Study On The P2X7 Mediates Morphological Changes And Receptor Expression In Microglial Cells

Purinergic receptors are divided into two major classes P1 and P2. The P1 receptors are responsive to adenosine, whereas the P2 receptors respond to a variety of nucleotides, including ATP. The P2 receptors are subdivided in two mechanistically distinct subclasses, the metabotropic G protein-coupled P2Y receptors ( P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11-13) which were concerned with activation of phopholipase C and formation of inositol triphosphate and the ionotropioc ligand-gated channel P2X receptors ( P2X1-7) which are permeable for potassium, sodium and calcium.Molecular cloning studies have identified seven P2X receptor subtypes, designated as P2X1-P2X7. P2X7 is also called P2Z. The properties of P2X7 receptors is distinguished from the other six subtypes. Electrophysiological experiments in vitro indicated that stimulation of extracellular ATP on P2X7 receptor provoked transient non-selective ion permeation. Repeatedly and persistent stimulation of ATP would lead to apoptosis in the end.The P2X receptors are widely distributed. Microglia have been shown to express multiple P2 receptor subtypes, including P2X7, P2Y2, and P2Y12, indicating that ATP may be a critical regulator of microglial cells.The concentration of extracellular ATP is too lower to activate microglial cells, although it is quite higher in cytoplasma. The latter is 1000 times more than the former, up to millimolar level. Under certain pathological conditions such as traumatic brain injury, ischemia and inflammation and so on, massive ATP released from lytic cells formed high concentration in the lesion locus and diffused to the adjacent regions, which would stimulate these two areas for a long time. Microglial cells would be activated in these conditions and expression of P2X7 receptors would be up regulated. Microglial cells became hypertrophic and proliferated after activation and form typical phagocytes. Many biological active substances such as active oxygen, nitrogen monoxidum, protease and some inflammatory substances which are harmful to surrounded cells released. On the other hand, transforming growth factor beta 1 and nerve growth factor released to facilitate nervous tissue regeneration and recovery. Investigations indicated that activated microglia cells played an important role in traumatic brain injury, ischemia, inflammation, disseminated sclerosis disease, protein viral disease, degenerative disease like Alzheimer and Parkinson's disease and the process of aging.Such pathological processes are related with activation of microglial cells, but the mechanism are not determined yet. According to the literatures and present study, P2X7 receptors are involved in the activation and apoptosis of microglial cells. With specific antagonists of the P2X7 receptors blocking the action of ATP, it would prevent activated microglial cells and astrocytes from damage on the surrounding tissues and cells and formation of glial scar. Thus it protected brain tissue from further injury and helped the regeneration of nerve fiber.To study on the P2X7 receptors mediate the morphological changes and receptor expression in microglial cells, we isolated microglial cells from cultures of newborn rat brains. Briefly, cerebral cortices were isolated from 1 or 2-day-old Sprague Dawley rats, stripped of the meninges. Glial cells were grown at 37℃in DMEM medium, supplemented with 0.6% glucose, 10% fetal bovine serum, 100μg/ml streptomycin, and 100 IU/ml penicillin, under a humidified 5% CO2 and 95% air atmosphere. Cells were cultured for 10-15 days and medium was replaced every 3 days. Microglial cells were harvested by gentle shaking for 15 minutes and seeded in 12-well plates overnight and pretreated with ATP at 0 mmol/L, 1 mmol/L,2 mmol/L, and 3 mmol/L, respectively. Microglia were fixed for 1 h in 4% paraformaldehyde in 0.05 mol/L PBS, and primary antibodies diluted by 500 times and monoclonal antibody ED1 were incubated at room temperature in wet box overnight. The second antibodies, donkey anti-rabbit IgG coupled to cyc3 diluted by 5000 times were incubated at room temperature for 1 h. After Hoechst nuclear staining for 2 min, microglial cells were observed in the fluorescent microscope. The purity of microglial cells was about 99%, as detected by staining with the ED1 antibody. Under different concentration of ATP treatment, the morphology of microglial cells changes gradually. Pseudopodium disappearing, cells shrinkage and the number of expression of P2X7 receptors were increasing with the concentration of ATP raised. The result indicated that with the stimulation of ATP P2X7 receptors mediated the changes of cell morphology and accumulation of receptor expression. Continuous stimulation of ATP would result in the apoptosis of microglial cells.Under treatment of extracellular ATP, P2X7 receptors may be involved in the process of activation and apoptosis of microglial cells. Specific agonist ATP and antagonist oATP were used to verify the effect of ATP on P2X7 receptors with flow cytometry. Two groups of primary microglial cells were pretreated respectively with ATP (3 mmol/L) and ATP (3 mmol/L) plus oATP (0.1 mmol/L) for 15 min, 30minutes, 60 min, 90 min, and 120 min. With annexin V/PI double staining, flow cytometry measured the percentage of apoptosis cells. Statistical analysis was performed to reveal the significance of differences between experimental groups, which indicated P2X7 receptors were definitely required for activation and apoptosis of microglial cells.Besides P2X7 receptors a variety of purinergic receptors were expressed on the microglial cells including P2Y2 and P2Y12 receptors. To exclude the influence of other receptors on morphology and receptor expression mediated by P2X7 receptors, we used plasmid which expressed P2X7 receptor for the first time. The clone of rat P2X7 receptor was inserted into pcDNA3-hi-EGFP vector with T4 ligase and transformed into E.coli DH5α. The positive recombinant plasmid was selected and identified via sequence assay and restrictive enzyme digestion. The recombinant plasmid was then transfected into HEK293 cells by Lipofectamine 2000. The cells containing stable transformants were identified by ISH and functional examination. The eukaryotic expression vector named pcDNA3-hi-EGFP-rP2X7 was successfully constructed and the stably transfected HEK293 cell line which expressed P2X7 was obtained.According to the previous reports, we have known that monoclonal cell model of P2X7 receptor was not used to analysis cell morphological changes and receptor expression. It is the first time that we use the monoclonal cell model of P2X7 receptor expressed on human HEK293 cell line to investigate those things. HEK293 cells in the 12-well plate were treated with ATP at the concentration of 0 mmol/L, 0.05 mmol/L, 0.1 mmol/L, 0.4mmol/L and 0.8 mmol/L respectively for 1 h. HEK293 cells were fixed for 1 h in 4% paraformaldehyde in 0.05 mol/L PBS, and primary antibodies diluted by 500 times were incubated at room temperature in wet box overnight. The second antibodies, donkey anti-rabbit IgG coupled to Cyc3 diluted by 5000 times were incubated at room temperature in wet box for 1 h. Then HEK293 cells were observed in the microscope. Under different concentration of ATP treatment, the morphology of microglial cells changes gradually. Cells shrinkage and the number of expression of P2X7 receptors were increasing with the concentration of ATP raised. The result indicated that ATP was really agonist of P2X7 and with the stimulation of ATP P2X7 receptors mediated the changes of cell morphology and accumulation of receptor expression. Continuous stimulation of ATP would result in the apoptosis of microglial cells. Furthmore, stimulation with ATP on the HEK293 cell model stably transfected with rat P2X7 receptor to some extent excluded the other purinergic receptors'interference.In this report stimulation on primary cultured microglial cell with ATP, analysis of P2X7 receptors mediated cell morphological changes and receptor expression, and study on the microglial cell apoptosis with ATP mediated by P2X7 through flow cytometry, and usage of HEK293 cell model stably transfected with rat P2X7 receptor to exclude interference of other purinergic receptor, we demonstrated that P2X7 receptors mediated activation and apoptosis of microglial cell by extracellular ATP. It helps further investigation the signaling and downstream processing of P2X7.