Exogenous ATP induces formation of membrane pore in PC12 cells
10.3969/j.issn.1000-4718.2014.09.011
- VernacularTitle:外源性 ATP 诱导 PC12细胞的膜孔形成
- Author:
Hui SHEN
;
Yaling YIN
;
Chaokun LI
;
Honggang ZHAO
;
Jie MA
;
Dongliang LI
- Publication Type:Journal Article
- Keywords:
Adenosine triphosphate;
Purinergic P2X7 receptor;
PC12 cells;
Membrane pore;
Brilliant blue G
- From:
Chinese Journal of Pathophysiology
2014;(9):1603-1609
- CountryChina
- Language:Chinese
-
Abstract:
AIM:To investigate the formation of membrane pore in PC 12 cells induced by exogenous adenosine triphosphate ( ATP) and to identify the key molecular targets .METHODS:PC12 cells were treated with different concen-trations of ATP to establish the injury model .The morphological change was observed under an inverted phase -contrast mi-croscope.The viability of the PC12 cells was measured by CCK-8 assay.Fluorescent dye YO-PRO-1 was used to detect the membrane permeability.The expression of P2X7 receptor and pannexin 1 (Panx1) at mRNA and protein levels was as-sessed by real-time PCR and Western blotting .RESULTS:After exposed to ATP (1 mmol/L, 3 mmol/L and 5 mmol/L) for 3 h, the PC12 cells became edematous , and the number of adherent cells decreased gradually in a dose-dependent man-ner .The cell viabilities in 3 mmol/L ATP group and 5 mmol/L ATP group were significantly decreased compared with con-trol group (P<0.05).YO-PRO-1 uptake in the PC12 cells exposed to ATP (0, 1, 3 and 5 mmol/L) for 15 min, 30 min and 60 min increased in a dose-dependent and time-dependent manner .The cell viability increased and the intracellular fluorescence intensity induced by ATP were significantly antagonized in brilliant blue G ( a P2X7 receptor inhibitor ) pre-treatment group (P<0.05), whereas it did not change in carbenoxolone (a Panx1 inhibitor) pretreatment group (P>0. 05).The expression of P2X7 receptor at mRNA and protein levels was significantly increased (P<0.05), but the expres-sion of Panx1 was not changed ( P>0.05) when PC12 cells were exposed to ATP for 3 h.CONCLUSION:Extracellular ATP at high concentration may induce membrane pore formation with the expression and activation of P 2X7 receptor in PC12 cells.
