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

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 P2X 7 receptors (P2X 7 Rs) are abundantly expressed in SN4741 cells, and both ATP-induced swelling and cell death are reversed by pretreatment with the P2X 7 Rs antagonist, KN62, or by knock-down of P2X 7 Rs with small interfering RNAs. Therefore, extracellular ATP release from injured tissues may act as an accelerating factor in necrotic SN4741 dopaminergic cell death via P2X 7 Rs. Parkinson disease (PD)2 is an idiopathic neurodegenerative disorder characterized by selective cell death of dopaminergic neurons in the substantia nigra (1). The symptoms of PD only become apparent when more than 50% of the dopaminergic neurons in the substantia nigra pars compacta are lost, which leads to an over 80% reduction in dopamine levels in the striatum (2). Epidemiological studies and pathological analyses demonstrate that sporadic PD with late onset occurs in 95% of patients, whereas the remaining 5% of PD cases are familial diseases with early onset (1, 2). Although the etiological causes of PD have not been fully elucidated, several factors have been suggested as causes of neuronal degeneration. These include environmental toxins, genetic factors, and mitochondrial dysfunction as well as proteasomal impairment and oxidative stress (3). Recently, however, there has been increasing recognition of the possible role of neuro-inflammation as a major factor in the pathogenesis of PD (4). The inflammatory component is an attractive target for therapeutic intervention. It is now generally accepted that high levels of extracellular ATP may be released under pathological conditions such as inflammation, trauma, and stress. The role of extracellular ATP and purinergic receptors in neurodegeneration is one of the focus areas of cell death research (5).P2X 7 receptors (P2X 7 Rs) are unusual purinergic receptors in that they can exist in two functional states: either as cationselective channels or as nonselective pores (6). The permeability transition of P2X 7 Rs from channel to pore occurs either upon sustained stimulation with high ATP concentrations or repeated pulses of ATP application (7). Seven members of the P2X receptor family have been cloned that share the same predicted structure with two transmembrane-spanning domains. These are a...

Targeting of the P2X7 receptor in pancreatic cancer and stellate cells

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P2X7 Nucleotide Receptor is Coupled to GSK-3 Inhibition and Neuroprotection in Cerebellar Granule Neurons

“…Likewise, nucleotides released after brain insults have been proposed as danger signals that display dual functions, first alerting tissue to trigger protective responses against damage, and then, upon sustained release and activation, can function as amplifiers to augment inflammation and toxicity. Focusing on P2X 7 receptors, sustained activation seems to be coupled to cell death, as was first reported in cells of blood lineage (Surprenant et al 1996;Di Virgilio 1999) and also in some neuronal and glial cells, such as cortical, dopaminergic neurons and cortical astrocytes (Fumagalli et al 2004;Kong et al 2005;Jun et al 2007). However, P2X 7 is not associated to cell death in other cells, such as cerebellar astrocytes (Delicado et al 2005), suggesting that the heterogeneity observed in P2X 7 -mediated actions can be cell and tissue specific and can depend on differences at P2X 7 receptor expression levels.…”

Effects of Extracellular ATP on Bovine Lung Endothelial and Epithelial Cell Monolayer Morphologies, Apoptoses, and Permeabilities

“…mRNA (1.5 g) was converted to cDNA by using reverse transcriptase (Reverse Transcriptase Systems; Promega, Madison, WI) according to the supplied protocol. A fixed volume of each sample cDNA was added to a Taq polymerase master mix (Promega), along with 5 M concentrations of the forward and reverse primers for bovine P2X 7 or ␤-actin. Primers were designed by performing a PubMed (National Center for Biotechnology Information, Bethesda, MD) search for coding sequences for these bovine genes.…”