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Keywords:

  • PAR1;
  • thrombin;
  • nucleotide release;
  • UDP-glucose;
  • 1321N1 human astrocytoma cells;
  • P2Y14 receptor

Background and purpose:

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.

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 Ca2+-dependent mechanism, engaging additional signalling independently of Gi 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.

British Journal of Pharmacology (2008) 153, 1528–1537; doi:10.1038/sj.bjp.0707692; published online 21 January 2008