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Endothelin receptor antagonists

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Abstract

Endothelin receptor antagonists (ERAs) have been developed to block the effects of endothelin-1 (ET-1) in a variety of cardiovascular conditions. ET-1 is a powerful vasoconstrictor with mitogenic or co-mitogenic properties, which acts through the stimulation of 2 subtypes of receptors [endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) receptors]. Endogenous ET-1 is involved in a variety of conditions including systemic and pulmonary hypertension (PH), congestive heart failure (CHF), vascular remodeling (restenosis, atherosclerosis), renal failure, cancer, and cerebrovascular disease. The first dual ETA/ETB receptor blocker, bosentan, has already been approved by the Food and Drug Administration for the treatment of pulmonary arterial hypertension (PAH). Trials of endothelin receptor antagonists in heart failure have been completed with mixed results so far. Studies are ongoing on the effects of selective ETA antagonists or dual ETA/ETB antagonists in lung fibrosis, cancer, and subarachnoid hemorrhage. While non-peptidic ET-1 receptor antagonists suitable for oral intake with excellent bioavailability have become available, proven efficacy is limited to pulmonary hypertension, but it is possible that these agents might find a place in the treatment of several cardiovascular and non-cardiovascular diseases in the coming future.

Introduction

The discovery of endothelin-1 (ET-1) is recent. In 1988, Yanagisawa reported the isolation, purification, and characterization of ET-1 from the culture supernatant of bovine aortic endothelial cells (Yanagisawa et al., 1988a). This report followed studies by Hickey et al. (1985) and Gillespie et al. (1986), which demonstrated the existence of a vasoconstrictor factor of endothelial origin. The initial observation that ET-1 had long lasting and 10 times more potent vasoconstrictor activity than angiotensin II quickly established its importance as a major regulator of blood pressure (Yanagisawa et al., 1988a, Clarke et al., 1989).

Local infusion of ET-1 into the healthy human forearm is associated with vasoconstriction and decreased forearm blood flow (Clarke et al., 1989). However, selective endothelin receptor subtype A (ETA) or dual ETA/endothelin receptor subtype B (ETB) receptor blockade has been reported either to increase forearm blood flow (Haynes & Webb, 1994, Haynes et al., 1996), or to leave forearm blood flow unchanged (Cardillo et al., 1999). Similarly, the dual ETA/ETB receptor antagonist bosentan has no systemic or pulmonary hemodynamic effect in normal anesthetized dogs (Teerlink et al., 1995) and does not affect whole lung hypoxic pulmonary vasoconstriction (Hubloue et al., 2003). The failure of endothelin receptor antagonists (ERAs) to disclose a vasoconstrictor effect of endogenous ET-1 appears related, at least in part, to the release of nitric oxide by the endothelium, which either counteracts the effect and/or inhibits the synthesis of ET-1 (Richard et al., 1995, Gellai et al., 1997, Hubloue et al., 2003).

Infusion of exogenous ET-1 reduces renal plasma flow and glomerular filtration and increases renal vascular resistance. These effects are partially or completely prevented by selective and non-selective ERAs (Schmetterer et al., 1998, Evans et al., 1999, Bohm et al., 2003, Vuurmans et al., 2004).

Freed et al. (1999) showed that infusion of SB-209670, a dual ERA, in healthy volunteers increases renal blood flow, suggesting a potential physiological role for endogenous endothelin in the control of renal vascular tone in humans. However, other investigators have been unable to demonstrate renal hemodynamic effects of selective and non-selective ERAs (Schmetterer et al., 1998, Binet et al., 2000, Bohm et al., 2003, Vuurmans et al., 2004).

Although, the most striking effect of endothelin described at the time of its isolation was its potent vasoconstrictor action, many other effects have been identified since then. ET-1 mediates fundamental cellular processes, such as cell proliferation (Janakidevi et al., 1992, Yang et al., 1999), fibrosis (Guarda et al., 1993, Mansoor et al., 1995, Hocher et al., 2000), and inflammation (Mullol et al., 1996, Ruetten & Thiemermann, 1997, Hocher et al., 2000).

Four structurally similar isopeptides named ET-1, ET-2, ET-3, and ET-4 (Inoue et al., 1989a, Saida et al., 1989) and several snake venom toxins called sarafotoxins have been identified as members of this family (Rubanyi & Polokoff, 1994). Endothelin-1, ET-2, and ET-3 are encoded by distinct genes located on chromosomes 6, 1, and 20, respectively (Inoue et al., 1989a). ET-1 is recognized as the major isoform of relevance in human cardiovascular physiology and pathophysiology.

The biologically active ET-1 is generated within the cell in a 2-step proteolytic process from a large precursor peptide of ∼ 200 amino acid residues called preproET-1 (Yanagisawa et al., 1988a). A neutral endopeptidase (Laporte et al., 1993) cleaves preproET-1 to generate a still inactive precursor big-ET-1, which is further converted to ET-1 by endothelin-converting enzymes (Takahashi et al., 1993, Xu et al., 1994, Emoto & Yanagisawa, 1995). This conversion is physiologically important because ET-1 is a much more potent vasoconstrictor than big-ET-1 (about 140-fold) (Rubanyi & Polokoff, 1994). Endothelin-1 is a 21-amino peptide forming a loop closed by 2 disulfur bonds (cyst-cyst) mainly synthetized by endothelial cells (Inoue et al., 1989b), as well as several other tissues including lung, heart, kidney, liver, brain, and some circulating cells (Nunez et al., 1990, Firth & Ratcliffe, 1992). Physiological stimuli for ET-1 expression and release from endothelial cells are ET-1 itself, angiotensin II, catecholamines, cardiotrophin-1, thrombin, growth factors, cytokines, free radicals, insulin, hypoxia, and shear stress (Emori et al., 1991, Masaki et al., 1991, Levin, 1995, Love & McMurray, 1996, Jougasaki et al., 2002). The synthesis of ET-1 is inhibited by agents including nitric oxide, natriuretic peptides, heparin, and prostaglandins (Boulanger & Luscher, 1990, Hu et al., 1992, Yokokawa et al., 1993, Prins et al., 1994).

Endothelial cells release ET-1 predominantly abluminally and ET-1 acts primarily as a local autocrine and paracrine factor rather than as an endocrine hormone (Wagner et al., 1992a). Only a minor portion is detected in the plasma, suggesting that circulating levels of ET-1 represent an overflow of endogenous tissue-bound ET-1 and thus do not allow for a true estimate of ET-1 activity.

The clearance of ET-1 from the circulation is very rapid. After intravenous injection of radioiodinated peptide in the rat the biological half-life is about 1 min, while the pressor effects are maintained for about 1 hr (Sirvio et al., 1990, Vierhapper et al., 1990). The majority of circulating ET-1 is retained by the lung (Anggard et al., 1989). The pulmonary circulation has the capacity to clear ET-1 from the circulation, a process mediated by the endothelin receptor B (Dupuis et al., 1996a). In a single pass through the lungs, the human pulmonary circulation extracts roughly 50% of circulating ET-1 (Dupuis et al., 1996b). There is normally no or minimal pulmonary arteriovenous ET-1 gradient, which is explained by a quantitatively similar production and release of ET-1 by the lung.

The endothelin receptors A and B (ETA, ETB) are members of the heptahelical G-protein-coupled receptor superfamily, range from 45 to 50 kDa in size (Levin, 1995) and are encoded by distinct genes located on chromosomes 4 and 13, respectively (Sakurai et al., 1992). They share ∼ 60% amino acid identity (Sakurai et al., 1992) and each type is highly preserved across mammalian species (85–90%) (Levin, 1995). Each receptor consists of a long extracellular amino-terminal domain, 7 loops of membrane-spanning domains, and an intracellular carboxy terminus. The C-terminal tail and the third cytoplasmic loop contain several putative phosphorylation sites. The Asp–Arg–Tyr motif in the second cytoplasmic loop is highly conserved and is thought to be involved in coupling to G-proteins (Bourne, 1997). Endothelin receptor A has 10 times more binding affinity for ET-1 and ET-2 than for ET-3 while ETB has equally potent affinities to all 3 endogenous endothelins (Watanabe et al., 1989, Takuwa et al., 1990, Williams et al., 1991).

In the systemic and pulmonary vessels, ETA receptors are located primarily on vascular smooth muscle cells (VSMCs) (Hosoda et al., 1991, MacLean et al., 1994) while ETB receptors are expressed on both endothelial cells (Ogawa et al., 1991) and VSMCs (Davenport et al., 1993). Both receptors expressed on VSMCs mediate vasoconstriction and cell proliferation (Clozel et al., 1992, Sumner et al., 1992, LaDouceur et al., 1993, Shetty et al., 1993, MacLean et al., 1994, Docherty & MacLean, 1998) while endothelial ETB receptors activate the release of vasodilating and anti-proliferative endothelium-derived factors, such as prostacyclin or nitric oxide (De Nucci et al., 1988, Clozel et al., 1992, Haynes & Webb, 1993, Sato et al., 1995, Muramatsu et al., 1999). Endothelin receptors have been identified in numerous tissues including lung, heart, kidney, intestine, adrenal gland, eye, and brain. The density of binding sites is especially high in lung and heart (Simonson & Dunn, 1990).

A third endothelin receptor, named ETC, has been cloned and characterized (Karne et al., 1993). This receptor is present in the cells of various species, but not in human tissues, and is specific for ET-3.

Endothelin-1 causes a biphasic increase in intracellular calcium [Ca++]i consisting of a rapid (2–5 s), transient increase followed by a lesser but sustained increase lasting up to 20 min in some experiments (Fig. 1) (Yanagisawa et al., 1988b, Simonson & Dunn, 1990). These short-term pathways of signal transduction mediate rapid biological actions of ET-1 such as contraction, relaxation, and secretion.

Receptor binding stimulates phospholipase C, which hydrolyzes phosphatidyl inositol 4,5 biphosphate to form the water-soluble inositol triphosphate (IP3) and the neutral diacylglycerol (DAG) (Resink et al., 1988). IP3 induces Ca++ mobilization from the sarcoplasmic reticulum through IP3 receptors leading to the first and transient [Ca++]i increase (Neylon, 1999) and opens store-operated Ca++ channels directly or indirectly by store depletion to further increase cytosolic Ca++. DAG opens receptor-operated Ca++ channels. The [Ca++]i elevation activates various ion channels resulting in calcium entry across the plasma membrane and the sustained increase of [Ca++]i (Van Renterghem et al., 1988, Chen & Wagoner, 1991). Elevation of [Ca++]i may open calcium-activated chloride channels leading to chloride efflux and cellular depolarization, which in turn causes activation of voltage-dependent Ca++ channels and an influx of Ca++ (Simonson & Dunn, 1992, Haynes & Webb, 1993). The voltage-gated K+ channels that contribute to determining membrane potential in pulmonary artery VSMCs (Archer et al., 2000) can also be inhibited by ET-1 (Shimoda et al., 1998). This leads to depolarization and influx of calcium through the voltage-dependent Ca++ channels (Weir & Archer, 1995). The [Ca++]i increase may also activate Na/H exchanger resulting in alkalinization of the cells and promoting Ca++ influx by activating the Na+/Ca++ exchanger (Grinstein & Rothstein, 1986, Koh et al., 1990, Lonchampt et al., 1991). The [Ca++]i increase may also trigger Ca++ release from the sarcoplasmic reticulum through ryanodine receptors and activation of calcium-activated potassium channels, which hyperpolarize the cells (Bialecki et al., 1989, Simpson & Ashley, 1989, Nelson et al., 1995). Therefore, the diversity of calcium signals evoked by ET-1 depends of the relative expression and distribution of these ion channels. Diacylglycerol is a second messenger that causes translocation of phosphokinases isoenzymes from the cytosol to a membrane fraction, thereby activating them (Newton & Keranen, 1994). Diacylglycerol and Ca++ activate protein kinase C, which triggers a negative feedback signal to dampen Ca++ signaling by ET-1 (Clerk et al., 1994, Simonson et al., 1996).

ET-1 also causes nuclear signal transduction cascades that activate gene transcription (Fig. 1). These pathways mediate long-term mitogenic, hypertrophic, and differentiation effects of ET-1. ET-1 stimulates protein tyrosine kinase activity by both protein kinase C-independent and -dependent pathways. The Ca++ influx stimulates the Ca++/calmodulin-dependent protein kinases and the protein tyrosine kinases. These pathways contribute to growth-promoting effects through induction of proto-oncogenes c-fos, c-myc, and c-jun, MAPK cascade, and DNA synthesis (Komuro et al., 1988, Zamora et al., 1993, Clerk et al., 2002).

The effects of the activation of ETB receptors are similar to those of activation of the ETA receptors in stimulating the activation of phospholipase C, generating inositol triphosphate and diacylglycerol, and mobilizing Ca++. However, ET-1 may also activate phospholipase A2 to release prostaglandin and thromboxane second messengers from arachidonic acid (Aramori & Nakanishi, 1992, Simonson & Dunn, 1992). Although both ETA and ETB receptors have been shown to stimulate the MAPK cascade, it is interesting that ET-1 is a more potent mitogen on VSMC expressing ETB receptors (Eguchi et al., 1994).

All these biological effects are in keeping with a major involvement of ET-1 in various cardiovascular and related conditions. Accordingly, the therapeutic potential of ERAs has been actively investigated in systemic and pulmonary hypertension (PH), congestive heart failure (CHF), vascular remodeling (restenosis, atherosclerosis), renal failure, neovascularized metastatic cancer, and subarachnoid hemorrhage. Indeed, it has only been 13 years between the discovery of the ET-1 system and the approval of the first dual ETA/ETB receptor antagonist bosentan, which is a very fast therapeutic application of a biological discovery.

Most preclinical and clinical pharmacological studies of ERAs have been reported in relation to their therapeutic potential in pulmonary arterial hypertension (PAH) and heart failure.

Section snippets

Endothelin receptor antagonists

The first report of an ERA was published only 2 years after the characterization of endothelin. This antagonist blocked some ET-1 responses but lacked specificity (Fabregat & Rozengurt, 1990). Shortly thereafter, a competitive endothelin antagonist (BE-18257B) was isolated from a natural byproduct of the fermentation of Streptomyces misakiensis (Ihara et al., 1991), and a structural analog of ET-1 that potentially inhibits ET-1-induced vasoconstriction was synthetized (Spinella et al., 1991).

Pulmonary arterial hypertension and endothelin receptor antagonists

Pulmonary arterial hypertension (PAH) is a syndrome of dyspnea, fatigue, chest pain, and syncope defined by an increase in pulmonary artery pressures and the absence of a known cause. The condition is either idiopathic, sporadic, familial, or occurs in association with a series of conditions which include collagen vascular disease, congenital left-to-right shunting, intake of appetite suppressant, human immunodeficiency virus infection, and portal hypertension (Simonneau et al., 2004).

The endothelin system in the heart

In the heart, endothelin-1 is produced by cardiac myocytes as well as by vascular endothelial cells (Yanagisawa et al., 1988a, Suzuki et al., 1993, Rubanyi & Polokoff, 1994). The ETA subtype accounts for more than 90% of ET receptors in isolated myocytes (Molenaar et al., 1993). In addition, ETA receptors predominate in the media of intramyocardial vessels (Davenport et al., 1993, Davenport et al., 1995). ETB receptors are localized to other cell types, such as endothelial cells (Tsukahara et

Acknowledgments

We are particularly grateful to the National Funds for Scientific Research (FNRS) of Belgium for research grant support (Grant 3.4516.02). Sophie Motte is research fellow “aspirant” of the FNRS. The authors are indebted to Dr. Nicolas Salomé whose help was instrumental in finalizing the manuscript.

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