L type Ca²+ channel blockers prevent oxaliplatin-induced cold hyperalgesia and TRPM8 overexpression in rats - PubMed (original) (raw)
L type Ca²+ channel blockers prevent oxaliplatin-induced cold hyperalgesia and TRPM8 overexpression in rats
Takehiro Kawashiri et al. Mol Pain. 2012.
Abstract
Background: Oxaliplatin is an important drug used in the treatment of colorectal cancer. However, it frequently causes severe acute and chronic peripheral neuropathies. We recently reported that repeated administration of oxaliplatin induced cold hyperalgesia in the early phase and mechanical allodynia in the late phase in rats, and that oxalate derived from oxaliplatin is involved in the cold hyperalgesia. In the present study, we examined the effects of Ca²⁺ channel blockers on oxaliplatin-induced cold hyperalgesia in rats.
Methods: Cold hyperalgesia was assessed by the acetone test. Oxaliplatin (4 mg/kg), sodium oxalate (1.3 mg/kg) or vehicle was injected i.p. on days 1 and 2. Ca²⁺ (diltiazem, nifedipine and ethosuximide) and Na⁺ (mexiletine) channel blockers were administered p.o. simultaneously with oxaliplatin or oxalate on days 1 and 2.
Results: Oxaliplatin (4 mg/kg) induced cold hyperalgesia and increased in the transient receptor potential melastatin 8 (TRPM8) mRNA levels in the dorsal root ganglia (DRG). Furthermore, oxalate (1.3 mg/kg) significantly induced the increase in TRPM8 protein in the DRG. Treatment with oxaliplatin and oxalate (500 μM for each) also increased the TRPM8 mRNA levels and induced Ca²⁺ influx and nuclear factor of activated T-cell (NFAT) nuclear translocation in cultured DRG cells. These changes induced by oxalate were inhibited by nifedipine, diltiazem and mexiletine. Interestingly, co-administration with nifedipine, diltiazem or mexiletine prevented the oxaliplatin-induced cold hyperalgesia and increase in the TRPM8 mRNA levels in the DRG.
Conclusions: These data suggest that the L type Ca²⁺ channels/NFAT/TRPM8 pathway is a downstream mediator for oxaliplatin-induced cold hyperalgesia, and that Ca²⁺ channel blockers have prophylactic potential for acute neuropathy.
Figures
Figure 1
The incidence of cold hyperalgesia (A) and expression of TRPM8 (B-E) following oxaliplatin or sodium oxalate treatment. Oxaliplatin (4 mg/kg) or sodium oxalate (1.3 mg/kg) was administered i.p. on days 1 and 2. A: The acetone test was performed on days 0, 3, 5, 8 and 15. B: On day 5 the rat L4-6 DRG treated with oxaliplatin was harvested and the mRNA expression of TRPM8 and G3PDH were determined by PCR. C: On day 5 the rat L4-6 DRG treated with sodium oxalate was harvested and the protein of TRPM8 and _β_-actin were determined by Western boltting. D, E: 500 μM of oxaliplatin (D) or sodium oxalate (E) was administered to cultured DRG cells for 3, 6 or 12 h. mRNA expression of TRPM8 and G3PDH was determined by PCR. Values are expressed as the mean ± SEM of 4-6 animals (A, B) or 4-6 wells (C, D). *p < 0.05, **p < 0.01 compared with vehicle or control group.
Figure 2
Increase in the intracellular Ca2+ following oxaliplatin or oxalate treatment in primary cultured DRG cells. Oxaliplatin (A: 100-500 μM) or sodium oxalate (B: 100-500 μM) was administered to cultured DRG cells. Nifedipine (C: 10-30 μM), diltiazem (D: 10-30 μM), ethosuximide (E: 1 mM) or mexiletine (F: 100 μ-1 mM) were co-administered with sodium oxalate (500 μM) to cells. Intracellular Ca2+ levels were determined based on Fura-2 fluorescence (340 nm/380 nm). Values are expressed as the mean of 4-8 wells.
Figure 3
NFAT nuclear translocation in primary cultured DRG cells. Oxaliplatin (A, B: 500 μM for 1-6 h) or sodium oxalate (C, D: 500 μM for 1-6 h) was administered to cultured DRG cells. E, F: Mexiletine (Mex, 1 mM), nifedipine (Nif, 30 μM), diltiazem (Dil, 30 μM) or vivit (2 μM) was co-administered with sodium oxalate (500 μM) to cells for 6 h. NFATc4 immunostaining (green) and nuclear staining with DAPI (blue). NFATc4 and DAPI-positive nuclei were visualized by fluorescence microscopy (A, C, E). The nuclear translocation of NFATc4 was calculated by comparing the ratio of nuclear NFATc4 immunofluorescence/total NFATc4 immunofluorescence (B, D, F). Values are expressed as the mean ± SEM of 24-33 cells. ††p < 0.01 compared with control group, **p < 0.01 compared with oxalate group.
Figure 4
Reversal of the oxalate-induced increase in TRPM8 mRNA in cultured DRG cells by Ca2+ and Na+ channel blockers. Mexiletine (Mex, 1 mM), nifedipine (Nif, 30 μM), diltiazem (Dil, 30 μM) or vivit (2 μM) was co-administered with sodium oxalate (500 μM) to cells for 12 h. The mRNA expression of TRPM8 and G3PDH were determined by PCR. Values are expressed as the mean ± SEM of 6 wells. ††p < 0.01 compared with control group, *p < 0.05, **p < 0.01 compared with oxalate group.
Figure 5
Reversal of the oxaliplatin-induced cold hyperalgesia in rats by Ca2+ and Na+ channel blockers. Oxaliplatin (4 mg/kg) was administered i.p. on days 1 and 2. Nifedipine (A: 10 and 30 mg/kg), diltiazem (B: 10 and 30 mg/kg), ethosuximide (C: 100 and 300 mg/kg) or mexiletine (D: 3-30 mg/kg) was orally co-administered with oxaliplatin. Acetone test was performed on days 0, 3, 5, 8 and 15. Values are expressed as the mean ± SEM of 6-10 animals. ††p < 0.01 compared with vehicle group, *p < 0.05, **p < 0.01 compared with oxaliplatin group.
Figure 6
Reversal of the oxaliplatin-induced increase of TRPM8 mRNA in rat DRG neurons by Ca2+ and Na+ channel blockers. Oxaliplatin (4 mg/kg) was administered i.p. on days 1 and 2. Mexiletine (Mex, 30 mg/kg), nifedipine (Nif, 30 mg/kg) or diltiazem (Dil, 30 mg/kg) was orally co-administered with oxaliplatin. The expression of TRPM8 and G3PDH mRNAs were determined by PCR on day 5. Values are expressed as the mean ± SEM of 5 animals. ††p < 0.01 compared with vehicle group, **p < 0.01 compared with oxaliplatin group.
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