Evidence supporting the Nucleotide Axis Hypothesis: ATP release and metabolism by coronary endothelium

The Nucleotide Axis Hypothesis, defined and supported herein, proposes that ATP stimulates the release of vasoactive mediators from endothelium includ ing ATP itself Here, we show rapid endothelium-dependent, agonist-stimulate d ATP elaboration in coronary vessels of guinea pigs. Measurement of extrac ellular ADP metabolism in intact vessels results in the time- and substrate -dependent formation of ATP in the coronary perfusate in amounts greater th an can be accounted for by release from endothelium alone. ATP formation by endothelial cells is saturable (K-M = 38.5 mu mol/l, where K-M is substrat e concentration at which rate is half-maximal.) and trypsin-sensitive, memb ranes from [gamma-P-32]ATP-labeled cells support ADP-dependent transphospho rylation by a 20-kDa protein, Western blots reveal the presence of a nucleo side diphosphate kinase (NDPK) of similar to 20 kDa in endothelial membrane s, and analysis of NDPK antibody binding by flow cytometry is consistent wi th the presence of an ecto-NDPK on cardiac endothelial cells. Sequencing of the endothelial cell ecto-NDPK reveals a predicted amino acid sequence wit h 85% identity to human Nm23-H1 and consistent with a protein whose propert ies may confer membrane association as well as sites of regulation of activ ity. Our data underscore the potential importance of a nucleotide axis in c ardiac blood vessels.