Indeed, in the mouse KM-1 cells ATP induced a sustained increase in the [Ca2+]i at a concentration of 4 mM, while it only induced a transient [Ca2+]i increase at a concentration of 0.5 mM ( Fig. 2A). BzATP, a potent P2X7R agonist, also induced a sustained increase in the [Ca2+]i of the KM-1 cells ( Fig. 2A). Pretreatment with A438079, a selective antagonist of P2X7R, clearly blocked the sustained [Ca2+]i increases induced by ATP and BzATP ( Fig. 2A), suggesting that P2X7R activation is involved in the [Ca2+]i increases induced by ATP. In contrast, no sustained [Ca2+]i increase was observed in the swine kidney macrophages stimulated with 4 mM ATP or 0.5 mM BzATP (Fig. 2B),

suggesting that these treatments did not sufficiently activate P2X7R in the swine kidney macrophages. An LPC-induced Akt targets sustained [Ca2+]i increase was detected in the swine kidney macrophages (Fig. 2B), which occurred in a P2X7R-independent

manner [13]. In addition, ADP, a selective agonist of P2Y receptors, induced a transient [Ca2+]i increase, and pretreatment with ADP desensitized the Ca2+ response of the swine kidney macrophages to 4 mM ATP (Fig. 2C). This suggests that the [Ca2+]i changes induced by ATP are mediated by P2Y receptors, but not by P2X7R, in swine kidney macrophages. We next examined whether extracellular ATP triggers the production and release of mature IL-1β (mIL-1β) in LPS-primed swine kidney macrophages. In response to LPS pretreatment, the 33-kDa precursor form of IL-1β (pro-IL-1β) was synthesized and accumulated in the cytosol of both the mouse KM-1 cells and swine kidney Bleomycin macrophages (Fig. 3A, lysates, second and fourth panels). Additional stimulation with ATP triggered the production and release of the 17-kDa form of mIL-1β in the LPS-primed KM-1 cells (Fig. 3A, sup, first panel). The ATP-induced release of mIL-1β from these cells was enhanced when ADP ribosylation factor they were incubated in Ca2+/Mg2+-free buffer (Fig. 3A, sup, first panel) because these divalent cations

steadily suppress the functions of P2X7R by directly modulating it [17]. Conversely, the ATP-induced release of mIL-1β from the KM-1 cells was inhibited by co-treatment with A438079 (Fig. 3B, sup). In contrast, no production or release of mIL-1β was detected in the LPS-primed swine kidney macrophages treated with ATP, even those incubated in Ca2+/Mg2+-free buffer (Fig. 3A, sup, third panel). The prolonged activation of P2X7R by ATP causes membrane pore dilation followed by cytolysis in monocytes/macrophages. Thus, we examined whether ATP triggers membrane pore formation in swine kidney macrophages. To this end, P2X7R-mediated pore formation and the resultant cytolysis were assessed using YO-PRO-1 and PI uptake, respectively. In LPS-primed mouse KM-1 cells, stimulation with 4 mM ATP elicited maximum YO-PRO-1 uptake within 20 min, which preceded PI uptake (Fig. 3C).