Comparative Sequences of Canine and Feline Endothelin-1
Endothelin-1 (ET-1, "mature ET-1") is a potent vasoconstrictor peptide that is made along with "big ET-1" from its precursor, preproET-1. Increased plasma concentrations of ET-1 and big ET-1 occur with various forms of cardiovascular disease in humans. Our laboratory is investigating plasma endothelins as diagnostic tests of cardiovascular disease in dogs and cats; however, commercial immunoassays designed specifically for use in dogs and cats are limited. Amino acid sequences of feline and canine big ET-1 were obtained and used to predict antibody cross-reactivity with immunoassay test kits from other species. Genomic DNA was extracted from peripheral blood and total RNA was extracted from canine and feline left ventricles for reverse transcription polymerase chain reaction (PCR) and PCR amplification of segments of the canine and feline preprohormone containing the big ET-1 sequences. The derived amino acid sequences were compared with known big ET-1 and ET-1 sequences of several other species, including human, mouse, and rat. Feline and canine big ET-1 had 87-97% and 89-100% homology, respectively, with that of other mammalian species. Canine ET-1 was identical to human, mouse, and rat ET-1. In contrast, the amino acid sequence of feline ET-1 was unique owing to a leucine for methionine substitution at position 7. It is highly likely that anti-human and anti-rodent ET-1 antibodies will cross-react with mature canine ET-1. In contrast, antibodies to mature ET-1 intended for use with feline tissues and antibodies to big ET-1 in either dogs or cats may have partial or no cross-reactivity depending on the peptide sequences used to produce the antibodies.
- "ET-1 is derived from a larger precursor (pre-pro-ET-1) that is cleaved into big ET-1 and then in the final active form (ET-1) (Suzuki et al., 1992 ). Therefore, the serum concentration of big ET-1 is proportional to that of ET-1 and the measurement of big ET-1 is preferable due to its longer half-life compared with ET-1 (O'Sullivan et al., 2007). The only limitation to the use of big ET-1 is its lower interspecies homology compared with ET-1 (Biondo et al., 2003). Nevertheless, Schellenberg et al. (2008) demonstrated a good performance in dogs for the kit employed in the present study based on antibodies against rat big ET-1. "
[Show abstract] [Hide abstract] ABSTRACT: Immunoassays for the measurement of concentrations of the cardiovascular peptides pro-atrial natriuretic peptide (proANP), brain natriuretic peptide (BNPPen and BNPPhoe), endothelin-1 (ET-1Bio, ET-1IBL and ET-1Phoe) and big endothelin-1 (Big-ETBio and Big-ETIBL) were validated in canine serum by determination of intra-assay variability and dilutional parallelism. Commercial kits that showed good results were further validated by determination of intra- and inter-assay variability, dilutional parallelism and spiking recovery. Assays for proANP, BNPPhoe, ET-1IBL and Big-ETIBL showed acceptable results in the preliminary validation and were fully validated. The intra- and inter-assay variability was acceptable for all four assays, linearity was demonstrated and recovery rates were acceptable. The performances of the different immunoassays varied considerably, underscoring the importance of validation. Of the assays studied, proANP, BNP(Phoe), ET-1IBL and Big-ETIBL produced precise, reproducible and accurate results and can be recommended for clinical application.0Comments 21Citations
- "Poor cross-reactivity of the anti-Big-ET antibody with canine Big-ET may be another reason for the poor results obtained with this kit. In contrast, canine and rat amino acid sequences for Big-ET show closer homology at the antibody-binding site than with the human sequences (Biondo et al., 2003). Better performance of the rat Big-ET kit could be expected and was confirmed in our study. "
[Show abstract] [Hide abstract] ABSTRACT: Living at 2300-m altitude combined with intermittent training at 3500 m leads to cardiovascular alterations in dogs, including increase in systemic and pulmonary artery pressure. Despite moderate to marked hypoxemia at these altitudes, erythrocytosis does not develop. To study humoral mechanisms of acclimatisation to high altitude, erythropoietin (EPO), endothelin-1 (ET-1), big endothelin (Big-ET) and vascular endothelial growth factor (VEGF) were measured in dogs living at 2300 m and intermittently ascending to 3500 m, and compared to the values obtained in control dogs living at 700-900 m. While the median EPO and ET-1 level in dogs at 2300 m did not differ from the one measured at 700-900 m, exposure from 2300 to 3500 m resulted in significantly elevated EPO and ET-1 levels. Big-ET levels were significantly higher at 2300 and 3500 m compared to dogs at low altitude, but did not differ between 2300 and 3500 m. VEGF was significantly elevated in dogs at 2300 m compared to dogs at low altitude. The increases in EPO, VEGF, ET-1 and Big-ET are thought to reflect the effect of hypoxia on a cellular level in these dogs. Obviously, the mild elevation of EPO levels observed at 3500 m was not sufficient to cause erythrocytosis. Elevations of the vasoconstrictors Big-ET and ET-1 may play some, but not a central role in hypoxic vasoconstriction in these dogs. Finally, serum VEGF measurement may be a sensitive and useful test to assess hypoxic stress in dogs.0Comments 11Citations
- "Canine ET-1 is homologous with human ET-1 (Vollmar et al., 1995). Canine and human Big-ET are not 100% homologous, but very similar (Biondo et al., 2003). To eliminate possible interfering effects due to different matrices, plasma samples were precipitated before measurement of ET-1 and Big-ET. "