Responses to extracellular ATP of lymphoblastoid cell lines from Duchenne muscular dystrophy patients

Abstract

We have observed a striking difference in the response to extracellular ATP in lymphoblastoid cell lines established from Duchenne muscular dystrophy patients and normal subjects. Duchenne muscular dystrophy cells stimulated by extracellular ATP underwent a large increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization, while normal cell lines were little or not at all responsive. These changes in intracellular ion homeostasis were due to activation of an ATP-gated membrane channel permeable to Na+ and Ca2+, with little or no contribution of Ca2+ release from intracellular stores. The channel was selectively activated by ATP, since other purine/pyrimidine nucleotides were ineffective, and it was inhibited by pretreatment with oxidized ATP, a compound previously reported to irreversibly inhibit P2 purinergic receptors. In the presence of extracellular ATP, lymphoblastoid cells established from Duchenne muscular dystrophy patients, but not from healthy controls, underwent rounding and swelling and eventually lysed. The results of this study suggest that lymphoblastoid cells isolated from Duchenne muscular dystrophy patients are eminently sensitive to stimulation by extracellular ATP.

Mutation of a Dibasic Amino Acid Motif Within the C Terminus of the P2X7 Nucleotide Receptor Results in Trafficking Defects and Impaired Function

“…Before treatment the medium was removed, and 50 l of the indicated concentration of the potent P2X 7 agonist BzATP, prepared in low divalent cation bathing solution (96 mM NaCl; 2 mM KCl; 0.1 mM CaCl 2 ; 5 mM HEPES, pH 7.55), was added to each well. These conditions are similar to those previously shown to facilitate agonist-dependent cell lysis in the presence of functional P2X 7 receptors (13,29,30). After a 6-h incubation at 37°C, 50 l of 2 mg/ml MTT in distilled water was added to each well, and the cells were incubated for 2 h at 37°C.…”

“…1, A and B). To screen for receptor function, we treated these transfected HEK cells with increasing concentrations of a P2X 7 agonist, Bz-ATP, and evaluated the capacity of this treatment to initiate a rapid low-divalent cation killing response, using conditions similar to those previously described to facilitate pore formation and cell lysis (13,29,30). Under these conditions, Bz-ATP treatment for 6 h caused a reduction in the viability of HEK cells expressing wild-type P2X 7 with an EC 50 between 30 and 100 M Bz-ATP (Fig.…”

Cytolytic P2X purinoceptors

“…All these observations suggested that susceptibility to ATP-mediated cytotoxicity was conferred by expression of a plasma membrane ATP-gated channel, and refractorines depended on the absence of such channel. Following studies demonstrated that ATP was cytotoxic not only to cells that express the typical`permeabilizing' P2Z receptor, such as macrophages, but also to those cells in which this nucleotide activates a cation-selective channel, with no evidence of permeabilization to larger hydrophylic solutes (Pizzo et al, 1991;Ferrari et al, 1994). This lead us to suggest that plasma membrane ATP-gated channels may form a new family of ion channels with different permeability and ion selectivity, but linked by the common property of causing cell death under conditions of sustained stimulation (Murgia et al, 1992b).…”

ATP receptors and giant cell formation

“…The most reliable and widely used are PPADS, a pyridoxalphosphate derivative [14], and oxidized ATP (oATP), an ATP derivative obtained by periodate oxidation [15]. We have previously extensively characterized the inhibitory activity of oATP on macrophages and lymphocytes [15,16]. oATP, at the optimal concentration of 300 µM, was a potent blocker of macrophage fusion.…”