Abstract
The endothelins (ETs) are a family of peptides that have the most potent and long-lasting vasoconstrictivity yet described. They have been shown to exhibit a wide variety of pharmacological activities and may play diverse pathophysiological roles in cardiovascular diseases. ET was first isolated from the supernatant of cultured porcine aortic endothelial cells and analysis of a human genomic library identified three structurally related ET isoforms ET-1, ET-2 and ET-3. Prior to the present study, no ET had ever been characterized structurally or biologically from any non-mammalian species. The present study is the first to purify and characterize structurally fish, amphibian and reptilian ETs. It also examined the effects of synthetic fish ET on the contractility of isolateci vascular and non-vascular smooth muscles from the rainbow trout, Oncorhynchus mykiss and the steroidogenic effects of frog ETs in the perifused frog adrenal gland. Therefore, the present work contributed to the understanding of the molecular and functional evolution of the ET family.|ET is generally secreted by a constitutive pathway so that steady-state concentrations in tissues are very low. In consequence, isolation of sufficient quantities of pure peptide for amino acid sequence analysis represents a technically challenging task. ET peptides were purified to apparent homogeneity from extracts of the kidney of the steelhead trout, Oncorhynchus mykiss, the stomach and liver of the European green frog, Rnnn ridibunda, and the kidney and lung of the American alligator, Alligator mississipiensis, by gel permeation chromatography and reverse-phase HPLC. Amino acid sequences were determined by automated Edman degradation and confirmed by mass spectrometry analysis. A single molecular form of trout ET was identified with four amino acid residue substitutions (Ala4-> Ser, Thr5 -»Ser, Phe6 ->Leu and Leu7 —»Met) compared with human ET-1. Two forms of endothelin were characterized from both frog and alligator: one is identical to human ET-1 and the other is identical to human ET-3 except for a single amino acid substitution (Tyr4 —>Phe), but a peptide corresponding to human ET-2 was not detected. The data are consistent with the hypothesis that three isoforms of ET in human arose from the duplication of an ancestral ET gene to give the ET-3 gene and a common ET-l/ET-2 gene, followed by a second duplication to give separate ET-1 and ET- 2 genes. The second duplication may have occurred relatively late in evolution, possibly after the divergence of mammals.|Trout ET and frog ET-3 were chemically synthesized using solid phase Fmoc chemistry and air oxidation was used to form disulfide bridges in the correct orientation. Trout ET potently contracted vascular rings prepared from trout efferent branchial artery (EBA; pD2 = 7.90 ± 0.06, n = 5), anterior cardinal vein (ACV; pD2 = 8.57 ± 0.25, n = 4), and rat abdominal aorta (AO; pD2 = 7.80 ± 0.20, n = 7). Trout and rat vessels were more sensitive to mammalian ET-1 than to trout ET (pD2 for human ET-1 in: EBA = 9.12 ± 0.14; ACV = 9.90 ± 0.15; AO = 8.86 ± 0.08), but there was no significant difference in the maximum tension produced by either peptide in these vessels. Trout ET produced concentration dependent contraction of isolated longitudinal smooth muscle strips from trout cardiac stomach (pD2 value: 7.52 ± 0.06; n = 8) and trout proximal intestine (pD2 value:7.80 ± 0.10; n = 8) and rat fundus (pD2 value: 7.80 ± 0.10; n = 6). There are no significant difference in the potency and maximal responses of trout ET and mammalian ET-1 in these preparations (pD2 value for mammalian ET-1 in: trout stomach = 7.98 ± 0.12, n = 8; trout intestine = 8.12 ± 0.12, n = 8; and rat fundus = 7.81 ± 0.10, n = 6), indicating that trout and rat gastrointestinal smooth muscle did not differentiate between trout ET and mammalian ET-1. The results suggest that the actions of trout ET in trout cardiovascular vessel and gastrointestinal smooth muscle are mediated through different ET receptor subtypes that may correspond to the well characterized mammalian ETa and ETb receptors, respectively.|These data demonstrate that the amino acid sequences of the ET family of peptides have been highly conserved during evolution and that the pathway of posttranslational processing of preproendothelin in the trout, frog and alligator is probably similar to that in mammals. Furthermore, the multiplicity of physiological process stimulated by ET in trout and frog suggests that the peptide of this family might have been incorporated into physiological regulation early on in vertebrate evolution.