Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), an inhibitor of P_2X‐purinoceptor‐mediated responses in rabbit vas deferens, was investigated for its ability to antagonize contractions evoked by α,β‐methylene ATP (α,β‐MeATP), carbachol and electrical field stimulation in the rabbit urinary bladder detrusor muscle. PPADS (1–30 μm) caused concentration‐dependent inhibition of contractions to the stable P_2X‐purinoceptor agonist, α,β‐MeATP, decreasing the maximum response to α,β‐MeATP (30 μm) at concentrations of 3–30 μm. The pD₂ value for α,β‐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 μm the negative log concentrations of α,β‐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). PPADS (1–30 μm) 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 μm, did not produce significantly greater antagonism than 10 μm. PPADS (30 μm) 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%. Radioligand binding studies carried out on rabbit bladder membranes with [³H]‐α,β‐methylene ATP ([³H]‐α,β‐MeATP) showed that PPADS concentration‐dependently inhibited the binding of [³H]‐α,β‐MeATP to P_2X‐purinoceptors, while the binding of [³H]‐quinuclidinyl benzilate to muscarinic cholinoceptors was not affected. Thus, PPADS (1–30 μm) antagonized responses mediated via P_2X‐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 P_2X‐purinoceptors.

1 Pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), a P₂-purinoceptor antagonist, was investigated for its ability to antagonize: (1) P_2X-purinoceptor-mediated contractions of the rabbit central ear artery and saphenous artery evoked by either α,β-methylene ATP (α,β-MeATP) or electrical field stimulation (EFS); (2) P_2Y-purinoceptor-mediated relaxations of the rabbit mesenteric artery; (3) endothelium-dependent and endothelium-independent, P_2Y-purinoceptor-mediated relaxations of the rabbit aorta. 2 α,β-MeATP (0.1–100 μm) caused concentration-dependent contractions of the rabbit ear and saphenous arteries. The negative log[α,β-MeATP] that produced a contraction equivalent to the EC₂₅ for noradrenaline (ear artery) or histamine (saphenous artery) in the absence of PPADS was 6.60 ± 0.18 (9) and 6.18 ± 0.17 (9) in the ear artery and saphenous artery, respectively. These effects of exogenous α,β-MeATP were concentration-dependently inhibited by PPADS (1–30 μm). In the ear artery, the negative log[α,β-MeATP] producing a contractile response equivalent to the EC₂₅ of noradrenaline, in the presence of PPADS at 1, 3 and 10 μm was 6.16 ± 0.18 (8), 5.90 ± 0.18 (8) and 4.72 ± 0.36 (8), respectively (P < 0.01). In the saphenous artery, the negative log[α,β-MeATP] values equivalent to the EC₂₅ for histamine in the presence of PPADS at concentrations of 1, 3, 10 and 30 μm were 5.90 ± 0.19 (8), 5.73 ± 0.16 (8), 4.99 ± 0.14 (8) and 4.51 ± 0.13 (8), respectively (P < 0.01). 3 PPADS at a concentration of 1 μm had no effect on contractions of the ear artery evoked by EFS (4–64 Hz; 1 μm phentolamine present). At higher concentrations (3–30 μm) it caused concentration-dependent inhibition of neurogenic contractions. In the saphenous artery, PPADS (1–30 μm) concentration-dependently inhibited contractions evoked by EFS at frequencies of 4, 8 and 16 Hz. Contractions evoked by EFS at frequencies of 32 and 64 Hz were significantly inhibited by PPADS only at concentrations of 10 and 30 μm. 4 PPADS (30 μm) had no effect on relaxations to 2-methylthio ATP (3 nm-3 μm) in rabbit mesenteric artery and to ATP (1 μm − 1 mm) in rabbit aorta (with endothelium intact or removed). In addition, PPADS (30 μm) had no significant influence on the contractile potency of noradrenaline and histamine in rabbit ear and saphenous artery, respectively. 5 In conclusion, these results support the evidence that PPADS is a selective antagonist of P_2X-purinoceptor-mediated responses.