Synergy between pairs of competitive antagonists at adult human muscle acetylcholine receptors

Abstract

Background: Synergistic neuromuscular blocking effects have been observed clinically with certain pairs of nicotinic acetylcholine receptor (nAChR) competitive antagonists. The mechanism for synergy has not been elucidated. We tested the hypothesis that synergy arises from a differential selectivity of antagonists for the two ligand binding sites on adult human nAChR.

Methods: We expressed nAChR in BOSC23 cells. We applied ACh with or without antagonists to outside-out patches and measured macroscopic currents at room temperature. We determined the IC(90) for (+)-tubocurarine, metocurine, pancuronium, vecuronium, cisatracurium, rocuronium, and atracurium. For 15 combinations of two antagonists, we determined the IC(90) for one antagonist in the presence of the IC(70) of a second antagonist. We constructed isobolograms for 90% inhibition. For single antagonists, we measured inhibition of receptors containing mutations in the epsilon- and delta-subunits to determine site selectivity.

Results: Two pairs of antagonists, metocurine+cisatracurium and cisatracurium+ atracurium exhibited additive inhibition. Ten combinations, including (+)-tubocurarine+ pancuronium and pancuronium+vecuronium, were highly synergistic such that the combination was two to three times more effective than expected for additivity. Three combinations were 1.5-1.6 times more effective than expected for additivity. Inhibition by (+)-tubocurarine and metocurine was sensitive to mutations in the epsilon-subunit only. Vecuronium was affected by the delta-subunit mutation only. Inhibition by other antagonists was decreased by mutations in either subunit.

Conclusions: Many combinations of antagonists exhibited synergistic effects on adult human nAChR. Synergy was observed with structurally similar and dissimilar antagonists. The degree of synergy did not always correlate well with site specificity assayed with mutants. In some, but not all cases, the synergy at the receptor level correlated with clinical determinations of synergy. We conclude that the synergistic actions of muscle relaxants can be partially explained by direct interactions with adult human nAChR.

Figures

Fig. 1

The hypothesis for the origin of synergy between (+)-tubocurarine and pancuronium. There are two distinct binding sites for ACh and competitive antagonists of the muscle nAChR. ACh must bind to both sites before the channel can open efficiently. (+)-tubocurarine has a higher affinity for the binding site at the αε interface than at the αδ interface. The hypothesis is that pancuronium has a higher affinity for the binding site at the αδ interface. In this case, the two antagonists would bind nearly independently and inhibition would be synergistic.

Fig. 2

A. An example of macroscopic currents activated by 300 μM ACh and inhibition by 50 nM (+)-tubocurarine. B. The concentration-response curve for (+)-tubocurarine. The IC90 value is obtained by determining the concentration at which the relative current is 0.1. The solid line is the fit to Equation 1; the gray lines are ±1 SD of the fit.

Fig. 3

Our approach to determining the isobologram for combination of (+)-tubocurarine and pancuronium. (A) The concentration-response curve of (+)-tubocurarine alone. The solid line is the fit to Equation 1; the gray lines are ±1 SD of the fit. The dashed line indicates the IC90. (B) The concentration-response curve of pancuronium alone. (C) The concentration-response curve of (+)-tubocurarine in the presence of 15 nM pancuronium. (D) The isobologram of the combination of (+)-tubocurarine and pancuronium. The solid squares indicate the IC90 values of each antagonist alone and provide the endpoints for the line of additivity. The open square is drawn at [pancuronium]=15 nM and [(+)-tubocurarine]= its IC90 determined in the presence of 15 nM pancuronium. The dashed curve is an isobologram assuming that the antagonists bind exclusively to different sites.

Fig. 4

The concentration response curves of A) (+)-tubocurarine with and without pancuronium, B) metocurine with and without cisatracurium. The data for pairs of antagonists are scaled to match the maximum value in the single antagonist curve (note right-hand axes). There is no shift in the apparent IC50 of (+)-tubocurarine when 15 nM pancuronium is present; this suggests that the antagonists bind independently. There is a 3-fold shift in the apparent IC50 of cisatracurium when 40 nM metocurine is present; this suggests that the antagonists compete for the same binding sites.

Fig. 5

The isobolograms for combinations of (A) vecuronium and pancuronium, (B) of (+)-tubocurarine and cisatracurium, and (C) of metocurine and cisatracurium. Closed symbols indicate data points derived from full concentration response curves (as in Figs. 3 and 4). The solid lines isobolograms that fit the closed symbol data point. Open symbols indicate concentration pairs that lie on the isobologram and should produce a relative current (Rel Curr) of 0.1. The insets show the measured relative current produced by these additional concentration pairs. None of the additional pairs produced a relative current significantly different from 0.1.

Fig. 6

The inhibition of mutant receptors α2βε(D59A)δ, α2βε(D173A)δ and α2βεδ(D180K) by antagonists. The indicated antagonist concentrations correspond to two times the IC50 for wild-type receptors. The dashed line indicates the fractional current, 0.33, obtained on wild-type receptors. Antagonist abbreviations are (+)-Tc ((+)-tubocurarine), Met (metocurine), Cisatr (cisatracurium), Pan (pancuronium), Vec (vecuronium) and Roc (rocuronium).

Fig. 7

Isobolograms predicted from Equation 2 assuming that antagonist1 binds to one site only (Lαδ1=∞) and the antagonist2 binds to the other site only (Lαε2=∞). Each axis give the fractional drug concentration relative to the ICx where x=50, 75, 90 or 95. At low receptor occupancy, ≤IC50, the difference between synergy and additivity is small and may be difficult to determine experimentally. The differences are more pronounced at higher occupancy levels.