In vitro neurotoxicity by ropivacaine is reduced by silencing Cav3.3 T-type calcium subunits in neonatal rat sensory neurons

Abstract

Neurotoxicity of local anaesthetics has been alerted by more and more peoples. Cav3.1 and Cav3.2 T-type calcium channels were closely related with local anaesthetics toxicity. However, the role of Cav3.3, another subtype of the T-type calcium channel, on the neurotoxicity induced by local anaesthetics remains unclear. CaMKII_γ is a kind of multifunctional kinase and associated with a variety of physiological and pathological process. T-type calcium channel is closely related with CaMKII_γ. Up-regulation CaMKII_γ can increase T-type currents at the dorsal root ganglia (DRG). On the contrary, down-regulation results in the T-type currents decrease. Is the relation between Cav3.3 T-type channel calcium and CaMKII_γ involved with the ropivacaine hydrochloride neurotoxicity? In this study, we generated pAd-Cav3.3 and pAd-shRNA adenovirus vector to up-regulate and down-regulate Cav3.3 mRNA expression of the DRG. The cells treated or untreated with ropivacaine hydrochloride (3 mM) for 4 h were used to evaluate the neurotoxicity. Cell viability, cell death rate and apoptosis rate, Cav3.3 and CaMKII_γ expression were detected with MTT method, Hoechst–PI, flow cytometry, qRT-PCR and western blotting. Results showed that the cell viability of the DRG treated with ropivacaine hydrochloride markedly decreased, death rate and apoptosis rate, Cav3.3 and CaMKII_γ mRNA and protein expression significantly increased. Cav3.3 overexpression aggravated DRG injury induced by ropivacaine hydrochloride and inhibition of Cav3.3 expression improved the cell damages. Cav3.3 can regulate CaMKII_γ mRNA and protein expression. In conclusion, Cav3.3 regulated CaMKII_γ in DRG, which was involved with the cell injury induced by ropivacaine hydrochloride.

Keywords:

• Local anaesthetics
• neurotoxicity
• DRG
• Cav3.3
• ropivacaine hydrochloride

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by the National Natural Science Foundation of China (Nos. 81471278, 81100831) and the Medical Scientific Research Projects of Foshan City, Guangdong Province (No. 2014AB00275).