The Effects And Mechanisms Of Formaldehyde And Menthol On TRPA1 And CGRP Expression In DRG Neurons

Transient receptor potential ankyrin 1 (TRPA1), a member of the transient receptor potential (TRP) family of ion channels expressed in nociceptive neurons, is activated by several reactive chemicals. Recently, it has been shown that formaldehyde (formalde) could activate TRPA1, whereas menthol could inhibit TRPA1. Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide which expressed in primary sensory neurons. However whether formalde or menthol affects TRPA1 and CGRP expression in primary dorsal root ganglion (DRG) sensory neurons remains unknown. In the present study, primary cultured newborn rat DRG neurons were used to assess the effects of formalde and menthol on TRPA1 and CGRP expression. The extracellular signal-regulated protein kinase (ERK1/2) signaling pathway involved in these effects was also determined. Furthermore, the interactions of the expression of TRPA1 and TRPV1 in DRG neurons in situ was investigated. At 48 hours of culture age, DRG neurons for TRPA1 and CGRP expression study, primary cultured DRG neurons were pretreated with TRPV1 receptor antagonist capsazepine (CPZ) (10μmol/L) for 30 minutes. After that, the DRG neuronal cultures were exposed to the following agents for an additional 24 hours:(1) formalde (10 μmol/L); (2) menthol (300 μmol/L); (3) ERK1/2 inhibitor PD98059 (10 μmol/L) 30 minutes before treatment with formalde (10 μmol/L); (4) ERK1/2 inhibitor PD98059 (10 μmol/L) 30 minutes before treatment with menthol (300 μmol/L); (5) The DRG neurons only treated with TRPV1 receptor antagonist CPZ (10 μmol/L) served as a control. After that, all above cultured DRG neurons were processed for real-time PCR analysis on the expression of TRPA1 mRNA and CGRP mRNA and Western blot assay on TRPA1 and CGRP protein levels and double fluorescent labeling of TRPA1 or CGRP and microtubule associated protein 2 (MAP2) on TRPA1 and CGRP expression in situ. At 48 hours of culture age, DRG neurons for TRPA1 and TRPV1 co-expression study were exposed to the following agents for an additional 24 hours: (1) formalde (10 μmol/L); (2) menthol (300 μmol/L); (3) ERK1/2 inhibitor PD98059 (10 μmol/L) 30 minutes before treatment with formalde (10 μmol/L); (4) ERK1/2 inhibitor PD98059 (10 μmol/L) 30 minutes before treatment with menthol (300 μmol/L); (5) The DRG neurons continuously cultured in culture medium served as a control. The results showed that formalde administration increased the TRPA1 mRNA and CGRP mRNA levels, TRPA1 and CGRP protein levels, and the percentage of TRPA1-positive and CGRP-positive neurons. Menthol incubation decreased TRPA1 mRNA and CGRP mRNA levels, TRPA1 and CGRP protein levels, and the percentage of TRPA1-positive and CGRP-positive neurons. The effects of formalde, but not menthol, on CGRP expression alterations could be blocked by treatment with ERK1/2 inhibitor PD98059. PD98059 did not affect TRPA1 expression in the presence of formalde or menthol. TRPA1 and TRPV1 were highly co-expressed in a subset of DRG neurons. Formalde could elevate the proportion of TRPA1-positive neurons in TRPV1-positive neurons. Menthol could decrease the proportion of TRPA1-positive neurons in TRPV1-positive neurons. These findings provide experimental evidence for further study the significance of the functions of TRPA1 and CGRP alterations during nociceptive processing.