Expression and function of the P2X7 receptor in rat C6 glioma cells
Introduction
Extracellular adenosine triphosphate (ATP) serves important roles in purinergic-mediated signaling as a neurotransmitter and a modulator of cellular functional responses. ATP produces its effects via the activation of P2 receptors comprising the metabotropic P2YR and the ionotropic P2XR receptor families [1], [2]. The P2XR family includes subtypes P2X1R–P2X7R which are coupled to non-selective cationic channels passing Na+ and Ca2+ influx and K+ efflux. A particularly unique member of the P2XR family is the subtype P2X7R that, when activated by ATP binding, forms a large pore allowing permeability to large hydrophilic molecules. A number of studies have reported that P2X7R expression is up-regulated under pathological conditions and can mediate cellular inflammatory responses [3], [4], [5], [6].
Gliomas are the most common primary brain tumors in adults and the rat C6 glioma model system has been widely used to elucidate mechanisms underlying the aggressive nature of these brain tumors [7]. Recent evidence suggests that ATP signaling may be involved in glioma development and that P2 receptors might provide novel therapeutic tools in the treatment of brain cancer [8], [9]. Several purinergic receptors of the P2YR family are expressed in C6 cells including P2Y1, P2Y2 and P2Y12 [10], [11] and P2YR have been reported to mediate changes in intracellular Ca2+ levels [Ca2+]i following ATP application [11] and to regulate glioma cell proliferation [12]. At present, however, no studies have reported involvement of P2XR in the progression of brain tumors. In this work we document expression and functional responses of the subtype P2X7R in C6 glioma cells.
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Cell culture
Glioma C6 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells from passage number 39–59 were used in this work. Cells were cultured in Kaighn’s modification of Ham’s F12 medium (F12K) with 2 mM l-glutamine modified by ATCC to contain 1.5 g/l sodium bicarbonate [13]. The medium was then supplemented with 15% horse serum, 2.5% fetal bovine serum, 0.5 μg/ml fungizone (Invitrogen: GIBCO, Grand Island, NY) and 0.02 mg/ml gentamicin (Invitrogen: GIBCO). Cells were
Intracellular calcium levels are altered in response to P2X7R agonist BzATP
Initial experiments examined effects of the specific P2X7R agonist, BzATP, to increase mobilization of [Ca2+]i in C6 glioma cells. A representative response elicited by BzATP (300 μM) is presented in Fig. 1A and shows the ligand induces a rapid rise in [Ca2+]i (measured as ratio F340/380) followed by a slowly decreasing component (mean response from n = 38 cells). The [Ca2+]i increase induced by BzATP was largely inhibited in the presence of the P2X7R antagonist OxATP (300 μM) (Fig. 1B; n = 30
Discussion
This study provides the first report for expression, at both mRNA and protein levels, of the purinergic subtype receptor, P2X7R, in C6 glioma. Activation of this receptor using the specific ligand BzATP induced a rapid transient increase in [Ca2+]i which was sensitive to pretreatment of cells with the P2X7R antagonist, OxATP. Functional responses mediated by P2X7R in C6 cells were also demonstrated in this work. These included increased expression of pro-inflammatory factors, enhanced migration
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