Korean J Pain.  2023 Jul;36(3):335-346. 10.3344/kjp.23039.

Perampanel ameliorates nitroglycerin-induced migraine through inhibition of the cAMP/ PKA/CREB signaling pathway in the trigeminal ganglion in rats

• Zhai Q
• Wang K
• Zhang D
• Chen J
• Dong X
• Pan Y

• KMID: 2544245
• DOI: http://doi.org/10.3344/kjp.23039

Abstract

Background
Perampanel, a highly selective glutamate AMPA receptor antagonist, is widely used to treat epilepsy. Since the existence of common pathophysiological features between epilepsy and migraine, the aim of this study was to investigate whether perampanel could exert an antimigraine effect.
Methods
Nitroglycerin (NTG) was used to induce a migraine model in rats, and the model animals were pretreatment with 50 μg/kg and 100 μg/kg perampanel. The expression of pituitary adenylate-cyclase-activating polypeptide (PACAP) was quantified by western blot and quantitative real-time PCR in the trigeminal ganglion, and rat-specific enzyme-linked immunosorbent assay in serum. Western blot was also conducted to explore the effects of perampanel treatment on the phospholipase C (PLC)/protein kinase C (PKC) and protein kinase A (PKA)/ cAMP-responsive-element-binding protein (CREB) signaling pathways. Moreover, the cAMP/PKA/CREB-dependent mechanism was evaluated via in vitro stimulation of hippocampal neurons. The cells were treated with perampanel, antagonists and agonists for 24 hours and cell lysates were prepared for western blot analysis.
Results
Perampanel treatment notably increased the mechanical withdrawal threshold and decreased head grooming and light-aversive behaviors in NTG-treated rats. It also decreased PACAP expression and affected cAMP/ PKA/CREB signaling pathway. However, PLC/PKC signaling pathway may not be involved in this treatment. In in vitro studies, perampanel notably decreased PACAP expression by inhibiting cAMP/PKA/CREB signaling pathway.
Conclusions
This study shows that perampanel inhibits the migraine-like pain response and that this beneficial effect might be attributable to regulation of the cAMP/PKA/CREB signaling pathway.

Keyword

Chronic Pain; Cyclic AMP-Dependent Protein Kinases; Glutamic Acid; Hippocampus; Hyperalgesia; Migraine Disorders; Nitroglycerin; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Receptors, AMPA

Figure

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  Fig. 1 Experimental design of the study and the anatomical location of trigeminal ganglions. NTG: nitroglycerin, i.p.: intraperitoneal.

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  Fig. 2 Effects of perampanel treatment on photophobia. (A) Time spent in the light compartment, Kruskal–Wallis statistic = 24.30, ***P < 0.001; (B) Latency to re-enter the light compartment, Kruskal–Wallis statistic = 16.33, ***P = 0.001; (C) Transition, Kruskal–Wallis statistic = 20.55, ***P < 0.001. Control, n = 8; NTG, n = 8; PER 50 μg/kg + NTG, n = 8; PER 100 μg/kg + NTG, n = 8. Data are presented as means ± SEM. NTG: nitroglycerin, PER: perampanel. $$P < 0.01 vs. NTG group; **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05 vs. NTG group in corresponding days.

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  Fig. 3 Effects of perampanel treatment on NTG-induced migraine-like pain and nociceptive behaviors. (A) Head grooming, subgroups, F (3, 28) = 25.95, P < 0.001; time, F (4, 112) = 0.295, P = 0.880; subgroups × time, F (12, 112) = 0.629, P = 0.814. Control, n = 8; NTG, n = 8; PER 50 μg/kg + NTG, n = 8; PER 100 μg/kg + NTG, n = 8. In periorbital withdrawal thresholds test, NTG were decreased periorbital withdrawal thresholds, while perampanel treatment, at both doses of 50 and 100 μg/kg significantly increased the thresholds. (B) Periorbital withdrawal thresholds, subgroups, F (3, 28) = 23.28, P < 0.001; time, F (4, 112) = 4.397, P = 0.002; subgroups × time, F (12, 112) = 0.9606, P = 0.491. Data are presented as means ± SEM. NTG: nitroglycerin, PER: perampanel. $P < 0.05, $$P < 0.01, $$$P < 0.001 vs. NTG group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. NTG group in corresponding days.

• 

  Fig. 4 Effects of perampanel treatment on eating disorders. (A) Body weight, subgroups, F (3, 28) = 4.720, P = 0.009; time, F (4, 112) = 67.16, P < 0.001; subgroups × time, F (12, 112) = 0.652, P = 0.793; (B) Food intake, subgroups, F (3, 28) = 4.311, P = 0.013; time, F (4, 112) = 5.916, P < 0.001; subgroups × time, F (12, 112) = 1.637, P = 0.091. Control, n = 8; NTG, n = 8; PER 50 μg/kg + NTG, n = 8; PER 100 μg/kg + NTG, n = 8. Data are presented as means ± SEM. NTG: nitroglycerin, PER: perampanel.

• 

  Fig. 5 Effects of perampanel treatment on PACAP level in NTG-induced migraine. (A) Western blot showing the protein levels of PACAP; (B) Western bolt analysis of PACAP expression in TG. Kruskal–Wallis statistic = 12.15, **P = 0.007; (C) qRT-PCR analysis of PACAP gene expression in TG. Kruskal–Wallis statistic = 14.16, **P = 0.003, P < 0.001; (D) Elisa analysis of PACAP level in serum. Kruskal–Wallis statistic = 9.828, *P = 0.020. Control, n = 4; NTG, n = 6; PER 50 μg/kg + NTG, n = 6; PER 100 μg/kg + NTG, n = 6. Data are presented as mean ± SEM. PACAP: pituitary adenylate-cyclase-activating polypeptide, NTG: nitroglycerin, TG: trigeminal ganglion, PER: perampanel. Significant differences: *P < 0.05, ***P < 0.001.

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  Fig. 6 Effects of perampanel on PKA/CREB and PLC/PKC signaling pathways in NTG-induced migraine. (A) Western blot showing the protein levels of PKA, CREB and p-CREB; (B) Western blot analysis of the expression of PKA in TG. Kruskal–Wallis statistic = 17.20, ***P < 0.001; (C) Western blot analysis of the expression of P-CREB in TG. Kruskal–Wallis statistic = 11.52, **P = 0.009; (D) Western blot analysis of the expression of CREB in TG. Kruskal–Wallis statistic = 9.125, *P = 0.028; (E) Western blot showing the protein levels of PLC and PKC. (F) Western blot analysis of the expression of PLC in TG. Kruskal–Wallis statistic = 3.631, P = 0.304; (G) Western blot analysis of the expression of PKC in TG. Kruskal–Wallis statistic = 7.030, P = 0.071. Control, n = 4; NTG, n = 6; PER 50 μg/kg + NTG, n = 6; PER 100 μg/kg + NTG, n = 6. Data are presented as mean ± SEM. PKA: protein kinase A, CREB: cAMP-responsive-element-binding protein, PLC: phospholipase C, PKC: protein kinase C, NTG: nitroglycerin, TG: trigeminal ganglion, PER: perampanel. Significant differences: *P < 0.05, **P < 0.01.

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  Fig. 7 Effects of perampanel on cAMP/PKA/CREB signaling pathways in vitro. (A) Western blot showing the protein levels of PACAP in HT-22 cell incubated with forskolin 10 µM for 24 hours; (B) Western blot analysis of the expression of PACAP. Kruskal–Wallis statistic = 7.538, *P = 0.011. Control, n = 4; Forskolin 10 µM, n = 4; PER 100 μM, n = 4; (C) Western blot showing the protein levels of PACAP in HT-22 cell incubated with 8-bromo-cAMP sodium salt 0.1 mM for 24 hours; (D) Western blot analysis of the expression of PACAP. Kruskal–Wallis statistic = 16.33, P = 0.001. Control, n = 4; 8-bromo-cAMP sodium salt 0.1 mM, n = 4; PER 100 μM, n = 4; (E) Western blot showing the protein levels of PACAP in HT-22 cell incubated with SQ22536 100 μM for 24 hours; (F) Western blot analysis of the expression of PACAP. Kruskal–Wallis statistic = 9.269, ***P < 0.001. Control, n = 4; SQ22536 100 μM, n = 4; PER 100 μM, n = 4. (G) Western blot showing the protein levels of PACAP in HT-22 cell incubated with H-89 10 µM for 24 hours; (H) Western blot analysis of the expression of PACAP. Kruskal–Wallis statistic = 9.846, ***P < 0.001. Control, n = 4; H-89 10 µM, n = 4; PER 100 μM, n = 4. (I) Western blot showing the protein levels of PACAP in HT-22 cell incubated with KG501 25 µM for 24 hours; (J) Western blot analysis of the expression of PACAP. Kruskal–Wallis statistic = 8.769, **P = 0.001. Control, n = 4; KG501 25 µM, n = 4; PER 100 μM, n = 4. Data are presented as mean ± SEM. PKA: protein kinase A, CREB: cAMP-responsive-element-binding protein, PER: perampanel. Significant differences: *P < 0.05, **P < 0.01.

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