IDENTIFICATION OF AN ECTO-NUCLEOSIDE DIPHOSPHOKINASE AND ITS CONTRIBUTION TO INTERCONVERSION OF P2 RECEPTOR AGONISTS

The P2Y(4) receptor is selectively activated by UTP, Although addition of neither ATP nor UDP alone increased intracellular Ca2+ in 1321N1 h uman astrocytoma cells stably expressing the P2Y(4) receptor, combined addition of these nucleotides resulted in a slowly occurring elevatio n of Ca2+. The possibility that the stimulatory effect of the combined nucleotides reflected formation of UTP by an extracellular transphosp horylating activity was investigated, Incubation of cells with [H-3]UD P or [H-3]ADP under conditions in which cellular release of ATP occurr ed or in the presence of added ATP resulted in rapid formation of the corresponding triphosphates, Transfer of the gamma-phosphate from [gam ma-P-33]ATP to nucleoside diphosphates confirmed that the extracellula r 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 UD P were effective substrates for transphosphorylation. Since ecto-nucle otidase(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 K-m a nd V-max values, Kinetic analyses of the ecto-nucleoside diphosphokina se and ecto-nucleotidase activities indicated that the rate of extrace llular transphosphorylation exceeds that of nucleotide hydrolysis by u p to 20-fold. Demonstration of the existence of a very active ecto-nuc leoside diphosphokinase together with previous observations that stres s induced release of ATP occurs from most cell types indicates that tr ansphosphorylation is physiologically important in the extracellular m etabolism of adenine and uridine nucleotides, Since the P2Y receptor c lass of signaling proteins differs remarkably in their respective spec ificity for adenine and uridine nucleotides and di- and triphosphates, these results suggest that extracellular interconversion of adenine a nd uridine nucleotides plays a key role in defining activities in nucl eotide-mediated signaling.