1321N1 Cells Research Articles

Mechanisms of ATP release from 1321N1 cells

Astrocytes are an important source of extracellular ATP in the brain. Regulated release of ATP from astrocytes occurs in response to diverse metabolic, mechanical, or inflammatory stimuli. Non‐junctional hemichannels composed of connexin 43 have been shown to be a major conduit for release of ATP from murine astrocytes in response to hypoxia, but the role of non‐junctional connexin hemichannels in G‐protein coupled receptor (GPCR) mediated ATP release is not determined. Previously, using 1321N1 human astrocytes we found that rapid accumulation of active GTP‐bound Rho in response to GPCR activation greatly enhances Ca2+ dependent ATP release. GPCR induced ATP release from 1321N1 cells is attenuated by carbenoxolone, flufenamic acid, and probenecid. These structurally unrelated compounds are non‐selective inhibitors of non‐junctional hemichannels. Here we investigate the contribution of connexin 43 non‐junctional hemichannels to GPCR induced ATP release from 1321N1 astrocytes by connexin 43 overexpression and connexin 43 knockdown.

Expression of corticosteroid-binding globulin in human astrocytoma cell line.

Glial tumor cells are known to be sensitive to glucocorticoids (GC) in vivo and in vitro. Here we studied the expression of corticosteroid-binding globulin (CBG) in the low-grade malignant human astrocytoma cell line 1321N1. CBG was observed in cytoplasm of most of these cells with immunocytochemistry. RT-PCR revealed the presence of the respective mRNA. Only scattered cells contained nuclear immunoreactivity for glucocorticoid receptor as visualized by double immunostaining. Immunoreactive CBG could be recovered from the supernatant of cultures that had been exposed to 10(-5) M cortisol. Our observations indicate the endogenous expression of CBG in 1321N1 cells which may occur independently from classical glucocorticoid receptor pathways. Cortisol seems to facilitate liberation of CBG in a paracrine manner, perhaps through membrane action of the steroid. Effects of adrenal steroids on proliferation and apoptosis of certain glial tumors may in part depend on these mechanisms.

Antidepressants Inhibit P2X4 Receptor Function: a Possible Involvement in Neuropathic Pain Relief

BackgroundNeuropathic pain is characterized by pain hypersensitivity to innocuous stimuli (tactile allodynia) that is nearly always resistant to known treatments such as non-steroidal anti-inflammatory drugs or even opioids. It has been reported that some antidepressants are effective for treating neuropathic pain. However, the underlying molecular mechanisms are not well understood. We have recently demonstrated that blocking P2X4 receptors in the spinal cord reverses tactile allodynia after peripheral nerve injury in rats, implying that P2X4 receptors are a key molecule in neuropathic pain. We investigated a possible role of antidepressants as inhibitors of P2X4 receptors and analysed their analgesic mechanism using an animal model of neuropathic pain.ResultsAntidepressants strongly inhibited ATP-mediated Ca2+ responses in P2X4 receptor-expressing 1321N1 cells, which are known to have no endogenous ATP receptors. Paroxetine exhibited the most powerful inhibition of calcium influx via rat and human P2X4 receptors, with IC50 values of 2.45 μM and 1.87 μM, respectively. Intrathecal administration of paroxetine produced a striking antiallodynic effect in an animal model of neuropathic pain. Co-administration of WAY100635, ketanserin or ondansetron with paroxetine induced no significant change in the antiallodynic effect of paroxetine. Furthermore, the antiallodynic effect of paroxetine was observed even in rats that had received intrathecal pretreatment with 5,7-dihydroxytryptamine, which dramatically depletes spinal 5-hydroxytryptamine.ConclusionThese results suggest that paroxetine acts as a potent analgesic in the spinal cord via a mechanism independent of its inhibitory effect on serotonin transporters. Powerful inhibition on P2X4 receptors may underlie the analgesic effect of paroxetine, and it is possible that some antidepressants clinically used in patients with neuropathic pain show antiallodynic effects, at least in part via their inhibitory effects on P2X4 receptors.

Changes In Antioxidant Status in HepG2, 1321N1 and HEK 293 Cells In Response To Cadmium Induced-oxidative Stress

Cadmium (Cd) has been implicated in a number of pathological diseases and oxidative stress has been proposed as one of its mechanisms of toxicity. In this present study, the toxic effect of 5, 10 and 50 μM of cadmium as cadmium chloride was investigated in three human cell lines: human hepatocellular liver carcinoma cells (HepG2), human astrocytoma cells (1321N1) and human embryonic kidney cells (HEK 293). Antioxidant defence responses of these cells were determined in order to evaluate the extent of oxidative stress induced by this metal after 24hrs exposure. Results indicate that CdCl2 decreases the viability of all three cells in a dose-dependent manner in agreement with earlier reports of Fotakis and Timbrell (2006). However it is clear that HepG2 cells are more sensitive (EC50 = 13.96 μM) than 1321N1 (EC50 = 19.92 μM) and HEK293 (EC50 = 221.8 μM) cells. This may be related to increased uptake, as it has been observed that Cd preferentially accumulates in liver cells in vivo. Increase LDH leakage has earlier been reported to be associated with CdCl2 induced toxicity (Bradley et al., 1987). Table 1 shows a significant increase in LDH leakage in all the three cells at 50 μM. An increase in Reactive Oxygen Species (ROS) was also significant in HepG2 cells but was not significant in 1321N1 and HEK 293 cells .This implies that ROS production may not be involve in CdCl2 induced toxicity in 1321N1 and HEK 293 cells. The results also show that CdCl2 does not deplete GSH in HepG2 cells but does deplete GSH in 1321N1 and HEK 293 cells (Table 1). There was a significant increase in malondialdehyde MDA levels at 50 μM exposure in all the three cells. The results also show a significant increase in the activities of the antioxidant enzymes; GST, GPx, SOD and catalase in the three cell lines at 50 μM CdCl2 with the exception of glutathione reductase in HepG2 cells and catalase in 1321N1 cells. Overall this suggests an adaptive response by the three cells to the CdCl2 insult and this is investigated further by Western blot analysis.

Curcumin Inhibits Glyoxalase 1—A Possible Link to Its Anti-Inflammatory and Anti-Tumor Activity

BackgroundGlyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor.Methodology/Principal FindingsCultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki = 5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-α, IL-6, IL-8, IL-1β) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1.Conclusions/SignificanceThe results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.

Characterization of a Multiple Ligand-Gated Ion Channel Cellular Membrane Affinity Chromatography Column and Identification of Endogenously Expressed Receptors in Astrocytoma Cell Lines

Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors.

Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing the P2Y-like receptor GPR17 is described. Cellular membranes from 1321N1 cells transiently transfected with GPR17 vector [GPR17(+)] and from the same cell line transfected with the corresponding empty vector [GPR17(−)] were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6-mm-i.d. glass columns to create GPR17(+)–IAM and GPR17(−)–IAM stationary phases. Frontal chromatography experiments on both GPR17(+)–IAM and GPR17(−)–IAM demonstrated the presence of a specific interaction with GPR17 only in the former that was maximized by increasing the membrane/IAM ratio. GPR17(+)–IAM was used in frontal affinity chromatography experiments to calculate the dissociation constants ( K d) of three ligands—the antagonist cangrelor (formerly AR-C69931MX, a P2Y 12/P2Y 13 antagonist), MRS2179 (a P2Y 1 receptor antagonist), and the agonist UDP—all of which have been reported to also interact with GPR17. Immobilized GPR17 retained its ability to specifically bind the three analytes, as demonstrated by the agreement of the calculated K d values with previously reported data. Preliminary ranking experiments suggest the application of GPR17(+)–IAM in ranking affinity studies for the selection of new potential candidates.

The sensitization of glioma cells to cisplatin and tamoxifen by the use of catechin

Telomerase expression strongly correlates with the grade of malignancy in glioma with inhibition illustrating a definite increase in chemosensitivity. This study was designed to investigate the effects of a green tea derivative, epigallocatechin-3-gallate (EGCG); together with either cisplatin or tamoxifen in glioma, and to investigate whether these effects are mediated through telomerase suppression. EGCG showed a significant cytotoxic effect on 1321N1 cells after 24 h and on U87-MG cells after 72 h (P < 0.001) without significantly affecting the normal astrocytes. Treatment with EGCG inhibited telomerase expression significantly (P < 0.01) and enhanced the effect of cisplatin and tamoxifen in both 1321N1 (P < 0.01) and U87-MG (P < 0.001) cells. EGCG, as a natural product has enormous potential to be an anti-cancer agent capable of enhancing tumour cell sensitivity to therapy.

3H]A-804598 ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1β, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC50=10nM), mouse (IC50=9nM) and human (IC50=11nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1β and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([3H]A-804598; 8.1Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [3H]A-804598 labeled a single class of high affinity binding sites (Kd=2.4nM and apparent Bmax=0.56pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [3H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r=0.95, P<0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and [3H]A-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors.

Rapid resensitization of purinergic receptor function in human platelets.

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y(1) and P2Y(12) purinergic receptors. Recently, we demonstrated that both receptors desensitize and internalize in human platelets by differential kinase-dependent mechanisms. To demonstrate whether responses to P2Y(1) and P2Y(12) purinergic receptors resensitize in human platelets and determine the role of receptor traffic in this process. These studies were undertaken either in human platelets or in cells stably expressing epitope-tagged P2Y(1) and P2Y(12) purinergic receptor constructs. In this study we show for the first time that responses to both of these receptors can rapidly resensitize following agonist-dependent desensitization in human platelets. Further, we show that in human platelets or in 1321N1 cells stably expressing receptor constructs, the disruption of receptor internalization, dephosphorylation or subsequent receptor recycling is sufficient to block resensitization of purinergic receptor responses. We also show that, in platelets, internalization of both these receptors is dependent upon dynamin, and that this process is required for resensitization of responses. This study is therefore the first to show that both P2Y(1) and P2Y(12) receptor activities are rapidly and reversibly modulated in human platelets, and it reveals that the underlying mechanism requires receptor trafficking as an essential part of this process.

Thrombin‐promoted release of UDP‐glucose from human astrocytoma cells

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.

Thrombin mediated regulation of CCN1 regulates cell proliferation in an integrin dependent manner

We previously reported that PAR1 (activated by thrombin) and the M3 muscarinic (activated by carbachol) receptors differentially effect AP‐1 mediated gene expression and DNA synthesis in 1321N1 astroglial cells. Microarray experiments were used to explore genes that were differently regulated by thrombin verses carbachol and revealed that the secreted protein cysteine‐rich 61 (Cyr61/CCN1) was robustly and selectively induced by thrombin. Thrombin but not carbachol activate RhoA therefore we postulated that Rho signaling mediates CCN1 expression. This was supported by experiments demonstrating that the C3 exoenzyme attenuates thrombin‐induced CCN1 expression and that other GPCR agonists that activate Rho also induce CCN1 expression while Gq coupled receptor agonists do not. Inhibition of CCN1 expression with siRNA decreased thrombin‐stimulated DNA synthesis by ~50% implicating CCN1 signaling in thrombin mediated proliferation. Currently integrins are the only known receptor for CCN1. Data shows that CCN1 is present on the cell surface of 1321N1 cells, that 1321N1 cells bind to CCN1 in an α6β1, α5β1, and β1 integrin dependent manner, and that functional inhibitory antibodies to these integrins attenuate thrombin mediated DNA synthesis. We suggest that CCN1 represents an intermediate by which G12/13 and Rho coupled GPCRs utilize integrins to elicit additional signals involved in cell proliferation.NIH 5R01‐GM36927

Nerve Growth Factor-Inducing Activity of Hericium erinaceus in 1321N1 Human Astrocytoma Cells

Neurotrophic factors are essential to maintain and organize neurons functionally; thereby neurotrophic factor-like substances or their inducers are expected to be applied to the treatment of neurodegenerative diseases such as Alzheimer's disease. In the present study, we firstly examined the effects of ethanol extracts of four edible mushrooms, Hericium erinaceus (Yamabushitake), Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Agaricus blazei (Himematsutake), on nerve growth factor (NGF) gene expression in 1321N1 human astrocytoma cells. Among the four mushroom extracts, only H. erinaceus extract promoted NGF mRNA expression in a concentration-dependent manner. In addition, secretion of NGF protein from 1321N1 cells was enhanced by H. erinaceus extracts, and the conditioned medium of 1321N1 cells incubated with H. erinaceus extract enhanced the neurite outgrowth of PC12 cells. However, hericenones C, D and E, constituents of H. erinaceus, failed to promote NGF gene expression in 1321N1 cells. The enhancement of NGF gene expression by H. erinaceus extracts was inhibited by the c-jun N-terminal kinase (JNK) inhibitor SP600125. In addition, H. erinaceus extracts induced phosphorylation of JNK and its downstream substrate c-Jun, and increased c-fos expression, suggesting that H. erinaceus promotes NGF gene expression via JNK signaling. Furthermore we examined the efficacy of H. erinaceus in vivo. ddY mice given feed containing 5% H. erinaceus dry powder for 7 d showed an increase in the level of NGF mRNA expression in the hippocampus. In conclusion, H. erinaceus contains active compounds that stimulate NGF synthesis via activation of the JNK pathway; these compounds are not hericenones.

Slow spontaneous [Ca2+]i oscillations reflect nucleotide release from renal epithelia

Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca(2+) concentration [Ca(2+)](i) through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca(2+)](i) oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin-Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca(2+)](i) increases in renal epithelia. Spontaneous [Ca(2+)](i) increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 +/- 6.7% (n = 23) of the cells showed spontaneous [Ca(2+)](i) increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca(2+)](i) oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca(2+)](i) oscillatory activity. The association between spontaneous [Ca(2+)](i) increases and nucleotide signalling was further tested in 132-1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca(2+)](i) increases. Transfection with either hP2Y(6) or hP2Y(2) receptors revealed a striking degree of oscillations. Similar spontaneous [Ca(2+)](i) increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y(2) knock out mice (0.050 +/- 0.020 events per second, n = 8) compared to the wild type (0.147 +/- 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca(2+)](i) oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function.

5,19-cyclo-9β,10ξ-androstane-3,17-dione promotes neurotrophic factor biosynthesis in 1321N1 human astrocytoma cells and improves passive avoidance learning impairment

Since neurotrophic factors are essential for neurons to form neuronal networks and maintain neuronal functions, neurotrophic factor-like substances or inducers of neurotrophic factors can be useful for the treatment of serious neuronal diseases such as Alzheimer's and Parkinson's diseases. In the present study, we examined an effect of 5,19-cyclo-9β,10ξ-androstane-3,17-dione (CAD) on neurotrophic factor synthesis in glial cells and scopolamine-induced impairment of learning in mice. 1321N1 human astrocytoma cells promoted secretion of certain neurotrophic factors in response to CAD with no cytotoxicity, which caused dramatic neurite outgrowth in rat pheochromocytoma (PC12) cells. In fact, CAD significantly enhanced nerve growth factor (NGF) secretion and its gene expression in 1321N1 cells, in a time and concentration-dependent manner. Because second messengers such as cAMP, inositol 1,4,5-trisphosphates and Ca 2+ induce NGF gene expression, we measured activities of adenylyl cyclase and phospholipase C and intracellular Ca 2+ concentration in 1321N1 cells. However, CAD changed neither second messenger levels. CAD enhanced the gene expression of proto-oncogene, c-fos that is one of the components of transcription factor (AP-1). In addition to those above, the in vivo effects of CAD were also examined. Although injection of muscarinic receptor antagonist scopolamine impaired passive avoidance learning in mice, pretreatment with CAD significantly reversed the adverse effect in a dose-dependent manner. Taking these results together, CAD has enormous therapeutic potential for serious neuronal diseases.

The control of phosphatidylinositol 3,4-bisphosphate concentrations by activation of the Src homology 2 domain containing inositol polyphosphate 5-phosphatase 2, SHIP2

Activation of class Ia PI3K (phosphoinositide 3-kinase) produces PtdInsP3, a vital intracellular mediator whose degradation generates additional lipid signals. In the present study vanadate analogues that inhibit PTPs (protein tyrosine phosphatases) were used to probe the mechanisms which regulate the concentrations of these molecules allowing their independent or integrated function. In 1321N1 cells, which lack PtdInsP3 3-phosphatase activity, sodium vanadate or a cell permeable derivative, bpV(phen) [potassium bisperoxo(1,10-phenanthroline)oxovanadate (V)], increased the recruitment into anti-phosphotyrosine immunoprecipitates of PI3K activity and of the p85 and p110a subunits of class Ia PI3K and enhanced the recruitment of PI3K activity stimulated by PDGF (platelet-derived growth factor). However, neither inhibitor much increased cellular PtdInsP3 concentrations, but both diminished dramatically the accumulation of PtdInsP3 stimulated by PDGF or insulin and markedly increased the control and stimulated concentrations of PtdIns(3,4)P2. These actions were accounted for by the ability of PTP inhibitors to stimulate the activity of endogenous PtdInsP3 5-phosphatase(s), particularly SHIP2 (Src homology 2 domain containing inositol polyphosphate 5-phosphatase 2) and to inhibit types I and II PtdIns(3,4)P2 4-phosphatases. Thus bpV(phen) promoted the translocation of SHIP2 from the cytosol to a Triton X-100-insoluble fraction and induced a marked (5-10-fold) increase in SHIP2 specific activity mediated by enhanced tyrosine phosphorylation. The net effect of these inhibitors was, therefore, to switch the signal output of class I PI3K from PtdInsP3 to PtdIns(3,4)P2. A key component controlling this shift in the balance of lipid signals is the activation of SHIP2 by increased tyrosine phosphorylation, an effect observed in HeLa cells in response to both PTP inhibitors and epidermal growth factor.

NAADP + is an agonist of the human P2Y 11 purinergic receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP +) is an intracellular second messenger releasing Ca 2+ from intracellular stores in different cell types. In addition, it is also active in triggering [Ca 2+] i increase when applied extracellularly and various underlying mechanisms have been proposed. Here, we used hP2Y 11-transfected 1321N1 astrocytoma cells to unequivocally establish whether extracellular NAADP + is an agonist of the P2Y 11 receptor, as previously reported for β-NAD + [I. Moreschi, S. Bruzzone, R.A. Nicholas, et al., Extracellular NAD + is an agonist of the human P2Y11 purinergic receptor in human granulocytes, J. Biol. Chem. 281 (2006) 31419–31429]. Extracellular NAADP + triggered a concentration-dependent two-step elevation of [Ca 2+] i in 1321N1-hP2Y 11 cells, but not in wild-type 1321N1 cells, secondary to the intracellular production of IP 3, cAMP and cyclic ADP-ribose (cADPR). Specifically, the transient [Ca 2+] i rise proved to be related to IP 3 overproduction and to consequent Ca 2+ mobilization, while the sustained [Ca 2+] i elevation was caused by the cAMP/ADP-ribosyl cyclase (ADPRC)/cADPR signalling cascade and by influx of extracellular Ca 2+. In human granulocytes, endogenous P2Y 11 proved to be responsible for the NAADP +-induced cell activation (as demonstrated by the use of NF157, a selective and potent inhibitor of P2Y 11), unveiling a role of NAADP + as a pro-inflammatory cytokine. In conclusion, we provide unequivocal evidence for the activation of a member of the P2Y receptor subfamily by NAADP +.

P2Y2 nucleotide receptors inhibit trauma‐induced death of astrocytic cells

Nucleotides as well as other neurotransmitters are known to be released to the extracellular space upon injury. To determine whether nucleotides acting on P2Y(2) nucleotide receptors promote protective or degenerative events after trauma in astrocytic cells, a well-established model of in vitro brain trauma was applied to 1321N1 cells expressing recombinant P2Y(2) nucleotide receptors (P2Y(2)R-1321N1). Cellular death was examined by measuring DNA fragmentation and caspase activation. Fragmented DNA was observed 48 h post-injury in 1321N1 cells, while P2Y(2) nucleotide receptor expressing cells did not show DNA fragmentation. A laddering pattern of fragmented DNA following injury was observed upon inhibition of P2Y(2) nucleotide receptors with suramin. Time-dependent increases of cleaved caspase-9, a mitochondrial-associated caspase, correlated with injury-induced cellular death. A decreased bax/bcl-2 gene expression ratio was observed in P2Y(2)R-1321N1 cells after traumatic injury, while untransfected 1321N1 cells showed a significant time-dependent increase of the bax/bcl-2 gene expression ratio. Activation of protein kinases was assessed to determine the signaling pathways involved in cell death and survival responses following traumatic injury. In P2Y(2)R-1321N1 and 1321N1 cells p38 phosphorylation was stimulated in a time-dependent manner but the phosphatidylinositol 3-kinase-dependent activation of extracellular signal-regulated kinase 1/2 and protein kinase B (PKB)/Akt was only observed in P2Y(2)R-1321N1 cells after injury. The stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling pathway was not activated by traumatic injury in either astrocytic cell line. Inhibition of p38 kinase signaling pathway by treatment with PD1693, a MKK3/6 inhibitor, abolished the expression of cleaved caspase-9, the increase in the bax/bcl-2 gene expression ratio, as well as the fragmentation of DNA that followed injury of 1321N1 cells. Taken together, our results demonstrate a novel role for P2Y(2) nucleotide receptors and extracellular nucleotides in mediating survival responses to glial cells undergoing cellular death induced by trauma.

Increased production of lipocalin-type prostaglandin D synthase in leptomeningeal cells through contact with astrocytes

Lipocalin-type prostaglandin D synthase (L-PGDS) is dominantly expressed in the leptomeninges surrounding the brain and secreted into the cerebrospinal fluid as beta-trace, a major cerebrospinal fluid protein. To examine the interaction between the leptomeninges and the brain parenchyma, we co-cultured rat leptomeningeal cells with cells dissociated from the neonatal rat cortex and found that the production of L-PGDS was remarkably increased after the co-cultivation. A similar increase in L-PGDS production was observed by the co-culturing of the leptomeningeal cells with cells dissociated from astrocyte-rich cultures or with 1321-N1 astrocytoma cells. When a crude membrane fraction prepared from 1321-N1 cells was added to leptomeningeal cell cultures, L-PGDS gene expression was slowly increased up to 48 h after the addition. These results indicate that leptomeningeal cells enhance their L-PGDS production by a slow activation of L-PGDS gene expression through their contact with astrocytes.

The P2Y2 nucleotide receptor requires interaction with αv integrins to access and activate G12

The P2Y2 nucleotide receptor (P2Y2R) interacts with alpha v integrins to activate G(o) and induce chemotaxis in human 1321N1 astrocytoma cells. In this study, it was determined that the P2Y2R also requires interaction with alpha v integrins to activate G12 and associated signaling pathways that control chemotaxis in 1321N1 cells. Mutation of the Arg-Gly-Asp (RGD) integrin-binding sequence in the first extracellular loop of the human P2Y2R to Arg-Gly-Glu (RGE), which prevents integrin interaction, did not inhibit G(q) or ERK1/2 signaling by the P2Y2R agonist UTP but completely inhibited activation of G12 and G12-mediated events, including Rho activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation and chemotaxis towards UTP. The involvement of G12 in all these events was verified by using a dominant negative G alpha12 construct. G12 activation by the P2Y2R also was inhibited by anti-alpha v beta5 integrin antibodies and alpha v integrin antisense oligonucleotides, suggesting that alpha v integrin activity and expression are required for the P2Y2R to activate G12. Co-immunoprecipitation experiments confirmed that G alpha12 protein associates with the wild-type P2Y2R and with alpha v integrins but not with the RGE mutant P2Y2R or with alpha3 integrins. Collectively, these results suggest that alpha v integrin complexes provide the P2Y2R with access to G12, thereby allowing activation of this heterotrimeric G protein that controls actin cytoskeletal rearrangements required for chemotaxis.