endothelin-1 and endothelin-(16-21)

A antiserum raised against the C-terminal hexapeptide ET16-21 common to ET-1, -2 and -3 was produced and characterized with respect to its binding properties for ET-1, -2, -3, ET16-21, the C-terminal octapeptide ET14-21, its derivative Phe21-ET14-21 and human big-ET-1. The antibody reacted with the peptides with decreasing binding affinities in the order: ET-1 greater than ET-2 greater than or equal to ET16-21 = ET 14-21 much greater than Phe21-ET14-21. It showed no crossreactivity with human big-ET-1. Similar results were obtained using [125I]ET-1, -2 or -3 as tracer. Substitution of Trp21 by Phe decreased the binding affinity of ET14-21 about 10 fold. Thus, the immunologically recognized sequence of the peptides is C-terminal and Trp21 seems to be important for high binding affinities. The significant differences in binding affinity observed for ET-1, -2, -3 and ET16-21 are consistent with an interaction of the C-terminal part of the endothelins with the bicyclic N-terminal part.

Topics: Amino Acid Sequence; Animals; Antibody Affinity; Cross Reactions; Endothelin-1; Endothelins; Humans; Immune Sera; Molecular Sequence Data; Peptide Fragments; Protein Precursors; Rabbits

1. The rat isolated superior mesenteric bed, perfused with Krebs-Henseleit solution containing 10 microM indomethacin and precontracted with 100 microM methoxamine, was used to study the vasorelaxation produced by some fragments of endothelin-1, by two alanyl-substituted analogues, and by human and porcine proendothelins. The systemic cardiovascular effects of some of these peptides were also studied in anaesthetized rats. 2. Endothelin (16-21) was an endothelium-dependent vasodilator about 10 times less potent than acetylcholine and its amide was about 500 times less potent than the free acid. Human proendothelin-1 and porcine proendothelin-1 also caused endothelium-dependent relaxations but neither [Ala3,11]endothelin-1 nor [Ala1,3,11,15]endothelin-1 produced significant reductions in the methoxamine-induced tone. Sodium nitroprusside was approximately 200 times less potent than acetylcholine in the presence of the endothelium and was the only vasorelaxant to be active after destruction of the endothelium by perfusion with 0.3% CHAPS; in the absence of the endothelium it was 3.7 times more potent as a vasodilator than in its presence. 3. Porcine proendothelin-1 had weak contractile activity in the isolated mesenteric bed but porcine endothelin (22-39), endothelin (16-21) and endothelin (16-21) amide did not have vasoconstrictor actions. 4. Endothelin-3, [Ala3,11]endothelin-1, [Ala1,3,11,15]endothelin-1 and endothelin (16-21) all had systemic blood pressure effects in the anaesthetized rat. Although endothelin (16-21) did not cause vasoconstriction in vitro, it increased mean arterial pressure in vivo but was at least 100 times less potent than endothelin-3. Despite causing no vasorelaxation in vitro, [Ala1,3,11,15]endothelin-1 and [Ala3,11]endothelin-1 induced short-lived systemic depressor responses which were followed by pressor responses. Endothelin-3 was more potent as a systemic depressor agent than as a pressor agonist and, as a vasodepressor agent, it was 3-4 times more potent than either of the alanyl-substituted analogues. Endothelin-3, [Ala3,11]endothelin-1 and [Ala1,3,11,15]endothelin-1 were equipotent vasopressor agents. Porcine endothelin (22-39) had no systemic blood pressure effects. 5. This study shows directly that the presence of both disulphide bonds is required in the endothelins for them to be able to cause endothelium-dependent relaxation in the mesenteric vascular bed and that this response is mediated by different receptors from

Topics: Acetylcholine; Animals; Blood Pressure; Endothelin-1; Endothelins; Endothelium, Vascular; In Vitro Techniques; Male; Mesenteric Arteries; Nitroprusside; Peptide Fragments; Perfusion; Protein Precursors; Rats; Rats, Inbred Strains; Vasoconstrictor Agents; Vasodilator Agents