ATP‐Stimulated Ca²⁺ Influx and Phospholipase D Activities of a Rat Brain‐Derived Type‐2 Astrocyte Cell Line, RBA‐2, Are Mediated Through P2X₇ Receptors

Abstract

This study characterizes and examines the P2 receptor-mediated signal transduction pathway of a rat brain-derived type 2 astrocyte cell line, RBA-2. ATP induced Ca 2ϩ influx and activated phospholipase D (PLD). The ATP-stimulated Ca 2ϩ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS), in a concentration-dependent manner. The agonist 2Ј-and 3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate (BzATP) stimulated the largest increases in intracellular Ca 2ϩ concentrations ([Ca 2ϩ ] i ); ATP, 2-methylthioadenosine triphosphate tetrasodium, and ATP␥S were much less effective, whereas UTP, ADP, ␣,␤-methylene-ATP, and ␤,␥-methylene-ATP were ineffective. Furthermore, removal of extracellular Mg 2ϩ enhanced the ATP-and BzATP-stimulated increases in [Ca 2ϩ ] i . BzATP stimulated PLD in a concentration-and time-dependent manner that could be abolished by removal of extracellular Ca 2ϩ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca 2ϩ mobilization agent, ionomycin, in an extracellular Ca 2ϩ concentration-dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP-stimulated PLD activity. Taken together, these data indicate that activation of the P2X 7 receptors induces Ca 2ϩ influx and stimulates a Ca 2ϩ -dependent PLD in RBA-2 astrocytes. Furthermore, protein kinase C regulates this PLD. Key Words: ATP-2Ј-and 3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate -Ca 2ϩ influx-Phospholipase D -P2X 7 receptor-Type-2 astrocytes.

Characterization ofN-(Adamantan-1-ylmethyl)-5-[(3R-aminopyrrolidin-1-yl)methyl]-2-chloro-benzamide, a P2X7Antagonist in Animal Models of Pain and Inflammation

“…As shown in Fig. 2A, BzATP increases calciumdependent Fluo-4 fluorescence in SH-SY5Y cells (Larsson et al, 2002) and RBA-2 cells (Sun et al, 1999) in a concentration-dependent manner. The EC 50 value for BzATP on SH-SY5Y cells was 67.1 Ϯ 4.3 M (n ϭ 8), whereas on RBA-2 cells, the EC 50 value was 13.3 Ϯ 1.1 M (n ϭ 5).…”

Activation of P2X7 receptors causes isoform-specific translocation of protein kinase C in osteoclasts

“…In general, involvement of PKC in P2X7 signaling has been inferred from the effects of PKC inhibitors on downstream responses, such as the activation of PLD. For example, both staurosporine and prolonged treatment with phorbol ester inhibit BzATP-stimulated PLD activity in astrocytic cells (Sun et al, 1999). Similarly, in rat submandibular-gland ductal cells, P2X7 receptors were found Membrane localization of EGFP-tagged PKC isozymes was quantified from fluorescence intensity profiles of confocal images as described in the Results.…”

Lipid metabolism modulation by the P2X7 receptor in the immune system and during the course of infection: new insights into the old view

“…[49]). The activation of PLD by P2X7R has been described in other cell types, including lymphocytes [48], rat parotid acinar cells [36], rat brain-derived type 2 astrocyte cells [33], primary astrocytes and microglia [30,33,35], as well as macrophages [32,34,50]. In primary and macrophage cell lines, PLD has been described as part of an important killing mechanism against Mycobacterium [34] and Chlamydia [50,51], as discussed below.…”

“…Later, the activation of P2X7R was directly associated with several cellular pathways, including the following: caspase-1 activation [18] and IL-1β maturation [19]; shedding of membrane proteins [20]; recruitment and activation of protein kinases, such as, the mitogen-activated protein kinase [21], protein kinase C (PKC) [22], Src [23], and glycogen synthase kinase 3 [24]; recruitment and activation of phosphatases [15]; and membrane blebbing via the activity of ROCK [25] and the activation of epithelial membrane proteins [26]. Despite this wide assortment of functions, this review will focus on the ability of P2X7R to modulate lipid pathways through the activation of different enzymes, such as, phospholipase A 2 (PLA2) [27,28], phospholipase C (PLC) [29][30][31], phospholipase D (PLD) [32][33][34][35][36][37][38][39], and sphingomyelinases [27,36]. Additionally, it will discuss how these enzymes modify arachidonic acid and its analogues [40][41][42][43][44] as well as ceramides [27,45,46].…”