CONTRACTILE ACTIVITY OF ENDOTHELIN PRECURSORS IN THE ISOLATED GALLBLADDER OF THE GUINEA-PIG - PRESENCE OF AN ENDOTHELIN-CONVERTING ENZYME

1 We have compared the activities of the endothelin precursors (human big ET-1(1-38), porcine big ET-1(1-39), big ET-2(1-38) and big ET-3(1-41 amide)) and their respective mature 21 amino acid peptides as contractors of isolated gallbladder strips of the guinea-pig. We have also used different protease inhibitors and/or epithelial cell removal to investigate the nature and the location of the endothelin-converting enzyme (ECE) activity responsible for the conversion of porcine big ET-1(1-39) in this isolated preparation. In addition, we have conducted binding studies to investigate whether porcine big ET-1(1-39) interacts directly with ET receptors. 2 ET-1, ET-2 and ET-3 were equipotent at causing 50% of the contractions of the isolated strips (22.9 +/- 6.8, 39.5 +/- 9.9 and 35.9 +/- 3.7 (x 10(-9) M), respectively), as estimated with contractions to 3 x 10(-7) M taken as 100%. Big ET-1(1-38) was equipotent to ET-1 while big ET-1(1-39) was one fifth as potent as ET-1, and big ET-2(1-38) was one fifth as potent as ET-2. Precursors of ET-1 and ET-2 induced similar contractions at 3 x 10(-7) M. Conversely, the contraction induced by big ET-3(1-41 amide) (3 x 10(-7) M) was only 14% of that induced by the same concentration of ET-3 (287 +/- 37% of 5 mu M histamine). 3 The kinetics of the responses induced by single additions of 3 x 10(-7) M of the endothelin isopeptides were compared. A single addition of 3 x 10(-7) M ET-1, ET-2 or ET-3 produced contractions that reached a steady state in 28.2 +/- 4.2, 21.1 +/- 1.3 and 24.0 +/- 3.8 min, respectively and took 2.7 +/- 0.4, 2.1 +/- 0.1 and 1.6 +/- 0.1 min to reach half of this steady-state response. 4 Contractions induced by 3 x 10(-7) M big ET-1(1-38) or big ET-1(1-39) reached a plateau in 38.5 +/- 3.6 and 35.6 +/- 3.3 min, respectively, and half of these responses were attained in 12.0 +/- 2.5 and 7.1 +/- 1.1 min. Thus, these contractions developed more slowly than those induced by ET-1. Contractions induced by 3 x 10(-7) M big ET-2(1-38) were also much slower to develop than those to ET-2, for these took 49 +/- 2 min to reach plateau and 19.4 +/- 2.1 min to attain half that response. Contractions induced by 3 x 10(-7) M big ET-3(1-41 amide) amide took 50.2 +/- 3.7 min to reach a plateau and 27.3 +/- 3.0 min to reach half of this response. 5 Phosphoramidon (0.1, 1 and 3 x 10(-4) M) inhibited contractions induced by big ET-1(1-39). For instance, the contractions induced by 3 x 10(-7) M big ET-1(1-39) were inhibited by 10(-4) M or 3 x 10(-4) M of phosphoramidon by 62.8 +/- 6.7% or 74.5 +/- 4.6%, respectively. Similarly, contractions induced by ET-2(1-38) were inhibited by 91.3 +/- 5.4% and the small response induced by big ET-3(1-41 amide) was abolished by 3 x 10(-4) M phosphoramidon. Conversely, the neutral endopeptidase (EC 24..11) inhibitor DL-thiorphan (3 x 10(-4) M) had no effect. Captopril (10(-5) M), pepstatin A (10(-5) M), phenylmethylsulphonylfluoride (PMSF, 10(-3) M), aprotinin (10(-5) M), E-64 (10(-5) M), cystatin (10(-6) M), leupeptin (10(-4) M), chymostatin (10(-4) M), or bestatin (10(-5) M) did not inhibit but rather increased to a similar, but small degree the contractions induced by 3 to 30 x 10(-9) M big ET-1(1-39) Only captopril (10(-5) M) or leupeptin (10(-4) M) increased the contraction induced by 3 x 10(-7) M big ET-1(1-39). Phosphoramidon (10(-4) M), pepstatin (10(-5) M) or PMSF (10(-3) M) did not affect contractions induced by ET-1. 6 Removal of the epithelium increased by 70% the size of the contraction induced by 5 mu M histamine (1.08 +/- 0.05 g; n = 160 to 1.84 +/- 0.14 g; n = 12) but did not affect, in absolute terms, the contraction induced by ET-1 (as a % of the response to histamine, these responses were, of course, apparently depressed). Epithelium removal did, however, increase the size of the contractions induced by 3 to 30 x 10(-9) M big ET-1(1-39) which was very similar to the effect of the protease inhibitors. 7 In competition binding studies on membranes prepared from the guinea-pig gallbladder, 10(-11) M ET-1 inhibited by 76.9 +/- 3.1% the binding of [I-125]-ET-1 while porcine big ET-1(1-39) caused no inhibition (0.7 +/- 3.0; n = 3). ET-1 (10(-6) M) inhibited binding by 95.7 +/- 1.1% (n = 3) while at this much higher concentration, big ET-1(1-39) inhibited binding by only 16.8 +/- 4.2% (n = 3). This clearly suggests that big ET-1(1-39) does not bind directly to ET receptors. 8 Thus, a phosphoramidon-sensitive endothelin-converting enzyme (ECE), different from neutral endopeptidase (NEP; EC 24.11) and not located on the epithelium, converts big ET-1 into ET-1 in the gallbladder of the guinea-pig. This ECE appears to act preferentially on big ET-1 or big ET-2 over big ET-3.