title: Pharmacological analysis of ecto-ATPase inhibition: evidence for combined enzyme inhibition and receptor antagonism in P2X-purinoceptor ligands.
abstract: 1. Previous studies have shown that suramin and FPL 66301 are competitive antagonists at the P2X-purinoceptor in the rabbit ear artery. Those studies employed alpha,beta-methylene ATP, a poorly hydrolysable ATP analogue, as the agonist. In this study these compounds have been tested using ATP as the agonist. 2. Suramin, in the concentration range 30-1000 microM, potentiated the contractile effects of ATP, producing a 3-fold leftward shift of the ATP E/[A] curves. FPL 66301, in the concentration range 100-1000 microM, produced a significant but small (approximately 3-fold) rightward shift of the ATP curves. These results are in marked contrast with previous studies using alpha,beta-methylene ATP in which 30-fold rightward shifts were achieved using the same concentration ranges of suramin and FPL 66301. 3. Suramin and FPL 66301 were tested as ecto-ATPase inhibitors in a human blood cell assay. Suramin inhibited the enzyme with a pIC50 of 4.3, FPL 66301 with a pIC50 of 3.3. 4. The pharmacological data were analysed using a theoretical model describing the action of a compound with dual enzyme inhibitory and receptor antagonistic properties on the effects of an agonist susceptible to enzymatic degradation. The model was found to fit the data well using the known pKB estimates for suramin and FPL 66301 and similar relative (but not absolute) pK1 estimates to those obtained for the compounds in the enzyme assay. 5. From this analysis it was concluded that the limited shifts of ATP E/[A] curves produced by suramin and FPL 66301 were the result of 'self-cancellation' of the potentiating (enzyme inhibitory) and rightward-shifting (receptor antagonistic) properties.6. The analysis also indicated that the presence of ecto-ATPase activity in the rabbit ear artery preparation has a marked effect on the apparent potency of ATP. The experimental p[A50] was 3.4,whereas the 'true' value, that is the value which would be obtained in the absence of ecto-ATPase activity, was 6.0, some 400-fold higher.7 Two conclusions are drawn from this study. Firstly, caution must be exercised in the use of suramin and FPL 66301 as tools for receptor classification. Absence of overt antagonism by these compounds when metabolically unstable agonists are used could lead to erroneous claims for receptor subtypes.Secondly, the agonist potency order currently used to designate P2X- purinoceptors may require modification.
authors.affiliation: Department of Pharmacology, Fisons R&D Labs, Loughborough, Leicestershire.
authors.forename: B E
authors.initials: BE
authors.lastname: Crack
authors.forename: M W
authors.initials: MW
authors.lastname: Beukers
authors.forename: K C
authors.initials: KC
authors.lastname: McKechnie
authors.forename: A P
authors.initials: AP
authors.lastname: Ijzerman
authors.forename: P
authors.initials: P
authors.lastname: Leff
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000097:blood / Q000494:pharmacology; D000818:Animals; D001158:Arteries / Q000187:drug effects; D004431:Ear, External / Q000098:blood supply; D066298:In Vitro Techniques; D008024:Ligands; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D011817:Rabbits; D012039:Regional Blood Flow / Q000187:drug effects; D013498:Suramin / Q000494:pharmacology*
chemical_list: C092578:FPL 66301; D008024:Ligands; D058919:Purinergic P2 Receptor Antagonists; D013498:Suramin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase