Extracellular ATP Mediates Necrotic Cell Swelling in SN4741 Dopaminergic Neurons through P2X7 Receptors

doi:10.1074/jbc.M707915200

Extracellular ATP Mediates Necrotic Cell Swelling in SN4741 Dopaminergic Neurons through P2X₇ Receptors*

^‡Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, San-31, Hyoja-Dong, Nam-Gu, Pohang 790-784, Republic of Korea and the ^§Department of Life Sciences, College of Natural Sciences, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Gu, Seoul 20-750, Republic of Korea

1 To whom correspondence should be addressed: Dept. of Life Science, POSTECH, San-31, Hyoja-Dong, Nam-Gu, Pohang 790-784, Republic of Korea. Tel.: 82-54-279-2297; Fax: 82-54-279-2199; E-mail: ktk{at}postech.ac.kr.

Abstract

Extracellular ATP has recently been identified as an important regulator of cell death in response to pathological insults. When SN4741 cells, which are dopaminergic neurons derived from the substantia nigra of transgenic mouse embryos, are exposed to ATP, cell death occurs. This cell death is associated with prominent cell swelling, loss of ER integrity, the formation of many large cytoplasmic vacuoles, and subsequent cytolysis and DNA release. In addition, the cleavage of caspase-3, a hallmark of apoptosis, is induced by ATP treatment. However, caspase inhibitors do not overcome ATP-induced cell death, indicating that both necrosis and apoptosis are associated with ATP-induced cell death and suggesting that a necrotic event might override the apoptotic process. In this study we also found that P2X₇ receptors (P2X₇Rs) are abundantly expressed in SN4741 cells, and both ATP-induced swelling and cell death are reversed by pretreatment with the P2X₇Rs antagonist, KN62, or by knock-down of P2X₇Rs with small interfering RNAs. Therefore, extracellular ATP release from injured tissues may act as an accelerating factor in necrotic SN4741 dopaminergic cell death via P2X₇Rs.

Footnotes

↵2 The abbreviations used are: PD, Parkinson disease; siRNA, small interfering RNA; P2X₇R, P2X₇ receptor; AM, acetoxymethyl ester; FACS, fluorescence-activated cell sorter; PI, propidium iodide; FITC, fluorescein isothiocyanate; RT, reverse transcription; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ER, endoplasmic reticulum; HEK, human embryonic kidney; ATPγS, adenosine 5′-O-(thiotriphosphate).
↵* This work was supported by Brain Neurobiology Research Program Grant M10412000023-06N1200-02310 and Korea Science and Engineering Foundation Grant R15-2004-033-06001-0 funded by the Korea government (Ministry of Science and Technology). This work was also supported by the Brain Korea 21 Program of the Korean Ministry of Education. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.
- Received September 21, 2007.
- Revision received October 16, 2007.