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- Discussion and conclusions
- Conflict of interest
ATP and other nucleotides are released from cells in a regulated manner to accomplish extracellular signalling functions through activation of P2X and P2Y purinergic receptors (Burnstock and Williams, 2000; Burnstock, 2006). P2X receptors, comprising seven species (P2X1–P2X7), are ATP-gated ion channels. P2Y receptors belong to the superfamily of G-protein-coupled receptors. At least eight P2Y receptor species have been identified, seven of which are activated by adenine and/or uridine nucleoside di- and triphosphates. The P2Y1, P2Y12 and P2Y13 receptors are activated by ADP. The P2Y2 receptor is activated by both ATP and UTP, and the P2Y4 (human) and P2Y6 receptors are activated by UTP and UDP, respectively. Unlike other P2 receptors, the P2Y14 receptor is activated by UDP-sugars, most potently by UDP-glucose, and is not activated by di- or triphosphonucleotides (Chambers et al., 2000).
Studies using heterologously expressed P2Y14 receptor have revealed a previously unnoticed accumulation of endogenous receptor agonist in extracellular solutions. Taking advantage of the selectivity of UDP-glucose pyrophophorylase in catalyzing the UDP-glucose-dependent conversion of pyrophosphate to UTP, UDP-glucose was identified and quantified with nanomolar sensitivity in the extracellular medium of many tissues and cell lines, including 1321N1 human astrocytoma cells (Lazarowski et al., 2003b). However, whether extracellular accumulation of UDP-glucose reflects a regulated mechanism of nucleotide release is not well defined. With the exception of nerve terminals and other specialized tissues that release ATP from secretory granules through Ca2+-regulated exocytosis, how nucleotides are released from most cell types is poorly understood. A major limitation in this understanding was, until recently, the paucity of pharmacological tools to induce nucleotide release in a regulated manner.
Thrombin and other serine proteases activate a family of four G-protein-coupled receptors, referred to as protease-activated receptors (PAR1–PAR4). Recently, Joseph et al. (2003) illustrated that activation of 1321N1 cells with thrombin resulted in enhanced release of ATP in a Ca2+-dependent manner. Subsequently, we observed that addition of thrombin to 1321N1 cells resulted in enhanced extracellular accumulation of UDP-glucose, in addition to ATP release (Lazarowski, 2006). However, whether thrombin-increased UDP-glucose accumulation reflected PAR-promoted UDP-sugar release and whether the signalling pathways were potentially involved in such a release were not addressed. In the present study, we investigated potential mechanisms involved in thrombin-elicited UDP-glucose release in 1321N1 cells. As the P2Y14 receptor is abundantly expressed in brain astrocytes (Moore et al., 2003), illustration of the regulated release of UDP-glucose by 1321N1 astrocytoma cells would provide support for the physiological significance of the P2Y14 receptor in the brain.
Discussion and conclusions
- Top of page
- Discussion and conclusions
- Conflict of interest
The major finding of the present study is that UDP-glucose release from 1321N1 astrocytes reflects a receptor-regulated, Ca2+-dependent event, which requires the integrity of the secretory pathway. It was previously observed that 1321N1 cells release UDP-glucose constitutively (Lazarowski et al., 2003b), and that addition of thrombin to these cells resulted in increased accumulation of this nucleotide-sugar in the extracellular medium (Lazarowski, 2006). Our present work demonstrates that increased UDP-glucose accumulation in the medium of thrombin-stimulated cells reflected receptor-promoted release of this UDP-sugar rather than inhibition of nucleotide hydrolysis (Figure 1). We illustrated that thrombin promoted both UDP-glucose release and second messenger production with a potency consistent with this protease acting on PAR1 (Trejo, 2003). We also demonstrated that the PAR1-selective peptide TFLLRNPNDK elicited second messenger production and UDP-glucose release in 1321N1 cells with an efficacy similar to that of thrombin (Figure 3). Finally, Reverse transcription-PCR analysis confirmed expression of PAR1 transcripts in these cells. Our data strongly suggest that thrombin-promoted UDP-glucose release in 1321N1 cells was mediated by activation of PAR1.
Recently, we illustrated that elevation of Ca2+ (with ionomycin) was enough to induce nucleotide release from airway epithelial goblet cells, an event associated with Ca2+-triggered exocytosis of mucin granules (Kreda et al., 2007). Consistent with the involvement of Ca2+ in nucleotide release, thrombin-evoked UDP-glucose release from 1321N1 cells was inhibited by BAPTA (added to cells as BAPTA-AM; Figure 2). However, a surprising finding of our current study was that agonist-promoted nucleotide release inversely correlated with the receptors' ability to evoke Gq/PLC signalling. The muscarinic M3 receptor is abundantly expressed on 1321N1 cells (Stephan and Sastry, 1992), and activation of these Gq-coupled/Ca2+-mobilizing receptors did not result in UDP-glucose release. These results are in line with previous studies illustrating that carbachol evoked only minor ATP release from 1321N1 cells, relative to thrombin (Joseph et al., 2003). One possible explanation for our data is that UDP-glucose release in thrombin-stimulated 1321N1 cells reflected signalling downstream of PAR1/Gq that differed, spatially and/or temporally, from M3-receptor-evoked Gq signalling. Alternatively, as PAR1 couples to Gq, G12/13 and Gi in 1321N1 cells, whereas muscarinic receptors on 1321N1 cells couple only to Gq (Majumdar et al., 1998), G12/13 and/or Gi effectors may participate in thrombin-promoted nucleotide release from these cells. Our data do not support the involvement of Gi in PAR-stimulated UDP-glucose release. Pertussis toxin, which inhibits Gi signalling in 1321N1 cells (Parr et al., 1994), and wortmannin, an inhibitor of PI3-kinase (PI3-kinase-γ isoform is activated by Gi (Rickert et al., 2000)), had no effect on PAR1-stimulated UDP-glucose release (Figure 4b). It is well established that PAR1 coupling to G12/13 leads to RhoGEF-mediated activation of Rho GTPases. A well-characterized downstream effector of Rho in 1321N1 cells is ROCK, which regulates morphologic changes. In 1321N1 cells, thrombin (but not carbachol) promotes myosin light change phosphorylation and cell shape changes, for example, cell rounding (Majumdar et al., 1998; Coleman and Olson, 2002). Although thrombin-elicited cell rounding was abolished in the presence of the ROCK inhibitor Y27632 (Figure 4a), thrombin-promoted UDP-glucose release was not affected by ROCK inhibitors (Figure 4b). Possibly, PAR-promoted nucleotide release could reflect involvement of effectors downstream of G12/13 other than ROCK, for example, TKs, A-kinase anchoring protein, Ras GTPase activating protein, cadherin and PLC-ɛ (Kurose, 2003).
Apart from the signalling involved, regulated release of nucleotides is considered to occur in the following two possible modes: (i) cytosolic nucleotide release through channels or transporters and (ii) exocytotic release of nucleotide-enriched vesicles (Lazarowski et al., 2003a; Kreda et al., 2007). Candidate transporters mediating UDP-glucose release from the cytosol of 1321N1 cells are SLC35 translocators, which transport nucleotide-sugars across subcellular membranes (Hirschberg et al., 1998; Ishida and Kawakita, 2004). SLC35 translocators transport UDP-glucose and other UDP-sugars from the cytosol to the ER/Golgi, using luminal UMP as the antiporter substrate (Hirschberg et al., 1998; Ishida and Kawakita, 2004). However, UDP-sugar/UMP translocators does not appear to be expressed/inserted in the plasma membrane of 1321N1 cells, as addition of UMP to the extracellular medium failed to increase UDP-glucose release (Figure 5). Moreover, the robust ecto-5′-nucleotidase activity present on 1321N1 cells (Figure 1b) makes it unlikely that endogenous UMP (for example, generated from released UTP; Lazarowski et al., 1997) accumulates on 1321N1 cell surfaces. Furthermore, the fact that UDP-glucose release from thrombin-stimulated 1321N1 cells was accompanied by enhanced ATP release suggests that a non-selective mechanism was involved. We cannot rule out that channels or transporters, other than SLC35, facilitated cytosolic nucleotide release from 1321N1 cells. For example, connexin and pannexin hemichannels have been proposed as ATP channels in several types of cells (Cotrina et al., 1998; Stout et al., 2002; Bao et al., 2004; De Vuyst et al., 2005; Eltzschig et al., 2006; Pelegrin and Surprenant, 2006; Huang et al., 2007). The potential contribution of connexin hemichannels to nucleotide release from astrocytes is unclear (Scemes et al., 2000; Coco et al., 2003; Bowser and Khakh, 2007).
Neurons, chromaffin cells, platelets, mast cells and pancreatic acinar cells package ATP in synaptic vesicles, chromaffin granules or dense core granules, which, upon stimulation with, for instance Ca2+ mobilizing agonists, fuse with the plasma membrane and release their contents into the extracellular space, a process commonly referred to as regulated exocytosis (Dean et al., 1984; Evans et al., 1992; Gualix et al., 1999; Sorensen and Novak, 2001). Coco et al. (2003) have illustrated that an ATP-rich fraction from astrocyte homogenates co-sedimented with secretogranin II-containing vesicles on sucrose density gradients, and that mechanically induced ATP release was Ca2+-dependent and was inhibited by tetanus neurotoxin and the v-ATPase inhibitor bafilomycin A1. These findings support the hypothesis that ATP release in mechanically stimulated astrocytes occur via regulated exocytosis (Coco et al., 2003). Our observation that UDP-glucose is released concomitantly with ATP from PAR-stimulated 1321N1 astrocytoma cells supports the involvement of vesicles in nucleotide release from astrocytes. UDP-glucose is utilized in the lumen of the ER and Golgi for quality control of glycoproteins. UDP-glucose is the glucose donor substrate for glucosylation of denatured domains of newly synthesized glycoproteins (Hirschberg et al., 1998). Like UDP-sugars, ATP is also transported to and used as an energy source within the ER/Golgi (Hirschberg et al., 1998). Nucleotides imported to the lumen of the ER and Golgi reach concentrations up to 20-fold higher than their cytosolic levels, and are not transported back to the cytosol (Hirschberg et al., 1998). Therefore, they are likely to be delivered as cargo molecules and released from cells during glycoprotein secretion. Our data illustrating that UDP-glucose release was inhibited by BFA (Figure 5b) further suggest (although do not prove) that nucleotides were released from the secretory pathway.
However, the hypothesis that nucleotide release is associated with agonist-promoted vesicle exocytosis has been difficult to test in 1321N1 cells, using styryl fluorophores, for example, FM 1-43 (Kreda et al., 2007; Tatur et al., 2008). Unlike most cells, 1321N1 cells displayed a steady, robust increase of FM 1-43- (or its analogues FM 2-10 or FM 1-64) associated fluorescence in the absence of stimuli, which precluded using these protocols to assess thrombin-promoted exocytosis in these cells (Kreda SM, unpublished data). Lowering the temperature has been used to assess the contribution of exocytosis to nucleotide release in several cell types. However, incubating 1321N1 cells at 16 °C markedly inhibited agonist-promoted [3H]inositol phosphate formation (>95% inhibition, data not shown), discouraging us from assessing the effect of temperature changes on agonist-evoked UDP-glucose release.
Regardless of the cellular pathways and mechanism(s) regulating UDP-glucose release from astrocytes, an understanding of the physiological processes regulated by the UDP-glucose-sensing P2Y14 receptor in glial cells and astrocytes is now emerging (Fumagalli et al., 2003; Lee et al., 2003; Moore et al., 2003; Skelton et al., 2003; Bianco et al., 2005; Abbracchio and Verderio, 2006; Kobayashi et al., 2006). P2Y14 receptor transcripts (Chambers et al., 2000), as well as P2Y14 receptor-associated immunoreactivity (Moore et al., 2003), are abundantly detected through several regions of the brain. Immunohistochemistry analysis of post-mortem human brain suggests that P2Y14 receptor localizes specifically to astrocytes. Functional evidence of P2Y14 receptor expression in astrocytes has been suggested by studies illustrating UDP-glucose-promoted Ca2+ mobilization in primary cultures of rat glial cells and cortical astrocytes (Fumagalli et al., 2003; Bianco et al., 2005). Expression of P2Y14 receptor mRNA in the rat brain is upregulated by immunological challenge (Moore et al., 2003; Bianco et al., 2005), suggesting that the receptor is involved in reactive astrogliosis.
PAR-activated astrocytes (Wang and Reiser, 2003) may be an additional source of regulated release of UDP-glucose. The interstitial fluid volume in the brain has been estimated to be approximately 200 μl g−1 (Friden et al., 2007). As there are 1–5 trillion cells in the adult human brain (∼1.4 kg weight), a conservative estimation of the volume of the interstitial fluid surrounding the cells would yield 200 nl per 106 cells. UDP-glucose released to the bulk medium following thrombin stimulation represented approximately 3 pmol per 106 cells (Figure 2). Therefore, on the basis of these assumptions, UDP-glucose concentration in the undiluted extracellular milieu of PAR-stimulated astrocytes could approach a value of 10–20 μM, which is in the range for promoting robust P2Y14 receptor activation (Chambers et al., 2000; Lazarowski et al., 2003b).
In summary, our study illustrates, for the first time, the occurrence of Ca2+-dependent release of UDP-glucose in receptor-stimulated 1321N1 astrocytes. Thus, UDP-glucose release reflects a physiologically regulated mechanism of nucleotide release, as opposed to nucleotide leakage from damaged cells. Demonstration of the regulated release of UDP-glucose, the most potent and selective naturally occurring P2Y14 receptor agonist, provides compelling evidence that, in addition to its well-established role in metabolic reactions, this nucleotide-sugar plays important roles in intercellular signalling.