AUTNEU-01644; No of Pages 7 Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
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The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation Bo Fan 1, Shuangmei Liu 1, Changshui Xu 1, Jun Liu 1, Fanjun Kong 1, Guilin Li, Chunping Zhang, Yun Gao, Hong Xu, Shicheng Yu, Chaoran Zheng, Lichao Peng, Miaomiao Song, Bing Wu, Qiulan Lv, Lifang Zou, Mofeng Ying, Xi Zhang, Shangdong Liang ⁎,1 Department of Physiology, Medical School of Nanchang University, Nanchang 330006, PR China
a r t i c l e
i n f o
Article history: Received 29 November 2013 Received in revised form 10 March 2014 Accepted 24 March 2014 Available online xxxx Keywords: PC12 cell Adenosine triphosphate P2X7 receptor Oxygen–glucose deprivation Intracellular Ca2 +
a b s t r a c t Adenosine triphosphate (ATP) plays an important role in signal transmission via acting on P2X receptors. P2X7 receptor is involved in pathophysiological changes of ischemic diseases. The PC12 cell line is a popular model system to study sympathetic neuronal function. In this study, the effects of P2X7 on the viability or [Ca2+]i in PC12 cells after exposure to oxygen–glucose deprivation (OGD) were investigated. The results showed that the viability of PC12 cells was decreased under the condition of OGD. BzATP, a P2X7 agonist, decreased the viability, while P2X7 antagonist oxATP or P2X7 siRNA reversed the viability of PC12 cells under the condition of OGD. The expression levels of P2X7 mRNA and protein in PC12 cells were up-regulated under the condition of OGD or BzATP treatment. The expression levels of P2X7 mRNA and protein were significantly decreased in OGD PC12 cells, which were pretreated with oxATP or P2X7 siRNA. It was also found that oxATP or P2X7 siRNA effectively suppressed the increase of [Ca2+]i induced by OGD. P2X7 agonist ATP or BzATP enhanced the [Ca2+]i rise induced by OGD in PC12 cells. The [Ca2+]i peak induced by ATP or BzATP in OGD group was decreased by ERK inhibitor U0126. Therefore, P2X7 antagonists or P2X7 siRNA could depress the sympathetic neuronal damage induced by ischemia. © 2014 Elsevier B.V. All rights reserved.
1. Introduction P2X7 receptor is a member of ATP-gated non-selective cation channels, which has a unique and important physiological function (Sperlagh et al., 2006; Liang et al., 2010; Skaper et al., 2010; Burnstock et al., 2011; Zhang et al., 2013). As an ionotropic receptor, P2X7 receptor is characterized by a low affinity for the endogenous ligand ATP and by two states of permeability (Arbeloa et al., 2011; Burnstock et al., 2011). Upon transient stimulation, P2X7 receptor agonists at the low concentration cause selective cationic inflow of Na+, K+, and Ca2 +. When activated by agonists at relatively high concentrations, sustained stimulation drives transition that leads to the formation of a nonselective pore permeable to aqueous solutes of molecular mass up to 700 Da (Sperlagh et al., 2006; Skaper et al., 2010). A common feature of both conductance states is the elevation of [Ca2+]i, which modulates cell functions. Studies have shown that P2X7 receptor has been implicated in the neuronal
⁎ Corresponding author at: Department of Physiology, Medical School of Nanchang University, Nanchang, Jiangxi 330006, PR China. Tel./fax: +86 791 86360552. E-mail address: [email protected] (S. Liang). 1 Joint first authors.
injury after ischemic diseases (Milius et al., 2008; Vasileiou et al., 2010; Arbeloa et al., 2011; Kong et al., 2013; Tu et al., 2013). During the ischemic heart diseases, the activation of cardiac afferents evokes sympathetic reflex responses (Shao et al., 2007; Zhang et al., 2007, 2008; Li et al., 2010, 2011; Kong et al., 2013; Liu et al., 2013; Tu et al., 2013). In the pathological conditions of myocardial ischemia, damaged cells release ATP into the extracellular space (Shao et al., 2007; Zhang et al., 2007, 2008; Fu and Longhurst, 2010; Li et al., 2010, 2011; Kong et al., 2013; Liu et al., 2013; Tu et al., 2013). ATP may act as an excitotoxin and result in neuronal injury (Shao et al., 2007; Zhang et al., 2007, 2008; Li et al., 2010, 2011; Arbeloa et al., 2011; Burnstock et al., 2011; Liu et al., 2013; Tu et al., 2013). In vitro studies also show that the application of exogenous ATP is toxic to primary neuronal cultures and causes injury (Arbeloa et al., 2011; Burnstock et al., 2011; Xu et al., 2013). Oxygen–glucose deprivation (OGD) also occurs in ischemia and a variety of pathologic conditions (Lecht et al., 2012). Hypoxia induces [Ca2 +]i elevation in PC12 cells, which also is involved in neuronal injury (Meng et al., 2008; Arbeloa et al., 2011). In previous studies, we found that P2X7 receptor is involved in the pathological injury of sympathetic nerve during myocardial ischemia (Kong et al., 2013; Liu et al., 2013; Tu et al., 2013). The aim of this research was to investigate the effects of P2X7 antagonism or
http://dx.doi.org/10.1016/j.autneu.2014.03.006 1566-0702/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
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P2X7-siRNA treatment upon the cell viability or [Ca2+]i when PC12 cells were exposed to OGD. 2. Materials and methods
detail, the P2X 7 siRNA and lipo2000 were diluted in the OptiMEM® medium without serum. Final concentration of P2X7 siRNA was 100 nM. The cells were cultured for 24 h at 37 °C. Total RNA was extracted and the efficiency of knockdown was verified by Real-time PCR.
2.1. Cell cultures PC12 cell line was originally obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The PC12 cells were seeded in 25 cm2 polystyrene flasks in Dulbecco's modified Eagle's medium (DMEM) (high glucose) (HyClone, USA), containing 5% heatinactivated fetal bovine serum (Gibco, USA) and 10% horse serum (Gibco, USA). The cells were incubated under a humidified atmosphere containing 5% CO2 (normal culture incubator) at 37 °C, and culture medium was replaced every 48 h. PC12 cells were plated in 6-well plates containing 2 ml of complete medium, at a density of 1.0 × 105/ml cells. When cell density reached 70%–80%, we established the model of “oxygen–glucose deprivation (OGD)” (Lecht et al., 2012). 2.2. Drugs and groups 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP, Sigma-Aldrich) and oxidized ATP (oxATP, Sigma) were dissolved in distilled water and used at a concentration of 300 nM and 30 μM, respectively. ATP (100 μM, Sigma) and ERK inhibitor U0126 (10 μM, Sigma) were used. According to the experimental purpose, PC12 cells were randomly divided into the following groups. (1) Control group; (2) oxygen– glucose deprivation (OGD) group; (3) OGD + oxATP group; (4) OGD + BzATP group; (5) OGD + P2X7 siRNA group; (6) OGD + U0126 group. OGD + scramble siRNA group was only used for testing the interference efficiency of P2X7 siRNA. 2.3. Oxygen–glucose deprivation (OGD) For OGD treatment (Hillion et al., 2005; Larsen et al., 2007; Lecht et al., 2012), the culture media were changed to a deoxygenated glucose-free Earle's balanced salt solution at pH 7.4. 10% fetal bovine serum was added. Then the cells were transferred to a sealed hypoxic box containing a mixture of 95% N2 and 5% CO2 at 37 °C for 2 h. After OGD, the cells were cultured in full culture medium with oxygen for 24 h and treated with indicated drugs. Oxygenated glucose-containing Earle's balanced salt solution was used as control.
2.6. Measurement of intracellular Ca2+ Intracellular Ca2+ was monitored using the fluorescent Ca2+sensitive dye, Fura 2-acetoxymethyl ester (Fura 2-AM) (Shi et al., 2009). Cells were divided into control group, OGD group, OGD + BzATP group, OGD + oxATP group, OGD + P2X7 siRNA group, and OGD + U0126 group. PC12 cells cultured with OGD were seeded in 6-well plates and incubated with oxATP (300 nM), BzATP (30 μM) (Morigiwa et al., 2000), P2X7 siRNA (50 nm) and ERK inhibitor U0126 (10 μM) respectively. [Ca2+]i was measured after treatment with drugs for 2 h. After digestion, cell suspensions were centrifuged for 5 min at 4 °C, 1000 rpm, and the pellet was resuspended in 50 ml medium. Incubation was continued for 3 h in the container containing magnetic puddler at 37 °C. After 3 h, abandon the liquid after being centrifuged for 5 min at 4 °C, 1000 rpm, wash once with Spinner medium, then the cells were incubated for 10 min in 20 ml Spinner medium containing 10% FBS at 37 °C as the method mentioned above. The cells were loaded for 30 min in 20 μl 1 mM Fura-2/AM, washed twice with Spinner medium and centrifuged for 5 min at 4 °C, 1000 rpm. 2 ml KRBH liquid was added and beated repeatedly. The suspension was transferred to a quartz cuvette and tested at excitation wavelengths of 340 and 380 nm in LS50B fluorescence spectrophotometer. 1 μl MnCl2 (100 mM) was added, and then 2 μl diethylene-triamine-penta-acetic acid (DTPA, 100 mM) to minus the extracellular spontaneous fluorescence. After measuring the basal fluorescence (R), 100 μM ATP or 100 μM BzATP was added to the suspension to observe the fluorescence. 10% Triton-100 was added, which resulted in maximum fluorescence (Rmax). Then, 40 μl EGTA (0.4 M) and 40 μl Tris (3 M) were added to couple calcium ions. The resultant fluorescence signal was designated as Rmin. Intracellular Ca2+ concentration was determinated in a fluorescence spectrophotometer with double wavelengths. The calculation equation is: [Ca2 +]i = 224 nM × [(R − Rmin) / (Rmax − R)] / (S1 / S2), where 224 is the Kd value of Fura-2/AM; S1 and S2 were the fluorescence intensities of zero Ca2 + (EGTA, 0.4 M) and saturated Ca2 + (Triton-100,10%) in 380 nm place, respectively.
2.4. MTS for cell viability PC12 cells were seeded into 96-well plastic plates with 0.1 ml culture medium at a density of 1 × 105 cells/ml. After growth for 48 h, the cells were treated with OGD for 4 h. After 48 h of reperfusion, the 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium (MTS) (purchased from Promega, Madison WI) colorimetric assay was performed to determine cell viability (McNeill-Blue et al., 2006). Each group contained 10 samples and 20 μl MTS were pipetted into each well of the 96-well assay plate within 100 μl culture medium. The plate was incubated at 37 °C for 3 h in a humidified, 5% CO2 atmosphere. The absorbance at 490 nm was recorded using a 96-well plate reader. Background absorbance was subtracted using a set of wells containing medium only. 2.5. Small interfering RNA (siRNA) knockdown experiment With siRNA technology to knockdown the P2X7 receptor of OGD PC12 cells (Hillion et al., 2005; Larsen et al., 2007), a P2X7 siRNA fragment was transfected into cultured cells using lipo2000. PC12 cells were divided into control group, OGD group, OGD + NC group and OGD + P2X7 siRNA group. The P2X7 siRNA cis-chain: 5′-GGAUGGACCCACAAAG UAAdTdT-3′; trans-chain: 5′-dTdTCCUACCUGGGUGUUUCAUU-3′. In
2.7. Western blotting analysis The level of P2X7 protein was determined by western-blotting. The cells were solubilized in lysis buffer. After incubating on ice for 30 min, the lysates were clarified by centrifugation at 14 000 g for 15 min. The supernatant was taken and the protein concentration was determined using the Bradford protein assay system (Bio-Rad). Protein was mixed with 5 × SDS sample buffer. Total protein (20 μg) was electrophoresed on 5% SDS-PAGE gel and 10% SDS-PAGE gel and then transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat dry milk for 2 h on a table concentrator at room temperature. The experiments were performed using the following primary antibodies: monoclonal anti-P2X 7 (1:1000) and anti-βactin (1:1000). A secondary goat anti-mouse (polyclonal) or goat anti-rabbit (polyclonal) antibody (both 1:1000) was used. Then using the enhanced chemiluminescence (ECL) kit (Shanghai Pufei Biotech. CO.), chemiluminescent signals were collected on an autoradiography film. The quantity of band intensity was carried out using Image Pro-Plus software. Protein expression levels were represented as densitometric ratios of the targeted protein to β-actin.
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
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2.8. Statistical analysis The data are expressed as the mean ± the standard error of the mean (SEM). All statistical analyses were performed using SPSS 17.0 software. Statistical significance was assessed using the one-way analysis of variance for multiple group comparisons followed by Tukey's post hoc test. A p-value of less than 0.05 was considered to be statistically significant. 3. Result 3.1. The growth and the viability of PC12 cells after OGD treatment 3.1.1. Effects of OGD on the growth of PC12 cells The cultured PC12 cells with or without oxygen and glucose deprivation (OGD) were observed (Fig. 1). The morphous of PC12 cells cultured in normal condition showed larger oval, fullness, three-dimensional sense of strong and clear halos. The PC12 cells treated with OGD displayed smaller neurons, short protrusion, thin and wrinkled shape. 3.1.2. Effects of P2X7 antagonist or P2X7 knockdown on the viability of PC12 cells after OGD To determine the effects of P2X7 antagonist or P2X7 knockdown on the injury responses of OGD treatment in PC12 cells, the cell viability was tested using MTS assay. The optical density (OD) values of MTS as the cell viability in control group, OGD group, OGD + oxATP group, OGD + BzATP group, OGD + P2X7 siRNA group, and OGD + ERK inhibitor U0126 group were measured (n = 10 for each group; Fig. 2). Compared with the control group, the viability of PC12 cells in OGD group was decreased (p b 0.05; Fig. 2). After being treated with oxATP (300 nM), P2X7 siRNA, and U0126 (10 μM), the viability of PC12 cells in OGD group was reversed in comparison with that in OGD group and OGD + BzATP (30 μM) group (p b 0.05; Fig. 2). P2X7 antagonist oxATP or P2X7 knockdown can increase the viability of OGD cells. 3.2. Changes of P2X7 expression in PC12 cells treated with OGD 3.2.1. Expression of P2X7 mRNA in PC12 cells treated with OGD The expression of P2X7 mRNA in PC12 cells was studied by Real-time PCR. The average relative quantification (RQ) value of P2X7 mRNA in OGD group was thought as 1, which was higher than that in control group (p b 0.05) (Fig. 3). The RQ values in OGD + P2X7 siRNA group and OGD + oxATP (300 nM) group were significantly lower than those in OGD group, OGD + scramble siRNA group and OGD + BzATP (30 μM) group (p b 0.05; Fig. 3). Interference efficiency of P2X7 siRNA was 55.9%. No difference was found in OGD group, OGD + scramble siRNA group and OGD + BzATP group (p N 0.05; Fig. 3) (n = 3 for
A
Fig. 2. Effects of P2X7 on OGD PC12 cell viability. The cell viability was tested by MTS assay. Histogram showed the optical density (OD) values of cell viability in control group, OGD group, OGD + oxATP group, OGD + BzATP group, OGD + P2X7 siRNA group, and OGD + U0126 group. Compared with the viability of PC12 cells in control group, the viability of OGD group cells was decreased. The viability of PC12 cells in OGD group after being treated with oxATP (300 nM), P2X7 siRNA, and U0126 (10 μM) was increased in comparison with that in OGD group and OGD + BzATP (30 μM) group. The values are means ± SEM (n = 10). *p b 0.05 vs. control group; #p b 0.05 vs. OGD group.
each group). P2X7 antagonist oxATP or P2X7 knockdown can decrease the expression of P2X7 mRNA in PC12 cells treated with OGD. 3.2.2. Expression of P2X7 protein in PC12 cells treated with OGD The stain values (average optical density, AOD) of P2X7 protein expression (normalized to each β-actin internal control) were tested by western blotting. In OGD group or OGD + BzATP (30 μM) group, the expression of P2X7 protein was significantly increased in comparison with that in control group. After P2X7 siRNA, oxATP or U0126 treatment in OGD group, the expression of P2X7 protein was decreased in comparison with that in OGD group or OGD + BzATP group (p b 0.05; Fig. 4) (n = 5 for each group). Our results suggested that P2X7 protein expression could be effectively reduced by P2X7 siRNA, P2X7 antagonist oxATP (300 nM) or ERK inhibitor U0126 (10 μM) in OGD group. 3.3. P2X7 receptor and alteration of [Ca2+]i in PC12 cells treated with OGD 3.3.1. Effects of ATP on the [Ca2+]i responses in PC12 cells treated with OGD ATP (100 μM) could increase the peak of [Ca2+]i in PC12 cells. The alteration value of [Ca2 +]i after ATP stimuli was shown as the difference-value (D-value) between the peak value of [Ca2 +]i and the initial value of [Ca2 +]i. There was a high initial value of [Ca2 +]i
B
Fig. 1. The growth of PC12 cells after cultured with or without oxygen–glucose deprivation (OGD). The PC12 cells in control group and OGD group were shown in (A) and (B) as the arrows indicated. The PC12 cells in control showed larger oval, fullness, three-dimensional sense of strong and clear halos. The PC12 cells in OGD displayed smaller neurons, short protrusion, thin and wrinkled shape. Scale bar indicates 100 μm.
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
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about 49.9% (p b 0.05, n = 5; Fig. 5A, B). These results indicated that P2X7 activation contributed to the [Ca2+]i rise. 3.3.2. Effects of BzATP on the [Ca2+]i responses of PC12 cells treated with OGD BzATP (30 μM) induced a [Ca2+]i rise in PC12 cells. There is a high initial value of [Ca2+]i concentration in OGD + BzATP group in comparison with control group (n = 5). The [Ca2+]i peak induced by BzATP stimuli in OGD + oxATP group was reduced to 68.9% in comparison with that in OGD group (p b 0.05, n = 5). The [Ca2 +]i peak induced by BzATP stimuli in OGD + P2X7 siRNA group was significantly inhibited to 70.7% (p b 0.05, n = 5). The [Ca2 +]i peak induced by BzATP stimuli in OGD treatment was partially inhibited by ERK inhibitor U0126 (10 μM) about 55.1% (p b 0.05, n = 5; Fig. 6A, B). Fig. 3. Effects of P2X7 antagonist or P2X7 knockdown on the expression of P2X7 mRNA in OGD PC12 cells. The expression of P2X7 mRNA was studied by Real-time PCR. The average relative quantification (RQ) value of P2X7 mRNA in OGD group was thought as 1. The RQ values in OGD + P2X7 siRNA group and OGD + oxATP (300 nM) group were significantly decreased in comparison with those in OGD group, OGD + scramble siRNA group and OGD + BzATP (30 μM) group. Histogram showed the RQ values of P2X7 mRNA in control group, OGD group, OGD + scramble siRNA group, OGD + P2X7 siRNA group, OGD + BzATP and OGD + oxATP group. The values are means ± SEM (n = 3 for each group), *p b 0.05 vs. control group; #p b 0.05 vs. OGD group.
concentration in OGD + BzATP (30 μM) group in comparison with that in control group. The D-value in OGD group was higher than that in control group, while the D-value in OGD + oxATP group, OGD + P2X7 siRNA group, and OGD + ERK inhibitor U0126 group was lower than that in OGD group or OGD + BzATP group (n = 5 for each group). The peak [Ca2+]i was partially inhibited by oxATP (300 nM) and P2X7 siRNA about 28.8% and 37.4%, respectively (p b 0.05, n = 5). The peak [Ca2 +]i was partially inhibited by ERK inhibitor U0126 (10 μM) by
A Control
OGD
OGD+ BzATP
OGD+ siRNA
4. Discussion ATP plays an important role in signal transmission via acting on purinergic receptors (Kennedy, 2005; Abbracchio et al., 2009; Burnstock et al., 2011; Sperlagh et al., 2012). ATP, released during myocardial ischemia, can lead to abnormal cardiac function in myocardial ischemic injury (Vassort, 2001; Shao et al., 2007; Zhang et al., 2007, 2008; Erlinge and Burnstock, 2008; Li et al., 2010, 2011; Kong et al., 2013; Liu et al., 2013; Tu et al., 2013). The accumulation of ATP can also initiate neuronal damage. The PC12 (rat pheochromocytoma) cell line, a popular model system to study sympathetic neuronal function, was used in this experiment (Xu et al., 2013). Our results showed that the viability of PC12 cells was decreased under the condition of oxygen–glucose deprivation. The viability of PC12 cells treated with BzATP was decreased. However, the viability of PC12 cells was increased after being pretreated with oxATP or P2X7 siRNA. These results suggested that OGD or P2X7 agonist BzATP decreased the viability of PC12 cells, and
OGD+ oxATP
OGD+ U0126
P2X7 (69Kd)
β -actin (42Kd)
B
Fig. 4. Effects of P2X7 antagonist or P2X7 knockdown on the expression of P2X7 protein in OGD PC12 cells. The stain values (average optical density, AOD) of P2X7 protein expression (normalized to each β-actin internal control) were tested by western blotting. A. Photos displayed the expression of P2X7 protein in each group. B. Histogram showed the expression values of P2X7 protein in control group, OGD group, OGD + BzATP group, OGD + P2X7 siRNA group, OGD + oxATP group or OGD + U0126 group. The expression of P2X7 protein in OGD group or OGD + BzATP (30 μM) group was significantly enhanced in comparison with that in control group. After being treated with siRNA-P2X7, oxATP or U0126 in OGD groups, the expression of P2X7 protein was lower than that in OGD group or OGD + BzATP group. The values are means ± SEM (n = 5 for each group), *p b 0.05 vs. control group; #p b 0.05 vs. OGD group.
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
B. Fan et al. / Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
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Fig. 5. Effects of P2X7 agonist ATP on the [Ca2+]i responses in OGD PC12 cells. A. Histogram showed the change of peak [Ca2+]i in each group. B. The photos showed the effects of ATP on the [Ca2+]i responses of PC12 cells treated with OGD in each group. The peak of [Ca2+]i in PC12 cells was increased by ATP (100 μM) stimuli. The difference-value (D-value) in OGD group or OGD + BzATP (30 μM) group was higher than that in control group. The D-value in OGD + oxATP group, OGD + P2X7 siRNA group, and OGD + ERK inhibitor U0126 group was decreased in comparison with that in OGD group or OGD + BzATP group. The values are means ± SEM (n = 5 for each group), *p b 0.05 vs. control group; #p b 0.05 vs OGD group.
P2X7 antagonist oxATP or P2X7 siRNA can reverse the viability of PC12 cells after OGD. Thus, P2X7 receptor participated in the pathophysiological process of neuronal damage induced by OGD. P2X7 receptor is a member of P2X family (non-selective cation channels), which plays important roles in several kinds of physiological functions and pathological process (North, 2002; Abbracchio et al., 2009; Burnstock et al., 2011). P2X receptors can be activated by ATP released from damaged cells. Purinergic signaling was involved in the
regulation of sympathetic and myocardial functions (Vassort, 2001; Gourine et al., 2009). The accumulation of ATP may up-regulate and activate P2X receptors, including P2X7 and initiate neuronal injury (Milius et al., 2008; Burnstock et al., 2011; Eyo et al., 2013; Xu et al., 2013). Our works showed that the expressions of P2X7 mRNA and protein in PC12 cells after OGD treatment were increased in comparison with those in control group, suggesting that P2X7 in PC12 cells was related to OGD. OxATP as a P2X7 antagonist can inhibit the expression of P2X7 receptor
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
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Fig. 6. Effects of P2X7 agonist BzATP on the [Ca2+]i responses in PC12 cells treated with OGD. A. Histogram showed the change of [Ca2+]i peak in each group. B. The photos showed the effects of BzATP on the [Ca2+]i responses of PC12 cells treated with OGD in each group. A [Ca2+]i rise in PC12 cells was induced by BzATP (30 μM) stimuli. The initial value of [Ca2+]i concentration in OGD group or OGD + BzATP group was higher than that in control group. The [Ca2+]i peak induced by BzATP stimuli in OGD + oxATP group, OGD + P2X7 siRNA group or ERK inhibitor U0126 (10 μM) group was significantly decreased in comparison with that in OGD group. The values are means ± SEM (n = 5 for each group), *p b 0.05 vs. control group; #p b 0.05 vs OGD group.
in pathological conditions (Cotrina and Nedergaard, 2009). Our results showed that the expression values of both P2X7 mRNA and protein were significantly increased when OGD PC12 cells exposed to BzATP (a selective P2X7 agonist). This response was inhibited in PC12 cells pretreated with oxATP or transfected with P2X7-siRNA in our experiment. Down-regulation of P2X7 receptor may prevent the neuronal damage caused by OGD.
It was reported that traumatic damage to the spinal cord induced ATP release and overactivation of P2X7 receptor in spinal neurons (Wang et al., 2004). Overactivation of P2X7 receptor can cause neuronal injury by calcium (Ca2 +) overload. In the present study, the [Ca2 +]i peak after OGD treatment was increased in comparison with that in control. The [Ca2 +]i peak induced by ATP or BzATP stimuli in OGD group was increased. When PC12 cells in OGD were co-treated with
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
B. Fan et al. / Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
oxATP or P2X7 siRNA, the [Ca2+]i peak induced by ATP or BzATP stimuli was significantly decreased in comparison with that in OGD group. It suggested that the activation of P2X7 receptor was involved in the enhanced [Ca2 +]i peak after OGD treatment. P2X7 antagonist oxATP and P2X7 siRNA treatment inhibited the [Ca2+]i concentration in PC12 cells after OGD. These results further indicated that down-regulation of P2X7 expression may attenuate the neuronal damage following OGD. P2X receptors mediated the activation of extracellular signalregulated kinases 1 and 2 (ERK1/2) (Gendron et al., 2003; Neary et al., 2003). BzATP (as a P2X7 agonist) and/or oxATP (as a P2X7 antagonist) have been used in experiments to activate mitogen-activated protein kinases (MAPKs) related to P2X7 receptor (Gendron et al., 2003). The family of mitogen-activated protein kinases (MAPKs) is divided into extracellular signal-regulated kinases (ERK1/2), c-Jun amino-terminal kinases (JNKs) and p38 MAPKs (Gendron et al., 2003; Neary et al., 2003). Our data showed that the [Ca2+]i concentration in OGD group treated with ERK inhibitor U0126 was significantly decreased in comparison with that in OGD group. These data demonstrated that ERK signal transduction pathway was involved in the regulation mechanism of P2X7 receptor on the cell viability and [Ca2+]i change in PC12 cells after OGD. 5. Conclusion In summary, our results showed that oxATP and P2X7 siRNA may inhibit the up-regulation of P2X7 receptor induced by OGD in PC12 cells and reduce P2X7 receptor-mediated the neuronal injury induced by OGD. ERK pathway may participate in the effect of P2X7 receptor on the cell viability and [Ca2+]i change affected by OGD. P2X7 antagonists or P2X7 siRNA could inhibit the sympathetic neuronal damage following ischemia. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the grant (Nos.: 81260187, 30860333, 81171184, 31060139, 30860086, 30660048, 81100829 and 81200853) from the National Natural Science Foundation of China, the grant (Nos.: 2010BSA09500 and 20111BBG70009-1) from the Technology Pedestal and Society Development Project of Jiangxi Province, the grant (Nos.: 0640042 and 2008GZY0029) from the Natural Science Foundation of Jiangxi Province, the grant (No.: 20114BAB215022) from the Youth Science Foundation of Jiangxi Province, the grant (Nos.: 2007-60, GJJ08049 and GJJ13155) from the Educational Department of Jiangxi Province, the grant (No.: GJJ11058) from the Youth Science Foundation of the Educational Department of Jiangxi Province. References Abbracchio, M.P., Burnstock, G., Verkhratsky, A., Zimmermann, H., 2009. Purinergic signalling in the nervous system: an overview. Trends Neurosci. 32 (1), 19–29. Arbeloa, J., Pérez-Samartín, A., Gottlieb, M., Matute, C., 2011. P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia. Neurobiol. Dis. 45, 954–961. Burnstock, G., Krügel, U., Abbracchio, M.P., Illes, P., 2011. Purinergic signalling: from normal behaviour to pathological brain function. Prog. Neurobiol. 95, 229–274. Cotrina, M.L., Nedergaard, M., 2009. Physiological and pathological functions of P2X7 receptor in the spinal cord. Purinergic Signal 5, 223–232. Erlinge, D., Burnstock, G., 2008. P2 receptors in cardiovascular regulation and disease. Purinergic Signal 4, 1–20. Eyo, U.B., Miner, S.A., Ahlers, K.E., Wu, L.J., Dailey, M.E., 2013. P2X7 receptor activation regulates microglial cell death during oxygen–glucose deprivation. Neuropharmacology 73C, 311–319. Fu, L.W., Longhurst, J.C., 2010. A new function for ATP: activating cardiac sympathetic afferents during myocardial ischemia. Am. J. Physiol. Heart Circ. Physiol. 299, H1762–H1771.
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Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
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The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation Bo Fan 1, Shuangmei Liu 1, Changshui Xu 1, Jun Liu 1, Fanjun Kong 1, Guilin Li, Chunping Zhang, Yun Gao, Hong Xu, Shicheng Yu, Chaoran Zheng, Lichao Peng, Miaomiao Song, Bing Wu, Qiulan Lv, Lifang Zou, Mofeng Ying, Xi Zhang, Shangdong Liang ⁎,1 Department of Physiology, Medical School of Nanchang University, Nanchang 330006, PR China
a r t i c l e
i n f o
Article history: Received 29 November 2013 Received in revised form 10 March 2014 Accepted 24 March 2014 Available online xxxx Keywords: PC12 cell Adenosine triphosphate P2X7 receptor Oxygen–glucose deprivation Intracellular Ca2 +
a b s t r a c t Adenosine triphosphate (ATP) plays an important role in signal transmission via acting on P2X receptors. P2X7 receptor is involved in pathophysiological changes of ischemic diseases. The PC12 cell line is a popular model system to study sympathetic neuronal function. In this study, the effects of P2X7 on the viability or [Ca2+]i in PC12 cells after exposure to oxygen–glucose deprivation (OGD) were investigated. The results showed that the viability of PC12 cells was decreased under the condition of OGD. BzATP, a P2X7 agonist, decreased the viability, while P2X7 antagonist oxATP or P2X7 siRNA reversed the viability of PC12 cells under the condition of OGD. The expression levels of P2X7 mRNA and protein in PC12 cells were up-regulated under the condition of OGD or BzATP treatment. The expression levels of P2X7 mRNA and protein were significantly decreased in OGD PC12 cells, which were pretreated with oxATP or P2X7 siRNA. It was also found that oxATP or P2X7 siRNA effectively suppressed the increase of [Ca2+]i induced by OGD. P2X7 agonist ATP or BzATP enhanced the [Ca2+]i rise induced by OGD in PC12 cells. The [Ca2+]i peak induced by ATP or BzATP in OGD group was decreased by ERK inhibitor U0126. Therefore, P2X7 antagonists or P2X7 siRNA could depress the sympathetic neuronal damage induced by ischemia. © 2014 Elsevier B.V. All rights reserved.
1. Introduction P2X7 receptor is a member of ATP-gated non-selective cation channels, which has a unique and important physiological function (Sperlagh et al., 2006; Liang et al., 2010; Skaper et al., 2010; Burnstock et al., 2011; Zhang et al., 2013). As an ionotropic receptor, P2X7 receptor is characterized by a low affinity for the endogenous ligand ATP and by two states of permeability (Arbeloa et al., 2011; Burnstock et al., 2011). Upon transient stimulation, P2X7 receptor agonists at the low concentration cause selective cationic inflow of Na+, K+, and Ca2 +. When activated by agonists at relatively high concentrations, sustained stimulation drives transition that leads to the formation of a nonselective pore permeable to aqueous solutes of molecular mass up to 700 Da (Sperlagh et al., 2006; Skaper et al., 2010). A common feature of both conductance states is the elevation of [Ca2+]i, which modulates cell functions. Studies have shown that P2X7 receptor has been implicated in the neuronal
⁎ Corresponding author at: Department of Physiology, Medical School of Nanchang University, Nanchang, Jiangxi 330006, PR China. Tel./fax: +86 791 86360552. E-mail address: [email protected] (S. Liang). 1 Joint first authors.
injury after ischemic diseases (Milius et al., 2008; Vasileiou et al., 2010; Arbeloa et al., 2011; Kong et al., 2013; Tu et al., 2013). During the ischemic heart diseases, the activation of cardiac afferents evokes sympathetic reflex responses (Shao et al., 2007; Zhang et al., 2007, 2008; Li et al., 2010, 2011; Kong et al., 2013; Liu et al., 2013; Tu et al., 2013). In the pathological conditions of myocardial ischemia, damaged cells release ATP into the extracellular space (Shao et al., 2007; Zhang et al., 2007, 2008; Fu and Longhurst, 2010; Li et al., 2010, 2011; Kong et al., 2013; Liu et al., 2013; Tu et al., 2013). ATP may act as an excitotoxin and result in neuronal injury (Shao et al., 2007; Zhang et al., 2007, 2008; Li et al., 2010, 2011; Arbeloa et al., 2011; Burnstock et al., 2011; Liu et al., 2013; Tu et al., 2013). In vitro studies also show that the application of exogenous ATP is toxic to primary neuronal cultures and causes injury (Arbeloa et al., 2011; Burnstock et al., 2011; Xu et al., 2013). Oxygen–glucose deprivation (OGD) also occurs in ischemia and a variety of pathologic conditions (Lecht et al., 2012). Hypoxia induces [Ca2 +]i elevation in PC12 cells, which also is involved in neuronal injury (Meng et al., 2008; Arbeloa et al., 2011). In previous studies, we found that P2X7 receptor is involved in the pathological injury of sympathetic nerve during myocardial ischemia (Kong et al., 2013; Liu et al., 2013; Tu et al., 2013). The aim of this research was to investigate the effects of P2X7 antagonism or
http://dx.doi.org/10.1016/j.autneu.2014.03.006 1566-0702/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
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P2X7-siRNA treatment upon the cell viability or [Ca2+]i when PC12 cells were exposed to OGD. 2. Materials and methods
detail, the P2X 7 siRNA and lipo2000 were diluted in the OptiMEM® medium without serum. Final concentration of P2X7 siRNA was 100 nM. The cells were cultured for 24 h at 37 °C. Total RNA was extracted and the efficiency of knockdown was verified by Real-time PCR.
2.1. Cell cultures PC12 cell line was originally obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The PC12 cells were seeded in 25 cm2 polystyrene flasks in Dulbecco's modified Eagle's medium (DMEM) (high glucose) (HyClone, USA), containing 5% heatinactivated fetal bovine serum (Gibco, USA) and 10% horse serum (Gibco, USA). The cells were incubated under a humidified atmosphere containing 5% CO2 (normal culture incubator) at 37 °C, and culture medium was replaced every 48 h. PC12 cells were plated in 6-well plates containing 2 ml of complete medium, at a density of 1.0 × 105/ml cells. When cell density reached 70%–80%, we established the model of “oxygen–glucose deprivation (OGD)” (Lecht et al., 2012). 2.2. Drugs and groups 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP, Sigma-Aldrich) and oxidized ATP (oxATP, Sigma) were dissolved in distilled water and used at a concentration of 300 nM and 30 μM, respectively. ATP (100 μM, Sigma) and ERK inhibitor U0126 (10 μM, Sigma) were used. According to the experimental purpose, PC12 cells were randomly divided into the following groups. (1) Control group; (2) oxygen– glucose deprivation (OGD) group; (3) OGD + oxATP group; (4) OGD + BzATP group; (5) OGD + P2X7 siRNA group; (6) OGD + U0126 group. OGD + scramble siRNA group was only used for testing the interference efficiency of P2X7 siRNA. 2.3. Oxygen–glucose deprivation (OGD) For OGD treatment (Hillion et al., 2005; Larsen et al., 2007; Lecht et al., 2012), the culture media were changed to a deoxygenated glucose-free Earle's balanced salt solution at pH 7.4. 10% fetal bovine serum was added. Then the cells were transferred to a sealed hypoxic box containing a mixture of 95% N2 and 5% CO2 at 37 °C for 2 h. After OGD, the cells were cultured in full culture medium with oxygen for 24 h and treated with indicated drugs. Oxygenated glucose-containing Earle's balanced salt solution was used as control.
2.6. Measurement of intracellular Ca2+ Intracellular Ca2+ was monitored using the fluorescent Ca2+sensitive dye, Fura 2-acetoxymethyl ester (Fura 2-AM) (Shi et al., 2009). Cells were divided into control group, OGD group, OGD + BzATP group, OGD + oxATP group, OGD + P2X7 siRNA group, and OGD + U0126 group. PC12 cells cultured with OGD were seeded in 6-well plates and incubated with oxATP (300 nM), BzATP (30 μM) (Morigiwa et al., 2000), P2X7 siRNA (50 nm) and ERK inhibitor U0126 (10 μM) respectively. [Ca2+]i was measured after treatment with drugs for 2 h. After digestion, cell suspensions were centrifuged for 5 min at 4 °C, 1000 rpm, and the pellet was resuspended in 50 ml medium. Incubation was continued for 3 h in the container containing magnetic puddler at 37 °C. After 3 h, abandon the liquid after being centrifuged for 5 min at 4 °C, 1000 rpm, wash once with Spinner medium, then the cells were incubated for 10 min in 20 ml Spinner medium containing 10% FBS at 37 °C as the method mentioned above. The cells were loaded for 30 min in 20 μl 1 mM Fura-2/AM, washed twice with Spinner medium and centrifuged for 5 min at 4 °C, 1000 rpm. 2 ml KRBH liquid was added and beated repeatedly. The suspension was transferred to a quartz cuvette and tested at excitation wavelengths of 340 and 380 nm in LS50B fluorescence spectrophotometer. 1 μl MnCl2 (100 mM) was added, and then 2 μl diethylene-triamine-penta-acetic acid (DTPA, 100 mM) to minus the extracellular spontaneous fluorescence. After measuring the basal fluorescence (R), 100 μM ATP or 100 μM BzATP was added to the suspension to observe the fluorescence. 10% Triton-100 was added, which resulted in maximum fluorescence (Rmax). Then, 40 μl EGTA (0.4 M) and 40 μl Tris (3 M) were added to couple calcium ions. The resultant fluorescence signal was designated as Rmin. Intracellular Ca2+ concentration was determinated in a fluorescence spectrophotometer with double wavelengths. The calculation equation is: [Ca2 +]i = 224 nM × [(R − Rmin) / (Rmax − R)] / (S1 / S2), where 224 is the Kd value of Fura-2/AM; S1 and S2 were the fluorescence intensities of zero Ca2 + (EGTA, 0.4 M) and saturated Ca2 + (Triton-100,10%) in 380 nm place, respectively.
2.4. MTS for cell viability PC12 cells were seeded into 96-well plastic plates with 0.1 ml culture medium at a density of 1 × 105 cells/ml. After growth for 48 h, the cells were treated with OGD for 4 h. After 48 h of reperfusion, the 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium (MTS) (purchased from Promega, Madison WI) colorimetric assay was performed to determine cell viability (McNeill-Blue et al., 2006). Each group contained 10 samples and 20 μl MTS were pipetted into each well of the 96-well assay plate within 100 μl culture medium. The plate was incubated at 37 °C for 3 h in a humidified, 5% CO2 atmosphere. The absorbance at 490 nm was recorded using a 96-well plate reader. Background absorbance was subtracted using a set of wells containing medium only. 2.5. Small interfering RNA (siRNA) knockdown experiment With siRNA technology to knockdown the P2X7 receptor of OGD PC12 cells (Hillion et al., 2005; Larsen et al., 2007), a P2X7 siRNA fragment was transfected into cultured cells using lipo2000. PC12 cells were divided into control group, OGD group, OGD + NC group and OGD + P2X7 siRNA group. The P2X7 siRNA cis-chain: 5′-GGAUGGACCCACAAAG UAAdTdT-3′; trans-chain: 5′-dTdTCCUACCUGGGUGUUUCAUU-3′. In
2.7. Western blotting analysis The level of P2X7 protein was determined by western-blotting. The cells were solubilized in lysis buffer. After incubating on ice for 30 min, the lysates were clarified by centrifugation at 14 000 g for 15 min. The supernatant was taken and the protein concentration was determined using the Bradford protein assay system (Bio-Rad). Protein was mixed with 5 × SDS sample buffer. Total protein (20 μg) was electrophoresed on 5% SDS-PAGE gel and 10% SDS-PAGE gel and then transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat dry milk for 2 h on a table concentrator at room temperature. The experiments were performed using the following primary antibodies: monoclonal anti-P2X 7 (1:1000) and anti-βactin (1:1000). A secondary goat anti-mouse (polyclonal) or goat anti-rabbit (polyclonal) antibody (both 1:1000) was used. Then using the enhanced chemiluminescence (ECL) kit (Shanghai Pufei Biotech. CO.), chemiluminescent signals were collected on an autoradiography film. The quantity of band intensity was carried out using Image Pro-Plus software. Protein expression levels were represented as densitometric ratios of the targeted protein to β-actin.
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
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2.8. Statistical analysis The data are expressed as the mean ± the standard error of the mean (SEM). All statistical analyses were performed using SPSS 17.0 software. Statistical significance was assessed using the one-way analysis of variance for multiple group comparisons followed by Tukey's post hoc test. A p-value of less than 0.05 was considered to be statistically significant. 3. Result 3.1. The growth and the viability of PC12 cells after OGD treatment 3.1.1. Effects of OGD on the growth of PC12 cells The cultured PC12 cells with or without oxygen and glucose deprivation (OGD) were observed (Fig. 1). The morphous of PC12 cells cultured in normal condition showed larger oval, fullness, three-dimensional sense of strong and clear halos. The PC12 cells treated with OGD displayed smaller neurons, short protrusion, thin and wrinkled shape. 3.1.2. Effects of P2X7 antagonist or P2X7 knockdown on the viability of PC12 cells after OGD To determine the effects of P2X7 antagonist or P2X7 knockdown on the injury responses of OGD treatment in PC12 cells, the cell viability was tested using MTS assay. The optical density (OD) values of MTS as the cell viability in control group, OGD group, OGD + oxATP group, OGD + BzATP group, OGD + P2X7 siRNA group, and OGD + ERK inhibitor U0126 group were measured (n = 10 for each group; Fig. 2). Compared with the control group, the viability of PC12 cells in OGD group was decreased (p b 0.05; Fig. 2). After being treated with oxATP (300 nM), P2X7 siRNA, and U0126 (10 μM), the viability of PC12 cells in OGD group was reversed in comparison with that in OGD group and OGD + BzATP (30 μM) group (p b 0.05; Fig. 2). P2X7 antagonist oxATP or P2X7 knockdown can increase the viability of OGD cells. 3.2. Changes of P2X7 expression in PC12 cells treated with OGD 3.2.1. Expression of P2X7 mRNA in PC12 cells treated with OGD The expression of P2X7 mRNA in PC12 cells was studied by Real-time PCR. The average relative quantification (RQ) value of P2X7 mRNA in OGD group was thought as 1, which was higher than that in control group (p b 0.05) (Fig. 3). The RQ values in OGD + P2X7 siRNA group and OGD + oxATP (300 nM) group were significantly lower than those in OGD group, OGD + scramble siRNA group and OGD + BzATP (30 μM) group (p b 0.05; Fig. 3). Interference efficiency of P2X7 siRNA was 55.9%. No difference was found in OGD group, OGD + scramble siRNA group and OGD + BzATP group (p N 0.05; Fig. 3) (n = 3 for
A
Fig. 2. Effects of P2X7 on OGD PC12 cell viability. The cell viability was tested by MTS assay. Histogram showed the optical density (OD) values of cell viability in control group, OGD group, OGD + oxATP group, OGD + BzATP group, OGD + P2X7 siRNA group, and OGD + U0126 group. Compared with the viability of PC12 cells in control group, the viability of OGD group cells was decreased. The viability of PC12 cells in OGD group after being treated with oxATP (300 nM), P2X7 siRNA, and U0126 (10 μM) was increased in comparison with that in OGD group and OGD + BzATP (30 μM) group. The values are means ± SEM (n = 10). *p b 0.05 vs. control group; #p b 0.05 vs. OGD group.
each group). P2X7 antagonist oxATP or P2X7 knockdown can decrease the expression of P2X7 mRNA in PC12 cells treated with OGD. 3.2.2. Expression of P2X7 protein in PC12 cells treated with OGD The stain values (average optical density, AOD) of P2X7 protein expression (normalized to each β-actin internal control) were tested by western blotting. In OGD group or OGD + BzATP (30 μM) group, the expression of P2X7 protein was significantly increased in comparison with that in control group. After P2X7 siRNA, oxATP or U0126 treatment in OGD group, the expression of P2X7 protein was decreased in comparison with that in OGD group or OGD + BzATP group (p b 0.05; Fig. 4) (n = 5 for each group). Our results suggested that P2X7 protein expression could be effectively reduced by P2X7 siRNA, P2X7 antagonist oxATP (300 nM) or ERK inhibitor U0126 (10 μM) in OGD group. 3.3. P2X7 receptor and alteration of [Ca2+]i in PC12 cells treated with OGD 3.3.1. Effects of ATP on the [Ca2+]i responses in PC12 cells treated with OGD ATP (100 μM) could increase the peak of [Ca2+]i in PC12 cells. The alteration value of [Ca2 +]i after ATP stimuli was shown as the difference-value (D-value) between the peak value of [Ca2 +]i and the initial value of [Ca2 +]i. There was a high initial value of [Ca2 +]i
B
Fig. 1. The growth of PC12 cells after cultured with or without oxygen–glucose deprivation (OGD). The PC12 cells in control group and OGD group were shown in (A) and (B) as the arrows indicated. The PC12 cells in control showed larger oval, fullness, three-dimensional sense of strong and clear halos. The PC12 cells in OGD displayed smaller neurons, short protrusion, thin and wrinkled shape. Scale bar indicates 100 μm.
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
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about 49.9% (p b 0.05, n = 5; Fig. 5A, B). These results indicated that P2X7 activation contributed to the [Ca2+]i rise. 3.3.2. Effects of BzATP on the [Ca2+]i responses of PC12 cells treated with OGD BzATP (30 μM) induced a [Ca2+]i rise in PC12 cells. There is a high initial value of [Ca2+]i concentration in OGD + BzATP group in comparison with control group (n = 5). The [Ca2+]i peak induced by BzATP stimuli in OGD + oxATP group was reduced to 68.9% in comparison with that in OGD group (p b 0.05, n = 5). The [Ca2 +]i peak induced by BzATP stimuli in OGD + P2X7 siRNA group was significantly inhibited to 70.7% (p b 0.05, n = 5). The [Ca2 +]i peak induced by BzATP stimuli in OGD treatment was partially inhibited by ERK inhibitor U0126 (10 μM) about 55.1% (p b 0.05, n = 5; Fig. 6A, B). Fig. 3. Effects of P2X7 antagonist or P2X7 knockdown on the expression of P2X7 mRNA in OGD PC12 cells. The expression of P2X7 mRNA was studied by Real-time PCR. The average relative quantification (RQ) value of P2X7 mRNA in OGD group was thought as 1. The RQ values in OGD + P2X7 siRNA group and OGD + oxATP (300 nM) group were significantly decreased in comparison with those in OGD group, OGD + scramble siRNA group and OGD + BzATP (30 μM) group. Histogram showed the RQ values of P2X7 mRNA in control group, OGD group, OGD + scramble siRNA group, OGD + P2X7 siRNA group, OGD + BzATP and OGD + oxATP group. The values are means ± SEM (n = 3 for each group), *p b 0.05 vs. control group; #p b 0.05 vs. OGD group.
concentration in OGD + BzATP (30 μM) group in comparison with that in control group. The D-value in OGD group was higher than that in control group, while the D-value in OGD + oxATP group, OGD + P2X7 siRNA group, and OGD + ERK inhibitor U0126 group was lower than that in OGD group or OGD + BzATP group (n = 5 for each group). The peak [Ca2+]i was partially inhibited by oxATP (300 nM) and P2X7 siRNA about 28.8% and 37.4%, respectively (p b 0.05, n = 5). The peak [Ca2 +]i was partially inhibited by ERK inhibitor U0126 (10 μM) by
A Control
OGD
OGD+ BzATP
OGD+ siRNA
4. Discussion ATP plays an important role in signal transmission via acting on purinergic receptors (Kennedy, 2005; Abbracchio et al., 2009; Burnstock et al., 2011; Sperlagh et al., 2012). ATP, released during myocardial ischemia, can lead to abnormal cardiac function in myocardial ischemic injury (Vassort, 2001; Shao et al., 2007; Zhang et al., 2007, 2008; Erlinge and Burnstock, 2008; Li et al., 2010, 2011; Kong et al., 2013; Liu et al., 2013; Tu et al., 2013). The accumulation of ATP can also initiate neuronal damage. The PC12 (rat pheochromocytoma) cell line, a popular model system to study sympathetic neuronal function, was used in this experiment (Xu et al., 2013). Our results showed that the viability of PC12 cells was decreased under the condition of oxygen–glucose deprivation. The viability of PC12 cells treated with BzATP was decreased. However, the viability of PC12 cells was increased after being pretreated with oxATP or P2X7 siRNA. These results suggested that OGD or P2X7 agonist BzATP decreased the viability of PC12 cells, and
OGD+ oxATP
OGD+ U0126
P2X7 (69Kd)
β -actin (42Kd)
B
Fig. 4. Effects of P2X7 antagonist or P2X7 knockdown on the expression of P2X7 protein in OGD PC12 cells. The stain values (average optical density, AOD) of P2X7 protein expression (normalized to each β-actin internal control) were tested by western blotting. A. Photos displayed the expression of P2X7 protein in each group. B. Histogram showed the expression values of P2X7 protein in control group, OGD group, OGD + BzATP group, OGD + P2X7 siRNA group, OGD + oxATP group or OGD + U0126 group. The expression of P2X7 protein in OGD group or OGD + BzATP (30 μM) group was significantly enhanced in comparison with that in control group. After being treated with siRNA-P2X7, oxATP or U0126 in OGD groups, the expression of P2X7 protein was lower than that in OGD group or OGD + BzATP group. The values are means ± SEM (n = 5 for each group), *p b 0.05 vs. control group; #p b 0.05 vs. OGD group.
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
B. Fan et al. / Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
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Fig. 5. Effects of P2X7 agonist ATP on the [Ca2+]i responses in OGD PC12 cells. A. Histogram showed the change of peak [Ca2+]i in each group. B. The photos showed the effects of ATP on the [Ca2+]i responses of PC12 cells treated with OGD in each group. The peak of [Ca2+]i in PC12 cells was increased by ATP (100 μM) stimuli. The difference-value (D-value) in OGD group or OGD + BzATP (30 μM) group was higher than that in control group. The D-value in OGD + oxATP group, OGD + P2X7 siRNA group, and OGD + ERK inhibitor U0126 group was decreased in comparison with that in OGD group or OGD + BzATP group. The values are means ± SEM (n = 5 for each group), *p b 0.05 vs. control group; #p b 0.05 vs OGD group.
P2X7 antagonist oxATP or P2X7 siRNA can reverse the viability of PC12 cells after OGD. Thus, P2X7 receptor participated in the pathophysiological process of neuronal damage induced by OGD. P2X7 receptor is a member of P2X family (non-selective cation channels), which plays important roles in several kinds of physiological functions and pathological process (North, 2002; Abbracchio et al., 2009; Burnstock et al., 2011). P2X receptors can be activated by ATP released from damaged cells. Purinergic signaling was involved in the
regulation of sympathetic and myocardial functions (Vassort, 2001; Gourine et al., 2009). The accumulation of ATP may up-regulate and activate P2X receptors, including P2X7 and initiate neuronal injury (Milius et al., 2008; Burnstock et al., 2011; Eyo et al., 2013; Xu et al., 2013). Our works showed that the expressions of P2X7 mRNA and protein in PC12 cells after OGD treatment were increased in comparison with those in control group, suggesting that P2X7 in PC12 cells was related to OGD. OxATP as a P2X7 antagonist can inhibit the expression of P2X7 receptor
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
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Fig. 6. Effects of P2X7 agonist BzATP on the [Ca2+]i responses in PC12 cells treated with OGD. A. Histogram showed the change of [Ca2+]i peak in each group. B. The photos showed the effects of BzATP on the [Ca2+]i responses of PC12 cells treated with OGD in each group. A [Ca2+]i rise in PC12 cells was induced by BzATP (30 μM) stimuli. The initial value of [Ca2+]i concentration in OGD group or OGD + BzATP group was higher than that in control group. The [Ca2+]i peak induced by BzATP stimuli in OGD + oxATP group, OGD + P2X7 siRNA group or ERK inhibitor U0126 (10 μM) group was significantly decreased in comparison with that in OGD group. The values are means ± SEM (n = 5 for each group), *p b 0.05 vs. control group; #p b 0.05 vs OGD group.
in pathological conditions (Cotrina and Nedergaard, 2009). Our results showed that the expression values of both P2X7 mRNA and protein were significantly increased when OGD PC12 cells exposed to BzATP (a selective P2X7 agonist). This response was inhibited in PC12 cells pretreated with oxATP or transfected with P2X7-siRNA in our experiment. Down-regulation of P2X7 receptor may prevent the neuronal damage caused by OGD.
It was reported that traumatic damage to the spinal cord induced ATP release and overactivation of P2X7 receptor in spinal neurons (Wang et al., 2004). Overactivation of P2X7 receptor can cause neuronal injury by calcium (Ca2 +) overload. In the present study, the [Ca2 +]i peak after OGD treatment was increased in comparison with that in control. The [Ca2 +]i peak induced by ATP or BzATP stimuli in OGD group was increased. When PC12 cells in OGD were co-treated with
Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
B. Fan et al. / Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
oxATP or P2X7 siRNA, the [Ca2+]i peak induced by ATP or BzATP stimuli was significantly decreased in comparison with that in OGD group. It suggested that the activation of P2X7 receptor was involved in the enhanced [Ca2 +]i peak after OGD treatment. P2X7 antagonist oxATP and P2X7 siRNA treatment inhibited the [Ca2+]i concentration in PC12 cells after OGD. These results further indicated that down-regulation of P2X7 expression may attenuate the neuronal damage following OGD. P2X receptors mediated the activation of extracellular signalregulated kinases 1 and 2 (ERK1/2) (Gendron et al., 2003; Neary et al., 2003). BzATP (as a P2X7 agonist) and/or oxATP (as a P2X7 antagonist) have been used in experiments to activate mitogen-activated protein kinases (MAPKs) related to P2X7 receptor (Gendron et al., 2003). The family of mitogen-activated protein kinases (MAPKs) is divided into extracellular signal-regulated kinases (ERK1/2), c-Jun amino-terminal kinases (JNKs) and p38 MAPKs (Gendron et al., 2003; Neary et al., 2003). Our data showed that the [Ca2+]i concentration in OGD group treated with ERK inhibitor U0126 was significantly decreased in comparison with that in OGD group. These data demonstrated that ERK signal transduction pathway was involved in the regulation mechanism of P2X7 receptor on the cell viability and [Ca2+]i change in PC12 cells after OGD. 5. Conclusion In summary, our results showed that oxATP and P2X7 siRNA may inhibit the up-regulation of P2X7 receptor induced by OGD in PC12 cells and reduce P2X7 receptor-mediated the neuronal injury induced by OGD. ERK pathway may participate in the effect of P2X7 receptor on the cell viability and [Ca2+]i change affected by OGD. P2X7 antagonists or P2X7 siRNA could inhibit the sympathetic neuronal damage following ischemia. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the grant (Nos.: 81260187, 30860333, 81171184, 31060139, 30860086, 30660048, 81100829 and 81200853) from the National Natural Science Foundation of China, the grant (Nos.: 2010BSA09500 and 20111BBG70009-1) from the Technology Pedestal and Society Development Project of Jiangxi Province, the grant (Nos.: 0640042 and 2008GZY0029) from the Natural Science Foundation of Jiangxi Province, the grant (No.: 20114BAB215022) from the Youth Science Foundation of Jiangxi Province, the grant (Nos.: 2007-60, GJJ08049 and GJJ13155) from the Educational Department of Jiangxi Province, the grant (No.: GJJ11058) from the Youth Science Foundation of the Educational Department of Jiangxi Province. References Abbracchio, M.P., Burnstock, G., Verkhratsky, A., Zimmermann, H., 2009. Purinergic signalling in the nervous system: an overview. Trends Neurosci. 32 (1), 19–29. Arbeloa, J., Pérez-Samartín, A., Gottlieb, M., Matute, C., 2011. P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia. Neurobiol. Dis. 45, 954–961. Burnstock, G., Krügel, U., Abbracchio, M.P., Illes, P., 2011. Purinergic signalling: from normal behaviour to pathological brain function. Prog. Neurobiol. 95, 229–274. Cotrina, M.L., Nedergaard, M., 2009. Physiological and pathological functions of P2X7 receptor in the spinal cord. Purinergic Signal 5, 223–232. Erlinge, D., Burnstock, G., 2008. P2 receptors in cardiovascular regulation and disease. Purinergic Signal 4, 1–20. Eyo, U.B., Miner, S.A., Ahlers, K.E., Wu, L.J., Dailey, M.E., 2013. P2X7 receptor activation regulates microglial cell death during oxygen–glucose deprivation. Neuropharmacology 73C, 311–319. Fu, L.W., Longhurst, J.C., 2010. A new function for ATP: activating cardiac sympathetic afferents during myocardial ischemia. Am. J. Physiol. Heart Circ. Physiol. 299, H1762–H1771.
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Please cite this article as: Fan, B., et al., The role of P2X7 receptor in PC12 cells after exposure to oxygen–glucose deprivation, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.006
