Inhibitory effects of some purinergic agents on ecto-ATPase activity and pattern of stepwise ATP hydrolysis in rat liver plasma membranes

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Abstract

Inhibitory effects of various purinergic compounds on the Mg2+-dependent enzymatic hydrolysis of [3H]ATP in rat liver plasma membranes were evaluated. Rat liver enzyme ecto-ATPase has a broad nucleotide-hydrolyzing activity, displays Michaelis–Menten kinetics with Km for ATP of 368±56 μM and is not sensitive to classical inhibitors of the ion-exchange and intracellular ATPases. P2-antagonists and diadenosine tetraphosphate (Ap4A) progressively and non-competitively inhibited ecto-ATPase activity with the following rank order of inhibitory potency: suramin (pIC50, 4.570)>Reactive blue 2 (4.297)≫Ap4A (3.268)>pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (2.930). Slowly hydrolyzable P2 agonists ATPγS, ADPβS, α,β-methylene ATP and β,γ-methylene ATP as well as the diadenosine polyphosphates Ap3A and Ap5A 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 [3H]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 [3H]ATP dephosphorylation still occurs in the presence of ecto-ATPase inhibitors suramin, Ap4A and PPADS, but the appearance of the ultimate reaction product, adenosine, was significantly delayed. In contrast, hydrolysis of [3H]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.

Keywords

Liver plasma membrane
Rat
Ecto-ATPase
P2 receptor
Inhibition

Keywords

ADPβS, adenosine 5′-O-(2-thiodiphosphate)
Ap3A, diadenosine triphosphate
Ap4A, diadenosine tetraphosphate
Ap5A, diadenosine pentaphosphate
ATPγS, adenosine 5′-O-(3-thiotriphosphate)
α,β-MeATP, α,β-methylene ATP
β,γ-MeATP, β,γ-methylene ATP
p-CMBS, p-chloromercuriphenyl sulfonate
PPADS, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid
RB2, Reactive blue 2 (Cibacron blue 3GA)
TLC, thin-layer chromatography

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1

Present address: MediCity Research Laboratory, University of Turku, Tykistökatu 6A, FIN-20520, Turku, Finland.