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Effects of reactive blue 2 and twelve structurally related compounds were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP; mediated by P2X-receptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y-receptors), and the degradation of ATP by rat vas deferens tissue. All compounds, except acid blue 41 and acid blue 129 (at up to 100 microM), shifted the concentration-response curve of alpha,beta-MeATP in the rat vas deferens to the right. Most increased, but uniblue A greatly decreased, the maximum of the curve. In the case of cibacron blue 3GA and reactive blue 19, of which three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from unity. The apparent Kd values of the effective substances ranged between 0.7 and 111 microM. Most compounds increased the contraction of the rat vas deferens elicited by high K+. In the guinea-pig taenia coli, all compounds, except uniblue A and reactive blue 19 (at up to 100 microM), shifted the concentration-response curve of ADPbetaS to the right in a parallel manner. In the case of acid blue 129 and acid blue 80, of which three concentrations were tested, the slope of the Arunlakshana-Schild regression did not differ from unity. The apparent Kd values of the effective substances were between 0.7 and 69 microM. Most compounds also reduced the relaxation of the guinea-pig taenia coli elicited by noradrenaline. The removal of ATP from the medium by vas deferens tissue was decreased only by reactive blue 2, cibacron blue 3GA, uniblue A and reactive blue 19, with IC25% values between 17 and 62 microM. The structure-activity relationships for P2X- and P2Y-receptor blockade in this series are strikingly dissimilar. In reactive blue 2 and its isomers, for example, both the 1-amino-anthraquinone-2-sulphonate core and the 'side-chain' of the molecule are involved in P2X-receptor binding; P2Y-receptor affinity, in contrast, resides largely or totally in the anthraquinone core. The most promising antagonists are uniblue A which is P2X- versus P2Y-selective and acid blue 129 which is P2Y- versus P2X-selective, both with few, if any, non-P2-receptor effects at concentrations blocking the respective P2-subtype.

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Effects of Evans blue and four derivatives as well as of trypan blue and four derivatives, mostly smaller fragments but two compounds with an additional ethylene bridge in the center of the molecule, were studied on contractions of the rat vas deferens elicited by alpha, beta-methylene ATP (alpha, beta-MeATP; mediated by P2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S; mediated by P2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. All compounds shifted the concentration-response curve of alpha, beta-MeATP in the rat vas deferens to the right, and most compounds increased the maximum of the curve. Each member of the Evans blue series was similar in potency to the corresponding member of the trypan blue series. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent Kd values were between 0.8 and 385 microM. In the guinea-pig taenia coli, only the members of the trypan blue group were relatively potent, shifting the concentration-response curve of ADP beta S to the right in a surmountable manner. In 2 of 3 cases where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent Kd values in the trypan blue group were between 5.2 and 324 microM. The removal of ATP from the medium by vas deferens tissue was decreased mainly by the members of the Evans blue group, with IC25% values between 13 and 158 (in 1 case > 1000) microM. The results indicate that the position of the sulphonate residues at the terminal naphthalene rings of these compounds hardly influences P2X purinoceptor affinity but greatly influences P2Y affinity and ecto-nucleotidase blockade. Among active compounds, apparent purinoceptor affinity and ecto-nucleotidase blockade increase with the size of the molecules up to Evans blue and trypan blue themselves; introduction of a central ethylene bridge does not result in a further gain in potency. NH01, the desmethyl derivative of Evans blue, seems to be interesting because it is the compound with the highest P2X- versus P2Y-selectivity presently available.