Abstract
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/106 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 P2Y14 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 P2Y14receptor. 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.
Footnotes
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↵1 An equilibrium constant of 0.67 for the reaction in the direction of UTP synthesis was reported (Hansen et al., 1966).
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↵2 In contrast to most glycosyltransferases, only UDP-xylose:proteoglycan core protein xylosyltransferase has been confirmed to be secreted both by cultured cells and in vivo (Gotting et al., 1998; Kuhn et al., 2001).
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This work was supported by United States Public Health Service grants HL34322 and GM38213.
- Abbreviations:
- UDP-glucose
- uridine 5′-diphosphoglucose
- HPLC
- high-performance liquid chromatography
- NTPppase
- nucleoside triphosphate pyrophosphatase
- PPi
- pyrophosphate
- DMEM
- Dulbecco's modified Eagle's medium
- ER
- endoplasmic reticulum
- Received October 28, 2002.
- Accepted February 3, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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