Extracellular Nucleotide Hydrolysis and the Integration of Signaling

The experiments presented here were undertaken from the point of view that the reaction sequence ATP ⇒ ADP ⇒ AMP ⇒ adenosine, occurring extracellularly, is a key sequence contributing to regulation of the time course of cell and tissue response during crisis or signaling events. Adenine nucleotides are released into the pericellular space during platelet thrombus formation, neurotransmission, catecholamine release from the adrenal medulla, strenuous exercise, and shock. Since the extent of phosphorylation can profoundly modify the physiological effects of extracellular adenine nucleotides, it is important to discover how the time course of their hydrolysis after release is controlled. Plasma membrane 5′-nucleotidase is known to be inhibited by ATP and ADP. This property suggests the possibility of a particularly interesting feature of the regulation of the time course of the extracellular conversion of ATP or ADP to adenosine. When the extracellular concentration of ATP or ADP is low, it should be quickly degraded all the way to adenosine. When a bolus of ATP or ADP of high enough concentration to inhibit 5′-nucleotidase appears, the rate of adenosine appearance should be diminished. We have examined the time course of this reaction sequence during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This arrangement permits study of the reactions in incubations with volume-to-cell-surface ratios approaching those of small blood vessels. Reaction rates are proportional to cell number, and the kinetics of the ectoenzymes are reproducible for at least 8 hr. When endothelial cells are presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP develop before significant conversion of AMP to adenosine occurs. Further, there is a paradoxical dependence of the initial rate of adenosine appearance on the initial ATP concentration: the higher the initial ATP concentration, the slower is the conversion of AMP to adenosine during early time points. The behavior of the system was modeled assuming one enzyme for each nucleotidase reaction and testing for inhibition of each reaction by its product and for inhibition of 5′-nucleotidase by ATP and/or ADP. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. K MS estimated in this way agreed well with those obtained from initial velocity measurements: for the ATPase, 300 μM; the ADPase, 240 μM; and 5′-nucleotidase, 26 μM. Maximum velocities varied from cell batch to cell batch but stayed in approximately the proportion ATPase : ADPase : AMPase = 6 : 1.5 : 1. The data for the rate of appearance of adenosine can only be fit if the model incorporates inhibition of 5′-nucleotidase by ATP or ADP. The KI for ADP is 5 μM. Product inhibition does not contribute significantly to the regulation of the pathway. These kinetic properties tend to maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would tend to create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an antiaggregatory milieu).