Dinucleotide polyphosphates contribute to purinergic signalling via inhibition of adenylate kinase activity

Abstract

Dinucleoside polyphosphates are well described as direct vasoconstrictors and as mediators with strong proliferative properties, however, less is known about their effects on nucleotide-converting pathways. Therefore, the present study investigates the effects of Ap4A (diadenosine tetraphosphate), Up4A (uridine adenosine tetraphosphate) and Ap5A (diadenosine pentaphosphate) and the non-selective P2 antagonist suramin on human serum and endothelial nucleotide-converting enzymes. Human serum and HUVECs (human umbilical vein endothelial cells) were pretreated with various concentrations of dinucleotide polyphosphates and suramin. Adenylate kinase and NDP kinase activities were then quantified radiochemically by TLC analysis of the ATP-induced conversion of [3H]AMP and [3H]ADP into [3H]ADP/ATP and [3H]ATP respectively. Endothelial NTPDase (nucleoside triphosphate diphosphohydrolase) activity was additionally determined using [3H]ADP and [3H]ATP as preferred substrates. Dinucleoside polyphosphates and suramin have an inhibitory effect on the serum adenylate kinase [pIC50 values (−log IC50): Ap4A, 4.67±0.03; Up4A, 3.70±0.10; Ap5A, 6.31±0.03; suramin, 3.74±0.07], as well as on endothelial adenylate kinase (pIC50 values: Ap4A, 4.17±0.07; Up4A, 2.94±0.02; Ap5A, 5.97±0.04; suramin, 4.23±0.07), but no significant effects on serum NDP kinase, emphasizing the selectivity of these inhibitors. Furthermore, Ap4A, Up4A, Ap5A and suramin progressively inhibited the rates of [3H]ADP (pIC50 values: Ap4A, 3.38±0.09; Up4A, 2.78±0.06; Ap5A, 4.42±0.11; suramin, 4.10±0.07) and [3H]ATP (pIC50 values: Ap4A, 3.06±0.06; Ap5A, 3.05±0.12; suramin, 4.14±0.05) hydrolyses by cultured HUVECs. Up4A has no significant effect on the endothelial NTPDase activity. Although the half-lives for Ap4A, Up4A and Ap5A in serum are comparable with the incubation times of the assays used in the present study, secondary effects of the dinucleotide metabolites are not prominent for these inhibitory effects, since the concentration of metabolites formed are relatively insignificant compared with the 800 μmol/l ATP added as a phosphate donor in the adenylate kinase and NDP kinase assays. This comparative competitive study suggests that Ap4A and Ap5A contribute to the purinergic responses via inhibition of adenylate-kinase-mediated conversion of endogenous ADP, whereas Up4A most likely mediates its vasoregulatory effects via direct binding-mediated mechanisms.