Exogenous ATP Induced The Formation Of Membrane Pore In PC12 Cells

BackgroundATP (Adenosine triphosphate, ATP) is an important energy regulator. It is a long time that ATP is taken as the physical form of cellular energy supply and use. However, more and more recent experiments have shown that high concentrations of extracellular ATP is a cell death factor. Recent literature indicates membrane permeability has undergone a qualitative change with the increase of the concentration and duration of action of ATP, molecules (<900D) and ions enters the cell, and then cells become swelling, vacuolization, apoptosis and necrosis. However, the effect and mechanisms of high concentrations of extracellular ATP in pheochromocytoma cell line (PC 12 cell) was rare.ObjectivesTo investigate the formation of membrane pore induced by exogenous ATP and to identify the key molecular targets in PC 12 cells.MethodsPC12 cells was induced with nerve growth factor (NGF) to differentiate into neurons. (1)PC 12 cells in the logarithmic growth phase were randomly divided into four groups:normal control group (0 mmol/L ATP group),1 mmol/L ATP group,3 mmol/L ATP group,5 mmol/L ATP group.Cell morphology was observed by an inverted microscope, and cell viability was measured by CCK-8 assay,after exposing to ATP for 3 h.The expression of P2X7 receptors and Panx 1 at mRNA and protein levels was assessed by Real-Time PCR and Western Blotting.Fluorescence intensity (485 nm excitation,516 nm emission) of each group cells was monitored after exposed to different concentrations of ATP and 2 μmol/L YO-PRO-1 for lh.(2)PC12 cells in the logarithmic growth phase randomly divided into 7 groups:normal control group,ATP injury group, Brilliant Blue G (BBG,P2X7 receptor antagonist) group,BBG+ATP group,carbenoxolone (CBX,pannexin 1 antagonist) group,CBX+ATP group,BBG+CBX+ATP group. Cell viability was measured by CCK-8 assay,after exposing to ATP for 3 h.Fluorescence intensity was detected by the Reader.Results1. The membrane pore formation induced by ATP in PC12 cellsThe body of PC 12 cells became edema,the number of adherent cells decreased gradually after exposed to ATP(1 mmol/L,3 mmol/L,5 mmol/L) for 3 h in a dose-dependent manner. The cell viability of 3 mmol/L or 5 mmol/L ATP group was significantly decreased, compared with the control group (P<0.05).2. ATP induced membrane pore formation in PC 12 cellsYO-PRO-1 uptake in PC12 cell exposed to ATP(0,1,3,5 mmol/L) for 15,30,60 min respectively increased in a dose-dependent and time-dependent manner.3. The effect of BBG or CBX on the ATP-induced cell viability and membrane pore formation in PC 12 cell.Cell viability increased and intracellular fluorescence intensity induced by ATP antagonised significantly (P<0.05) in BBG pretreatment group, whereas did not change in CBX pretreatment group (P> 0.05).4. The P2X7 receptor and pannexin 1 (Panx 1) mRNA expression of PC 12 cell with extracellular ATP in different concentrationThe gene and protein expression of P2X7 receptor were significantly increased (P <0.05), but those of Panx 1 were little change(P> 0.05) when PC 12 cells exposed to ATP for 3 h.ConclusionsExtracellular ATP in high concentration may induce membrane pore formation in a dose and time dependent, which may be related mainly with P2X7 receptor expression and activation in PC12 cells.