1 The effect of pyridoxalphosphate-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 > alpha,beta-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 mu 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 mu 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 mu M) antagonized the relaxant effects to alpha,beta-methylene ATP in a surmountable manner. An extensive shift of the CRC was produced only by 30 mu 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 mu 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 mu M) obtained in the enzyme assay was much higher than the functional EC(50)-value (0.45 mu M) of ATP. PPADS (3-100 mu M) was only weakly active in inhibiting ecto-nucleotidase activity leaving a residual activity of 81.8 +/- 5.1% at 100 mu 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 > > alpha,beta-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 mu 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 mu M vs. 1-10 (30) mu 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.
