ACETYLATED ENDOTHELIN-1 IS A CONSTRICTOR IN GUINEA-PIG LUNG VASCULATURE BUT NOT IN ISOLATED VASCULAR STRIPS

We examined the relative importance of the two amino groups of endothelin-1 in mediating pulmonary vasoconstrictor activity. Complete acetylation of prefolded endothelin-1 (fAcET-1[AcK9]) yielded a product with vasoconstrictor properties (EC50 = 0.52 +/- 0.04 nM) in isolated Ringer-perfused guinea pig lungs similar to native endothelin-1 (EC50 = 0.31 +/- 0.05 nM). However, fAcET-1[AcK9] exhibited a marked reduction in potency when assessed by contraction of isolated guinea pig pulmonary artery strips or by contraction of carotid artery or aortic strip preparations. fAcET-1[AcK9] at concentrations up to 100 nM failed to induce appreciable contraction of any vascular strip preparation. In contrast, endothelin-1 had an EC50 of 1.46 +/- 0.32 to 1.88 +/- 0.19 nM in various vessel preparations. The differences in response to fAcET-1[AcK9] in the intact lung vs. strip preparation suggest different receptor populations in the two preparations. The importance of specific amino groups for contractile activity in the vascular strip preparation was explored by acetylation of individual sites (amino terminus or lysine sidechain) or both sites during peptide synthesis to produce AcET-1, ET-1[AcK9], and AcET-1[AcK9], respectively. The order of potency was endothelin-1 >> ET-1[AcK9] > AcET-1 > AcET-1[AcK9]. These results suggest that chemical modifications (e.g., biotinylation) should be made preferentially at the lysine-9 sidechain in order to retain maximal biological activity in vascular strip preparations. We conclude: 1) that the endothelin receptor population in the intact lung and isolated vascular strip preparations may differ; 2) that the amino groups of endothelin-1 are important for full agonist activity in vascular strip preparations, but not the intact pulmonary vascular bed; and 3) that the lysine-9 sidechain is a preferential site for chemical modification compared to the amino terminus.