The
P2Y4 receptor is selectively activated by
UTP. Although addition of neither
ATP nor
UDP alone increased intracellular Ca2+ in 1321N1 human
astrocytoma cells stably expressing the
P2Y4 receptor, combined addition of these
nucleotides resulted in a slowly occurring elevation of Ca2+. The possibility that the stimulatory effect of the combined
nucleotides reflected formation of
UTP by an extracellular transphosphorylating activity was investigated. Incubation of cells with [3H]
UDP or [3H]
ADP under conditions in which cellular release of
ATP occurred or in the presence of added
ATP resulted in rapid formation of the corresponding triphosphates. Transfer of the gamma-
phosphate from [gamma-33P]
ATP to
nucleoside diphosphates confirmed that the extracellular enzymatic activity was contributed by a
nucleoside diphosphokinase. The majority of this activity was associated with the cell surface of 1321N1 cells, suggesting involvement of an ectoenzyme. Both
ADP and
UDP were effective substrates for transphosphorylation. Since
ecto-nucleotidase(s) has been considered previously to be the primary
enzyme(s) responsible for metabolism of extracellular
nucleotides, the relative rates of hydrolysis of
ATP,
ADP,
UTP, and
UDP also were determined for 1321N1 cells. All four
nucleotides were hydrolyzed with similar Km and Vmax values. Kinetic analyses of the ecto-
nucleoside diphosphokinase and
ecto-nucleotidase activities indicated that the rate of extracellular transphosphorylation exceeds that of
nucleotide hydrolysis by up to 20-fold. Demonstration of the existence of a very active ecto-
nucleoside diphosphokinase together with previous observations that stress-induced release of
ATP occurs from most cell types indicates that transphosphorylation is physiologically important in the extracellular metabolism of
adenine and
uridine nucleotides. Since the P2Y receptor class of signaling
proteins differs remarkably in their respective specificity for
adenine and
uridine nucleotides and di- and triphosphates, these results suggest that extracellular interconversion of
adenine and
uridine nucleotides plays a key role in defining activities in
nucleotide-mediated signaling.