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 P2y-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 P2-purinoceptor blockade and ectonucleotidase 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>a,,B-methylene ATP with the respective pD2-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 yM) caused a consistent dextral shift of the concentrationresponse curve (CRC) of 2-methylthio ATP and ATP resulting in a biphasic Schild plot. A substantial shift was only observed at 100 JM PPADS, the respective pA2-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 pM) antagonized the relaxant effects to a,fi-methylene ATP in a surmountable manner. An extensive shift of the CRC was produced only by 30 gM PPADS (pA2 = 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 uM).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 Km-value (366 pM) obtained in the enzyme assay was much higher than the functional EC50-value (0.45 pM) of ATP. PPADS (3-100 gM) was only weakly active in inhibiting ecto-nucleotidase activity leaving a residual activity of 81.8 ± 5.1% at 100 pM.Enzyme inhibition by PPADS was concentration-independent and non-competitive. 6 In rat duodenum, the rank order of potency was: 2-methylthio ATP > ATP> >ca,,B-methylene ATP, the respective pD2-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 gM) in a concentration-dependent manner, and Schild analysis gave a pA2-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 P2y-purinoceptors were different from those recently described at the P2k-subtype: inhibition of P2y-purinoceptor-mediated responses was observed at higher concentrations (3-100 gM vs. 1-10 (30) gM). Furthermore, we conclude th...

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1 We have compared the binding properties of the enantiomers of phenglutarimide (1) and of six related compounds to M, receptors in NB-OK-1 cells, M2 receptors in rat heart, M3 receptors in rat pancreas and the M4 receptors of rat striatum, with their functional (antimuscarinic) properties in rabbit vas deferens (MI/M4-like), guinea-pig atria (M2) and guinea-pig ileum (M3) receptors. The binding properties of the enantiomers of three of the compounds were also measured on cloned human ml-m4 receptors expressed by CHO cells, using [3H] 3 The pA2 values at the inhibitory heteroreceptors in the rabbit vas deferens, and at the guinea-pig atria and ileum for the seven more potent enantiomers were compatible with the previous classification of these receptors as MI/M4-like, M2 and M3, respectively. 4 Replacement of the phenyl by a thienyl ring or of the diethylamino by a piperidino group in the phenglutarimide molecule did not affect markedly the potencies of the high affinity enantiomer. In contrast, replacement of the phenyl by a cyclohexyl ring decreased 20 fold the active enantiomers potency. Methylation of the piperidine-2,6-dione nitrogen also reduced markedly the eutomers' affinities, more on the M, than on the other subtypes. 5 The selectivity profiles (recognition of four receptor subtypes) of six of the seven less active enantiomers were different from the corresponding more active enantiomers selectivity profiles, suggesting that the preparations used in this study were pure. However, we cannot not exclude the hypothesis that the batch of (S)-thienglutarimide used in this study was contaminated by less than 0.02% of the eutomer. 6 In contrast with the eutomer binding site, replacement of the phenyl ring by a thienyl or cyclohexyl ring did not affect binding of the low affinity enantiomers to the muscarinic receptor or the [3H]-NMSreceptor complex. The replacement of the diethylamino group by a piperidine ring, and N-methylation of the piperidine-2,6 dione moiety increased slightly these enantiomers' potencies. 7 The muscarinic receptors were extremely stereoselective, and had up to 20 000 fold lower affinity for the less active enantiomers. However, the stereochemical requirements of the muscarinic receptor subtypes were different for the enantiomers of compounds 1-7, being most stringent at M, receptors. 8 The weaker enantiomers behaved as competitive antagonists in pharmacological studies, at least in the concentration-range investigated.

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