Brief activation of the ATP-sensitive P2X(7) receptor (P2X(7)R) stimulates the maturation and release of interleukin 1beta (IL-1beta)in macrophages, whereas prolonged agonist activation induces the formation of cytolytic pores in cell membranes. The present study investigated potential downstream mechanisms associated with native human P2X(7)R activation in lipopolysaccharide and interferon-gamma differentiated THP-1 cells. 2,3-O-(4-Benzoylbenzoyl)-ATP (BzATP)-induced pore formation (EC(50) = 35 microM) was blocked by a selective P2X(7)R antagonist, 1[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) (IC(50) = 44 nM) and by pyridoxal phosphate-6-azophenyl-2-4-disulfonic acid (PPADS) (IC(50) = 344 nM). KN-62 and PPADS also blocked BzATP-induced IL-1beta release (EC(50) = 617 microM) with IC(50) values of 75 and 3500 nM, respectively. The selective p38 mitogen-activated protein kinase (MAPK) inhibitor, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB 202190), potently inhibited BzATP-induced pore formation (IC(50) = 75 nM) but did not alter P2X(7)-mediated calcium influx or IL-1beta release. SB 202190 and KN-62 also attenuated BzATP-mediated activation of phosphorylated p38 MAPK (pp38 MAPK). Two caspase inhibitors, YVAD (caspase 1) and DEVD (caspase 3), attenuated both BzATP-induced pore formation and IL-1beta release in a concentration-dependent fashion. Neither DEVD nor p38-MAPK inhibitors blocked cell membrane pore formation evoked by maitotoxin or by activation of human P2X(2a) receptors. These results indicate that P2X(7)R-mediated pore formation results from a coordinated cascade involving both the p38 MAPK and caspase pathways that is distinct from other cytolytic pore-forming mechanisms. In contrast, P2X(7)R-mediated IL-1beta release is dependent on caspase activity but not p38 MAPK. Taken together, these results support the hypothesis that downstream cellular signaling mechanisms, rather than channel dilation, mediate cytolytic pore formation after prolonged agonist activation, which underlies P2X(7) receptors.