Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8

Abstract

Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with K (m) values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X(1-7) and P2Y(2,4,11) receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y(1,12,13) receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y(6) receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-8.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5'-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

Figure 1

Substrate specificity of plasma membrane bound NTPDases. The assays for the enzymatic activity were carried out with protein extracts from transiently transfected COS-7 cells in the presence of 0.5 mM adenine or uracil nucleotide with either 1 mM CaCl₂ (open bars) or 1 mM MgCl₂ (solid bars), as described under Materials and methods. The average ± SEM of two to five experiments performed in triplicate is shown.

Figure 2

The effect of pH on plasma membrane NTPDases. Enzyme activity assays with protein extracts from transiently transfected COS-7 cells were carried out in 50 mM BisYTris, 50 mM Tris, 50 mM Glycine, 2 mM CaCl₂ at the indicated pH. Reaction were initiated with 0.5 mM ATP (•) or ADP (□). A representative of at least three independent experiments performed in triplicate is shown.

Figure 3

Profiles of nucleotide hydrolysis by plasma membrane NTPDases. Reactions were initiated by the addition of protein extracts from COS-7 cells transfected with plasmid encoding an NTPDase to a medium containing 0.5 mM ATP and/or UTP, 5 mM CaCl₂ and 80 mM Tris, pH 7.4. Tris was replaced by 80 mM MES pH 6.4 for mouse NTPDase8. A sample of protein extracts was added to obtain 24 nmol/min of activity with ATP as a substrate of human NTPDases 1, 2, 3 and rat NTPDase8, and of 10–12 nmol/min of all mouse NTPDases. This amount of activity was doubled when both substrates (ATP and UTP) were added together. Controls with protein extracts from COS-7 cells not expressing NTPDase activities were performed and their activity subtracted from the activity of samples containing NTPDases. Aliquots were taken at the indicated time points and the reaction was stopped immediately by the addition of an equal volume of ice-cold 1 M perchloric acid. These samples were prepared and analyzed for nucleotide contents by HPLC, as described under Materials and methods. Data from a representative experiment performed in triplicate is given. (a) ATP hydrolysis by human and murine NTPDases: ATP (•), ADP (▪), AMP (≆). (b) UTP hydrolysis by NTPDases: UTP (○), UDP (□), UMP (▵). (c) Simultaneous hydrolysis of ATP and UTP by NTPDases: ATP (•), ADP (▪), AMP (≆), UTP (○), UDP (□), UMP (▵). *From Lavoie et al. [7]. #Reprinted from Bigonnesse et al. [8], Copyright 2004 American Chemical Society.