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

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Abstract
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 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 P2X‐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 P2X‐purinoceptors, while the binding of [3H]‐quinuclidinyl benzilate to muscarinic cholinoceptors was not affected. Thus, PPADS (1–30 μm) 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.