The P2X(7) purinoceptor is a ligand-gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype which includes release of biologically active inflammatory cytokine, interleukin (IL)-1beta following activation, and unique ion channel biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant-induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1beta, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggests a selective effect of the gene deletion on release of IL-1beta and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1. In addition, we show that this receptor is upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients. We hypothesise that the P2X(7) receptor, via regulation of mature IL-1beta production, plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin. Drugs which block this target may have the potential to deliver broad-spectrum analgesia.

We have isolated a 1785-bp complementary DNA (cDNA) encoding the murine P2X U receptor subunit from NTW8 mouse microglial cells. The encoded protein has 80% and 85% homology to the human and rat P2X U subunits, respectively. Functional properties of the heterologously expressed murine P2X U homomeric receptor broadly resembled those of the P2X U receptor in the native cell line. However, marked phenotypic differences were observed between the mouse receptor, and the other P2X U receptor orthologues isolated with respect to agonist and antagonist potencies, and the kinetics of formation of the large aqueous pore.z 1998 Federation of European Biochemical Societies.

1 We have utilized the human monocytic cell line, THP-1, and freshly isolated adherent human monocytes with the compounds pyridoxalphosphate-6-azophenyl-2',4'-disuphonic acid (PPADS), oxidized ATP, and 1-(N,O-bis{5-isoquinolinesufonyll}-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN-62) to pharmacologically characterize the P2 receptor involved in ATP-induced release of interleukin 1b (IL-1b). We have also investigated the involvement of P2 receptors in lipopolysaccharide (LPS)-induced IL-1b release from both cell types. 2 ATP caused release of IL-1b from LPS primed THP-1 cells in both a time-and concentrationdependent manner, with a minimal e ective ATP concentration of 1 mM. Stimulation of cells with 5 mM ATP resulted in detectable concentrations of IL-1b in cell supernatants within 30 min. 3 The ATP analogue benzoylbenzoyl ATP (DBATP), a P2X 7 receptor agonist, was approximately 10 fold more potent than ATP at eliciting IL-1b release. 4 KN-62 (1 mM), PPADS (100 mM) or oxidized ATP (100 uM) signi®cantly inhibited 5 mM ATPinduced IL-1b release by 81, 90 and 66% respectively, but failed to signi®cantly inhibit LPS-induced IL-1b release in both THP-1 cells and in freshly isolated human monocytes. 5 In both THP-1 cells and freshly isolated human monocytes, addition of the ATP degrading enzyme apyrase (0.4 U ml 71 ) to cell supernatants prior to LPS activation failed to signi®cantly inhibit the LPS-induced IL-1b release. In addition there was no correlation between extracellular ATP concentrations and IL-1b release in THP-1 cells when studied over a 6 h time period. 6 In conclusion our data con®rm the involvement of P2X 7 receptors in ATP-induced IL-1b release in human monocytes. However no evidence was obtained which would support the involvement of either endogenous ATP release or P2X 7 receptor activation as the mechanism by which LPS-induces IL-1b release in either the THP-1 cell line or in freshly isolated human monocytes.

1 We have used whole-cell patch clamping methods to study and characterize the cytolytic P2X 7 (P2Z) receptor in the NTW8 mouse microglial cell line. 2 At room temperature, in an extracellular solution containing 2 mM Ca 2+ and 1 mM Mg 2+ , 2'-and 3'-O-(4-benzoylbenzoyl)-adenosine-5'-triphosphate (Bz-ATP; 300 mM), or ATP (3 mM), evoked peak whole cell inward currents, at a holding potential of 790 mV, of 549+191 and 644+198 pA, respectively. Currentvoltage relationships generated with 3 mM ATP reversed at 4.6 mV and did not display strong recti®cation. 3 In an extracellular solution containing zero Mg 2+ and 500 mM Ca 2+ (low divalent solution), brief (0.5 s) application of these agonists elicited larger maximal currents (909+138 and 1818+218 pA, Bz-ATP and ATP, respectively). Longer application of ATP (1 mM for 30 s) produced larger, slowly developing, currents which reached a plateau after approximately 15 ± 20 s and were reversible on washing. Under these conditions, in the presence of ATP, ethidium bromide uptake could be demonstrated. Further applictions of 1 mM ATP produced rapid currents of the same magnitude as those observed during the 30 s application. Subsequent determination of concentration-e ect curves to Bz-ATP, ATP and 2-methylthio-ATP yielded EC 50 values of 58.3, 298 and 505 mM, respectively. These a ects of ATP were antagonized by pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS; 30 mM) but not suramin (100 mM). 4 In low divalent solution, repeated application of 1 mM ATP for 1 s produced successively larger currents which reached a plateau, after 8 applications, of 466% of the ®rst application current. PPADS (30 mM) prevented this augmentation, while 5-(N,N-hexamethylene)-amiloride (HMA) (100 mM) accelerated it such that maximal augmentation was observed after only one application of ATP in the presence of HMA. At a bath temperature of 328C, current augmentation also occurred in normal divalent cation containing solution. 5 These data demonstrate that mouse microglial NTW8 cells possess a purinoceptor with pharmacological characteristics resembling the P2X 7 receptor. We suggest that the current augmentation phenomenon observed re¯ects formation of the large cytolytic pore characteristic of this receptor. We have demonstrated that pore formation can occur under normal physiological conditions and can be modulated pharmacologically, both positively and negatively.

Generation and deposition of the amyloid b (Ab) peptide following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 and c-secretase is central to the aetiology of Alzheimer's disease. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Ab, is an attractive therapeutic approach for the treatment of Alzheimer's disease. We have designed a selective non-peptidic BACE-1 inhibitor, GSK188909, that potently inhibits b-cleavage of APP and reduces levels of secreted and intracellular Ab in SHSY5Y cells expressing APP. In addition, we demonstrate that this compound can effectively lower brain Ab in vivo. In APP transgenic mice, acute oral administration of GSK188909 in the presence of a p-glycoprotein inhibitor to markedly enhance the exposure of GSK188909 in the brain decreases b-cleavage of APP and results in a significant reduction in the level of Ab40 and Ab42 in the brain. Encouragingly, subchronic dosing of GSK188909 in the absence of a p-glycoprotein inhibitor also lowers brain Ab. This pivotal first report of central Ab lowering, following oral administration of a BACE-1 inhibitor, supports the development of BACE-1 inhibitors for the treatment of Alzheimer's disease.