Identification of a
G protein-coupled receptor activated by
UDP-glucose led us to develop a sensitive and specific assay for
UDP-glucose mass and to test whether this
sugar nucleotide is released as an extracellular signaling molecule. Mechanical stimulation of 1321N1 human
astrocytoma cells by a change of medium resulted in an increase in extracellular levels of both
ATP and
UDP-glucose. Whereas
ATP levels peaked within 10 min and subsequently returned to resting extracellular levels of 3 nM,
UDP-glucose levels attained a steady state that exceeded that of resting
ATP levels by 3- to 5-fold for at least 3 h. Similar rates of basal release of
UDP-glucose and
ATP (72 and 81 fmol/min/10(6) cells) combined with a rate of
UDP-glucose metabolism approximately three times lower than
ATP hydrolysis account for the elevated extracellular
UDP-glucose levels on resting cells. A medium change also resulted in rapid appearance of
UDP-glucose on the
luminal surface of highly differentiated polarized human airway epithelial cells but at levels 2- to 3-fold lower than
ATP. However,
nucleotide sugar levels increased 3- to 5-fold over the ensuing 2 h, whereas
ATP levels decayed to a resting level; consequently, resting extracellular
UDP-glucose levels exceeded those of
ATP by 5- to 10-fold.
UDP-glucose also was observed at levels that equaled or exceeded those of
ATP in the extracellular medium of Calu-3 airway epithelial, COS-7, CHO-K1, and C6
glioma cells. Consistent with the observation of significant extracellular
UDP-glucose levels, expression of the
UDP-glucose-activated P2Y(14) receptor in COS-7 cells resulted in
G protein-promoted
inositol phosphate accumulation that was partially reversed by enzymatic removal of
UDP-glucose from the medium. Taken together, these results indicate constitutive release of
UDP-glucose from physiologically relevant tissues and suggest that
UDP-glucose acts as an autocrine activator of the P2Y(14) receptor. Because cellular
UDP-glucose is concentrated in the lumen of the endoplasmic reticulum, we speculate that
UDP-glucose release may occur as a result of vesicle transport during trafficking of
glycoproteins to the plasma membrane.