Primary rat microglia stimulated with either ATP or 2-and 3-O-(4-benzoylbenzoyl)-ATP (BzATPActivated microglia have been observed in patients suffering from both acute (stroke) and chronic (Alzheimer's disease) neurological disorders (1, 2). Microglia are believed to contribute to the progression of Alzheimer's disease (AD) 1 because these cells can release pro-inflammatory substances known to induce neurotoxicity (3). Reactive oxygen intermediates (ROIs), one of several pro-inflammatory substances released by microglia (4), are likely to play a very important role in AD because hallmark modifications of ROI damage such as lipid peroxidation and nitrotyrosine conjugates are characteristic of post-mortem AD brains (3). Hence, pro-inflammatory stimuli that promote microglial ROI production might contribute to the pathogenesis of AD.ATP is an important messenger in the brain and can be released from cells by both lytic and non-lytic mechanisms (5). ATP evokes a variety of biological responses in microglia (6 -9).The effects of ATP are mediated through interactions with the P2 purinoceptors, broadly classified into P2Y metabotropic and P2X ionotropic receptors (10). The P2Y receptors are G proteincoupled and P2X receptors are ligand-gated ion channels (10). Whereas the P2Y receptors are responsible for Ca 2ϩ release predominantly from intracellular stores, P2X receptors are responsible for Ca 2ϩ influx from extracellular sources. Microglia possess both P2Y and P2X receptors (11-13). The P2X 7 receptor is highly expressed by cells of the macrophage lineage, such as dendritic cells, alveolar macrophages, and microglia. Activation of the P2X 7 receptor is unique in triggering the formation of large nonselective membrane pores, permeable to molecules up to 900 Da which ultimately results in death of the cell (9, 14). ATP and ATP analogs have been used to characterize the role of P2 receptors in microglial activation. Micromolar concentrations of ATP are required to activate the P2Y receptors, whereas millimolar (1-5 mM) concentrations of ATP are required to activate the P2X receptors. The ATP analog BzATP is a selective agonist at the P2X receptor and does not bind P2Y receptors (15,16). Oxidized ATP (oATP) is a specific antagonist of P2X 7 that binds irreversibly to the receptor and prevents its activation by ATP (17). In this study, these pharmacological tools were used to determine the purinergic receptors involved in O 2. production in microglia.The P2X 7 receptor plays a role in the generation of superoxide in microglia. Our studies elucidate a putative signal transduction pathway that mediates this response. These studies also demonstrate that BzATP-and ATP-activated microglia can mediate neurotoxicity. Finally, a distinct alteration was detected in the staining pattern for P2X 7 receptor in a transgenic mouse model of AD, suggesting that P2X 7 receptor activation could play a contributing role in AD. MATERIALS AND METHODSReagents-Reagents not specified otherwise were obtained from Sigma. PD98059, SB203580, LY2940...

Current data suggest that apoptosis controls neutrophil numbers in tissues. We analyzed roles for and the sites of action for the cAMP-dependent protein kinases (cAPKs) in apoptosis induced in human neutrophils by in vitro storage, cycloheximide (CHX) exposure, and anti-Fas exposure. Treatment with 8-chlorophenylthiocAMP (8-CPT-cAMP) prolonged the time required for 50% of the cells to exhibit apoptotic morphology (t 50 ) from 16.3 to 41.8 h (in vitro culture), from 2.4 to 7.8 h (CHX), and from 4.8 to 6.5 h (anti-Fas). CHX ؎ 8-CPTcAMP did not significantly alter resting intracellular calcium levels and H-89, a selective inhibitor of cAPK, had no effect on apoptosis in the absence of the analogue. In contrast, site-selective cAMP analogues that specifically activated the type I cAPK, but not type II cAPK, synergistically attenuated apoptosis. Exposure to 8-CPT-cAMP delayed, in parallel, the activity of caspase-3 (CPP-32␤), whereas mitogen-activated protein kinase kinase (MAPKK) inhibitor, PD98059, had no effect on CHX-induced apoptosis ؎ 8-CPT-cAMP. Together these results indicate that type I cAPK activation is necessary and sufficient to mediate cAMP-induced delay in human neutrophil apoptosis induced by several mechanisms and suggest that one of the major sites of cAPK action is upstream of caspase-3 (CPP-32␤) activation.Human neutrophils are short-lived cells that play important roles in both host defense and acute inflammation in humans (1, 2). Recent data suggest that apoptosis, a physiologic mechanism for cell death, regulates both production and survival of neutrophils, representing a basic biological mechanism for this cell type (3-6). Various inflammatory mediators are known to accelerate or delay apoptosis in neutrophils (7-9), but little about the intracellular signaling pathways that regulate this process (10) is understood. Recent evidence indicates that a family of cysteine proteases, the caspases, are critical components of apoptosis cascades, are primary determinants for the orderly disassembly of structural components, and incapacitate repair and homeostatic mechanisms in cells (10). Roles for caspase proteases in apoptosis have been analyzed in several cell types but have not been investigated in neutrophils.Intracellular signaling mechanisms typically involve protein kinases as major regulators of cellular processes (11). The cAMP-dependent protein kinases (cAPK), 1 one of the best understood classes of protein kinases, are tetramers composed of two regulatory (R) and two catalytic (C) subunits and are functionally regulated by cAMP (12-14). Binding of cAMP to the R subunit releases the catalytically active C subunit from the R subunit, resulting in protein phosphorylations. This activates downstream signaling and transcription events, which likely account for recognized roles for cAMP in neutrophil chemotaxis (15), neutrophil respiratory burst activity (16), and more recently, apoptosis in neutrophils and other cell types (17)(18)(19)(20). The cAPK exists as two major isoforms, type I and type ...

Beta-amyloid (A beta) accumulation is believed to contribute to neuronal cell death in Alzheimer's disease. To understand the role of cAMP in the regulation of A beta induced cell death, we used 8-chlorophenylthio-cAMP (8-CPT-cAMP, a cAMP analog) to raise intracellular cAMP levels. Exposure of rat cortical neurons to A beta(25-35) resulted in a gradual increase in lactate dehydrogenase (LDH) over 48 h, which was preceded by a transient elevation in caspase-3-like activity. In the presence of 8CPT-cAMP, both caspase-3 activity and LDH release was significantly reduced. These data suggest that elevation of intracellular cAMP levels attenuate A beta-induced neurotoxicity and may delay or prevent the onset of A beta-induced neurodegeneration.

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