5‐HT antagonists do not vasodilate the normal pulmonary circulation but can attenuate hypoxia‐ or disease‐induced pulmonary vasoconstriction where tone is increased (Bonvallet et al., 1994; Barnes and Liu, 1995; Murray et al., 2011). An intact endothelium is vital for maintaining the low tone of the pulmonary circulation. Endothelium dysfunction, as seen in PAH, can shift the response of circulating mediators to vasoconstriction since many of the endogenous mediators that stimulate release of NO and PGI2 via GPCRs on PAEC lead to vasoconstriction if they directly act on PASMC, via Gαi/Gαq/11/Gα12/13 (Morrell et al., 2009). Once endothelial‐dependent relaxation is attenuated, the expression and activity of GPCRs on PASMC drive remodelling and increased vasoconstriction of the pulmonary artery; PASMC are an important cellular target for PAH.

Table 1.

GPCR expression and function in pulmonary vascular cells

Endogenous ligand GPCR Cell type G‐protein Cell specific response References
Angiotensin‐II AT1 PASMC Gαq/11 Proliferation/Vasoconstriction/Anti‐apoptosis (Yamada et al., 1996; Morrell et al., 1999)
AT2 PAEC Gαi Vasodilation/ anti‐proliferation/ Apoptosis (Lee et al., 2010a,b; Bruce et al., 2015)
ANG 1–7 MAS PAEC Gαq Vasodilation (Shenoy et al., 2010)
Endothelin‐1 ETA PASMC Gαq Proliferation/ Vasoconstriction (MacLean et al., 1994; Shichiri et al., 1997; Mcculloch et al., 1998; Sakai et al., 2016)
ETB PASMC Gαq Proliferation/ Vasoconstriction/ Anti‐apoptosis
ETB PAEC Gαq Vasodilation/ Anti‐apoptosis
NA/adrenline β2‐AR PASMC Gαs Vasodilation (Boe and Simonsson, 1980; Leblais et al., 2008)
α1‐AR PASMC Gαq / Gα12/13 Vasoconstriction
α2‐AR PAEC Gαq Vasodilation
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=294 http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=13 PAEC Gαq Vasodilation (Norel et al., 1996)
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=15 PAEC Gαq Vasodilation
M3 PASMC Gαq Vasoconstriction
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=649 http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=42 PAEC Gαq Vasodilation/ Apoptosis (Taraseviciene‐Stewart et al., 2005)
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2168 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=66 PAEC Gαq Vasodilation (Smith et al., 2006)
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=683 http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=47 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=11 PASMC Gαs Vasodilation (Upton et al., 2001)
PAEC Gαq Vasodilation
VIP VPAC1 PASMC Gαs Vasodilation (Busto et al., 2000)
VPAC2 PASMC Gαs Vasodilation
Ca2+, Mg2+, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=710 CaS PASMC Gαq, Gαi Proliferation/ Vasoconstriction (Li et al., 2011)
CGRP CGRP PASMC Gαs Vasodilation/ anti‐proliferation (Chattergoon et al., 2005)
CGRP PAEC Gαq
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2098 http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=360 1 PAEC Gαq Vasodilation (Pedersen et al., 2000)
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=361 PASMC Gαq Vasoconstriction
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1204 http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=262 PASMC Gαq Vasoconstriction (Ortiz et al., 1992)
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2153 http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=365 PASMC Gαq Vasoconstriction (MacLean et al., 2000a,b)
Adenosine A2A PASMC Gαs Vasodilation/ Apoptosis (Morgan et al., 1991; Xu et al., 2011; Huang et al., 2015)
A2B PASMC Gαs Vasodilation
Oxytocin, Vasopressin OT PASMC Gαi, Gαq Vasoconstriction (Roberts et al., 1992; unpublished data)
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1713, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1712, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1734, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1749 http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=324 , http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=325 , http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=326 PASMC Gαq Vasoconstriction (McCormack et al., 1989; Chootip et al., 2002)
PAEC Gαq Vasodilation
5‐HT 5‐HT1B PASMC Gαi Proliferation/ Vasoconstriction/ Anti‐apoptosis (Morecroft and MacLean, 1998; Hoyer et al., 2002; Liu et al., 2013)
5‐HT2A PASMC Gαq / Gα12/13 Proliferation/ Vasoconstriction/ Anti‐apoptosis
5‐HT2B PASMC Gαq Proliferation/ Vasoconstriction/ Anti‐apoptosis
Prostacyclin IP PASMC Gαs Vasodilation/ anti‐proliferation (Shaul et al., 1991)
PGE1/2 EP1 PASMC Gαq Vasoconstriction (Hirata and Narumiya, 2011)
EP2 PASMC Gαs Vasodilation
EP3 PASMC Gαi Vasoconstriction
EP4 PASMC Gαs Vasodilation
PGD2 DP1 PASMC Gαs Vasodilation
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4482 FP and TP PASMC Gαq Proliferation/ Vasoconstriction (Cogolludo, 2003)
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2452 http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=275 PAEC Gαi PAEC barrier protection (Ancellin and Hla, 1999; Garcia et al., 2001; Birker‐Robaczewska et al., 2008)
SIP2 PASMC Gαq / Gα12/13 Proliferation
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=277 PAEC Gαq / Gαi / Gα12/13 PAEC barrier dysfunction
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4453 PAR1 PAEC Gαq / Gα12/13 PAEC barrier dysfunction (Sacks et al., 2008)
PAR1/PAR2/PAR3 PASMC Gαq / Gα12/13 Proliferation/ Vasoconstriction
Apelin; Elabela/Toddler apelin PAEC Gαq /Gαi/o Vasodilation (Japp et al., 2008; Yang et al., 2015)
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1504 http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=305 PASMC Gαi/o Proliferation/ Vasoconstriction (Crnkovic et al., 2014)
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=306 PASMC Gαi/o Vasoconstriction
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=307 PASMC Gαi/o Vasoconstriction
Oestradiol GPER PAEC Gαi/Gαs Vasodilation (Alencar et al., 2017)
PASMC Gαi / Gαs Vasodilation/ Anti‐proliferative
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PASMC, Pulmonary artery smooth muscle cells; PAEC, endothelial cells.

PAH‐PASMC have decreased cAMP and increased [Ca2+]i compared with control‐PASMC (Zhang et al., 2007; Murray et al., 2011), which can be attributed at least in part, to the altered expression and/or activity of GPCRs in PASMC; circulating or tissue levels of endogenous GPCR agonists are seen in PAH (Table 2). A number of GPCR agonists and antagonists, as reviewed in Table 2, have been shown to reverse or blunt PAH both clinically and preclinically by restoring the balance of second messengers in PASMC; the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=345 and http://www.guidetopharmacology.org/GRAC/FamilyIntroductionForward?familyId=21s are the targets of drugs currently approved to treat PAH (Lau et al., 2017). GPCRs whose altered expression or activity contribute to the imbalance of pulmonary vascular tone with PAH are outlined below: a Gαi/Gαq/G12/13 versus Gαs shift is evident with PAH.

Table 2.

GPCRs targeted clinically/preclinically in PAH

GPCR Expression In PAH Level of endogenous ligand Drug Effect References
Clinical/preclinical Increased 6MWD and improved PA remodelling ETA/B ↑ ↑ Bosentan Antagonist (clinical) (Rubin et al., 2002)
ETA http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3951 Antagonist (clinical) (Galie et al., 2008)
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7352 (Pulido et al., 2013)
*http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3950 (Barst et al., 2004)
VPAC2 ↑ ↓ Aviptadil Agonist (clinical, in vivo) (Petkov et al., 2003; Leuchte et al., 2008)
IP ↓ ↓ Epoprostenol Analogue (clinical) (Barst et al., 1996)
Iloprost (Olschewski et al., 2002)
Treprostinil (Simonneau et al., 2002)
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1967 (Galiè et al., 2002)
Selexipag Agonist (clinical) (Simonneau et al., 2012)
Preclinical Improved Pulmonary artery remodelling & Right heart function – – – http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5852 Agonist (in vitro, in vivo) (Fuchikami et al., 2017)
UT – – http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2167 Antagonist (in vivo, in vitro) (Mei et al., 2011)
CaS ↑ – http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=716 NAM (in vivo, in vitro) (Tang et al., 2016)
Apelin ↓ ↓ Apelin Agonist (clinical) (Brash et al., 2015)
Apelin/ ELA Agonist (in vitro, in vivo) (Yang et al., 2017)
A2B – – http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2844 Agonist (clinical) (Morgan et al., 1991; Rossi et al., 2018)
A2A – LASSBio‐1359 Agonist (in vitro, in vivo) (Alencar et al., 2013)
AT1 ↑ ↑ http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=590 Antagonist (in vitro, in vivo) (Morrell et al., 1999; de Man et al., 2012aa, b)
AT2 ↓ C‐21 Agonist (in vitro, in vivo) (Bruce et al., 2015)
5‐HT1B ↑ ↑ GR127935 Antagonist (in vitro, in vivo) (Keegan et al., 2001)
SB216641 (Hood et al., 2017)
5‐HT2A ↑ Ketanserin Antagonist (in vitro) (McGoon and Vlietstra, 1987; Frishman et al., 1995)
5‐HT2A/B Terguride Antagonist (in vitro, in vivo) (Launay et al., 2002; Dumitrascu et al., 2011)
5‐HT2B PRX‐08066 Antagonist (in vitro, in vivo) (Porvasnik et al., 2010)
S1P2 ↑ ↑ http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2917 Antagonist (in vitro, in vivo) (Chen et al., 2014a,b)
β2‐AR – – Bisoprolol/ Carvedilol Antagonist (in vivo, in vitro) (Perros et al., 2017)
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7246 Agonist (in vivo, in vitro) (Perros et al., 2015)
B2 – – B9972 Agonist (in vitro, in vivo) (Taraseviciene‐Stewart et al., 2005)
M3 – – C1213 Agonist (in vivo, in vitro) (Ahmed et al., 2016)
GPER – – http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1014 Agonist (in vitro, in vivo) (Alencar et al., 2017)
MAS ↓ – ACE‐2 and Ang 1–7 Agonists (in vitro, in vivo) (Shenoy et al., 2010)
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(–) No reported data; (↑) Up‐regulated; (↓) Down‐regulated; (*) Withdrawn from clinical use; (6MWD) 6 min walking distance; (PA) pulmonary artery; (RH) right heart; (AR) adrenoceptor.

GPCRs that contribute to the imbalance of pulmonary vascular tone with PAH

Prostanoid receptors

Prostanoid receptors, which include http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=338 , http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=339, http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=58, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=344, IP and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=346, are activated by http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1881, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1882, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1883, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1884, PGI2 and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4483 / http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4482 respectively. IP, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=341, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=343 and DP1 are Gαs‐coupled and therefore increase cAMP and are vasodilatory in PASMC, whereas http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=340, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=342, and FP and TP are Gαq/11 or Gαi‐coupled so increase [Ca2+]i or decrease cAMP and lead to vasoconstriction of PASMC (Hirata and Narumiya, 2011). The main eicosanoids, produced via metabolism of arachidonic acid, in the pulmonary circulation are PGI2 and PGE2, which are vasodilators, and PGF2α and TXA2, which are vasoconstrictors; PGI2 synthase predominates in PAEC and directs metabolism towards PGI2, which acts on PASMC to keep normal pulmonary tone low. This homeostatic balance is however dysregulated in PAH, which results in decreased levels of PGI2‐ and increased levels of TXA2‐ in lungs and urine of PAH patients (Christman et al., 1992). Increasing PGI2 production by overexpressing PGI synthase in mice prevents the development of PAH (Geraci et al., 1999). Intravenous prostacyclin (http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1915) and more stable, inhaled and/or orally active prostacyclin analogues, such as http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5820 and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1895, are approved in the UK for PAH and improve haemodynamics and exercise tolerance, long‐term survival in patients and importantly reduce the need for lung transplantation (McLaughlin et al., 2015).

All prostanoid receptors are expressed at the level of mRNA in the pulmonary circulation; however, the extent to which they control vascular tone is not fully understood (Hirata and Narumiya, 2011). In the human pulmonary artery, the IP receptor, a Gαs‐coupled GPCR whose activation increases cAMP, is highly expressed and functional and the primary therapeutic target of the prostacyclin analogues; the severity of hypoxic‐induced PAH is greater in IP receptor‐deficient mice (Hoshikawa et al., 2001; Falcetti et al., 2010). PAH is associated with reduced IP and DP receptor expression (both Gαs‐coupled) and increased EP3 receptor expression (Gαi‐coupled), which taken together could attenuate the vasodilatory effect of endogenous eicosanoids in PASMC (Table 2). Since each prostacyclin analogue has a different pharmacological profile, altered prostanoid receptor expression may even determine their full clinical response. For example, in addition to the IP receptor, iloprost has high affinity for EP1 receptors whose activation in PASMC would initiate vasoconstriction, whereas treprostinil has high affinity for DP1 and EP2 receptors whose activation would enhance PASMC vasodilatation (Whittle et al., 2012): reduced IP and DP expression could blunt the vasodilatory response of these analogues. Furthermore, prostacyclin analogues have also been shown to have prostanoid receptor independent effects, via KCNK3, clearing ET‐1 and PPAR‐γ (Olschewski et al., 2006; Falcetti et al., 2007). Highly selective IP receptor agonists, such as http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7552 and its active metabolite MRE‐269/ACT‐333679 have been developed and shown to reduce PASMC proliferation, inhibit PAH in models of the disease and relax isolated pulmonary artery (Morrison et al., 2012; Fuchikami et al., 2017). Selexipag decreases the risk of morbidity/mortality of PAH patients alone, or in combination with other therapies (McLaughlin et al., 2015).

A major problem with the use of most GPCR agonists is that their biological response can diminish over time, which requires that the dose needs to be increased to maintain efficacy (Lefkowitz, 1993). Such desensitization can be attributed to receptor phosphorylation and internalisation and reduced receptor expression. Desensitization and internalization of the IP receptor has been seen both in vitro and in vivo, which attenuates the vasoreactivity of prostacyclin analogues (Schermuly et al., 2007); recent advances in the pharmacology of the IP receptor could help reduce receptor desensitization and enhance the efficacy of drugs. Of interest, MRE‐269/ACT‐333679 has been shown to act as a full agonist in terms of vasodilatation and inhibition of PASMC proliferation, but a partial agonist in terms of recruitment of β‐arrestin and IP receptor internalisation. In vivo this pharmacological profile translates to sustained efficacy in animal models of PAH due to limited IP receptor desensitization (Morrison et al., 2012). Furthermore, an IP positive allosteric modulator (PAM) has been developed (IPPAM) (Yamamoto et al., 2017). PAMs are ligands that act at on allosteric sites to increase receptor function and potentiate the activity of the orthosteric ligand (Lefkowitz, 1993). PAMs have no intrinsic activity, increase selectivity and can also reduce receptor desensitization, therefore could have exciting therapeutic potential for PAH. IPPAM has been shown to enhance the effects of PGI2 in vitro; however, in vivo preclinical studies in models of PAH have yet to be completed (Yamamoto et al., 2017). Understanding the mechanism of the reduced expression of IP receptors with PAH and advances in drugs design continue to enforce the benefit of targeting this receptor in PAH.

Vasoactive intestinal peptide receptors

VIP and the related http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2257 are potent vasodilators of the pulmonary circulation and inhibit PASMC proliferation and platelet activation (Said, 2012). VIP has shown a protective role in the presence of pulmonary vasoconstrictors such as ET‐1 and attenuates or reverses the development of PAH in animal models (Boomsma et al., 1991; Hamidi et al., 2005; Hamidi et al., 2011). The effects of VIP and PACAP are mediated by VIP receptors (http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=371 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=372) and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=370, which are primarily Gαs‐coupled and expressed in PASMC (Busto et al., 2000); the VPAC2 receptor is highest expressed in human PASMC. PAH is associated with increased expression of VPAC1 and VPAC2 receptors (Petkov et al., 2003; unpublished data), which can be speculated to be a compensatory mechanism due to reduced serum VIP levels in PAH patients. One interesting observation, which could be important in relation to the increased VPAC receptor expression associated with PAH, is that VIP activates PLC and increases [Ca2+]i in stable cell lines overexpressing VPAC (MacKenzie et al., 1996). It would be interesting to determine if VIP‐mediated G protein‐dependent signalling differs in PAH‐PASMC.

Mice lacking the VIP gene develop a moderate form of PAH and right ventricular hypertrophy, which is attenuated by VIP treatment (Said et al., 2007). PAC1 KO mice develop PAH soon after birth, which suggest this receptor may also be key in the regulation of pulmonary vascular tone (Otto, 2004). However, VPAC2 KO mice do not develop PAH, and pulmonary remodelling has not been reported in VPAC1 KO mice, suggesting these receptors may have redundant roles in PASMC (Asnicar et al., 2002; Fabricius et al., 2011). VPAC2, but not VPAC1 selective agonists have been shown to improve right ventricular systolic pressure, in animal models of PAH, implying that the VPAC2 receptor could be a more promising target for PAH (Koga et al., 2014). Although original clinical trials with VIP (Aviptadil) showed reduced pulmonary vascular resistance and improved stroke volume (Petkov et al., 2003; Leuchte et al., 2008), additional trials showed no benefit (Said, 2012). Future work needs to fully dissect VPAC1, VPAC2 and PAC1 receptor‐dependent signalling in the pulmonary circulation and develop more specific and stable agonists that can be tested in the clinic: conjugating VIP to nanoparticles or co‐administration of VIP with a neutral endopeptidases inhibitor has been shown to prevent VIP degradation and augment its effects (Leuchte et al., 2015; Athari et al., 2016).

Endothelin receptors

ET‐1, which is produced and released predominantly by PAEC, is crucial for regulating pulmonary vascular tone and seen as a key mediator of PAH (Abman, 2009). ET‐1 mediates its action via two ET receptor subtypes: http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=219 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=220, which are Gαq‐coupled. PAEC express both ETA and ETB receptors, whereas PASMC predominately express ETA receptors (Table 1). ETB receptor activation in PAEC promotes vasodilatation by increased production of NO and PGI2 release, inhibits apoptosis and mediates the clearance of ET‐1 (Hirata et al., 1993). In contrast, ETA and ETB receptor activation in PASMC induces vasoconstriction of the pulmonary arteries (MacLean et al., 1994). Elevated levels of ET‐1 are observed in plasma and lungs of patients with PAH, and there is a direct correlation between ET‐1 concentrations and increased pulmonary vascular resistance (Giaid et al., 1993; Bauer, 2002). The expression and distribution of both ETA and ETB receptors are increased in the PAH‐PASMC and increased ETA receptor‐mediated vasoconstriction has been shown in both the large and small pulmonary arteries (Li et al., 1994); increased ETA and ETB receptors in PASMC contributes to increased tone of the PAH‐PASMC (MacLean et al., 1994). Dual endothelin receptor antagonists, such as http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3494, are approved for PAH and shown to improve time to clinical worsening (Rubin et al., 2002). Drugs selective for the ETA receptors were developed in order to preserve ETB receptor endothelial‐dependent vasodilatation and ET‐1 clearance, while inhibiting vasoconstriction and proliferation mediated by the ETA receptors: PAH is more severe in ETB deficient rats (Ivy et al., 2002; Wilkins, 2004). Ambrisentan, a potent ETA antagonist improves exercise capacity and haemodynamics and is utilized for initial combination therapy although longer studies are required to assess effect on mortality. Recent crystal structure identification of the ET receptor may help facilitate new rational drug design (Shihoya et al., 2016). Of interest, functional autoantibodies for ET‐1 have been shown to circulate and contribute to pathophysiology of disease by stimulating the receptor and have been associated with scleroderma‐induced PAH (Becker et al., 2014). Neutralisers of these autoantibodies have been implicated as a viable treatment, which could be therapeutically relevant for PAH in the future.

5‐HT receptors

The neurotransmitter 5‐HT (serotonin), which is synthesized in PAEC from L‐trytophan by tryptophan hydroxylase, is a potent pulmonary vasoconstrictor and mitogen that increases pulmonary artery remodelling and increases pulmonary vascular resistance (MacLean and Dempsie, 2010). PAH patients exhibit elevated levels of 5‐HT in plasma (Hervé et al., 1995) and increased hypoxia‐induced vascular tone and remodelling can be enhanced by 5‐HT (Eddahibi et al., 2000). Seven 5‐HT receptor families, six of which are GPCRs, mediate the response to 5‐HT; http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2 (Gαi), http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=6 (Gαq), http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=7 (Gαq) and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=12 (Gαs) receptors have been shown to be expressed in the pulmonary circulation (Ullmer et al., 1995; Morecroft and MacLean, 1998): 5‐HT1B and 5‐HT2B receptors, both of which would increase [Ca2+]i, are up‐regulated in biopsies from PAH patients and animal models of the disease (Launay et al., 2002). The 5‐HT1B receptor is a key mediator of 5‐HT‐induced vasoconstriction and proliferation in small and large human pulmonary arteries: RhoA activation and subsequent nuclear translocation of phosphorylated http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=514 and activity of GATA4 are key downstream pathways in PASMC activated by 5‐HT (Hoyer et al., 1994; MacLean et al., 1996). Inhibition of the 5‐HT1B receptor expression (5‐HT1B −/− mice) or activity (5‐HT1B antagonist‐http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=14) attenuated the chronic hypoxia associated vascular remodelling (Keegan et al., 2001). 5‐HT1B receptor antagonists have begun uncovering novel 5‐HT signalling pathways that elucidate the aberrant redox signalling in PAH remodelling (Hood et al., 2017). 5‐HT2B KO mice are protected from the development of hypoxia‐induced PAH and antagonists prevent pulmonary remodelling, which highlights this receptor as an additional target (Launay et al., 2002; Blanpain et al., 2003; West et al., 2016). Furthermore, 5‐HT2A receptors mediate contraction and proliferation of PASMC via a Gαq‐mediated increase [Ca2+]i and PKC activation (MacLean et al., 2000b), although only at 5‐HT concentrations above the normal physiological range; 5‐HT2A inhibits KV and hKV1.5 currents (Morecroft et al., 1999; MacLean et al., 2000b; Cogolludo, 2006). Unfortunately, 5‐HT receptor antagonists such as PRX‐8006 (5‐HT2B‐anatgonist), http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=88 (5‐HT2A‐anatagonist) or http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=56 (dual 5‐HT2A/B antagonist) have not shown much success clinically, with studies either having to be discontinued, due to the lack of specificity to these receptors in the pulmonary circulation; 5‐HT2A mediates systemic vasoconstriction (McGoon and Vlietstra, 1987; Dumitrascu et al., 2011).

One additional aspect of 5‐HT receptors, which still makes them relevant targets for PAH, is that their expression or function can regulate or be regulated by known risk factors of PAH. Appetite suppressants, a pharmacological risk factor for PAH, can increase 5‐HT and a dexfenfluramine metabolite is an agonist of 5‐HT2A and 5‐HT2B receptors (Eddahibi et al., 2001; MacLean et al., 2004). The sex hormone oestrogen, via decreasing miR96, up‐regulates the 5‐HT1B receptor, which implicates this GPCR in the female predominance of PAH (White et al., 2011). A mutation in the 5‐HT2B receptor itself, which reduces NOS activation, has been reported in PAH; however more importantly, 5‐HT receptor‐mediated signalling has been shown to interact with http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1794 signalling (mutations in which underlie most cases of heritable PAH) (Dempsie and MacLean, 2008). 5‐HT‐mediated pulmonary remodelling and vasoconstriction is enhanced in BMPR2‐deficient mice (Long et al., 2006). 5‐HT inhibits BMPR2‐mediated Smad1/5 and Id3 activation to increase pulmonary artery remodelling (Long et al., 2006; West et al., 2016). Fully elucidating the role of 5‐HT receptors in the predisposition to PAH may highlight novel pathways leading to the development of the disease. More recently, the field has moved onto targeting tryptophan hydroxylase or the 5‐HT transporter (MacLean et al., 2004; Morecroft et al., 2007); however, new pharmacological developments such as new selective antagonists, multi‐receptor antagonists or even negative allosteric modulators of the 5‐HT receptors could restore their potential clinical utility.

Angiotensin II and MAS receptors

The renin angiotensin system (RAS) is a key regulator of vascular endothelial function and has been implicated in the remodelling of the pulmonary artery and the right ventricle seen with PAH (Morrell et al., 1995). Increased renin, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1613 and ANG‐II have all been associated with PAH (Morrell et al., 1999; de Man et al., 2012a, b). The actions of ANG‐II is mediated by angiotensin receptors 1 (http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=34, Gαq/11) and 2 (http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=35, Gαi); AT1 receptor expression is increased with PAH, whereas AT2 receptors are decreased (Morrell et al., 1999; de Man et al., 2012a, b). AT1 receptors enhance the proliferation of PASMC via Gαq/11 dependent activation of MAPK, receptor TKs, non‐receptor TK and increasing ROS (Morrell et al., 1999; Heeneman et al., 2000; Mehta and Griendling, 2007). Although AT1 antagonists, such as losartan, have been shown to prevent the progression of MCT‐induced PAH (de Man et al., 2012a, b), their clinical utility is controversial, due to systemic side effects. In contrast, AT2 receptor activation, due to their expression in PAEC, counteracts this proliferation by increased NO and prostacyclin production. AT2 agonists have been shown to reduced pulmonary artery pressure, fibrosis, inflammation and improve right ventricular function in experimental models of PAH; however, their response may be blunted due to the decrease in AT2 receptor expression with the disease (Bruce et al., 2015).

http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1614, a more recently discovered component of the RAS system, has also been identified as a novel target in PAH (Shenoy et al., 2010). ACE2 catalyses ANG‐II to http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5820, which acts on the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=150 (class A orphan GPCR). MAS1 is expressed in PAEC and similar to AT2 receptors can counteract the proliferative and vasoconstrictive of the ACE‐ANG‐II‐AT1 axis (Shenoy et al., 2010); MAS1 is down‐regulated with PAH. Treatment with ANG (1–7) prevented the remodelling of the pulmonary artery and right ventricular hypertrophy in a model of PAH, an effect that could be blocked by the MAS inhibitor A‐779 (Shenoy et al., 2010). Recently, it has been shown that the beneficial effects of AT2 receptor agonists, such as C21, may also be through ACE2‐ANG‐(1–7)‐MAS axis since it increases ACE2 expression and its beneficial effects in PAH were blocked in part by A‐7799. These data suggest that the RAS is worth revisiting as a therapeutic option for PAH.

Apelin receptor

More recently the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=36, a GPCR with a similar sequence to the AT1 receptor, has been identified to be an important in regulator of cardiovascular physiology and plays a role in the pathophysiology of PAH (Tatemoto et al., 1998; Japp et al., 2008; Kim, 2014). The endogenous ligands of apelin receptors, which are highly expressed in PAEC, are apelin family of peptides and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7930; both apelin and ELA have a comparable cardiovascular profile (Kang et al., 2013; Yang et al., 2017). Agonist‐induced apelin receptor signalling in PAEC activates both a G‐protein dependent (Gαq‐ and Gαi‐coupled) decrease in cAMP and increase in PKC activity and G‐protein independent induction of β‐arrestin (Yang et al., 2017). Apelin stimulation of apelin receptor leads to pulmonary vasodilatation, at least in part, by increasing endothelial NO via AMPK and Kruppel‐like factor 2 (Chandra et al., 2011; Yang et al., 2015). PAH‐patients and animal models of the disease have lower levels of apelin, ELA and apelin receptors, inhibiting their ability to counteract pulmonary vasoconstriction (Yang et al., 2017). Exogenous apelin (http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=599) and ELA (ELA‐13) peptides have been shown to reverse MCT‐induced remodelling of the PA and right ventricular hypertrophy, (Falcão‐Pires et al., 2009). Apelin infusion during right heart catheterisation increases cardiac output and decreases pulmonary vascular resistance in patients with PAH, which supports further investigation into the therapeutic relevance of the apelin receptor in PAH (Brash et al., 2015). Apelin restores BMPR2 signalling and PAEC function, making enhancing apelin receptor signalling an attractive target for heritable PAH (Alastalo et al., 2011). An interesting aspect of apelin receptor pharmacology is the availability of biased agonists, such as http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=9448. GPCR‐biased agonists have been used successfully to increase the beneficial effects of targeting GPCRs, but blunt side effects. CMF‐019 decreases cAMP (G protein dependent signalling) but does not induce β‐arrestin mediated internalization (G protein independent signalling) (Read et al., 2016). Ligand‐dependent trafficking of the apelin receptor, mediated via β arrestin, also contributes to differential signalling pathways and cellular functions (Lee et al., 2010a; Pope et al., 2016). Additional apelin ligand‐dependent signalling and trafficking could prevent receptor down‐regulation with chronic agonist use and thereby be harnessed to increase the responsiveness of PAH patients to apelin receptor‐targeted drugs.

Additional GPCRs, whose expression is increased in PAH‐PASMC

It is clear that altered GPCR expression associated with PAH, shifts the balance of Gαi/Gαq/Gα12/13 versus Gαs signalling, favouring vasoconstriction and proliferation of PASMC. In addition to those GPCRs discussed in detail above, the expression of the http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=12, the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=276, the http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=4 and the http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=59 (PAR1/2/3) are also increased with PAH and are important regulators of PASMC vasoconstriction, proliferation, migration and pulmonary vascular tone (Boe and Simonsson, 1980; Garcia et al., 1995; Eckhart et al., 1996; Hsiao et al., 2005; Birker‐Robaczewska et al., 2008; Molostvov et al., 2008; Sacks et al., 2008; Szczepaniak et al., 2010; Yamamura et al., 2012). Up‐regulation of these Gαq/Gα12/13/Gαi‐coupled receptors results in [Ca2+]i/PKC or ERK activation and decreased cAMP accumulation (Nakaki et al., 1990; Birker‐Robaczewska et al., 2008; Sacks et al., 2008; Li et al., 2011). Animal studies used to dissect the functional impact of CaS, SIP2 receptors and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=348 show that inhibiting the expression or function of the receptors attenuates or blocks the development of experimental PAH (Kwapiszewska et al., 2012; Chen et al., 2014b; Tang et al., 2016). Advances in pharmacology have allowed for the rational design of modulators (see Table 2) for these receptors, which by shifting the balance away from vasoconstriction and proliferation, could one day have clinical utility in PAH.

Summary: the future of GPCRs in PAH

GPCRs, by modulating second messengers, are important regulators of basal pulmonary vascular tone. Altered expression GPCRs and endothelial dysfunction shifts the balance of Gαi/Gαq/G12/13 versus Gαs‐dependent signalling, favouring vasoconstriction and proliferation of PASMC. Decreased Gαq‐coupled GPCRs in the endothelium and increased Gαi/Gαq/G12/13‐coupled GPCRs in PASMC is clearly associated with PAH (Figure 2), highlighting that altered expression of GPCRs is functionally relevant.

Figure 2.

Figure 2

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GPCRs that are dysregulated in PASMC in patients or animal models of PAH at the level of mRNA. (Red) Gαi/Gαq/Gα12/13‐coupled GPCRs that have been implicated in PASMC vasoconstriction and proliferation. (Blue) Gαs‐coupled GPCRs that have been implicated in PASMC vasodilation and reduced proliferation. MAS and apelin receptors have also been reportedly down‐regulated in PAH at the mRNA level in PAEC (not shown in figure above). It should be noted that the protein expression and efficiency of signalling by these GPCRs would be critical to determine the full physiological consequence of this ‘imbalance’. AR: adrenoceptor.

In addition to contributing to the imbalance in pulmonary vascular tone, GPCRs are also associated with risk factors of PAH (sex, drug/toxin exposure and crosstalk with signalling pathways responsible for the genetic predisposition), which further enforces their importance in the progression of the disease. Interestingly, sex differences have been shown in the responsiveness of PAH‐patients to GPCR agonists; females respond better to ET‐1 receptor antagonists and prostacyclin analogues; however, the reason for this is not fully understood (Marra et al., 2016). Recently, there has been extensive research into the role of the female sex hormone, oestrogen and its metabolites in the progression of PAH; http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=221 (GPCR that mediates the non‐genomic effects of oestrogen) is a novel target for PAH. The expression of GPER has been confirmed in PAEC and PASMC, and a GPER agonist has been shown to prevent pulmonary artery remodelling and right ventricular dysfunction in MCT‐induced PAH; however, the mechanism and site of action is still unclear (Alencar et al., 2017). Investigating the impact of sex on GPCR expression and function in cells, such as PASMC, could be important in uncovering additional targets in the female bias of the disease and differential response to drugs. Sex‐specific transcriptional profiles are evident in cultured cells and tissue (Shah et al., 2014). Our preliminary studies in isolated PASMC have shown female bias of a number of previously unrecognized GPCRs, which together could differentiate the control of pulmonary vascular tone between the sexes.

Although we have focused this review on GPCR targets that mediate PASMC‐dependent remodelling of the pulmonary artery with PAH, inflammation, adventitial thickening and right ventricular hypertrophy also characterize the disease. Right ventricular function is a key determinant of PAH severity and prognosis (Sandoval et al., 1994; van de Veerdonk et al., 2011). Altered adrenoceptor expression has been shown in the right ventricle of animal models of PAH: α1, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=28‐ and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=29‐adrenoceptors are decreased. Low‐dose noradrenaline, via β1‐adrenoreceptors, increases right ventricular contractility, right ventricle‐pulmonary artery coupling and cardiac output (Packer and Leier, 1987; Kerbaul et al., 2004). However, the effectiveness of these drugs could be blunted in the right ventricle due to down‐regulation of these receptors (Maron and Leopold, 2015). More recently, improvement in right ventricular function and remodelling by blockade of the adrenoceptors is gaining support (Bogaard et al., 2010). β‐Blockers such as http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7129 and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=551 have also successfully improved and reversed right ventricular function and remodelling in MCT‐induced PAH (de Man et al., 2012a, b; Perros et al., 2017), although these are still currently contraindicated for clinical use (Galiè et al., 2016). Since the structural changes in the pulmonary artery with PAH can also be attributed to proliferative, apoptosis‐resistant and migratory myofibroblasts in the adventitia, the GPCRs expressed and functional in these cells could also be useful pharmacological targets for the disease. To date, a number of GPCRs, including amlin, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=324, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=19, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=267, 5‐HT2A and ET receptors have been shown to regulate pulmonary fibroblast phenotype and fibrosis (Kim, 2014; Chen et al., 2014a; Qian et al., 2015). In addition, altered expression and function of a number of GPCRs have also been documented in a number of the inflammatory cells that infiltrate the pulmonary artery in PAH; http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=58, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=62, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=64, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=74 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=71 are targets for to inhibit the inflammation associated with the disease (Balabanian et al., 2002; Bull et al., 2004; Montani et al., 2011; Rabinovitch et al., 2014). Uncovering GPCRs, which also modulate right ventricular function, inflammation and fibroblast activation is an important direction for future therapeutic targets.

Changes in GPCR activity and expression at the cellular level associated with PAH, as outlined above and through work undertaken by our lab, correlate with altered signalling and the progression of the disease. However, it is important to acknowledge that a number of aspects of GPCR pharmacology may increase the complexity of their physiological role; constitutive activity of GPCRs, receptor desensitization, the significance of their ability to modulate more than one signalling pathway, the stoichiometry of the pathway and their localization in membrane microdomains need to be explored to understand their true therapeutic potential in the setting of PAH. The relative importance of G protein–dependent versus G protein–independent pathways on GPCR activation in PASMC needs to be fully dissected. GPCRs can couple to multiple Gα proteins and can also signal via MAPK, src and β arrestin. For example, several GPCRs expressed in PASMC, such as AT1, ETA and P2Y2, have all been shown to initiate cell migration and proliferation via β arrestin, independent of their respective G protein; however, such data are not available in PASMC (Morris et al., 2012; Kendall et al., 2014): highlighting the signalling pathways downstream of GPCR activation that are necessary and sufficient for their beneficial effects could provide a number of additional targets for PAH. Furthermore, we have previously shown that the cellular localization of GPCRs and their signalling components, for example, in lipid rich microdomains such as caveolae, is important for their physiological response (Ostrom and Insel, 2004). Since a number of these microdomains are increased in PAH‐PASMC (Patel et al., 2007), this could alter the response of GPCR agonists in diseased cells. For example, increased caveolae in PASMC could bring specific channels and GPCRs closer together and thereby contribute to heightened tone in the pulmonary artery; co‐localization of http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=542 and 5‐HT2A in caveolae leads to 5‐HT‐dependent inhibition of Kv current (Cogolludo et al., 2006). Altered GPCR localization with PAH remains to be fully explored.

Since the intracellular level, duration and function of second messengers is governed by an array of mediators downstream of GPCRs, it may be that in order to see the full beneficial effect of a GPCR, these components also need to be targeted. This is likely true for PAH, where the activity and expression of a number of http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=260, the main enzymes responsible for the degradation of cAMP, are increased (Maclean et al., 1997; Murray et al., 2007): PDE inhibitors could additively or synergistically increase the duration and degree of response to GPCR drugs that raise cAMP. Other components of the cAMP pathway such as ACs, multi‐drug resistant protein 4 and 5, A‐kinase anchor proteins and cAMP downstream targets are also dysregulated with PAH (Jourdan et al., 2001; Ostrom et al., 2002; Hara et al., 2011). A comprehensive analysis of the expression and activity of the various components of GPCR signalling pathways could uncover a series of diagnostic markers and/or targets for PAH. A pathway‐dependent approach to restore second messenger signalling in a pulmonary specific manner is the way forward to developing a successful therapeutic approach for the disease.

In summary, research into GPCRs in PAH have led to a better understanding of the complexity and multi‐faceted nature of the disease. Advances in GPCR pharmacology, such as allosteric modulators, biased agonists or neutralisers of autoantibodies, may offer a fresh approach to the therapeutic utility of the GPCRs shown to be successful preclinically (Table 2). Although major therapeutic advances have been made in the past 20 years with regard to PAH treatment, in part due to the approved drugs outlined above, new pulmonary specific targets are still required. As the field of GPCRs in PAH moves forward, it is important to remember that although a number have already been identified, since individual cells can express greater than a 100 different GPCRs, it is likely that many more associated with PAH are yet to be uncovered. New techniques identifying previously uncharacterized or even orphan GPCRs in cells have proven successful in providing insights into the pathophysiology of disease and identifying a vast array of new therapeutic targets (Insel et al., 2015). We have used a GPCR real‐time‐PCR array to profile GPCR expression in male and female control‐PASMC and PAH‐PASMC, which has uncovered ‘novel’ (normally expressed but not previously recognised) GPCRs in PASMC, including orphan GPCRs (Insel et al., 2015; unpublished data). Given this caveat, we believe that key GPCRs involved in ‘tipping the balance of pulmonary vascular tone’ have not yet been investigated and could offer promising new therapeutic targets for PAH.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a,b,c).

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by grants from the ATS and Wellcome Trust.

Iyinikkel, J. , and Murray, F. (2018) GPCRs in pulmonary arterial hypertension: tipping the balance. British Journal of Pharmacology, 175: 3063–3079. https://doi.org/10.1111/bph.14172.

This review is part of a series entitled ‘Non‐traditional/orphan GPCRs as novel therapeutic targets’.

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