Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto‐ATPase

Abstract

FPL 67156 (6‐N,N‐diethyl‐β,γ‐dibromomethylene‐ d ‐ATP), is a newly synthesized analogue of ATP. In a rabbit isolated tracheal epithelium preparation, measuring P 2U ‐purinoceptor‐dependent chloride secretion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP‐γ‐S. UTP agonist‐concentration effect (E/[A]) curves were shifted to the left by 5‐fold in the presence of 100 μ m FPL 67156. The differential effect of FPL 67156 on UTP and ATP‐γ‐S was hypothesized to be due to the greater susceptibility of UTP to enzymatic dephosphorylation and the ability of FPL 67156 to inhibit this process. FPL 67156 was tested as an ecto‐ATPase inhibitor in a human blood cell assay, measuring [γ 32 P]‐ATP dephosphorylation. The compound inhibited [γ 32 P]‐ATP degradation with a pIC 50 of 4.6. FPL 67156 was then tested for its effects on ATP and α,β‐methylene‐ATP responses at P 2X ‐purinoceptors in the rabbit isolated ear artery. In the concentration range 30 μ m ‐1 m m , the compound potentiated the contractile effects of ATP but not those of α,β‐methylene‐ATP. At 1 m m , FPL 67156 produced a 34‐fold leftward shift of ATP E/[A] curves. The effects of FPL 67156 on ATP E/[A] curves in the rabbit ear artery were analysed using a theoretical model (Furchgott, 1972) describing the action of an enzyme inhibitor on the effects of a metabolically unstable agonist. This analysis provided an estimate of the p K 1 for FPL 67156 as an ecto‐ATPase inhibitor of 5.2. Using appropriate assays, FPL 67156 was shown to have weak antagonist effects at P 2X ‐ and P 2T ‐purinoceptors (p A 2 ≅ 3.3 and 3.5 respectively), and weak agonist effects at P 2U ‐purinoceptors (p[ A 50 ] ≅ 3.5). The degree of potentiation of ATP and UTP effects elicited 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 have a major effect on the agonist potency orders currently used to designate purinoceptors. This study indicates FPL 67156 to be a potentially valuable probe in studies on the action of nucleotides and in the classification of purinoceptors.