Hydrolysis of P2-purinoceptor agonists by a purified ectonucleotidase from the bovine aorta, the ATP-diphosphohydrolase

• Published in: Biochemical pharmacology 1996-06, Vol.51 (11), p.1453-1460
• Main Authors: Picher, M, Sévigny, J, D'Orléans-Juste, P, Beaudoin, A R
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - analogs & derivatives; Adenosine Diphosphate - metabolism; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Animals; Aorta - enzymology; Apyrase - isolation & purification; Apyrase - metabolism; Binding, Competitive; Cattle; Cell Membrane - enzymology; Hydrolysis; Kinetics; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2 - metabolism
• ISSN: 0006-2952
• DOI: 10.1016/0006-2952(96)00086-X

Pharmacologists are becoming more and more aware of the possibility that certain ATP analogues currently used to classify the P2-purinoceptors are dephosphorylated by ectonucleotidases. In this study, we provide evidence that in the vascular system, these purine analogues are hydrolysed by an ATP-diphosphohydrolase (ATPDase). This enzyme is known as the major plasma membrane nucleotidase of endothelial and smooth muscle cells, and is believed to dephosphorylate extracellular triphospho- and diphosphonucleosides. Assays were conducted with a purified ATPDase from smooth muscle cells of bovine aorta. At a concentration of 250 microM, adenosine 5'-(alpha,beta-methylene) triphosphonate (alpha,beta-metATP), adenosine 5'-(beta,gamma-methylene) triphosphonate (beta,gamma-metATP), adenosine 5'-(alpha,beta-methylene) disphosphonate (alpha,beta-metADP), adenylyl 5'-(beta,gamma-imido) diphosphonate (beta,gamma-imidoATP) and adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) all resisted dephosphorylation, whereas 2-chloroadenosine triphosphate (2-chloroATP), 2-methylthioadenosine triphosphate (2-MeSATP) and 8-bromoadenosine triphosphate (8-bromo-ATP) were hydrolysed at 99, 63, and 20% of the rate of ATP hydrolysis, respectively. All the non-hydrolysable analogues tested, except alpha,beta-metADP, competed with ATP and ADP for the ATPDase catalytic site, reducing their hydrolysis by 35-50%. Apparent Km values for ATP and ADP were estimated at 14.1 and 12.0 microM, respectively, whereas apparent Km and Ki values for the purine analogues ranged from 12 to 28 microM. These results strongly support the view that (1) the ATPDase is expected to reduce substantially the P2-response induced by ATP, ADP, and some hydrolysable agonists; and (2) by competing with the hydrolysis of endogenously released ATP and ADP, non-hydrolysable analogues could alter the amplitude or direction of the cellular response induced by these natural substrates.