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Thrombin-promoted release of UDP-glucose from human astrocytoma cells.

ABSTRACT: 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.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.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.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.

SUBMITTER: Kreda SM

PROVIDER: S-EPMC2437904 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Thrombin-promoted release of UDP-glucose from human astrocytoma cells.

Kreda S M SM Seminario-Vidal L L Heusden C van Cv Lazarowski E R ER

British journal of pharmacology 20080121 7

<h4>Background and purpose</h4>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.<h4>Experimental approach</h4>UDP-glucose release and hydrolysis were measured using HPLC-b ...[more]

PMID: 18204471

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Project description:We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755-763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion channels (VSOAC) and that hypertonic stress strongly inhibits PAR1 gating of VSOAC. We tested the hypothesis that a VSOAC-type permeability might comprise a GPCR-regulated pathway for ATP export by determining whether PAR1-sensitive ATP release from 1321N1 cells is similarly potentiated by hypotonicity but suppressed by hypertonic conditions. Strong hypotonic stress by itself elicited ATP release and positively modulated the response to thrombin. Thrombin-dependent ATP release was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium. Neither the potency nor efficacy of thrombin as an activator of proximal PAR1 signaling was affected by hypotonicity or hypertonicity. 1,9-Dideoxyforskolin and carbenoxolone similarly attenuated PAR1-dependent ATP release and suppressed the PAR1-independent ATP elicited by strong hypotonic stress. Probenecid attenuated PAR1-stimulated ATP release under isotonic but not mild hypotonic conditions and had no effect on PAR1-independent release stimulated by strong hypotonicity. PAR1-dependent ATP export under all osmotic conditions required concurrent signaling by Ca(2+) mobilization and Rho-GTPase activation. In contrast, PAR1-independent ATP release triggered by strong hypotonicity required neither of these intracellular signals. Thus, we provide the new finding that GPCR-regulated ATP release from 1321N1 astrocytoma cells is remarkably sensitive to both positive and negative modulation by extracellular osmolarity. This supports a model wherein GPCR stimulation and osmotic stress converge on an ATP release pathway in astrocytes that exhibits several features of VSOAC-type channels.

Project description:UDP-glucose dehydrogenase (UGDH) generates essential precursors of hyaluronic acid (HA) synthesis, however mechanisms regulating its activity are unclear. We used enzyme histostaining and quantitative image analysis to test whether cytokines that stimulate HA synthesis upregulate UGDH activity. Fibroblast-like synoviocytes (FLS, from N = 6 human donors with knee pain) were cultured, freeze-thawed, and incubated for 1 hour with UDP-glucose, NAD+ and nitroblue tetrazolium (NBT) which allows UGDH to generate NADH, and NADH to reduce NBT to a blue stain. Compared to serum-free medium, FLS treated with PDGF showed 3-fold higher UGDH activity and 6-fold higher HA release, but IL-1beta/TGF-beta1 induced 27-fold higher HA release without enhancing UGDH activity. In selected proliferating cells, UGDH activity was lost in the cytosol, but preserved in the nucleus. Cell-free assays led us to discover that diaphorase, a cytosolic enzyme, or glutathione reductase, a nuclear enzyme, was necessary and sufficient for NADH to reduce NBT to a blue formazan dye in a 1-hour timeframe. Primary synovial fibroblasts and transformed A549 fibroblasts showed constitutive diaphorase/GR staining activity that varied according to supplied NADH levels, with relatively stronger UGDH and diaphorase activity in A549 cells. Unilateral knee injury in New Zealand White rabbits (N = 3) stimulated a coordinated increase in synovial membrane UGDH and diaphorase activity, but higher synovial fluid HA in only 2 out of 3 injured joints. UGDH activity (but not diaphorase) was abolished by N-ethyl maleimide, and inhibited by peroxide or UDP-xylose. Our results do not support the hypothesis that UGDH is a rate-liming enzyme for HA synthesis under catabolic inflammatory conditions that can oxidize and inactivate the UGDH active site cysteine. Our novel data suggest a model where UGDH activity is controlled by a redox switch, where intracellular peroxide inactivates, and high glutathione and diaphorase promote UGDH activity by maintaining the active site cysteine in a reduced state, and by recycling NAD+ from NADH.

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Thrombin-promoted release of UDP-glucose from human astrocytoma cells.

Publications

Thrombin-promoted release of UDP-glucose from human astrocytoma cells.

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