Inhibition of extracellular ATP‐mediated lysis of human macrophages by calmodulin antagonists

Abstract

Lysis of human culture-derived macrophages by extracellular ATP has recently been described, and treatment of macrophages with interferon-gamma rendered those cells significantly more sensitive to lysis. In addition, cell death occurred more rapidly in interferon (IFN)-treated cells than in untreated macrophages. In an attempt to identify the mechanism by which extracellular ATP affects macrophages, as well as to explore the differences between interferon-gamma-treated and untreated macrophages, selected metabolic inhibitors were included in the lytic assays. Of the compounds tested, three antagonists of calmodulin-linked pathways (trifluoperazine, KN-62, and calmidazolium) blocked the ATP-mediated lysis of both interferon-gamma-treated and colony-stimulating factor-treated macrophages in a dose-dependent manner. Early signals of the ATP ligation of the P2Z purinoceptors of human macrophages included increases in cytosolic [Ca2+] and depolarization of the plasma membrane. However, the inclusion of calmodulin antagonists in these assays did not abrogate either effect. These results suggest that the mechanism which mediates the efflux of 51Cr-labeled proteins from ATP-lysed macrophages is distinct from calcium mobilization and membrane depolarization, and may involve the generation of secondary pores/channels in the plasma membrane via a calmodulin-linked pathway.

P2X7Receptors Mediate ATP Release and Amplification of Astrocytic Intercellular Ca2+Signaling

“…Based on the agonist dose-response curve for antagonistpretreated cells, two types of antagonism were observed: a competitive antagonism provided by the fenamate FFA and a noncompetitive antagonism provided by the glycyrrhetinic acid derivative CBX and the quinine derivative MFQ. Moreover, our results showing that the long chain alcohols heptanol and octanol can block YoPro uptake without affecting BzATP-induced Ca 2ϩ influx suggest that these compounds like calmidazolium and KN-62 (Blanchard et al, 1995;Virginio et al, 1997;Chessell et al, 1998;Michel et al, 2000) can differentiate between "channel" and "pore" forms of the P2X 7 R. Differences in type of antagonism and potency of blockade observed here for the gap junction channel blockers are likely related to the distinct structures of these compounds. Compared with their action on gap junction channels, the blockade of P2X 7 R by FFA, CBX, and MFQ is either similar or more effective than what has been reported (Spray et al, 2002;Srinivas and Spray, 2003;Cruikshank et al, 2004).…”

ATP-Mediated Killing ofMycobacterium bovisBacille Calmette-Guérin Within Human Macrophages Is Calcium Dependent and Associated with the Acidification of Mycobacteria-Containing Phagosomes

“…Therefore, the mechanism by which ATP causes the killing of mycobacteria within host macrophages may provide a target for the design of novel immunotherapeutics in the treatment of tuberculosis, which has recently increased in incidence due to the HIV epidemic and the occurrence of multidrug resistant strains (17). Although Ca 2ϩ mobilization has been implicated in ATP-mediated host cell death (37,38), the pertinent source of Ca 2ϩ , the mechanism of ATP-stimulated BCG killing, and the precise relationship between macrophage death and the demise of the intracellular mycobacteria have not been fully elucidated.…”

Extracellular ATP in the lymphohematopoietic system: P2Z purinoceptors and membrane permeabilization

“…The molecular nature of P2Z purinoceptors is not fully understood and evidence exists indicating that fast-activating transmembrane cation currents and membrane permeabilization (pore formation) are indeed separate phenomena (21,22). The P2X 7 molecule shares several pharmacological and electrophysiological properties with P2Z purinoceptors such as the activation by ATP 4-and BzATP, and the induction of fast-activating cation current, leading some groups to name this molecules P2Z/P2X 7 .…”

“…The change in free intracellular Ca 2+ concentration is only a part of the complete cascade of the poorly characterized signal transduction pathways employed by all P2 purinoceptors. Macrophages have P2Z, P2Y, and P2U purinoceptors (3,17,18) and, besides the receptor-gated cation channels such as the ones that will be described later in this review, candidate intermediates include G protein, phospholipases A 2 and D, and calmodulin-regulated pathways (3,22,27,28). P2Y and P2U purinoceptors may induce the release of intracellular Ca 2+ stores, while P2Z has been shown to open G proteincoupled transmembrane Ca 2+ channels (29) and to activate phospholipase D in macrophages (30) and lymphocytes (31).…”