Contractile activity of endothelin precursors in the isolated gallbladder of the guinea‐pig: presence of an endothelin‐converting enzyme
Authors: Battistini Bruno , Woods Mandy , O'Donnell Luke J.D. , Warner Timothy D. , Corder Roger , Fournier Alain , Farthing Michael J.G. , Vane John R. 
Published: July 2012

1 We have compared the activities of the endothelin precursors (human big ET‐1–38, porcine big ET‐l1–39, big ET‐21–38 and big ET‐31–41 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‐l1–39 in this isolated preparation. In addition, we have conducted binding studies to investigate whether porcine big ET‐l1–39 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 (× 10−9M), respectively), as estimated with contractions to 3 × 10−7M taken as 100%. Big ET‐l1–38 was equipotent to ET‐1 while big ET‐1–39 was one fifth as potent as ET‐1, and big ET‐21–38 was one fifth as potent as ET‐2. Precursors of ET‐1 and ET‐2 induced similar contractions at 3 × 10−7 M. Conversely, the contraction induced by big ET‐31–41 amide (3 × 10−7 M) was only 14% of that induced by the same concentration of ET‐3 (287 ± 37% of 5 μm histamine).3 The kinetics of the responses induced by single additions of 3 × 10−7 M of the endothelin isopeptides were compared. A single addition of 3 × 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 × 10−7 M big ET‐l1–38 or big ET‐11–39 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 × 10−7 M big ET‐21.38 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 × 10−7 M big ET‐31–41 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× 10−4m) inhibited contractions induced by big ET‐11–39. For instance, the contractions induced by 3 × 10−7M big ET‐11–39 were inhibited by 10−4M or 3 × 10−4M of phosphoramidon by 62.8 ± 6.7% or 74.5 ± 4.6%, respectively. Similarly, contractions induced by ET‐21_38 were inhibited by 91.3 ± 5.4% and the small response induced by big ET‐31–41 amide was abolished by 3±10−4M phosphoramidon. Conversely, the neutral endopeptidase (EC 24.11) inhibitor DL‐thiorphan (3±10−4m) had no effect. Captopril (10−5M), pepstatin A (10−5M), phenylmethylsul‐phonylfluoride (PMSF, 10−3 m), aprotinin (10−5 m), E‐64 (10−5 M), cystatin (10−6 m), leupeptin (10−4 M), chymostatin (10−4m), or bestatin (10−5M) did not inhibit but rather increased to a similar, but small degree the contractions induced by 3 to 30 × 10−9 m big ET‐l1–39. Only captopril (10−5 M) or leupeptin (10−4M) increased the contraction induced by 3×10−7m big ET‐11–39. Phosphoramidon (10−4m), pepstatin (10−5M) or PMSF (10−3 M) did not affect contractions induced by ET‐l.6 Removal of the epithelium increased by 70% the size of the contraction induced by 5 μm 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 × 10−9M big ET‐11.39 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 [125I]‐ET‐1 while porcine big ET‐11–39 caused no inhibition (0.7 ±3.0; ai = 3). ET‐1 (10−6m) inhibited binding by 95.7 ± 1.1% (n = 3) while at this much higher concentration, big ET‐l1–39 inhibited binding by only 16.8 ± 4.2% (n = 3). This clearly suggests that big ET‐l1–39 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.