ANTAGONIST INHIBITION CURVES AND THE MEASUREMENT OF DISSOCIATION-CONSTANTS

1 Experiments carried out on guinea-pig isolated ileum with carbachol as agonist and diphenylacetoxyethyl- dimethyl-ethyl- ammonium (DADMEA) bromide as antagonist gave results which fit the theoretical relation between fractional inhibition (Q) of the effects of an agonist ([Al) and the concentration of a competitive antagonist ([B]): this also inv olves the Hill coefficient (logistic slope factor, P) for the agonist concentration-response curve and the degree of agonist stimulation, [A ]/[A](50), where [A](50) produces a half-maximum response. 2 Values of IC50 and an exponent, P', can be obtained by fitting Q to [B] using a logistic approximation to the relation. Both P' and IC50 should be gr eater with higher agonist stimulation but the increase in P' may be ma sked by errors in extreme values of Q. Estimates of IC50, however, inv ariably increased with higher agonist stimulation but with a steep con centration-response curve (P>1) and low agonist stimulation ([A]/[A](5 0) < 1), IC50 can be less than K-D. 3 K-D was calculated from the resu lts in three ways: (i) by a least-squares fit of Q to [B] using the va lues of P and [A]/[A](50) calculated from the control concentration-re sponse curve; (ii) from the value of IC50 for each line and the values of P and [A]/[A]50 and (iii) by using the agonist concentration-respo nse curve to calculate the dose-ratio and estimate of K-D for each res ponse in the presence of the antagonist. The methods gave similar resu lts (nM: 11 experiments), 12.4+/-1.1 (i), 11.7+/-0.9 (ii), 14.8+/-1.6 (iii) but there are advantages in using methods (i) or (ii) rather tha n (iii). 4 The method by which K-D is calculated is less important tha n the experimental design: the plan used in this work, with alternativ e small and large responses from the tissue, is very suitable for esti mating K-D with low concentrations of antagonists and small dose-ratio s. Although it is not a sensitive test for competitive behaviour becau se only a small range of concentrations of antagonist is tested, the e stimate of affinity should be free from complications involved in the use of higher concentrations of antagonist (and agonist) and the natur e of the antagonism can always be checked by doing further experiments in the presence of a known competitive antagonist.