The Endothelin-Like Domain Peptide (ELDP) co-synthesised with endothelin-1 from the EDN1 gene modulates ET-1 induced vasoconstriction and has potential for biomarker measurements.

J Yuzugulen1, IC Villar1, EG Wood1, JA Douthwaite1, NSA Patel1, J Jegard1, A Montoya2, P Cutillas2, H Gaertner3, I Rossitto-Borlat3, K Rose3, O Hartley3, A Ahluwalia1, R Corder1. 1William Harvey Research Institute, Barts & the London, Queen Mary University of London, London, EC1M 6BQ, UK, 2Barts Cancer Institute, Barts and the London, Queen Mary University of London, London, EC1M 6BQ, UK, 3Faculty of Medicine, University of Geneva, Geneva, Switzerland

Endothelin-1 (ET-1) is synthesised from the EDN1 gene as part of a 212 amino acid peptide precursor referred to as preproendothelin-1 (ppET-1). ET-1 is strongly implicated in cardiac and renal pathologies both as a potent vasoconstrictor, and as an inducer of tissue remodelling and fibrosis. Cardiac over expression of the human EDN1 gene in mice leads to a lethal heart failure, which is not blocked by ET antagonists (Yang et al., 2004). This implies other EDN1 gene derived peptides contribute to tissue responses, and produce pathological effects that are resistant to ET antagonist treatment. The aim of these investigations was to characterise other peptides derived from ppET-1 and to evaluate interactions with ET-1 responses.

Specific immunoassays and HPLC were used to identify the peptide products of ppET-1 in conditioned media samples from the human endothelial (EA.hy 926) and epithelial (A549) cell lines. Secretion of ppET-1 peptides was also investigated using primary cultures of human aortic endothelial and smooth muscle cells, and cardiomyocytes. Synthetic peptides corresponding to the identified peptides NT-proET-1 (ppET-1[18-50]), Endothelin-like Domain Peptide (ELDP, ppET-1[93-166]) and CT-proET-1 (ppET-1[169-212]) were chemically synthesized. Male Wistar rats (200 – 440 g) anaesthetized with sodium thiopentone (120 mg/kg i.p.) were used for in vivo studies. The clearance rates of NT-proET-1, ELDP and CT-proET-1 were determined after i.v. bolus injection (1 nmol/kg of each as a mixture of the three peptides). The effect of ELDP on blood pressure, and interaction with the pressor response to ET-1 was evaluated by i.v. bolus injection of ELDP (3 nmol/kg) followed 15 min later by i.v. bolus injection of ET-1 (0.3 nmol/kg). Interactions between ELDP and ET-1 were also investigated using rat isolated mesenteric resistance arteries following previously described methods (Chauhan et al., 2003).

NT-proET-1, ELDP and CT-proET-1 were secreted from all cell types investigated with levels of release correlating with ET-1. Arterial plasma levels after i.v. administration (1 nmol/kg) showed markedly different elimination rates for the three peptides (p <0.001). NT-proET-1 had the most rapid clearance (<5 min), and CT-proET-1 the slowest (8% of initial values 40 min after injection). ELDP alone (3 nmol/kg) had no effect on blood pressure in anaesthetized rats, but significantly increased the duration of the pressor response to ET-1 (0.3 nmol/kg, P <0.02). On rat mesenteric resistance arteries ELDP alone had no significant vasoconstrictor effect up to 10 nM, but after pre-treatment with ET-1 (1 – 3 nM) produced a concentration-dependent vasoconstriction. Pre-incubation of rat mesenteric resistance arteries with 10 nM ELDP increased the response of 1 nM ET-1 by ˜5 fold (P <0.002).

ProET-1 derived peptides may play an important role in vascular pathologies by modulating ET-1 responses. The slower systemic clearance of CT-proET-1 and ELDP indicate their potential as biomarkers of EDN1-linked pathologies.

References:

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