Korean J Physiol Pharmacol.  2016 Jan;20(1):101-109. 10.4196/kjpp.2016.20.1.101.

Brief low [Mg2+]o-induced Ca2+ spikes inhibit subsequent prolonged exposure-induced excitotoxicity in cultured rat hippocampal neurons

• Kim HJ
• Yang JS
• Yoon SH

• KMID: 2150479
• DOI: http://doi.org/10.4196/kjpp.2016.20.1.101

Abstract

Reducing [Mg2+]o to 0.1 mM can evoke repetitive [Ca2+]i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg2+]o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca2+ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg2+]o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca2+ channel antagonist nimodipine, which blocked 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca2+]i spikes. The intracellular Ca2+ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca2+]i spikes. While Go6976, a specific inhibitor of PKCalpha had no effect on the tolerance, both the PKCepsilon translocation inhibitor and the PKCzeta pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca2+]i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg2+]o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca2+]i spike-induced activation of PKCepsilon and PKCxi, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.

Keyword

Ca2+ spikes; Excitotoxicity; Low [Mg2+]o preconditioning

MeSH Terms

Animals
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Cell Death
Cycloheximide
N-Methylaspartate
Neurons*
Nimodipine
Phosphatidylinositol 3-Kinases
Phosphotransferases
Rats*
Receptors, AMPA
Seizures
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Cycloheximide
N-Methylaspartate
Nimodipine
Phosphatidylinositol 3-Kinases
Phosphotransferases
Receptors, AMPA

Figure

• 

  Fig. 1 Low [Mg2+]o-induced neuronal cell death is inhibited by brief exposure to low [Mg2+]o.(A) Neuronal death is induced by 0.1 mM [Mg2+]o, in a time-dependent manner. Hippocampal neurons were exposed to 0.1 mM [Mg2+]o-containing DMEM for the indicated times (0, 3 min, 5 min, 8 min, and 24 h) at day 13.5. (B) Low [Mg2+]o-induced neuronal death is inhibited by a low [Mg2+]o-induced preconditioning for 5 min. Hippocampal neurons were preconditioned with 0.1 mM [Mg2+]o medium for 3 or 5 min and then 24 h later exposed to the same medium for 24 h. Data are expressed as means±SEM. *p<0.05 relative to control, #p<0.05 relative to insult (ANOVA with Bonferroni's test).

• 

  Fig. 2 The preconditioning by brief exposure to a 0.1 mM [Mg2+]o medium for 5 min protects cells against prolonged exposureinduced exitotoxicity.Representative photographs show the same field of cultured rat hippocampal neurons before (left) and 24 h after a 5 min preconditioning and 24 h insult (right) in control (A, B), preconditioning only (C, D), insult (E, F) and preconditioning+insult (G, H). Neurons (identified by a light halo around the soma and long fine processes) grew on a bed of non-neuronal cells that formed a mosaic beneath them.

• 

  Fig. 3 Low [Mg2+]o-induced tolerance is mediated through Ca2+ signaling.(A) While NBQX inhibited low [Mg2+]o-induce [Ca2+]i spikes (Aa), D-AP5, nimodipine blocked the spikes (Ab-Ac). Ad, BAPTA-AM gradually inhibited the spikes. [Mg2+]o was reduced to 0.1 mM for the entire imaging period and drugs were added by superfusion at the times indicated by the horizontal bars. (B) While each treatment with D-AP5 and nimodipine during preconditioning markedly inhibited the low [Mg2+]o-induced tolerance, NBQX and BAPTA-AM moderately inhibited the tolerance. Neurons were exposed to 0.814 mM [Mg2+]o DMEM (control, n=10) or 0.1 mM [Mg2+]o DMEM without (n=4) or with NBQX (n=10), D-AP5 (n=8), nimodipine (n=4) and BAPTA-AM (n=4) for 5 min, and 24 h later exposed to 0.1 mM [Mg2+]o medium for following 24 h. Data are expressed as means±SEM. **p<0.01 relative to control, ##p<0.01 relative to insult, †p<0.05 relative to preconditioning+insult (ANOVA with Bonferroni's test).

• 

  Fig. 4 Low [Mg2+]o-induced tolerance is mediated through activation of certain forms of PKC.(A) Low [Mg2+]o-induced [Ca2+]i spikes are not affected by staurosporin (Aa), Gö6976 (Ab), the specific PKCε translocation inhibitor (Ac) and the PKCζ pseudosubstrate inhibitor (Ad). The [Mg2+]o was reduced to 0.1 mM for the entire imaging period and drugs were added by superfusion at the times indicated by the horizontal bars. (B) Low [Mg2+]o-induced tolerance is mediated through certain forms of PKC. Neurons were exposed to 0.814 mM [Mg2+]o DMEM (control, n=4) or 0.1 mM [Mg2+]o DMEM without (n=4) or with staurosporin (n=4), Gö6976 (n=4), PKCε translocation inhibitor (n=4), and PKCζ pseudosubstrate inhibitor (n=4) for 5 min, and 24 h later exposed to low [Mg2+]o medium or control medium for 24 h. Data are expressed as mean±SEM. **p<0.01 relative to control, ##p<0.01 relative to insult. †p<0.05 relative to preconditioning+insult (ANOVA with Bonferroni's test).

• 

  Fig. 5 Low [Mg2+]o-induced tolerance is mediated through activation of the JAK-2, MAPK kinase and CaMKII but not the PI3-kinase.Low [Mg2+]o-induced tolerance is affected by AG490, PD98059 and KN-62 but not by LY294,002. Neurons were exposed to 0.814 mM [Mg2+]o DMEM (control, n=8) or 0.1 mM [Mg2+]o DMEM without (n=4) or with LY294,002 (n=4), AG490 (n=8), PD98059 (n=4) or KN-62 (n=6) for 5 min, and then exposed 24 h later to 0.1 mM [Mg2+]o medium or control medium for 24 h. Data are expressed as means±SEM. **p<0.01 relative to control, ##p<0.01 relative to insult. †p<0.05 relative to preconditioning+insult (ANOVA with Bonferroni's test).

• 

  Fig. 6 Low [Mg2+]o-induced tolerance by de novo synthesis of proteins.Hippocampal neurons were exposed to 0.814 mM [Mg2+]o DMEM (control, n=4) or 0.1 mM [Mg2+]o DMEM without or with cycloheximide for 5 min, and then exposed to 0.814 mM [Mg2+]o DMEM (n=4) with or without cycloheximide (n=4) for 24 h before an exposure to 0.1 mM [Mg2+]o medium for 24 h. Data are expressed as mean±SEM. **p<0.01 relative to control, ##p<0.01 relative to insult. †p<0.05 relative to preconditioning+insult (ANOVA with Bonferroni's test).

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