Thrombin-promoted release of UDP-glucose from human astrocytoma cells.
OBJECTIVE
The P2Y14 receptor is activated by UDP-sugars, most potently by UDP-glucose, but not by free nucleotides, suggesting that UDP-glucose is the cognate agonist for this receptor. However, evidence for regulated release of UDP-glucose is scarce. In the present study, the occurrence of receptor-promoted release of UDP-glucose was investigated, using 1321N1 human astrocytoma cells.


METHODS
UDP-glucose release and hydrolysis were measured using HPLC-based techniques. Phospholipase C activation and actin cytoskeleton reorganization were assessed by measuring inositol phosphate formation and fluorescence confocal microscopy, respectively.


RESULTS
Thrombin and the protease-activating receptor-1 (PAR1) peptide TFLLRNPNDK (PAR1-AP) evoked the release of UDP-glucose and ATP, which was accompanied by enhanced inositol phosphate formation. Although carbachol promoted fourfold greater inositol phosphate formation than thrombin, it failed to promote nucleotide release. Thrombin-promoted nucleotide release was inhibited by BAPTA-AM, brefeldin A and cytochalasin D, and was insensitive to Pertussis toxin and PI3-kinase inhibitors. Thrombin, but not carbachol, induced actin cytoskeleton reorganization, a hallmark of Rho activation in 1321N1 cells. However, PAR-promoted UDP-glucose release was not affected by Rho kinase inhibition.


CONCLUSIONS
PAR1-evoked UDP-glucose release reflected a Ca2+-dependent mechanism, engaging additional signalling independently of G(i) and Rho kinase activation and requiring a functional actin cytoskeleton and Golgi structures. Our study demonstrates the occurrence of Ca2+ -dependent release of UDP-glucose from astrocytoma cells in response to a physiologically relevant stimulus, that is, a G-protein-coupled receptor agonist. Given the presence of P2Y14 receptors in astrocytes, UDP-glucose may have important autocrine/paracrine functions in the brain.
Kreda
S M
SM
Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina School of Medicine, Chapel Hill, NC, USA.
Seminario-Vidal
L
L
Heusden
C van
Cv
Lazarowski
E R
ER
D000199:Actins / Q000378:metabolism; D001254:Astrocytoma / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D003599:Cytoskeleton / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D044463:Receptor, PAR-1 / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism
D000199:Actins; C518528:P2Y14 receptor, human; D044463:Receptor, PAR-1; D018048:Receptors, Purinergic P2; D002217:Carbachol; D054460:rho-Associated Kinases; D010738:Type C Phospholipases; D013917:Thrombin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium; D014532:Uridine Diphosphate Glucose