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 sl opes 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 ma nner 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 monophasic ally inhibited 0.1 nM [I-125]-ET-1 (K-D 1.16+/-0.9 nM) but competed fo r 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 agains t 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 desensitiz ation experiments, S6b (300 nM) did not contract preparations which we re no longer responsive to ET-1 whereas a small contraction to ET-1 (3 00 nM) was obtained in preparations rendered unresponsive to S6b. 5 Me dial sections of non-diseased human aorta, which express only ET(A) re ceptors, 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 w as similar with 20-30% of each ligand dissociating at 4 h. 6 These dat a suggest that whilst BQ123, in common with other endothelin antagonis ts, is a much more potent blocker of S6b contractile responses than of ET-1 contractile responses, this is not reflected by the equal affini ty 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 possib le explanation is that ET-1 is activating an additional population of receptors which may have lower affinity for BQ123. This is suggested b y the discrepancy in receptor density identified by [I-125]-ET-1 and [ I-125]-S6b.