Extracellular adenosine 5′-phosphate (ATP) mediates a wide range of effects by acting on P2-receptors expressed on many tissues throughout the body. P2-receptors have been classified into 2 classes, P2X and P2Y, based on molecular structure of the cloned receptors and mechanism of signal transduction (Fredholm et al., 1994; Burnstock, 1996). Within the P2Y and P2X receptor families are a number of subtypes, designated P2Y1-P2Y7 and P2X1-P2X6, based on structure and pharmacological profiles of agonist and antagonist responses (Burnstock, 1996). Thus the P2U receptor is now classed as a P2Y2 receptor. Many cell types express the G-protein coupled, P2Y receptors, whose occupancy activates the phosphoinosi-tide specific phospholipase C signalling cascade and raises cytosolic [Ca2+] by releasing Ca2+ from internal stores (Dubyak & El-Moatassim, 1993). The P2X receptors are ligand gated cation channels which conduct Ca2+ from the extracellular medium. The P2Z receptor, which may constitute a third class of P2-receptor (Fredholm et al., 1994), is expressed on cells of immune and haemopoietic origin. The P2Z receptor is also a ligand-gated cation channel with a distinct agonist profile, but its function is less clear (Wiley & Dubyak, 1989; Wiley et al., 1990; 1992; Dubyak & El-Moatassim, 1993). Some evidence suggests that the P2Z receptor may mediate apoptosis of rat thymocytes and murine lymphocytes and macrophages, since extracellular ATP causes influx of Ca2+, release of lactic dehydrogenase (LDH) and cytolysis (Zheng et al., 1991; Di Virgilio, 1995). In human leukaemic lymphocytes, we have shown multiple effects of extracellular ATP mediated via occupancy of the P2Z receptor by the agonist, fully ionized ATP4- species. Thus extracellular ATP increases the permeability of lymphocytes to Ca2+, Ba2+ and ethidium+ (Wiley et al., 1990; 1993; 1994), stimulates phospholipase D activity (Gargett et al., 1996) and induces shedding of L-selectin (Ja-mieson et al., 1996). Recently, a P2Z receptor has been cloned from rat brain and the first 395 amino acids shown to have 35–40% homology with P2X receptors. This cloned receptor, designated P2X7 on the basis of this homology, contains a long carboxyl terminal domain not found in other P2X subtypes which confers the unique permeability of P2Z-channels to large cations such as fluorescent dyes (Surprenant et al., 1996).
At present, there are no specific antagonists of the P2Z receptor which are active in the nanomolar range. The best known inhibitor of P2-receptors is suramin, which inhibits not only the lymphocyte P2Z receptor (IC50 60 μM) but also P2X and P2Y receptors (Leff et al., 1990; Wiley et al., 1993). Suramin also inhibits receptors for several growth factors (eg bFGF, PDGF, IL-3 etc) and a wide variety of enzymes, including ecto-ATPase (Voogd et al., 1993; Crack et al., 1995). Oxidised ATP (2,3 dialdehyde ATP) inhibits the P2Z receptor but its effect is irreversible and requires exposure of cells to high concentrations of inhibitor (ca 300 μM for 60 min) (Murgia et al., 1993; Wiley et al., 1994). Amiloride analogues, such as hexamethylene amiloride (HMA), are the most potent known inhibitors of the P2Z-receptor with IC50s of 2–10 μM, but at maximal effective concentrations produce only incomplete block (70–85%) of cation fluxes through the P2Z ion channel (Wiley et al., 1996). The problems of antagonist potency and non-selectivity are further compounded by the co-expression of several P2 receptor subtypes on many cells. Thus mast cells express P2Y and P2Z receptors (Cockcroft & Gomperts; 1979; Osipchuck & Cahalan, 1992) and macro-phages P2Y and P2Z (Nuttle et al., 1993). However, lymphocytes from patients with chronic lymphocytic leukaemia only express P2Z receptors and since this receptor has been well characterized (Wiley et al., 1990; 1992; 1993; 1994), lymphocytes are an ideal cell type in which to evaluate the effect of antagonists.
In order to determine whether P2Z-stimulated responses in human lymphocytes were dependent on calmodulin-dependent protein kinase II (CaM-kinase II), a study was made of the isoquinolinesulphonamide, KN-62, a selective and potent inhibitor (IC50 0.9 μM) of the kinase (Tokumitsu et al., 1990; Hidaka & Kobayashi, 1992). A structural analogue, KN-04, which has no effect on CaM-kinase II at 100 μM (Ishikawa et al., 1990) was used as a control compound. In this paper, we present the unexpected finding that both KN-62 and KN-04 are potent inhibitors of P2Z-receptor mediated effects and are active in the low nanomolar range.