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...

Interaction of intracellular free calcium ([Ca 2؉ ] i ) and cAMP signaling mechanisms was examined in intact single megakaryocytes by using a combination of singlecell f luorescence microscopy to measure [Ca 2؉ ] i and f lash photolysis of caged Ca 2؉ , inositol 1,4,5-trisphosphate (IP 3 ), or cAMP to elevate rapidly the concentration of these compounds inside the cell.

(S)-N-[1-(3-Morpholin-4-ylphenyl)ethyl]-3-phenylacrylamide (2) was synthesized as an orally bioavailable KCNQ2 potassium channel opener. In a rat model of migraine, 2 demonstrated significant oral activity in reducing the total number of cortical spreading depressions induced by potassium chloride.

1. Inhibition of inositol 1,4,5-trisphosphate (IP3) receptor-mediated Ca2+ release by cGMP was examined in intact rat megakaryocytes, by using a combination of single cell fluorescence microscopy to monitor intracellular free calcium ([Ca2+]i) and flash photolysis of caged second messengers. 2. Sodium nitroprusside (SNP), a nitric oxide (NO) donor, and the hydrolysis-resistant cGMP analogue 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (pCPT-cGMP) inhibited Ca2+ release induced by photolysis of caged IP3. Neither of them affected the rate of Ca2+ removal from the cytoplasm following photolysis of caged Ca2+. 3. Photolysis of the caged NO donor 3-morpholinosydnonimine (SIN-1) during agonist-induced [Ca2+]i oscillations inhibited Ca2+ release without affecting the rate of Ca2+ uptake and/or extrusion. 4. We conclude that the inhibition of IP3-induced Ca2+ release is the principal mechanism of NO-cGMP-dependent inhibition of [Ca2+]i mobilization. 5. IPG, a specific peptide inhibitor of cGMP-dependent protein kinase (cGMP-PK), blocked the inhibitory effect of pCPT-cGMP, indicating that the inhibition of IP3-induced Ca2+ release by pCPT-cGMP is mediated by cGMP-PK. However, the simultaneous application of both IPG and IP20, a specific peptide inhibitor of cAMP-dependent protein kinase (cAMP-PK), was required to block the inhibitory effect of SNP. These data strongly suggest that NO-cGMP-dependent inhibition of [Ca2+]i mobilization is mediated via the activation of both cGMP-PK and cAMP-PK.

BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine] produced a concentration-dependent membrane hyperpolarization of cultured human bladder myocytes, assessed as either a reduction in fluorescence of the voltage-sensitive dye bis-(1,2-dibutylbarbituric acid)trimethine oxonol (EC 50 ϭ 1.26 Ϯ 0.6 M) or by direct electrophysiological measurement (EC 50 ϭ 1.49 Ϯ 0.08 M). BL-1249 also produced a membrane hyperpolarization of acutely dissociated rat bladder myocytes. Voltage-clamp studies in human bladder cells revealed that BL-1249 activated an instantaneous, noninactivating current that reversed near E K . The BL-1249-evoked outward K ϩ current was insensitive to blockade by glyburide, tetraethylammonium, iberiotoxin, 4-aminopyridine, apamin, or Mg 2ϩ . However, the current was inhibited by extracellular Ba 2ϩ

The formation of a reactive intermediate was found to be responsible for CYP3A4 metabolism-dependent inhibition (MDI) observed with (S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]-3-phenyl-acrylamide (1). Structure-3A4 MDI relationship studies culminated in the discovery of a difluoro analogue, (S)-N-[1-(4-fluoro-3-morpholin-4-ylphenyl)ethyl]-3-(4-fluoro-phenyl)acrylamide (2), as an orally bioavailable KCNQ2 opener free of CYP3A4 MDI.

No abstract

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-αq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-αq signaling complex, and define new mutations in both RGS and G-αq, including a unique hypo-adapation allele of G-αq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.