PPADS selectively antagonizes P2X‐purinoceptor‐mediated responses in the rabbit urinary bladder

Abstract

1 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. 2 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). 3 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. 4 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%. 5 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. 6 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.