PHARMACOLOGICAL-ANALYSIS AND BIOCHEMICAL-ANALYSIS OF FPL-67156, A NOVEL, SELECTIVE INHIBITOR OF ECTO-ATPASE

1 FPL 67156 (6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP), is a ne wly synthesized analogue of ATP. 2 In a rabbit isolated tracheal epith elium preparation, measuring P-2U-purinoceptor-dependent chloride secr etion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP-gamma-S. UTP agonist-concentration effect (E/[A]) cu rves were shifted to the left by 5-fold in the presence of 100 mu M FP L 67156. The differential effect of FPL 67156 on UTP and ATP-gamma-S w as hypothesized to be due to the greater susceptibility of UTP to enzy matic dephosphorylation and the ability of FPL 67156 to inhibit this p rocess. 3 FPL 67156 was tested as an ecto-ATPase inhibitor in a human blood cell assay, measuring [gamma(32)P]-ATP dephosphorylation. The co mpound inhibited [gamma(32)P]-ATP degradation with a pIC(50) of 4.6. 4 FPL 67156 was then tested for its effects on ATP and alpha,beta-methy lene-ATP responses at P-2X- purinoceptors in the rabbit isolated ear a rtery. In the concentration range 30 mu M -1 mM, the compound potentia ted the contractile effects of ATP but not those of alpha,beta-methyle ne-ATP. At 1 mM, FPL 67156 produced a 34-fold leftward shift of ATP E/ [A] curves. 5 The effects of FPL 67156 on ATP E/[A] curves in the rabb it ear artery were analysed using a theoretical model (Furchgott, 1972 ) describing the action of an enzyme inhibitor on the effects of a met abolically unstable agonist. This analysis provided an estimate of the pK(1) for FPL 67156 as an ecto-ATPase inhibitor of 5.2. 6 Using appro priate assays, FPL 67156 was shown to have weak antagonist effects at P-2X- and P-2T-purinoceptors (pA(2) approximate to 3.3 and 3.5 respect ively), and weak agonist effects at P-2U-purinoceptors (p[A(50)]approx imate to 3.5). 7 The degree of potentiation of ATP and UTP effects eli cited by FPL 67156 confirms previous results concerning the influence that ecto-ATPase has on the position of E/[A] curves for metabolically unstable agonists. The magnitude of this influence is predicted to ha ve a major effect on the agonist potency orders currently used to desi gnate purinoceptors. 8 This study indicates FPL 67156 to be a potentia lly valuable probe in studies on the action of nucleotides and in the classification of purinoceptors.