Endothelin-1 (ET-1): Potent Vasoconstrictor Peptide Signaling Through ETA and ETB Receptors with ERA Therapy in Pulmonary Arterial Hypertension Research

# Endothelin-1 (ET-1): Potent Vasoconstrictor Peptide Signaling Through ETA and ETB Receptors with ERA Therapy in Pulmonary Arterial Hypertension Research

Introduction

Endothelin-1 (ET-1) was identified in 1988 as the most potent vasoconstrictor peptide yet discovered — more potent than angiotensin II on a molar basis, and distinguished by producing an exceptionally prolonged contraction rather than the transient responses typical of other vasoconstrictors. This potency, combined with the observation that ET-1 is produced constitutively by vascular endothelial cells, immediately suggested a fundamental role in vascular homeostasis and disease.

Subsequent decades confirmed this: the endothelin axis is critically involved in blood pressure regulation, cardiac hypertrophy, renal sodium handling, and — most therapeutically relevant — pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Endothelin receptor antagonists (ERAs) have become standard-of-care in PAH, with three approved agents (bosentan, ambrisentan, macitentan) demonstrating improved exercise capacity, hemodynamics, and clinical outcomes.

The endothelin family comprises three 21-amino-acid peptides (ET-1, ET-2, ET-3) and two G-protein-coupled receptors (ETA/EDNRA and ETB/EDNRB). Their biology intersects with vasoconstriction, mitogenesis, fibrosis, neurohormonal activation, renal tubular function, and melanocyte biology — making them targets of broad research interest beyond their established clinical role.

Discovery

Yanagisawa et al. (1988) isolated endothelin-1 from conditioned medium of porcine aortic endothelial cells after observing a potent vasoconstricting activity that was distinct from known mediators. They characterized a 21-amino-acid peptide with two intramolecular disulfide bonds, cloned the precursor, and named it endothelin — from "endothelium-derived" (Nature, PMID 2451132).

The initial paper reported:

• •ET-1 contracts vascular smooth muscle strips at picomolar concentrations
• •It is produced by endothelial cells under basal conditions and upregulated by various stimuli
• •It has a much longer duration of action than other vasoconstrictors
• •The sequence suggested a novel peptide family

Within two years, ET-2 and ET-3 were identified as additional family members, and the two receptors (ETA and ETB) were cloned and characterized. The discovery launched intense pharmaceutical development — endothelin receptor antagonists became among the most competitive drug classes of the 1990s-2000s.

Gene Structure, Biosynthesis, and Protein Processing

The Endothelin Gene Family

• •EDN1 (chromosome 6p24.1): encodes ET-1 and its precursors; predominantly expressed in endothelial cells
• •EDN2 (chromosome 1p34): encodes ET-2; expression in intestine, heart, kidney
• •EDN3 (chromosome 20q13.32): encodes ET-3; expression in brain, gut, adrenal medulla

Biosynthesis: A Two-Step Cleavage Process

Step 1 — Prepro-endothelin → Big ET-1:

The EDN1 gene encodes prepro-ET-1 (212 amino acids), which undergoes signal peptide cleavage and furin/PC5 endoproteolytic processing to produce Big ET-1 (38 amino acids). Big ET-1 is the main secreted intermediate and has minimal vasoconstrictor activity on its own.

Step 2 — Big ET-1 → ET-1 (active peptide):

Endothelin-converting enzyme-1 (ECE-1) cleaves Big ET-1 at Trp21-Val22 to generate the mature 21-amino-acid ET-1. ECE-1 is a membrane-bound zinc metalloprotease (meprin/neprilysin family) expressed on endothelial cell surfaces, allowing local ET-1 generation at the point of action.

Phosphoramidon (a metalloprotease inhibitor) blocks ECE-1 activity and has been used experimentally to inhibit endogenous ET-1 production.

ET-1 Protein Structure

• •21 amino acids (sequence: CSCSSLMDKECVYFCHLDIIW)
• •Two disulfide bonds: Cys1-Cys15 and Cys3-Cys11 create a bicyclic structure
• •The constrained conformation is required for receptor binding
• •C-terminal tryptophan (Trp21) is essential for ETA receptor activation
• •N-terminal region determines ETB selectivity
• •Molecular weight: ~2.5 kDa
• •Highly stable due to disulfide cross-linking

ET-1, ET-2, and ET-3 share the N-terminal bicyclic disulfide structure but differ at positions 4-7 (determining receptor selectivity): ET-1 and ET-2 bind ETA with high affinity; ET-3 has low ETA affinity but equal ETB affinity to ET-1/2.

Regulation of ET-1 Production

ET-1 secretion is predominantly abluminal (toward smooth muscle, not into blood), making plasma ET-1 levels (typically 0.5-5 pg/mL) an imperfect measure of local vascular ET-1 activity.

Stimulants of ET-1 production:

• •Shear stress (paradoxically — high shear during flow stimulates ET-1, while laminar shear chronically suppresses via eNOS/NO)
• •Hypoxia: HIF-1α binds hypoxia response elements in the EDN1 promoter — critical for PAH pathobiology
• •TGF-β1: direct transcriptional induction
• •Angiotensin II: AT1R activation → ET-1 synthesis — RAAS/ET axis crosstalk
• •Insulin and IGF-1: stimulate ET-1 in insulin resistance states
• •Cytokines: IL-1β, TNF-α, IL-6 upregulate ET-1
• •Thrombin: endothelial activation promotes ET-1
• •LDL and oxidized LDL: atherogenic stimulus

Suppressors of ET-1:

• •Nitric oxide (NO): cGMP pathway suppresses EDN1 transcription — the vasoprotective endothelium produces NO to counter its own ET-1
• •Prostacyclin (PGI2): cAMP pathway suppresses ET-1
• •Atrial natriuretic peptide (ANP): cGMP-mediated suppression
• •Laminar shear stress: chronic flow pattern promotes eNOS expression, suppressing ET-1
• •Statins: HMG-CoA reductase inhibitors suppress ET-1 transcription (independent of cholesterol lowering)

Receptor Biology: ETA and ETB

ETA (EDNRA) Receptor

• •Gene: EDNRA, chromosome 4q31.22-q31.23
• •Expression: predominantly on vascular smooth muscle cells (VSMCs) throughout the vasculature; cardiac myocytes; fibroblasts; limited endothelial expression
• •Selectivity: ET-1 = ET-2 >> ET-3 (1000-fold preference for ET-1/2 over ET-3)
• •G-protein coupling: primarily Gq → PLC → IP3 + DAG → PKC + Ca²⁺ release; also G12/13 → RhoA/ROCK
• •Effects: vasoconstriction (calcium-dependent and -independent mechanisms), proliferation, hypertrophy, fibrosis, anti-apoptosis
• •Internalization: ETA undergoes slow recycling; ET-1 receptor complexes may persist intracellularly and continue signaling

Pharmacology: Selective ETA antagonists include ambrisentan (ETA Ki ~0.1 nM) and atrasentan (investigational for IgA nephropathy).

ETB (EDNRB) Receptor

• •Gene: EDNRB, chromosome 13q22.3
• •Expression: dual location — endothelial cells and VSMCs; also melanocytes, enteric neurons, kidney collecting duct, brain
• •Selectivity: ET-1 = ET-2 = ET-3 (non-selective; equal affinity for all three endothelins)
• •G-protein coupling: Gi → decreased cAMP; also Gq; Gs in some contexts
• •Endothelial ETB effects: vasodilatory — ETB on endothelium → eNOS activation → NO production + PGI2 → vasodilation and ET-1 clearance
• •Vascular smooth muscle ETB effects: vasoconstriction (similar to ETA but weaker)
• •Clearance function: ETB mediates approximately 80-90% of circulating ET-1 clearance in the lungs — acting as a "sponge" that removes ET-1 from circulation

ETB genetic disease: Loss-of-function EDNRB mutations cause Hirschsprung disease (colonic aganglionosis — ET-3/ETB signaling is essential for enteric neural crest cell migration) and piebaldism/Waardenburg syndrome type 4A (melanocyte development failure).

Pharmacology: Selective ETB antagonists include BQ-788 (research tool); ETB agonists include IRL-1620 (BQ-3020) and sarafotoxin S6c (from snake venom).

Dual (Non-Selective) ETA/ETB Antagonists

Bosentan (Tracleer) was the first approved ERA — a dual ETA/ETB antagonist that blocks both receptor subtypes. Macitentan (Opsumit) is a newer dual antagonist with improved tissue penetration and dissociation kinetics.

Signaling Pathways Downstream of ET-1

In Vascular Smooth Muscle Cells (VSMCs)

ETA → Gq → PLC → IP3 → Ca²⁺ release:

The primary acute vasoconstriction mechanism. IP3 opens ryanodine-sensitive ER calcium channels → intracellular Ca²⁺ surge → calmodulin-dependent myosin light chain kinase → actin-myosin cross-bridging → contraction.

ETA → G12/13 → RhoA/ROCK:

Rho-kinase (ROCK) phosphorylates and inactivates myosin light chain phosphatase, sustaining contraction even when calcium returns to baseline — "calcium sensitization." This explains ET-1's prolonged contractile effect relative to other vasoconstrictors.

ETA → ERK1/2 (MAPK):

Proliferative signaling; contributes to VSMC hypertrophy and medial thickening in PAH and systemic hypertension.

ETA → EGFR transactivation:

Metalloprotease-mediated HB-EGF shedding → EGFR → ERK → proliferation; important in cancer contexts.

In Cardiac Myocytes

ET-1 is a potent hypertrophic stimulus for cardiomyocytes:

• •Gq → PKC → NFAT/GATA4/MEF2C transcription factor activation → fetal gene program re-expression (β-MHC, ANF, BNP)
• •ET-1 contributes to pathological cardiac hypertrophy in heart failure, hypertension, and volume overload
• •Gq-mediated Ca²⁺ oscillations affect cardiac automaticity

In Renal Tubular Cells

The renal endothelin system regulates sodium and water excretion:

• •ET-1 produced by collecting duct cells acts locally on ETB → inhibits vasopressin-stimulated water reabsorption (aquaporin-2 trafficking)
• •Also inhibits ENaC-mediated sodium reabsorption
• •Collecting duct-specific ET-1 knockout mice develop salt-sensitive hypertension — ET-1/ETB in kidney is natriuretic and anti-hypertensive
• •Conversely, ETA on afferent arterioles contributes to vasoconstriction and GFR regulation

ET-1 in Pulmonary Arterial Hypertension: The Key Clinical Domain

Endothelin Dysregulation in PAH

PAH is characterized by:

• •Elevated plasma and pulmonary tissue ET-1 (2-10 fold above normal)
• •Increased EDN1 expression in pulmonary artery endothelial cells, particularly in plexiform lesions
• •Upregulated ETA receptor density on pulmonary artery smooth muscle cells
• •Downregulated ETB clearance function (reducing the physiological brake)
• •Net result: sustained unopposed ETA-mediated PASMC vasoconstriction and proliferation

Triggers of pulmonary ET-1 excess:

• •Hypoxia (major driver in PAH associated with lung disease)
• •BMPR2 haploinsufficiency: impaired BMP/SMAD1/5/8 signaling reduces ET-1 suppression
• •Inflammatory cytokines (IL-1β, TNF-α)
• •Shear stress from elevated pulmonary pressure
• •TGF-β (elevated in PAH matrix)

Bosentan (Tracleer) — First ERA, FDA Approved 2001

Bosentan is an oral dual ETA/ETB antagonist (ETA Ki ~4 nM; ETB Ki ~50 nM). BREATHE-1 trial (Rubin et al., 2002, NEJM,):

• •213 patients with WHO functional class III-IV PAH
• •Bosentan 125 mg or 250 mg BID vs. placebo for 16 weeks
• •Primary endpoint: 6MWD improved by +36 m vs. placebo (p<0.05)
• •PVR decreased, time to clinical worsening improved

Bosentan received FDA approval November 2001 — the first oral PAH therapy. Subsequently approved for pediatric PAH and for prevention of digital ulcers in systemic sclerosis.

Safety: Hepatotoxicity (elevated liver enzymes in ~11% at 250 mg dose) requires monthly LFT monitoring. Teratogenic (contraindicated in pregnancy). Strong drug interactions via CYP3A4/CYP2C9 induction.

Ambrisentan (Letairis) — Selective ETA, FDA Approved 2007

Ambrisentan is a highly selective ETA antagonist (ETA Ki ~0.1 nM; ETA/ETB selectivity ~4000-fold), a carboxylic acid derivative. ARIES-1 and ARIES-2 trials (Galiè et al., 2008, Circulation,):

• •202 and 192 patients respectively with PAH
• •Ambrisentan 5 or 10 mg QD vs. placebo for 12 weeks
• •6MWD improved +30-51 m vs. placebo across doses
• •Time to clinical worsening significantly delayed

Rationale for ETA selectivity: by sparing ETB, endothelial vasodilatory/clearance function is preserved; but clinical superiority over dual blockade is not established.

AMBITION trial (Galiè et al., 2015, NEJM, PMID 26308684): ambrisentan + tadalafil (PDE5i) combination vs. monotherapy — significantly reduced risk of first clinical failure event (primary endpoint). Now standard combination first-line therapy.

Macitentan (Opsumit) — Dual ERA with Tissue Penetrance, FDA Approved 2013

Macitentan is a non-sulfonamide dual ETA/ETB antagonist designed for enhanced tissue penetration and slower receptor dissociation kinetics (more sustained receptor occupancy). SERAPHIN trial (Pulido et al., 2013, NEJM, PMID 23984728):

• •742 patients, first event-driven trial in PAH
• •Macitentan 3 mg or 10 mg QD vs. placebo over long-term follow-up (median ~2 years)
• •Primary composite endpoint (morbidity/mortality): 38% relative risk reduction at 10 mg dose vs. placebo (HR 0.55, p<0.001)
• •Significant improvement in 6MWD and reduction in PVR
• •First PAH trial powered for morbidity/mortality outcome

Advantages over bosentan: lower hepatotoxicity risk (LFT monitoring not required by label), no teratogenicity data (but still Category X precaution), fewer drug-drug interactions.

ET-1 in Other Diseases

Systemic Sclerosis (Scleroderma)

Systemic sclerosis (SSc) is characterized by vasculopathy (Raynaud's, digital ulcers), fibrosis, and autoimmunity. ET-1 mediates:

• •Digital ischemia (vasoconstriction)
• •Digital ulcer formation and healing impairment
• •Skin and lung fibrosis (ETA→fibroblast TGF-β/collagen)

Bosentan reduces frequency of new digital ulcers in SSc (RAPIDS-2 trial); ambrisentan and macitentan are also used.

Chronic Kidney Disease and Diabetic Nephropathy

Renal ET-1 via ETA promotes:

• •Renal vasoconstriction (afferent arteriole)
• •Mesangial cell proliferation and matrix expansion → glomerulosclerosis
• •Proteinuria in diabetic nephropathy

Atrasentan (selective ETA antagonist) significantly reduced proteinuria in IgA nephropathy (PROTECT trial, 2023) — received FDA approval in 2024 for IgA nephropathy. Sparsentan (dual ETA/AT1R antagonist) also approved for IgA nephropathy.

Heart Failure

Plasma ET-1 and Big ET-1 are elevated in heart failure and correlate with severity (NYHA class) and prognosis. However, ERA trials in heart failure were largely disappointing:

• •REACH-1 and ENABLE trials with bosentan did not show benefit in systolic heart failure
• •Tezosentan (IV ERA) failed in acute decompensated heart failure (RITZ trials)
• •The failure may reflect counter-productive blockade of ETB-mediated compensatory vasodilation and ET-1 clearance in heart failure

Cancer

ET-1 drives tumor angiogenesis, invasion, and metastasis via ETA on tumor-associated endothelium and cancer cells. ETA receptor overexpression in ovarian, prostate, breast, and colorectal cancers. Atrasentan was tested in prostate cancer (Phase III SWOG S0421) without survival benefit. Newer antibody-drug conjugate approaches (anti-ETA-ADC) are in early development.

Endothelin and the CNS

ET-1 is produced by astrocytes and microglia and has roles in:

• •Ischemic stroke: ET-1 contributes to post-ischemic vasoconstriction; intracortical ET-1 injection creates reproducible focal ischemia models
• •Cerebral vasospasm after subarachnoid hemorrhage: ETB-selective agonists investigated therapeutically
• •Neuroinflammation: astrocyte ET-1 → ETA on neurons and oligodendrocytes

Research Tools and Models

Current Research Frontiers

Combination PAH Therapy Optimization

With bosentan/ambrisentan + PDE5i (tadalafil/sildenafil) now standard first-line, and prostacyclin analogs (selexipag, treprostinil) as add-on, the question is optimal triple therapy sequencing. TRITON and GRIPHON trials inform this. How ERAs interact with sotatercept (ActRIIA-Fc) mechanistically is a frontier question.

ETA Antagonism in Renal Disease Beyond IgA Nephropathy

Following atrasentan's success in IgA nephropathy, ETA antagonism is being evaluated in FSGS, diabetic kidney disease, and ADPKD. The challenge is separating beneficial ETA blockade from potentially counter-productive ETB interference.

ET-3/ETB Selective Approaches

ET-3 (ETB-selective) has largely been understudied relative to ET-1. ETB signaling in enteric nervous system, melanocyte development, renal collecting duct, and CNS represents biology with distinct therapeutic implications from the ETA-dominated cardiovascular field.

Endothelin-Converting Enzyme Inhibitors

ECE-1 inhibition upstream of ET-1 production could potentially reduce ET-1 while preserving the physiological ETB-mediated clearance and vasodilation — theoretically superior to receptor antagonism. ECE inhibitors remain in preclinical/early clinical development.

ET-1 in Pulmonary Fibrosis and ILD-PAH

In interstitial lung disease-associated PAH (Group 3), ET-1 is elevated and may contribute to both vascular and parenchymal fibrosis. Macitentan's tissue penetrance makes it a candidate for combined PAH + fibrosis indications — MARIGOLD trial in ILD-PAH is ongoing.

Conclusion

Endothelin-1, discovered in 1988 as a uniquely potent endothelium-derived vasoconstrictor, has proven to be one of the most therapeutically tractable vascular mediators of the modern era. The pathway from discovery → receptor cloning → antagonist pharmacology → clinical trials → FDA approvals in PAH, scleroderma, and IgA nephropathy represents a 35-year arc of sustained translational success.

For researchers, the endothelin system offers well-validated pharmacological tools (ERAs across multiple selectivity profiles), robust genetic models, and disease settings (PAH, renal disease, scleroderma, heart failure, cancer) where mechanistic questions remain open. The evolving combination therapy landscape in PAH — integrating ERAs with PDE5 inhibitors, prostacyclins, and now ActRIIA-Fc traps (sotatercept) — makes endothelin biology an active intersection of multiple signaling axes relevant to vascular medicine.

Key Research Citations

1. Yanagisawa M, et al. (1988). A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332(6163), 411-415. PMID: 2451132

2. Arai H, et al. (1990). Cloning and expression of a cDNA encoding an endothelin receptor. Nature, 348(6303), 730-732. PMID: 2175396

3. Sakurai T, et al. (1990). Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature, 348(6303), 732-735. PMID: 2175397

4. Rubin LJ, et al. (2002). Bosentan therapy for pulmonary arterial hypertension. New England Journal of Medicine, 346(12), 896-903. PMID: 11907289

5. Galiè N, et al. (2008). Ambrisentan for the treatment of pulmonary arterial hypertension: results of the ambrisentan in pulmonary arterial hypertension, randomized, double-blind, placebo-controlled, multicenter, efficacy (ARIES) study 1 and 2. Circulation, 117(23), 3010-3019. PMID: 18506008

6. Pulido T, et al. (2013). Macitentan and morbidity and mortality in pulmonary arterial hypertension. New England Journal of Medicine, 369(9), 809-818. PMID: 23984728

7. Galiè N, et al. (2015). Initial use of ambrisentan plus tadalafil in pulmonary arterial hypertension. New England Journal of Medicine, 373(9), 834-844. PMID: 26308684

8. Parving HH, et al. (2012). Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial. Lancet, 399, 1287-1299. [IgA nephropathy context]

10. Davenport AP, et al. (2016). Endothelin. Pharmacological Reviews, 68(2), 357-418. PMID: 26956245

---

This article is intended for Research Use Only (RUO). Endothelin receptor antagonists and related compounds described herein, outside of their specific approved indications, are for research applications only. All studies involving endothelin system modulation must comply with applicable institutional and regulatory guidelines. This content does not constitute medical advice.