Nimodipine promotes neurite outgrowth and protects against neurotoxicity in PC12 cells

Abstract

Objectives: Nimodipine is an L-type voltage-dependent calcium channel (VDCC) antagonist. However, the actions of nimodipine except calcium blocking are poorly understood. This study aimed to investigate the effect of nimodipine on neurite outgrowth and neuroprotection in vitro.

Materials and methods: After PC12 cells were treated with different concentrations of nimodipine, neurite outgrowth was estimated using the ImageJ software. Neuroprotective effects of nimodipine against H₂O₂ and calcium ionophore-induced neurotoxicity were investigated using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, the activation of extracellular signal-regulated kinase (ERK) and cyclic AMP-response element-binding protein (CREB) pathway was investigated for clarifying the action mechanism of nimodipine.

Results: Nimodipine treatment at doses of higher than 10 µM induced neurite outgrowth in the cells. Additionally, VDCC knockdown by siRNA significantly suppressed the nimodipine-induced neurite outgrowth in PC12 cells, suggesting that the drug promotes neurite outgrowth by binding to VDCC. H₂O₂ and calcium ionophore induce oxidative and calcium stress in PC12 cells. Nimodipine exhibited neuroprotective effects against H₂O₂- and calcium ionophore-induced neurotoxicity by increasing the mRNA expression levels of neurotrophic factors, calcium-binding proteins, and antioxidants that are transcribed by CREB activation.

Conclusion: This is the first report that nimodipine induces neurite outgrowth and exerts its neuroprotective activity through the ERK/CREB signaling pathway in PC12 cells.

Keywords: Calcium channels; MAP kinase signaling – system; Neuronal outgrowth; Neuroprotection; Nimodipine.

Figure 1

Effect of nimodipine on neurite outgrowth in PC12 cells. (a) Toxicity of nimodipine, as evaluated by MTT assay. (b) Phase-contrast microscopy of PC12 cells in the absence (upper panel) or presence (lower panel) of 20 µM nimodipine (left panel) or 50 µM nifedipine (right panel) for 72 hr. The scale bar represents 50 μm. (c) Estimated length of neurites (n = 100) and level of neurofilament-L expression induced by nimodipine or nifedipine. *P<0.05 relative to the control

Figure 2

Effect of L-type calcium channel (VDCC) knockdown on nimodipine-induced neurite outgrowth. Phase-contrast microscopy of control cells (a) and knockdown cells (b) after treatment with 20 µM nimodipine for 48 hr. The scale bar represents 50 μm. (c) Length of neurites (n=100) of control cells and knockdown cells after treatment with 20 µM nimodipine for 48 hr was measured in at least 5 randomly selected areas. (d) Level of L-type calcium channel (VDCC) expression, as evaluated by semi-quantitative RT-PCR. *P<0.05 relative to the control

Figure 3

Effect of nimodipine on various signaling pathways. After treatment of the PC12 cells with 20 μM nimodipine for 120 min, the cells were recovered and Western blotting was performed using specific antibodies against (a) ERK and p-ERK, (b) Akt and p-Akt, and (c) CREB and p-CREB. *P<0.05 relative to the control

Figure 4

Effects of MEK and PKC inhibitors on nimodipine-induced neurite outgrowth. PC12 cells were treated with 20 μM nimodipine in the absence (a), presence of 10 μM PD98059 (c), or 10 μM BIM-III (d). PC12 cells without nimodipine treatment are shown as control (b). After 72 hr, the lengths of the neurites (n=100) were measured. The scale bar represents 50 μm (e). Bars represent SD. *P<0.05 relative to the treatment with nimodipine alone

Figure 5

Effect of nimodipine on the expression of neurotrophic factors and their receptor (a–c), calcium-binding proteins (d–f), and antioxidants (g, h). After treatment of the PC12 cells with 20 μM nimodipine for 48 hr, the cells were recovered and semi-quantitative RT-PCR analysis was performed using specific primers. *P<0.05 relative to the control

Figure 6

Neuroprotective effect of nimodipine against calcium stress or oxidative stress. (a) PC12 cells were treated with nimodipine at the indicated concentrations, in the absence or presence of 0.1 μM A23187. After 24 hr, the viable cells were estimated by MTT assay. (b) PC12 cells were treated with nimodipine at the indicated concentrations, in the absence or presence of 72 mM H₂O₂. After 24 hr, the viable cells were estimated by MTT assay. *P<0.05 relative to the treatment with A23187 or H₂O₂ alone