Suramin is a slowly‐equilibrating but competitive antagonist at P2x‐receptors in the rabbit isolated ear artery

Abstract

1 The antagonist dynamics of suramin were investigated at P_2x-receptors in isolated rings of endothelium-denuded ear artery from New Zealand White (NZW) rabbits. 2 α,β-Methylene adenosine 5′-triphosphate (ATP) concentration-effect curves were constructed cumulatively in a paired curve design in the absence and presence of increasing concentrations of suramin, incubated for 45 min. The slope of the resulting Schild plot was significantly greater than unity (1.50 ± 0.08). 3 Assuming that slow equilibration by suramin explains the steep Schild plot, further experiments were conducted using short (15 min) and long (3 h) incubation times. The resulting Schild plot slopes were 1.66 ± 0.36 and 1.06 ± 0.13 respectively confirming the assumption. However, after 3 h incubation, suramin also caused depression of α,β-methylene ATP curves. 4 In an attempt to minimize the depressant effect of suramin, a kinetic study was designed to calculate the minimum incubation times for each concentration of suramin used in the Schild analysis to achieve effectively complete equilibrium. Theoretically fractional occupancy for the antagonist is given by (r − 1)/r, where r is the dose-ratio. A plot of (r − 1)/r against time allowed the apparent ‘on’ and ‘off’ rate constants to be calculated. 5 With the resulting rate constant estimates, an optimised antagonism study was carried out in which incubation times were chosen such that > 95% occupancy by suramin could be achieved without agonist curve depression at each concentration of suramin used. 6 Under these conditions, suramin fulfilled all criteria for simple competition: parallel rightward displacement of α,β-methylene ATP curves and a Schild plot slope of unity (1.00 ± 0.09). The resulting pK_B estimate was 4.79 ± 0.05. This estimate of affinity was shown to be independent of the agonist used in another experiment in which l-β,γ-methylene ATP was employed (pK_B = 5.17). 7 Under the same conditions, suramin was found to have no effect on KCl-induced contractions and only slight effects on phenylephrine- and histamine-induced responses. 8 This analysis provides the first evidence that suramin is a genuine competitive P_2x-receptor antagonist.