Research reportThe antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1αβ knockout mice
Introduction
P2X7 receptors belong to the family of ATP-activated ionotropic P2X receptors, which include seven homomeric receptor subtypes (P2X1–P2X7) [5], [29]. P2X7 receptors are selectively expressed on cells of hematopoietic lineage including mast cells, lymphocytes, erythrocytes, fibroblasts, and peripheral macrophages [39]. Within the CNS, functional P2X7 receptors are localized on microglia and Schwann cells, as well as on astrocytes [7], [10], [36]. High concentrations (>100 μM) of ATP are required to activate P2X7 receptors. Agonist binding stimulates an immediate cationic permeability and, following more prolonged activation, permeability of large (∼900 Da) molecules [29]. In addition to channel opening, activation of P2X7 receptors also leads to rapid membrane blebbing, as well as maturation and release of IL-1β [21], [31], [32]. Thus, P2X7 receptors represent a multifunctional pro-inflammatory purinoceptor that contributes to pain after an inflammatory insult [10].
Data from studies of P2X7 receptor knockout mice [6], [20] support this hypothesis since P2X7 knockout mice show less chronic inflammation and nerve injury induced pain relative to wild-type controls. The role of P2X7 receptors in mediating some forms of persistent pain in experimental models of nerve injury is further supported by data showing that selective P2X7 receptor antagonists reduce pain sensitivity in these models [15], [21], [25]. The ability of ATP, acting at P2X7 receptors, to stimulate the release of IL-1β suggests that this major pro-inflammatory cytokine, may mediate the nociceptive effects of P2X7 receptor activation [10], [11]. Endogenous IL-1 levels are increased in the CNS in response to trauma or inflammation associated with mechanical damage, ischemia, seizures, and hyperexcitability [3], [4], [40]. At the level of the spinal cord, blockade of IL-1 receptors with the IL-1 receptor antagonist (IL-1ra), generally results in reduced nociception in animal models of inflammation and nerve-injury induced pain [11], [24], [33], [34], [38]. Additionally, mice lacking genes for both IL-1α and IL-1β have been generated [14], [16] and, while overtly normal, show attenuated inflammatory and neuropathic pain responses relative to wild-type mice [13].
A common feature of many prototypic P2X7 receptor antagonists like PPADS and KN-62 is their differential affinity for human versus rodent P2X7 receptors [8], [15], [18], [23], [25]. For example, KN-62 [17] potently blocks human P2X7 receptors, but lacks activity at the rat receptor. The inability of these antagonists to block rodent P2X7 receptors is an obvious impediment to determining the physiological roles of P2X7 modulation in experimental models. More recently, several structurally distinct and competitive P2X7 receptor antagonists (e.g. A-740003 and A-438079) have been described that show less human versus rat species differences and have sufficient pharmacokinetic properties to enable their use for in vivo studies [15], [25].
The present studies were carried out to characterize the in vitro and in vivo activity of a structurally novel P2X7 antagonist, A-839977, and to determine the role of IL-1 and P2X7 in an inflammatory pain model, by evaluating the differential antinociceptive effects of A-839977 in IL-1αβ (−/−) and wild-type control mice.
Section snippets
Materials
The following were purchased from Sigma (St. Louis, MO): BzATP, 2,3-O-(4-benzoylbenzoyl)-ATP. Fluo-4 dye was purchased from TEF Labs (Austin TX) and YO-PRO-1(4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1[3-(trimethylammonio)propyl])-diiodide was purchased from Invitrogen (Eugene, OR). All cell culture medium and Dulbecco's phosphate buffered saline (DPBS), pH 7.4 were also obtained from Invitrogen (Grand Island, NY). In the behavioral studies, A-839977
A-839977 potently and selectively blocks mammalian P2X7 receptors
A-839977 potently blocked BzATP-evoked changes in intracellular calcium concentrations in 1321N1 cells stably expressing human (IC50 = 20 nM), rat (IC50 = 42 nM) or mouse (IC50 = 150 nM) P2X7 receptors (Fig. 2). The concentration of BzATP used in these experiments represented its approximate EC70 for each species. Human THP-1 cells differentiated with LPS and IFNγ into a macrophage phenotype express P2X7 receptors and receptor activation leads to IL-1β release as well as YO-PRO uptake [8], [15].
Discussion
The present data demonstrate that A-839977 is a potent antagonist of human, rat, and mouse P2X7 receptors as shown by blocking calcium influx. A-839977 also potently blocked consequences of P2X7 receptor activation in differentiated human THP-1 cells, including BzATP-evoked IL-1β release and prolonged agonist stimulated pore formation. A-839977 shows much less species differences compared to the human selective antagonist KN-62 [1] and other prototypic P2X receptor antagonists [9], [26].
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