CHARACTERIZATION OF ENDOTHELIN (ET) RECEPTORS IN THE ISOLATED GALL-BLADDER OF THE GUINEA-PIG - EVIDENCE FOR AN ADDITIONAL ET RECEPTOR SUBTYPE

1 We have characterized the receptors mediating contractions induced b y endothelin-1 (ET-1), ET-2, ET-3 and the ET(B)-selective receptor ago nists, sarafotoxin 6c (SX6c), IRL 1620, BQ-3020, [Ala (1,3,11,15)]ET-1 and ET (16-21) in strips of the isolated gall bladder of the guinea-p ig (GPGB). We used as antagonists BQ-123 (ET(A) receptor selective) an d PD 145065 (ET(A)/ET(B) receptor non-selective). 2 ET-1, ET-2 and ET- 3 (10(-10) M to 3 x 10(-7) M) caused similar slowly-developing concent ration-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 (1 0(-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 ind uced 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 h istamine 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 [Ala(1, 3,11,15)]ET-1 at 3 X 10(-7) M were even less than those produced by IR L 1620. ET (16-21) was inactive up to 10(-5) M. Addition of a concentr ation of 3 x 10(-7) M of ET-1 to tissues with developed contractions i nduced by the bolus addition of 3 x 10(-7) M SX6c caused a further con traction of the GPGB to the level observed with ET-1 alone at 3 x 10(- 7) M (n = 8). 4 BQ-123 (10(-5) M) did not affect the concentration-res ponse curve to ET-1 and the contraction induced by 3 x 10(-7) M was al so 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-1 at 3 x 10(-7) M was inhibit ed by 15% (n = 6; NS). A higher concentration of BQ-123 (10(-4) M) cau sed a significant shift to the right of the ET-1 concentration-respons e curve similar to that caused by PD 145065 (10(-5) 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 (u p to 10(-7) M) and inhibited the response to ET-1 at 3 x 10(-7) M by 5 2% (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%, respective ly (n = 5, 5, 3; P<0.05). PD 145065 (10(-6), 10(-5) or 10(-4) M) atten uated 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 dec reased 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 abo lished contractions to ET-3 (n = 4; up to 3 x 10(-7) M). 6 Contraction s induced by SX6c, especially those observed at concentrations lower t han 10(-8) M, were attenuated by BQ-123 (up to 10(-4) M). PD 145065 (1 0(-5) M) shifted to the right the concentration-response curve to SX6c and inhibited by 38% (P<0.05) the contractions induced by 3 x 10(-7) M. However, the contractions induced by a bolus addition of a high con centration of SX6c (3 x 10(-7) M) and the subsequent addition of an id entical concentration of ET-1 on top of SX6c were not affected by BQ-1 23 (10(-6) or 10(-5) M). 7 These results suggest that ET(B) receptors are involved in the contractions induced by endothelins in the GPGB. H owever, SX6c and other selective ET(B) agonists produced only half or less than half of the contractile response induced by non-selective ag onists. In addition, the responses to ET-1 but not to ET-3, were insen sitive to the antagonist action of BQ-123 at 10(-5) M whereas BQ-123 o r PD 145065 at 10(-5) M strongly antagonized contractions induced by E T-3. Finally, BQ-123 at 10(-4) M inhibited contractions to ET-1 and SX 6c. Thus, within the GPGB there may well be additional ET receptor(s) not conforming to the established ET(A)/ET(B) receptor subtype classif ication, as well as ET(B) receptors.