J Physiol Sci (2017) 67:235–245 DOI 10.1007/s12576-016-0460-5 ORIGINAL PAPER Nicotine inhibits activation of microglial proton currents via interactions with a7 acetylcholine receptors 1 1 Mami Noda AI Kobayashi Received: 8 March 2016 / Accepted: 12 May 2016 / Published online: 2 June 2016 The Author(s) 2016 Abstract Alpha 7 subunits of nicotinic acetylcholine Introduction receptors (nAChRs) are expressed in microglia and are involved in the suppression of neuroinﬂammation. Over the Nicotine, like several other abused drugs, is known to act past decade, many reports show beneﬁcial effects of on the reward system in the brain. It was shown that cues nicotine, though little is known about the mechanism. Here related to smoking induce not only a subjective emotional we show that nicotine inhibits lipopolysaccharide (LPS)- alteration but also sympathetic activation in smokers . induced proton (H ) currents and morphological change by Apart from the reward system, according to epidemiolog- using primary cultured microglia. The H channel currents ical studies since the early 1990s, smoking lowers the risk were measured by whole-cell patch clamp method under of neurodegenerative diseases such as Alzheimer’s disease voltage-clamp condition. Increased H current in activated (AD) and Parkinson’s disease (PD) [2–4]. A marked microglia was attenuated by blocking NADPH oxidase. decrease of nicotine receptor expression has been reported The inhibitory effect of nicotine was due to the activation in AD patients [5, 6], PD patients [7, 8], and other neu- of a7 nAChR, not a direct action on the H channels, rodegenerative disease patients [9–13] including aging and because the effects of nicotine was cancelled by a7 nAChR dementia , suggesting the importance of the nicotinic antagonists. Neurotoxic effect of LPS-activated microglia receptors in brain function. Chronic nicotine infusion due to inﬂammatory cytokines was also attenuated by pre- increases the basal level of acetylcholine (ACh) release in treatment of microglia with nicotine. These results suggest the cerebral cortex and enhances responses of cortical ACh that a7 nAChRs in microglia may be a therapeutic target in release, but not in aged animals . The lack of an effect neuroinﬂammatory diseases. of chronic nicotine in aged animals may be due to a decrease in nAChRs in the cerebral cortex during aging as Keywords Microglia Nicotine a7 nAChRs mentioned above. In the brain, glial cells are considered to be the Lipopolysaccharide NADPH oxidase Proton currents principal pathologic response element; both microglial cells [16–18] and astrocytes . Microglia are the pri- mary immune cells in the central nervous system (CNS) (see review ). Under pathological conditions such as ischemia, trauma, and stroke, they are rapidly activated, and secrete various cytokines, including tumor necrosis The original version of this article was revised due to a retrospective factor (TNF)-a and interleukin-1b [17, 21]. Even minor open access order. pathology, such as early-life stress, changes in microglial & Mami Noda function, such as increased motility, in adulthood . email@example.com Alternatively, microglia can exert neuroprotective func- tions by secreting growth factors or diffusible anti-in- Laboratory of Pathophysiology, Graduate School of ﬂammatory mediator (see review ). Over the past Pharmaceutical Sciences, Kyushu University, 3-1-1 decade, many reports show beneﬁcial effects of nicotine Maidashi, Higashi-ku, Fukuoka 812-8582, Japan 123 236 J Physiol Sci (2017) 67:235–245 (see review, [23, 24]), but there are few electrophysio- Electrophysiological measurements logical analysis on microglia to explain the effect. Voltage-gated H channels in all cells enable recovery Whole-cell recordings of microglial cells were made as from an acute acid load, though their expression is reported previously [34, 37] using an Axopatch-200B ampliﬁer (Axon Instruments), under voltage-clamp condi- mainly restricted to immune cells . H channels enable NADPH oxidase function by compensating cel- tions at holding potential of -60 mV. The voltage pulses of 1 s were applied from -100 to ?100 mV with a 20-mV lular loss of electrons with protons. It is known that NADPH oxidase-mediated brain damage in stroke can be interval. The proton currents were measured according to previous reports [28, 38, 39] using a patch pipette con- inhibited by the suppression of H channels . It is also known that mouse and human brain microglia, but taining (in mM): 2-morpholinoethanesulfonic acid , not neurons or astrocytes, expressed large H channels- 120; NMDG aspartate, 85; BAPTA, 1; MgCl , 3. The pH mediated currents, and H channels were required for was adjusted to 5.5 with 1 N CsOH. The pipette resistance NADPH oxidase-dependent ROS generation in brain was 6–9 MX. The external solution contained (in mM): microglia in situ and in vivo . Slowly activating NMDG-aspartate, 85; HEPES, 100; CaCl , 1; MgCl ,1. 2 2 outward H currents were measured in microglia during The pH was adjusted to 7.3 with 1 N CsOH. The osmo- larity was *310 mOsm. The temperature monitored in the membrane depolarization [27–29]. Under pathological conditions such as neurodegeneration, pH homeostasis is recording dishes was 37 C. reduced but H channels contribute to its recovery . It was suggested that intracellular acidosis plays an Drugs important role in the progression of AD . Production of ROS in neutrophil was decreased in H channels- The NADPH oxidase inhibitor diphenyleneiodonium (DPI; deﬁcient mice [32, 33]. Since H channels are crucial SIGMA) was dissolved in DMSO at 10 mM, and the for oxidative stress-related brain disorders, microglial H solutions were diluted into the control medium to prepare channels might be one of the targets for nicotine. We working solution (1 lM). The concentration of DMSO in therefore sought to examine the effect of nicotine on the medium was 0.01 %. As control for DPI application, microglial H channels and to investigate its potential the same amount of DMSO (0.01 %) was added as vehicle. (-)-Nicotine hydrogen tartrate salt, a-bungarotoxin-te- role in neuroprotection in inﬂammatory neuronal damage. tramethylrhodamine (a-Bgt) and methyllycaconitine (MLA) citrate salt hydrate were purchased from SIGMA. Immunocytochemistry Materials and methods Microglial cells Cultured mouse microglial cells were stained according to previous reports [41, 42]. Brieﬂy, murine microglial cells Microglial cells were isolated from the mixed cultures of seeded on the slide glass (4 9 10 cells/dish) were ﬁxed cerebrocortical cells from postnatal day 1–2 C57BL/6 with 4 % paraformaldehyde (PFA), then initially rinsed mice, as reported previously [34–36]. In brief, cortical three times before treated with a primary antibody against microglia (rabbit anti-mouse Iba-1, Wako Pure Chemical tissue was trypsinized for 2 min, dissociated with a ﬁre- polished pipette. Mixed glial cells were cultured for Industries, Osaka, Japan, 1:2000 in 10 % block ace) overnight at 4 C, and then incubated with the Alexa Fluor 9–12 days in Dulbecco’s modiﬁed Eagle’s medium (DMEM; Nissui) supplemented with 10 % fetal bovine 488-conjugated goat anti-rabbit IgG (1:1000) for 3 h at serum (FBS; Hyclone Laboratories, Inc), 2 mM L-glu- 18 C, Texas Red-conjugated phalloidin (2U/ml 11 % tamine, 0.2 % D-glucose, 5 lg/ml insulin, 0.37 % BSA) for 1 h at 18 C. A series of images were examined NaHCO 100 U/ml penicillin, 100 lg/ml streptomycin, with a confocal laser-scanning microscope (LSM510; Carl 3, with medium changes every 5 day. Microglial cells were Zeiss, Oberkochen, Germany). then separated from the underlying astrocytic layer by gentle shaking of the ﬂask for 2 h at 37 C in a shaker- Western blotting incubator (120 rpm). Microglial cells were then isolated Expression protein level of proton channel in cultured as strongly adhering cells after unattached cells were removed. The purity of microglia was [98 %, which was microglial cells was examined by Western blotting relative to b-actin. Cultured microglial cells (10 cells for control evaluated by staining with Iba-1, a marker for microglia/macrophage. and LPS or nicotine/LPS group, each) were plated and 123 J Physiol Sci (2017) 67:235–245 237 incubated for 24 h. After treatment of LPS (SIGMA) (Dojindo, Kumamoto, Japan) according to the manufac- (1 lg/ml) for 24 h with or without pretreatment with turer’s instructions. nicotine (1 lM) for 1 h, the cells were lysed. The total lysates derived from culture microglia were resolved in Statistical analysis 7.5 % sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis and transferred to a polyvinylidene diﬂuoride The results were expressed as the mean ±SEM. The data membrane. The membrane was blocked for 30 min in Tris- were compared with Student’s t test or one-way ANOVA buffered saline containing 0.1 % Tween 20 (TBS-T) and followed by Scheffe´ test using the software package Stat- 10 % non-fat dried milk. Then, the membranes were View 5.0j. Values of p\ 0.05 were considered statistically incubated with primary antibody (HVCN1 (K-11): sc- signiﬁcant. 136712, Santa Cruz Biotechnology) (1:500) and anti-b- actin (1:1000, Sigma) in TBS-T containing 10 % non-fat dried milk), overnight, at 4 C. After washing with TBS-T, Results the membrane was incubated with horseradish peroxidase- conjugated anti-rabbit IgG antibody (1:5000 in TBS-T Proton currents are increased in activated microglia containing 1 % non-fat dried milk) (Millipore) for 1 h at room temperature. The membrane was washed four times H currents in microglia were changed in morphology and with 1 % non-fat dried milk and then with TBS-T. HVCN1 functional state by LPS . First, to conﬁrm that H proteins were visualized using ECL plus Western blot currents are affected in activated microglia, cultured detection system (GE Healthscience) and analyzed using microglial cells were stimulated by Gram-negative bacte- an LAS-4000 imaging system. rial LPS (Fig. 1). LPS (100 ng/ml and 1 lg/ml) was applied for 24 h and H currents were recorded by whole- Collection of microglial conditioned medium cell patch clamp method at the holding potential of (MCM) -60 mV. The H currents at positive potentials were increased after treatment of LPS (1 lg/ml) (Fig. 1a). The Cultured microglial cells from C57/BL6 mice were plated current–voltage relationships showed that 1 lg/ml, but not on 24-well glass dishes and incubated with serum-free 100 ng/ml LPS, signiﬁcantly increased the amplitude of DMEM for 24 h according to a previous report . H currents at the end of a 1-s pulse between ?20 and Microglial cells were incubated with LPS (1 lg/ml) for ?100 mV of membrane voltage (Fig. 1b). The current 24 h. Nicotine (1 lM) was pre-treated for 1 h before amplitudes at ?100 mV showed that 1 lg/ml LPS showed application of LPS. To completely remove LPS and nico- signiﬁcant increase compared to those in control and tine from the medium, the cultures were rinsed three times 100 ng/ml LPS, suggesting a concentration-dependent for 5 min with PBS, and then incubated in fresh serum-free effect of LPS (Fig. 1c). DMEM for 24 h. The media was centrifuged (1200 9 g, 10 min) and the supernatants were collected. These were Increased proton currents in activated microglia is supposed to include LPS-stimulated inﬂammatory cytoki- attenuated by blocking NADPH oxidase nes from microglia, referred to as LPS-treated microglial conditioned medium (LPS–MCM). LPS–MCM was pre- It is known that LPS upregulates NADPH oxidase in served at -30 C until use. neutrophils  and H channels and NADPH are important in phagocytes [30, 46]. To test whether or not Neuronal cell culture from hippocampus and cortex NADPH oxidase is important for the LPS-induced H current, an inhibitor of NADPH oxidase, dibenziodolium Hippocampal and cortex neurons were obtained from chloride (DPI), was used. Pre-treatment with DPI (1 lM) embryonic day 14–16 C57BL/6 mice as described previ- for 1 h attenuated LPS (lg/ml)-induced H current ously [36, 43]. Brieﬂy, neurons were cultured at 37 Cina (Fig. 2a). DPI alone did not have any effect. The cur- 5% CO /95 % O incubator for 5–7 days with neurobasal rent–voltage relationships showed that 1 lg/ml LPS sig- 2 2 medium (GIBCO) containing 2 % B27 supplement niﬁcantly increased the amplitude of H currents at the (GIBCO) and 0.5 mM L-glutamine (GIBCO). end of a 1-s pulse between ?60 and ?100 mV of membrane voltage (Fig. 2b). The relative current ampli- Neuronal cell accounting tudes of H currents at ?100 mV showed a signiﬁcant increase by 1 lg/ml LPS, while pre-incubation with DPI The number of live neuronal cells with or without drug almost completely inhibited LPS-induced H currents application was checked by using a cell counting kit-8 (Fig. 2c). 123 238 J Physiol Sci (2017) 67:235–245 Fig. 1 Microglial H currents are signiﬁcantly increased by appli- absence of LPS (control, ﬁlled square), with 100 nM/ml (ﬁlled circle) cation of lipopolysaccharide (LPS) in a dose-dependent manner. and 1 lg/ml LPS (ﬁlled upright triangle) are shown. c The relative a Current traces from -100 to ?100 mV from the holding potential current amplitudes at ?100 mV in b are shown. *p \ 0.05, # ## of -60 mV for 1 s with or without (control) application of LPS (1 lg/ **p \ 0.01, ***p \ 0.005 compared to control. p \ 0.5, p \ 0.01 ml) for 24 h are shown. b Current–voltage (I–V) relationships in the compared to LPS (100 ng/ml) Fig. 2 NADPH oxidase inhibitor attenuates LPS-induced microglial vehicle, ﬁlled square), with 1 lg/ml LPS (ﬁlled circle), 1 lM DPI H currents. a Current traces from -100 to ?100 mV from the alone (ﬁlled upright triangle), and DPI ? LPS (ﬁlled downright holding potential of -60 mV for 1 s are shown. LPS (1 lg/ml) was triangle) are shown. c The relative current amplitudes at ?100 mV in applied for 24 h and NADPH oxidase inhibitor, dibenziodolium b are shown. *p \ 0.05 compared to control. p \ 0.5 compared to chloride (DPI, 1 lM), was pre-treated for 1 h prior to the application LPS (1 lg/ml) of LPS. b I–V relationships in the absence of LPS (control with 123 J Physiol Sci (2017) 67:235–245 239 Inhibition of LPS-induced proton currents The mechanism of inhibitory effects of nicotine and morphological change of microglia by nicotine on LPS-induced proton currents in microglia Next, we tested whether or not LPS-induced H current First, the expression level of proton channel was tested by is inhibited by nicotine. LPS-induced H currents were Western blotting. The expression of HVCN1 was signiﬁ- attenuated by pre-incubation of nicotine for 1 h in a cantly up-regulated by treatment of LPS (1 lg/ml) for dose-dependent manner (Fig. 3a). The current–voltage 24 h. The pre-treatment with nicotine (1 lM) before relationships for H current amplitudes at the end of a application of LPS did not affect the up-regulated expres- 1-s pulse recorded after LPS pretreatment were signif- sion of HVCN1 (Fig. 5). icantly reduced by 300 nM and 1 lM nicotine at posi- Next, to test the involvement of the nicotinic acetyl- tive membrane potential (Fig. 3b). The current choline receptor (nAChR), an a7 nAChR antagonist, amplitudes at ?40 mV showed signiﬁcant inhibition of methyllycaconitine (MLA) and a-bungarotoxin (a-Bgt) LPS-induced H currents by 300 nM and 1 lMnico- were applied. MLA (100 nM) or a-Bgt (100 nM) were tine, but not with 100 nM nicotine (Fig. 3c). The half applied 30 min before application of nicotine for 1 h, fol- inhibitory concentration (IC ) of nicotine was lowed by application of LPS (1 lg/ml) for 24 h. The 112.1 nM (Fig. 3d). inhibitory effects of nicotine on LPS-induced H currents The morphological change of microglia was also were cancelled by MLA or a-Bgt (Fig. 6). observed. Application of LPS for 24 h caused so-called activated shape of microglia; bigger cell bodies with Nicotine inhibits neurotoxic effect of activated retracted processes. However, pre-treatment of microglial microglia cells with nicotine prevented the morphological change of microglia (Fig. 4). LPS is known as a strong activator of microglia, leading production and release of pro-inﬂammatory cytokines and Fig. 3 Nicotine dose-dependently attenuates LPS-induced microglial square), with 1 lg/ml LPS (ﬁlled upright triangle), LPS with 100 nM H currents. a Current traces from -100 to ?100 mV from the (open diamond), 300 nM (ﬁlled downright triangle), and 1 lM Nic holding potential of -60 mV for 1 s are shown. Application of LPS (ﬁlled circle) are shown. c The relative current amplitudes at ?40 mV (1 lg/ml) was for 24 h and nicotine at concentration of 100 nM, in b are shown. d Dose-dependent effect of Nic on LPS-induced 300 nM, and 1 lM were pre-treated for 1 h before application of microglial H currents is shown. The half inhibitory concentration LPS. b I–V relationships in the absence of LPS (control, ﬁlled (IC ) of Nic is 112.13 nM. **p \ 0.01 compared to LPS (1 lg/ml) 123 240 J Physiol Sci (2017) 67:235–245 Fig. 4 Effect of nicotine on LPS-induced morphological change of Alexa Fluor 488; green), and anti-phalloidin (anti-F-actin antibody) microglia. a Effects of LPS and nicotine on cellular morphology of (labeled with Alexa Fluor 568; red) are shown. b Images in white microglia. Microglial cells were treated with LPS (1 lg/ml) for 24 h. squares in a are enlarged with different scale. More ﬁlopodia and Nicotine (1 lM) was pre-treated for 1 h before application of LPS. membrane rufﬂing (actin polymerization) are shown in LPS-treated Immunoﬂuorescence stained with anti-Iba-1 antibody (labeled with microglia applied to cultured neuronal cells. Due to LPS-induced inﬂammatory cytokines released from microglia, the number of living neuronal cell decreased signiﬁcantly by application of LPS–MCM. However, LPS–MCM with pre- treatment of nicotine rescued neuronal cell damage, keep- ing the number of living cell intact (Fig. 7). Discussion Our data suggest that nicotine inhibits H currents of microglia via interactions with a7 nAChRs. This means that a7 nAChRs agonists may have a therapeutic potential via regulation of microglial activation in neuroinﬂamma- tory diseases. First, we showed that LPS activated H currents of Fig. 5 Nicotine does not affect LPS-increased expression of H microglia. An LPS-sensitive H current was previously channels in microglia. (Upper panel) Western blotting of H channel, reported in microglia [27–29, 50] and dendritic cells . HVCN1, and b-actin in cultured microglia. Microglial cells were treated with 1 lg/ml LPS for 24 h, and nicotine (1 lM) was pre- In our study, the electrophysiological recording was per- treated for 1 h before application of LPS. HVCN1 protein is detected formed at 37 C because the voltage-gated H channel was at around 32 kDa in whole-cell lysate from microglia. (Lower panel) reported to be temperature sensitive . Relative expression levels of HVCN1 compared to b-actin are shown As mononuclear phagocytic cells, microglial cells in control, LPS, and Nic ? LPS. *p \ 0.05 compared to control express high levels of superoxide-producing NADPH oxi- dases . The sole function of members of the NADPH reactive oxygen species (ROS) [47–49]. To assess whether oxidase family is to generate reactive oxygen species nicotine suppresses the LPS-mediated neurotoxic effect via (ROS) and upregulate the production of TNF-a [53, 54] microglia, LPS-treated microglial conditioned medium that are believed to be important in CNS host defense . (LPS–MCM) with or without nicotine pre-treatment was However, ischemia can also lead to NADPH oxidase- 123 J Physiol Sci (2017) 67:235–245 241 Fig. 6 Nicotinic acetylcholine (nACh) receptor inhibitors cancel the nicotine, followed by LPS application. b I–V relationships in control effect of nicotine on LPS-increased proton current in microglia. a (ﬁlled square), with 1 lg/ml LPS (ﬁlled circle), LPS with Nic (ﬁlled Current traces from -100 to ?100 mV from the holding potential of upright triangle), pre-treated with MLA (ﬁlled downright triangle) -60 mV for 1 s are shown. Application of LPS (1 lg/ml) was for and a-Bgt (open diamond) are shown. c The relative current 24 h and nicotine (1 lM) was pre-treated for 1 h before application of amplitudes at ?100 mV in b are shown. **p \ 0.01, ***p \ 0.005 LPS. Methyllycaconitine (MLA, 100 nM) and a-bungarotoxin (a- compared to LPS ? Nic Bgt, 100 nM) were pre-treated for 30 min before application of oxidase-dependent H currents. NADPH oxidase is elec- trogenic , generating electron current (Ie) [57, 58] with voltage-dependency . Ie is compensated by H efﬂux mediated by voltage-gated H channels [59, 60], which may explain why phagocytes need H channels . Voltage-gated H channels was required for NADPH oxidase-dependent ROS generation in brain microglia. Therefore, blocking either NADPH oxidase or H channels is useful to reduce neurotoxic effects due to activation of microglia and ROS generation. Neutrophils exposed to LPS upregulates NDPH oxidase assembly . Since nicotine inhibits ﬁbrillar b amyloid peptide (1-42) (fAb )-induced NADPH oxidase activa- 1-42 tion , it is likely that nicotine inhibits LPS-induced Fig. 7 Nicotine inhibits neurotoxic effect of LPS-activated micro- glia. The conditioned medium from LPS-activated microglia (LPS– NADPH oxidase activation as well. MCM) has neurotoxicity due to inﬂammatory cytokines. However, The LPS-induced H currents of microglia were inhib- LPS–MCM from cells with nicotine (1 lM) pre-treatment signiﬁ- ited by nicotine (Fig. 3). Though LPS up-regulated cantly restored neuronal cells. **p \ 0.01 compared to control. ## expression of HVCN1, nicotine did not affect the LPS- p \ 0.01 compared to LPS–MCM increased expression of HVCN1 (Fig. 5). It is likely that nicotine affects the function of H channel, either single induced ROS production and inﬂict damage on native cells. channel conductance or open probability, not the signal Therefore, the function of NADPH oxidases in microglia is pathway on the way or during the transcription of H a double-edged sword. channel gene. This functional change of H channel should In our study, LPS-induced currents in microglia were be investigated in the future. The inhibitory effect of completely inhibited by the NADPH oxidase inhibitor DPI nicotine was also observed morphologically in LPS-treated (Fig. 2), suggesting that the currents were NADPH microglia (Fig. 4). The typical morphological change in 123 242 J Physiol Sci (2017) 67:235–245 Fig. 8 Proposed schema on inhibitory effects of nicotine on LPS- NOX-dependent ROS generation. Nicotine binds to a7 nAChR in 2? induced microglial activation. LPS, glycolipids found in the outer microglia, causing transient increase in intracellular Ca in membrane of some types of Gram-negative bacteria, bind to Toll-like phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3)-dependent receptor 4 (TLR4) and activate signaling pathways; extracellular manner , negatively modulates LPS-induced release of TNF-a. signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase Cholinergic protection via a7 nAChR and PI3K-Akt pathway in LPS- (MAPK), AP1, nuclear factor-jB (NF-jB), or IRFs (IRF3/IRF7), and induced neuroinﬂammation is also reported . Nicotine may inhibit hence production and release of pro-inﬂammatory cytokines, nitric LPS-induced NOX. On the other hand, nicotine inhibits H current oxide (NO) via inducible NO synthase and tumor necrosis factor-a without affecting LPS-increased expression of HVCN1. Presumably, (TNF-a). LPS also upregulates NADPH oxidase (NOX) assembly. a7 nAChR signaling inhibits function of HVCN1 either directly or by The voltage-gated H channel, HVCN1, enables NOX function by inhibiting NOX, hence attenuating ROS production and further compensating cellular loss of electrons with protons, which are stimulation of pro-inﬂammatory cytokines, NO and TNF-a required for phagocytosis. Furthermore, HVCN1 was required for 2? LPS-treated microglia is retracting processes and becoming The nicotine-induced Ca signals, which are dependent non-polar, assume a large, round, ﬂat shape, and gradually on phospholipase C and inositol 1,4,5-trisphosphate (IP ), develop many microspikes all over the cell body . modulated the release of TNF-a in response to either However, if the cells were pre-treated with nicotine, the activation of P2X receptors (positive modulation) or LPS morphological change was almost cancelled, suggesting (negative modulation) . The cholinergic inhibition of the NADPH oxidase-H channel cascade is also involved LPS-induced TNF-a release from microglia is mediated by in LPS-induced change in microglial morphology. the inhibition of p38 mitogen-activated protein kinase The question is how does nicotine inhibit the H cur- (MAPK) and p44/42 . On the other hand, anti-depres- ? ? rent? To test whether nicotine affects the H channel sants and local anesthetics inhibit the voltage-gated H directly or indirectly, we tested the involvement of a7 channels in microglial cell lines [38, 39]. In T cells, lido- nAChR. Both a-Bgt and MLA, a7 nAChR antagonists, caine down-regulates nuclear factor-jB signaling and cancelled the inhibitory effect of nicotine on LPS-induced inhibits cytokine production . Therefore, it is specu- ? ? H current (Fig. 6). This means that down-stream signal- lated that inhibition of the H channel results in the inhi- ing of the a7 nAChR mediates the inhibitory effect on bition of LPS-induced cytokine production, though the NADPH-H channel cascade. Nicotine can alkalinize precise signal pathway is not clear. intracellular solution , reducing H concentration. This Taken together, it is suggested that the inhibitory effect ? ? could reduce the outward H -current. However, it is unli- of nicotine on H current in LPS-stimulated microglia is kely because nicotine concentration was quite low and the mediated either by blocking NADPH oxidase or indirectly effect was blocked by a-Bgt and MLA. by a7 nACh signaling. Consequently, inhibiting H current 123 J Physiol Sci (2017) 67:235–245 243 acetylcholine receptor proteins in the cerebral cortex of Alzhei- reduces the production of ROS and subsequent formation mer patients. Brain Res Mol Brain Res 76:385–388 of pro-inﬂammatory cytokines NO and TNF-a. The func- 6. 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Uchida S, Hotta H, Misawa H, Kawashima K (2013) The missing Brown (University College London, UK) for reading the manuscript link between long-term stimulation of nicotinic receptors and the and useful comments. We also thank The Research Support Center, increases of acetylcholine release and vasodilation in the cerebral Research Center for Human Disease Modeling, Kyushu University cortex of aged rats. J Physiol Sci 63:95–101 Graduate School of Medical Sciences for Technical Assistance. 16. Kim SU, De Vellis J (2005) Microglia in health and disease. J Neurosci Res 81:302–313 Compliance with ethical standards 17. Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19:312–318 Conﬂict of interest The author(s) declare that they have no com- 18. Perry VH, Andersson PB, Gordon S (1993) Macrophages and peting interests. inﬂammation in the central nervous system. Trends Neurosci 16:268–273 19. 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The Journal of Physiological Sciences – Springer Journals
Published: Jun 2, 2016
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