DINUCLEOTIDE POLYPHOSPHATES CONTRIBUTE TO PURINERGIC SIGNALLING VIA INHIBITION OF ADENYLATE KINASE ACTIVITY: 2C.06

Abstract

Introduction: 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, Up4A and Ap5A and the non-selective P2 antagonist suramin on human serum and endothelial nucleotide-converting enzymes. Methods: Human serum and human umbilical vein endothelial cells (HUVEC) were pre-treated with various concentrations of dinucleotide polyphosphates and suramin. Adenylate kinase and NDP kinase activities were then quantified radiochemically by thin-layer chroma-tographic (TLC) analysis of ATP-induced conversion of [3H]-AMP and [3H]-ADP into high-energy[3H]-ATP, respectively. Endothelial NTPDase activity was additionally determined using [3H]-ADP and [3H]-ATP as preferred substrates. Results and Discussion: Dinucleoside polyphosphates and suramin have an inhibitory effect on the serum adenylate kinase as well as on endothelial adenylate kinase, 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 and [3H]-ATP) hydrolyses by cultured HUVEC. Up4A has no significant effect on the endothelial NTPDase activity. Although the half-lives for Ap4A, Up4A and Ap5A in serum are comparable to incu-bation times of the assays used in this study, secondary effects of the dinucleotide metabolites are not prominent for these inhibitory effects, since the concentration of metabolites formed are relatively insignificant compared to the 800 μmol/L of ATP added as a phosphate donor in the adenylate kinase and NDP kinase assay. Conclusion: This comparative competitive study suggests that Ap4A and Ap5A contribute to the purinergic responses via inhibition of adenylate-kinase mediated conversion of endoge-nous ADP, while Up4A most likely mediates its vasoregulatory effects via direct binding-mediated mechanisms.