Characterization of endothelin (ET) receptors in the isolated gall bladder of the guinea-pig: evidence for an additional ET receptor subtype.

Abstract

1. We have characterized the receptors mediating contractions induced by endothelin-1 (ET-1), ET-2, ET-3 and the ETB-selective receptor agonists, sarafotoxin 6c (SX6c), IRL 1620, BQ-3020, [Ala1,3,11,15]ET-1 and ET (16-21) in strips of the isolated gall bladder of the guinea-pig (GPGB). We used as antagonists BQ-123 (ETA receptor selective) and PD 145065 (ETA/ETB receptor non-selective). 2. ET-1, ET-2 and ET-3 (10(-10) M to 3 x 10(-7) M) caused similar slowly-developing concentration-dependent contractions of the GPGB. Contractile effects induced by ET-1, ET-2 or ET-3 (at 3 x 10(-7) M) were also similar (230 +/- 25, 241 +/- 7 and 287 +/- 37% of that to histamine at 5 x 10(-6) M, n = 7, 6, 12, respectively). However, the threshold concentration for ET-1 or ET-2 was 10(-10) M whereas it was 3 x 10(-9) M for ET-3. 3. SX6c (10(-10) M to 3 x 10(-7) M) also caused slowly-developing concentration-dependent contractions at a threshold concentration of 10(-10) M (n = 16). However, the contraction caused by SX6c at 3 x 10(-7) M was 116 +/- 9% of that to histamine at 5 x 10(-6) M, which was half of that induced by the same concentration of the ET isopeptides. The contraction induced by IRL 1620 at 3 x 10-7 M (n = 9) was 43 +/- 9% of that to histamine at 5 x 10-6 M, which was one fifth of that produced by the same concentration of ET-1. Contractions induced by BQ-3020 or [Ala1,3,11,15]ET-I at 3 x 10-7 M were even less than those produced by IRL 1620. ET (16-21) was inactive up to 10-5 M. Addition of a concentration of 3 x 10-7 M of ET-1 to tissues with developed contractions induced by the bolus addition of 3 x 10-7 M SX6c caused a further contraction of the GPGB to the level observed with ET-1 alone at 3 x 10-7M (n = 8).4. BQ-123 (10-5 M) did not affect the concentration-response curve to ET-1 and the contraction induced by 3 x 10- M was also not affected (n = 5; 239 +/- 19% of histamine at 5 x 10-6 M). PD 145065(10-5 M) shifted the ET-1 concentration-response curve to the right and the contraction induced by ET-1at 3 x 10-7 M was inhibited by 15% (n = 6; NS). A higher concentration of BQ-123 (10-4 M) caused a significant shift to the right of the ET-1 concentration-response curve similar to that caused by PD 145065 (10-s M) and caused a 24% (n = 6; NS) inhibition of the contractions induced by ET-1 at 3 x 10-7 M. PD 145065 (10-4 M) abolished contractions induced by ET-1 (up to 10- M) and inhibited the response to ET-1 at 3 x 10-7 M by 52% (n = 4; P< 0.05).5. Contractions induced by ET-3 were more sensitive to inhibition by the antagonists. BQ-123 (10-6,10-5 or 10-4 M) inhibited responses to 3 x 10-7 M ET-3 by 66, 71 and 83%, respectively (n = 5, 5, 3;P< 0.05). PD 145065 (10-6, 10-5 or 10-4 M) attenuated more strongly than did BQ-123 the contractions induced by ET-3. For instance, the contractions caused by ET-3 at 3 x 10-7 M were decreased by 73 and 80% (n = 5, 5; P<0.05) in the presence of PD 145065 (10-6 or 10-5 M, respectively). PD 145065(10-4 M) completely abolished contractions to ET-3 (n = 4; up to 3 x 10-7M).6. Contractions induced by SX6c, especially those observed at concentrations lower than 10-8 M, were attenuated by BQ-123 (up to 10-4 M). PD 145065 (10-5M) shifted to the right the concentration response curve to SX6c and inhibited by 38% (P<0.05) the contractions induced by 3 x 10-7M.However, the contractions induced by a bolus addition of a high concentration of SX6c (3 x 10-7 M)and the subsequent addition of an identical concentration of ET-1 on top of SX6c were not affected byBQ-123 (10-6 or 10-5 M).7. These results suggest that ETB receptors are involved in the contractions induced by endothelins in the GPGB. However, SX6c and other selective ETB agonists produced only half or less than half of the contractile response induced by non-selective agonists. In addition, the responses to ET-1 but not to ET-3, were insensitive to the antagonist action of BQ-123 at 10-5 M whereas BQ-123 or PD 145065 at 10-5 M strongly antagonized contractions induced by ET-3. Finally, BQ-123 at 10-4 M inhibited contractions to ET-1 and SX6c. Thus, within the GPGB there may well be additional ET receptor(s) not conforming to the established ETA/ETB receptor subtype classification, as well as ETB receptors.