PPADS SELECTIVELY ANTAGONIZES P(2X)-PURINOCEPTOR-MEDIATED RESPONSES IN THE RABBIT URINARY-BLADDER

1 Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), an inhibitor of P2X-purinoceptor-mediated responses in rabbit vas deferens, was investigated for its ability to antagonize contractions evoked by alpha,beta-methylene ATP (alpha,beta-MeATP), carbachol and electrical field stimulation in the rabbit urinary bladder detrusor muscle. 2 PPADS (1-30 mum) caused concentration-dependent inhibition of contractions to the stable P2X-purinoceptor agonist, alpha,beta-MeATP, decreasing the maximum response to alpha,beta-MeATP (30 muM) at concentrations of 3 - 30 muM. The pD2 value for alpha,beta-MeATP in the absence of PPADS was 6.52 +/- 0.10 (8). In the presence of PPADS at concentrations of 1, 3, 10 and 30 muM the negative log concentrations of alpha,beta-MeATP that cause the same contractile response as the pD2 value were significantly different from control, being respectively 6.17 +/- 0.09 (8), 5.64 +/- 0.12 (7), 5.15 +/- 0.23 (7) and 4.78 +/- 0.22 (5). 3 PPADS (1-30 muM) caused concentration-dependent inhibition of contractions to stimulation of intramural purinergic nerves (1 - 32 Hz). There was a greater inhibition at lower frequencies (1 - 8 Hz) than at higher frequencies (16 - 32 Hz). PPADS, 30 muM, did not produce significantly greater antagonism than 10 muM. 4 PPADS (30 muM) had no significant influence on the contractile potency of carbachol: the pD2 values of carbachol in the absence and presence of PPADS were not significantly different being 6.42 +/- 0.16 (5) and 6.33 +/- 0.18 (5), respectively. However, PPADS caused a small, but significant, suppression of the maximal response of carbachol, reducing it by approximately 9%. 5 Radioligand binding studies carried out on rabbit bladder membranes with [H-3]-alpha,beta-methylene ATP ([H-3]-alpha,beta-MeATP) showed that PPADS concentration-dependently inhibited the binding of [H-3]-alpha,beta-MeATP to P2X-purinoceptors, while the binding of [H-3]-quinuclidinyl benzilate to muscarinic cholinoceptors was not affected. 6 Thus, PPADS (1 - 30 muM) antagonized responses mediated via P2X-purinoceptors in the rabbit urinary bladder. It was selective for P2-purinoceptor-mediated contractions rather than those mediated via muscarinic receptors. Binding studies demonstrated that the antagonistic effect of PPADS is via a direct interaction with P2X-purinoceptors.