Inhibitory effects of some purinergic agents on ecto-ATPase activity and pattern of stepwise ATP hydrolysis in rat liver plasma membranes.
Inhibitory effects of various purinergic compounds on the Mg(2+)-dependent enzymatic hydrolysis of [(3)H]ATP in rat liver plasma membranes were evaluated. Rat liver enzyme ecto-ATPase has a broad nucleotide-hydrolyzing activity, displays Michaelis-Menten kinetics with K(m) for ATP of 368+/-56 microM and is not sensitive to classical inhibitors of the ion-exchange and intracellular ATPases. P2-antagonists and diadenosine tetraphosphate (Ap(4)A) progressively and non-competitively inhibited ecto-ATPase activity with the following rank order of inhibitory potency: suramin (pIC(50), 4.570)>Reactive blue 2 (4.297)&z.Gt;Ap(4)A (3. 268)>pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (2. 930). Slowly hydrolyzable P2 agonists ATPgammaS, ADPbetaS, alpha, beta-methylene ATP and beta,gamma-methylene ATP as well as the diadenosine polyphosphates Ap(3)A and Ap(5)A did not exert any inhibitory effects on the enzyme activity at concentration ranges of 10(-4)-10(-3) M. Thin-layer chromatography analysis of the formation of [(3)H]ATP metabolites indicated the presence of other enzyme activities on liver surface (ecto-ADPase and 5'-nucleotidase), participating in concert with ecto-ATPase in the nucleotide hydrolysis through the stepwise reactions ATP-->ADP-->AMP-->adenosine. A similar pattern of sequential [(3)H]ATP dephosphorylation still occurs in the presence of ecto-ATPase inhibitors suramin, Ap(4)A and PPADS, but the appearance of the ultimate reaction product, adenosine, was significantly delayed. In contrast, hydrolysis of [(3)H]ATP in the presence of Reactive blue 2 only followed the pattern ATP-->ADP, with formation of the subsequent metabolites AMP and adenosine being virtually eliminated. These data suggest that although nucleotide-binding sites of ecto-ATPase are distinct from those of P2 receptors, some purinergic agonists and antagonists can potentiate cellular responses to extracellular ATP through non-specific inhibition of the ensuing pathways of purine catabolism.
Yegutkin
G G
GG
Autonomic Neuroscience Institute, Royal Free and University College Medical School, University College London, Royal Free Campus, Rowland Hill Street, NW3 2PF, London, UK.
Burnstock
G
G
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D015226:Dinucleoside Phosphates / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D008297:Male; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013498:Suramin / Q000494:pharmacology*; D014227:Triazines / Q000494:pharmacology*
D015226:Dinucleoside Phosphates; D004791:Enzyme Inhibitors; D014227:Triazines; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; C020733:diadenosine tetraphosphate; C012842:Cibacron Blue F 3GA; D011732:Pyridoxal Phosphate; D013498:Suramin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase