Inhibitory action of PPADS on relaxant responses to adenine nucleotides or electrical field stimulation in guinea‐pig taenia coli and rat duodenum

1 The effect of pyridoxarphosphate‐6‐azophenyl‐2′, 4′‐disulphonic acid (PPADS) on the relaxant response to adenine nucleotides was examined in the carbachol‐contracted guinea‐pig taenia coli and rat duodenum, two tissues possessing P 2y ‐purinoceptors. In addition, in the taenia coli PPADS was investigated for its effect on relaxations evoked by adenosine, noradrenaline and electrical field stimulation. In order to assess the selectivity of PPADS between P 2 ‐purinoceptor blockade and ecto‐ nucleotidase activity, its influence on ATP degradation was studied in guinea‐pig taenia coli. 2 The resulting rank order of potency for the adenine nucleotides in guinea‐pig taenia coli was: 2‐methylthio ATP≫ ATP>>α, β‐methylene ATP with the respective pD 2 ‐values 7.96 ±0.08 (n = 23), 6.27 ±0.12 (n = 21) and 5.88 ±0.04 (n = 24). 3 In guinea‐pig taenia coli, PPADS (10–100 μ m ) caused a consistent dextral shift of the concentration‐response curve (CRC) of 2‐methylthio ATP and ATP resulting in a biphasic Schild plot. A substantial shift was only observed at 100 μ m PPADS, the respective pA 2 ‐values at this particular concentration were 5.26 ±0.16 (n = 5) and 5.15 ±0.13 (n = 6). Lower concentrations of PPADS (3–30 μ m ) antagonized the relaxant effects to α, β‐methylene ATP in a surmountable manner. An extensive shift of the CRC was produced only by 30 μ m PPADS (pA 2 = 5.97 ± 0.08, n = 6), and the Schild plot was again biphasic. 4 The relaxant responses to electrical field stimulation (80 V, 0.3 ms, 5 s, 0.5–16 Hz) in guinea‐pig taenia coli were concentration‐dependently inhibited by PPADS (10–100 μ m ). 5 In guinea‐pig taenia coli, the potency of ATP in inducing relaxation appeared to be independent of its rate of degradation by ecto‐nucleotidases, since the K m ‐value (366 μ m ) obtained in the enzyme assay was much higher than the functional EC 50 ‐value (0.45 μ m ) of ATP. PPADS (3–100 μ m ) was only weakly active in inhibiting ecto‐nucleotidase activity leaving a residual activity of 81.8 ±5.1% at 100 μ m . Enzyme inhibition by PPADS was concentration‐independent and non‐competitive. 6 In rat duodenum, the rank order of potency was: 2‐methylthio ATP > ATP≫α, β‐methylene ATP, the respective pD 2 ‐values being 6.98 ±0.04 (n = 76), 6.26 ±0.02 (n = 6) and 4.83 ±0.02 (n = 6). Among these agonists, 2‐methylthio ATP displayed the lowest apparent efficacy. 7 The CRC of 2‐methylthio ATP in rat duodenum was shifted to the right by PPADS (10–100 μ m ) in a concentration‐dependent manner, and Schild analysis gave a pA 2 ‐value of 5.09 ±0.06 (slope =1.02, n=14). 8 PPADS was without any effect on the carbachol‐induced contraction in guinea‐pig taenia coli or rat duodenum and on the relaxation to noradrenaline or adenosine in guinea‐pig taenia coli. 9 In conclusion, the antagonistic properties of PPADS at the taenia coli and rat duodenum P 2y ‐ purinoceptors were different from those recently described at the P 2x ‐subtype: inhibition of P 2y ‐ purinoceptor‐mediated responses was observed at higher concentrations (3–100 μ m vs. 1–10 (30) μ m ). Furthermore, we conclude that in addition to the classical P 2y ‐subtype, which is largely PPADS‐resistant, the guinea‐pig taenia coli may be endowed with a distinct relaxation‐mediating P 2 ‐purinoceptor subtype which is sensitive to PPADS.