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. 2008 Feb 29;40(1):11-8.
doi: 10.3858/emm.2008.40.1.11.

Activation of nicotinic acetylcholine receptor prevents the production of reactive oxygen species in fibrillar beta amyloid peptide (1-42)-stimulated microglia

Ju Hyun Moon  1 , Soo Yoon Kim, Hwan Goo Lee, Seung U Kim, Yong Beom Lee
Affiliations

Activation of nicotinic acetylcholine receptor prevents the production of reactive oxygen species in fibrillar beta amyloid peptide (1-42)-stimulated microglia

Ju Hyun Moon et al. Exp Mol Med. .

Abstract

Recent studies have reported that the cholinergic anti-inflammatory pathway regulates peripheral inflammatory responses via alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) and that acetylcholine and nicotine regulate the expression of proinflammatory mediators such as TNF-alpha and prostaglandin E2 in microglial cultures. In a previous study we showed that ATP released by beta-amyloid-stimulated microglia induced reactive oxygen species (ROS) production, in a process involving the P2X(7) receptor (P2X(7)R), in an autocrine fashion. These observations led us to investigate whether stimulation by nicotine could regulate fibrillar beta amyloid peptide (1-42) (fAbeta1-42)-induced ROS production by modulating ATP efflux-mediated Ca(2+) influx through P2X(7)R. Nicotine inhibited ROS generation in fAbeta(1-42)-stimulated microglial cells, and this inhibition was blocked by mecamylamine, a non-selective nAChR antagonist, and a-bungarotoxin, a selective alpha7 nAChR antagonist. Nicotine inhibited NADPH oxidase activation and completely blocked Ca(2+) influx in fAbeta(1-42)-stimulated microglia. Moreover, ATP release from fAbeta(1-42)-stimulated microglia was significantly suppressed by nicotine treatment. In contrast, nicotine did not inhibit 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP)-induced Ca(2+) influx, but inhibited ROS generation in BzATP-stimulated microglia, indicating an inhibitory effect of nicotine on a signaling process downstream of P2X(7)R. Taken together, these results suggest that the inhibitory effect of nicotine on ROS production in fAbeta1-42-stimulated microglia is mediated by indirect blockage of ATP release and by directly altering the signaling process downstream from P2X(7)R.

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Figures

Figure 1
Figure 1
Effects of nicotine on reactive oxygen species production in fAβ1-42-stimulated microglia. (A) Primary rat microglia ere plated onto coverslips (3×104 cells/coverslip), and then microglia were pretreated with nicotine for 30 min and stimulated with fAβ1-42(0.5 µM) for 2 h. The intracellular ROS production in microglial cells was determinded using 10 µM DCF as described in Materials and Methods. (B) DCF intensities of cells were counted using Imagegage 4.0 (Fujifilm). Fluorescence (DCF) images and differential interference contrast (DIC) images were taken using an IX71 confocal microscope (Olympus). Values are mean±SEM of 40-50 cells. *P <0.01 compared with Aβ1-42 alone. Scale bar, 20 µm.
Figure 2
Figure 2
Effects of nAChRs antagonists on fAβ1-42-induced ROS production from nicotine-treated microglia. (A) The cells were preincubated with nicotine (10 µM) for 30 min in the presence or absence of 10 µM mecamylamine (Mec) or 10 nM α-bungarotoxin (α-Bgt), and then treated with 0.5 µM fAβ1-42 for 2 h. The intracellular ROS production in microglial cells was determined using 10 µM DCF as described in Materials and Methods. (B) DCF intensities of cells were counted using Imagegage 4.0 (Fujifilm). Fluorescence (DCF) images and differential interference contrast (DIC) images were taken using an IX71 confocal microscope (Olympus). Values are mean±SEM of 40-50 cells. *P <0.01 compared with Aβ1-42plus nicotine. Scale bar, 20 µm.
Figure 3
Figure 3
Nicotine inhibits fAβ1-42-induced NADPH oxidase activation. NADPH oxidase was activated by fAβ1-42, as evidenced by the translocation of the p47phox, p67phox, Rac 1 subunits from the cytosol to the membrane; this translocation was inhibited by nicotine treatment. (A) The cells were treated with 10 µM nicotine for 30 min and stimulated with 0.5 µM fAβ1-42 for 90 min. Fractionated proteins were analyzed by SDS-PAGE and subjected to immunoblotting with anti-p47phox, anti-p67phox, anti-Rac 1 antibody. The blots were reprobed with antibodies against the calnexin membrane protein as loading controls to exhibit fractionation efficiency. (B) The histogram shows quantitation of p67phox, p47phox, Rac 1 levels expressed as the ratio of membrane fraction to total. The results represent the mean ± SEM of four to five separate experiments. *P < 0.01 compared with control, #P<0.01 compared with Aβ1-42 alone.
Figure 4
Figure 4
Effects of nicotine on Ca2+ influx in fAβ1-42- or BzATP-stimulated microglia and ATP efflux in fAβ1-42-stimulated microglia. Microglial cells were plated onto coverslips (3×104 cells/coverslip), and pretreated with nicotine (10 µM) (A-D), apyrase (5 U/ml), or oATP (100 µM) for 30 min (A), and then treated with 0.5 µM fAβ1-42 (A and B) or 300 µM BzATP (C and D). (A and C) Intracellular Ca2+ concentration was measured by Fluo-3 as described in Materials and Methods, and represented by the ratio between the fluorescence intensity after treatment (F) and fluorescence in the resting state (F0). (B) Microglial cells (3×104 cells/well) were plated into 96 well plate, and pretreated with nicotine (10 µM) for 30 min, and then treated with 0.5 µM fAβ1-42. ATP concentrations in the culture supernatants were determined at 1 h after fAβ1-42 stimulation. Values are mean ± SEM of triplicate samples. *P < compared with Aβ1-42. (D) Microglial cells were plated onto coverslips (3×104 cells/coverslip), and pretreated with nicotine (10 µM) for 30 min, and then treated with 300 µM BzATP. Intracellular ROS levels were assayed 2 h after BzATP stimulation using 10 µM DCF. Fluorescence (DCF) images were taken using an IX71 confocal microscope (Olympus). Scale bar, 20 µm. DCF intensities of cells were counted using Imagegage 4.0 (Fujifilm). Values are mean ± SEM of 40-50 cells. *P < 0.01 compared with BzATP alone.
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