Activation of alpha7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures

Abstract

BACKGROUND: Nicotinic acetylcholine (Ach) receptors are ligand-gated pentameric ion channels whose main function is to transmit signals for the neurotransmitter Ach in peripheral and central nervous system. However, the alpha7 nicotinic receptor has been recently found in several non-neuronal cells and described as an important regulator of cellular function. Nicotine and ACh have been recently reported to inhibit tumor necrosis factor-alpha (TNF-alpha) production in human macrophages as well as in mouse microglial cultures. In the present study, we investigated whether the stimulation of alpha7 nicotinic receptor by the specific agonist nicotine could affect the functional state of activated microglia by promoting and/or inhibiting the release of other important pro-inflammatory and lipid mediator such as prostaglandin E2. METHODS: Expression of alpha7 nicotinic receptor in rat microglial cell was examined by RT-PCR, immunofluorescence staining and Western blot. The functional effects of alpha7 receptor activation were analyzed in resting or lipopolysaccharide (LPS) stimulated microglial cells pre-treated with nicotine. Culture media were assayed for the levels of tumor necrosis factor, interleukin-1beta, nitric oxide, interleukin-10 and prostaglandin E2. Total RNA was assayed by RT-PCR for the expression of COX-2 mRNA. RESULTS: Rat microglial cells express alpha7 nicotinic receptor, and its activation by nicotine dose-dependently reduces the LPS-induced release of TNF-alpha, but has little or no effect on nitric oxide, interleukin-10 and interleukin-1beta. By contrast, nicotine enhances the expression of cyclooxygenase-2 and the synthesis of one of its major products, prostaglandin E2. CONCLUSIONS: Since prostaglandin E2 modulates several macrophage and lymphocyte functions, which are instrumental for inflammatory resolution, our study further supports the existence of a brain cholinergic anti-inflammatory pathway mediated by alpha7 nicotinic receptor that could be potentially exploited for novel treatments of several neuropathologies in which local inflammation, sustained by activated microglia, plays a crucial role.

Figure 1

α7 nAChR subunit is expressed in rat microglial cultures. Semiquantitative RT-PCR analysis of α7 nAChR mRNA expression in rat microglial cells (A) and in PC12 cells and rat hippocampus (C). A 754-bp band corresponding to α7 nAChR was specifically amplified (acc. number S53987; amplified region: 906–1660). Expression of β-actin is shown as internal control. No contamination of genomic DNA was present as shown in panel B (-RT: RNA from microglia that was reverse transcribed without the enzyme and amplified for α7 subunit).

Figure 2

Western blot and fluorescent immunostaining of α7 nAChR in rat microglial cultures. A: Proteins from microglial cultures and PC12 cells were analysed by western blot (50 ug/lane) using specific polyclonal anti AChRα7 antibodies. B: microglial cells were pre-incubated in the absence (B, left panel) or presence of 500 μM nicotine (B, right panel) for 10 min and then incubated with FITC-labeleled-α-Bgtx (1.5 μg/ml) for 15 min at 4°C. A strong binding of α-Bgtx was observed on the cell surface of microglial cells. Nicotine pre-treatment resulted in a marked reduction of the intensity of binding.

Figure 3

Effects of specific α7 nAChR agonist and antagonist on TNF-α production by activated rat microglial cultures. Microglial cells were subcultured for 24 h in 10% FCS-containing medium, which was replaced with fresh medium before stimulation. Nicotine (0.1–1 μM) and/or α-Bgtx were added 30 min before LPS stimulation (10 ng/ml). Supernatants were collected after 4 h and analyzed for TNF-α content. Data are shown as mean ± SEM for 3 independent experiments, run in duplicate. *p < 0.03 vs LPS.

Figure 4

Effect of specific α7 nAChR agonist and antagonist on PGE₂ synthesis by activated rat microglial cultures. Microglial cells were subcultured as in Fig. 3, and nicotine (0.1–1 μM) added 30 min before LPS stimulation (10 ng/ml). Supernatants were collected after 24 h and analyzed for PGE₂ content. Data, with induction expressed as a percentage of LPS-induced PGE₂ production, are shown as mean ± SEM for 5 independent experiments, run in duplicate. The levels of PGE₂ were undetectable in basal conditions, and were 24 ± 6 ng/mg protein after LPS-stimulation for 24 h. *p < 0.05 vs LPS; **p < 0.02 vs LPS.

Figure 5

Semiquantitative RT-PCR analysis of COX-2 mRNA. Representative semi-quantitative RT-PCR analysis of COX-2 mRNA in microglial cultures, subcultured as in Fig. 3, pre-treated with nicotine (Nic, 0.1–1 μM) for 30 min and stimulated for 7 h (A, upper panels) or 24 h (B upper panels) with LPS (10 ng/ml). The amount of COX-2 mRNA, expressed as the ratio of densitometric measurement of the sample to the corresponding internal standard (β-actin), is shown in the lower panels. Data are shown as mean ± SEM for 3 to 4 independent experiments, with the exception of 1 μM nicotine, panel A (n = 2); all run in duplicate. * p < 0.05 vs fcs; **p < 0.05 vs fcs.