Premium
Thrombin‐promoted release of UDP‐glucose from human astrocytoma cells
Author(s) -
Kreda S M,
SeminarioVidal L,
Heusden C,
Lazarowski E R
Publication year - 2008
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0707692
Subject(s) - bapta , cytoskeleton , chemistry , pertussis toxin , biochemistry , thrombin , microbiology and biotechnology , inositol , thrombin receptor , receptor , biology , g protein , cell , platelet , immunology
Background and purpose: The P2Y 14 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. Experimental approach: 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. Key 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 and implications: PAR1‐evoked UDP‐glucose release reflected a Ca 2+ ‐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 Ca 2+ ‐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 P2Y 14 receptors in astrocytes, UDP‐glucose may have important autocrine/paracrine functions in the brain. British Journal of Pharmacology (2008) 153 , 1528–1537; doi: 10.1038/sj.bjp.0707692 ; published online 21 January 2008