Nicotine inhibits activation of microglial proton currents via interactions with α7 acetylcholine receptors

Abstract

Alpha 7 subunits of nicotinic acetylcholine receptors (nAChRs) are expressed in microglia and are involved in the suppression of neuroinflammation. Over the past decade, many reports show beneficial effects of nicotine, though little is known about the mechanism. Here we show that nicotine inhibits lipopolysaccharide (LPS)-induced proton (H⁺) currents and morphological change by using primary cultured microglia. The H⁺ channel currents were measured by whole-cell patch clamp method under voltage-clamp condition. Increased H⁺ current in activated microglia was attenuated by blocking NADPH oxidase. The inhibitory effect of nicotine was due to the activation of α7 nAChR, not a direct action on the H⁺ channels, because the effects of nicotine was cancelled by α7 nAChR antagonists. Neurotoxic effect of LPS-activated microglia due to inflammatory cytokines was also attenuated by pre-treatment of microglia with nicotine. These results suggest that α7 nAChRs in microglia may be a therapeutic target in neuroinflammatory diseases.

Keywords: Lipopolysaccharide; Microglia; NADPH oxidase; Nicotine; Proton currents; α7 nAChRs.

Fig. 1

Microglial H⁺ currents are significantly increased by application of lipopolysaccharide (LPS) in a dose-dependent manner. a Current traces from −100 to +100 mV from the holding potential of −60 mV for 1 s with or without (control) application of LPS (1 μg/ml) for 24 h are shown. b Current–voltage (I–V) relationships in the absence of LPS (control, filled square), with 100 nM/ml (filled circle) and 1 μg/ml LPS (filled upright triangle) are shown. c The relative current amplitudes at +100 mV in b are shown. *p < 0.05, **p < 0.01, ***p < 0.005 compared to control.^# p < 0.5, ^## p < 0.01 compared to LPS (100 ng/ml)

Fig. 2

NADPH oxidase inhibitor attenuates LPS-induced microglial H⁺ currents. a Current traces from −100 to +100 mV from the holding potential of −60 mV for 1 s are shown. LPS (1 μg/ml) was applied for 24 h and NADPH oxidase inhibitor, dibenziodolium chloride (DPI, 1 μM), was pre-treated for 1 h prior to the application of LPS. b I–V relationships in the absence of LPS (control with vehicle, filled square), with 1 μg/ml LPS (filled circle), 1 μM DPI alone (filled upright triangle), and DPI + LPS (filled downright triangle) are shown. c The relative current amplitudes at +100 mV in b are shown. *p < 0.05 compared to control. ^# p < 0.5 compared to LPS (1 μg/ml)

Fig. 3

Nicotine dose-dependently attenuates LPS-induced microglial H⁺ currents. a Current traces from −100 to +100 mV from the holding potential of −60 mV for 1 s are shown. Application of LPS (1 μg/ml) was for 24 h and nicotine at concentration of 100 nM, 300 nM, and 1 μM were pre-treated for 1 h before application of LPS. b I–V relationships in the absence of LPS (control, filled square), with 1 μg/ml LPS (filled upright triangle), LPS with 100 nM (open diamond), 300 nM (filled downright triangle), and 1 μM Nic (filled circle) are shown. c The relative current amplitudes at +40 mV in b are shown. d Dose-dependent effect of Nic on LPS-induced microglial H⁺ currents is shown. The half inhibitory concentration (IC₅₀) of Nic is 112.13 nM. **p < 0.01 compared to LPS (1 μg/ml)

Fig. 4

Effect of nicotine on LPS-induced morphological change of microglia. a Effects of LPS and nicotine on cellular morphology of microglia. Microglial cells were treated with LPS (1 μg/ml) for 24 h. Nicotine (1 μM) was pre-treated for 1 h before application of LPS. Immunofluorescence stained with anti-Iba-1 antibody (labeled with Alexa Fluor 488; green), and anti-phalloidin (anti-F-actin antibody) (labeled with Alexa Fluor 568; red) are shown. b Images in white squares in a are enlarged with different scale. More filopodia and membrane ruffling (actin polymerization) are shown in LPS-treated microglia

Fig. 5

Nicotine does not affect LPS-increased expression of H⁺ channels in microglia. (Upper panel) Western blotting of H⁺ channel, HVCN1, and β-actin in cultured microglia. Microglial cells were treated with 1 μg/ml LPS for 24 h, and nicotine (1 μM) was pre-treated for 1 h before application of LPS. HVCN1 protein is detected at around 32 kDa in whole-cell lysate from microglia. (Lower panel) Relative expression levels of HVCN1 compared to β-actin are shown in control, LPS, and Nic + LPS. *p < 0.05 compared to control

Fig. 6

Nicotinic acetylcholine (nACh) receptor inhibitors cancel the effect of nicotine on LPS-increased proton current in microglia. a Current traces from −100 to +100 mV from the holding potential of −60 mV for 1 s are shown. Application of LPS (1 μg/ml) was for 24 h and nicotine (1 μM) was pre-treated for 1 h before application of LPS. Methyllycaconitine (MLA, 100 nM) and α-bungarotoxin (α-Bgt, 100 nM) were pre-treated for 30 min before application of nicotine, followed by LPS application. b I–V relationships in control (filled square), with 1 μg/ml LPS (filled circle), LPS with Nic (filled upright triangle), pre-treated with MLA (filled downright triangle) and α-Bgt (open diamond) are shown. c The relative current amplitudes at +100 mV in b are shown. **p < 0.01, ***p < 0.005 compared to LPS + Nic

Fig. 7

Nicotine inhibits neurotoxic effect of LPS-activated microglia. The conditioned medium from LPS-activated microglia (LPS–MCM) has neurotoxicity due to inflammatory cytokines. However, LPS–MCM from cells with nicotine (1 μM) pre-treatment significantly restored neuronal cells. **p < 0.01 compared to control. ^## p < 0.01 compared to LPS–MCM

Fig. 8

Proposed schema on inhibitory effects of nicotine on LPS-induced microglial activation. LPS, glycolipids found in the outer membrane of some types of Gram-negative bacteria, bind to Toll-like receptor 4 (TLR4) and activate signaling pathways; extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK), AP1, nuclear factor-κB (NF-κB), or IRFs (IRF3/IRF7), and hence production and release of pro-inflammatory cytokines, nitric oxide (NO) via inducible NO synthase and tumor necrosis factor-α (TNF-α). LPS also upregulates NADPH oxidase (NOX) assembly. The voltage-gated H⁺ channel, HVCN1, enables NOX function by compensating cellular loss of electrons with protons, which are required for phagocytosis. Furthermore, HVCN1 was required for NOX-dependent ROS generation. Nicotine binds to α7 nAChR in microglia, causing transient increase in intracellular Ca²⁺ in phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3)-dependent manner [69], negatively modulates LPS-induced release of TNF-α. Cholinergic protection via α7 nAChR and PI3K-Akt pathway in LPS-induced neuroinflammation is also reported [70]. Nicotine may inhibit LPS-induced NOX. On the other hand, nicotine inhibits H⁺ current without affecting LPS-increased expression of HVCN1. Presumably, α7 nAChR signaling inhibits function of HVCN1 either directly or by inhibiting NOX, hence attenuating ROS production and further stimulation of pro-inflammatory cytokines, NO and TNF-α