Failure of BQ123, a more potent antagonist of sarafotoxin 6b than of endothelin-1, to distinguish between these agonists in binding experiments

1 In homogenates of human saphenous vein, [I-125]-ET-1 and [I-125]-S6b each labelled a single population of high affinity binding sites with K-D values of 0.64+/-0.11 nM and 0.55+/-0.08 nM respectively. Hill slopes were close to one. However, the density of receptors labelled by [I-125]-ET-1 was significantly greater than that by [I-125]-S6b (187.6+/-23.0 compared to 91.7+/-23.6 fmol mg(-1) protein, P<0.02). 2 BQ123, an ET(A)-selective antagonist, inhibited specific [I-125]-ET-1 and [I-125]-S6b binding with equal affinity. BQ123 competed in a biphasic manner for both [I-125]-ET-1 (0.1 nM) and [I-125]-S6b (0.1 nM) with ET(A) K-D values of 0.55+/-0.17 nM and 0.52+/-0.02 nM and ET(B) K-D values of 14.4+/-2.60 mu M and 11.2+/-0.31 mu M respectively. S6b monophasically inhibited 0.1 nM [I-125]-ET-1 (K-D 1.16+/-0.9 nM) but competed for 0.25 nM [I-125]-ET-1 in a bipahasic manner (K-D high affinity site 1.99+/-0.84 nM, K-D low affinity site 0.68+/-0.63 mu M, ratio 67%:33%). 3 BQ123 antagonized the vasoconstrictor responses of ET-1 with a pK(B) value of 6.47 whereas BQ123 exhibited 50 fold higher affinity against S6b-mediated vasoconstriction with a pK(B) value of 8.18. Regression slopes were 0.80+/-0.13 and 1.08+/-0.11 respectively. 4 In desensitization experiments, S6b (300 nM) did not contract preparations which were no longer responsive to ET-1 whereas a small contraction to ET-1 (300 nM) was obtained in preparations rendered unresponsive to S6b. 5 Medial sections of non-diseased human aorta, which express only ET(A) receptors, were used to compare dissociation rates of the two agonists. The time course for the dissociation of [I-125]-ET-1 and [I-125]-S6b was similar with 20-30% of each ligand dissociating at 4 h. 6 These data suggest that whilst BQ123, in common with other endothelin antagonists, is a much more potent blocker of S6b contractile responses than of ET-1 contractile responses, this is not reflected by the equal affinity of BQ123 determined in competition binding experiments against both [I-125]-ET-1 and [I-125]-S6b. This discrepancy in antagonist potency is probably not due to a marked difference in the rate of dissociation of [I-125]-ET-1 and [I-125]-S6b from endothelin receptors. One possible explanation is that ET-1 is activating an additional population of receptors which may have lower affinity for BQ123. This is suggested by the discrepancy in receptor density identified by [I-125]-ET-1 and [I-125]-S6b.