Release of cellular UDP-glucose as a potential extracellular signaling molecule.

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.

Novel epinephrine and cyclic AMP-mediated activation of BCAM/Lu-dependent sickle (SS) RBC adhesion.

The vasoocclusive crisis is the major clinical feature of sickle cell anemia, which is believed to be initiated or sustained by sickle (SS) red blood cell (RBC) adhesion to the vascular wall. SS RBCs, but not unaffected (AA) RBCs, adhere avidly to multiple components of the vascular wall, including laminin. Here we report a novel role for epinephrine and cyclic adenosine monophosphate (cAMP) in the regulation of human SS RBC adhesiveness via the laminin receptor, basal cell adhesion molecule/Lutheran (BCAM/Lu). Our data demonstrate that peripheral SS RBCs contain greater than 4-fold more cAMP than AA RBCs under basal conditions. Forskolin or the stress mediator epinephrine further elevates cAMP in SS RBCs and increases adhesion of SS RBCs to laminin in a protein kinase A (PKA)-dependent manner, with the low-density population being the most responsive. Epinephrine-stimulated adhesion to laminin, mediated primarily via the beta 2-adrenergic receptor, occurred in SS RBC samples from 46% of patients and was blocked by recombinant, soluble BCAM/Lu, implicating this receptor as a target of cAMP signaling. Thus, these studies demonstrate a novel, rapid regulation of SS RBC adhesion by a cAMP-dependent pathway and suggest that components of this pathway, particularly PKA, the beta 2-adrenergic receptor, and BCAM/Lu, should be further explored as potential therapeutic targets to inhibit SS RBC adhesion.