Background and Goal of Study: Local anesthetics have been shown to be neurotoxic even at normal clinical dose. Recently, we reported that bupivacaine induced neuronal apoptosis via blocking T-type Ca2+ channels, but the detailed mechanisms are still not clear. Since mitochondria play key roles in activating apoptosis in mammalian cells, we investigated the role of mitochondria in apoptosis induced by bupivacaine via blocking T-type Ca2+ channels. Materials and Methods: Human neuroblastoma SH-SY5Y cells were treated with bupivacaine, NNC550396 (T-type Ca2+ channel inhibitor), EGTA (a divalent cation chelater) and CaCl2, then activation of caspase-3 was determined. In order to further illustrate whether mitochondria contribute to apoptosis, alteration of mitochondrial membrane potential (MMP) and production of intracellular reactive oxygen species (ROS) in bupivacaine-treated cells were also analyzed by flow cytometry. Results and Discussion: Bupivacaine activated caspase-3 in a time-dependent manner. EGTA further enhanced bupivacaine-induced caspase-3 activation at 16 h after treatment. CaCl2, however, significantly decreased bupivacaine-induced caspase-3 activation. NNC55-0396 not only activated caspase-3 in a timedependent manner but also significantly enhanced bupivacaine-induced activation of caspase-3. At 24 h after treatment, bupivacaine significantly reduced MMP. Bupivacaine-induced reduction in MMP was greatly increased by NNC55-0396 and EGTA, but significantly inhibited by CaCl2. Both bupivacaine and NNC550396 significantly increased level of intracellular ROS. Furthermore, a combination of bupivacaine and NNC55-0396 greatly increased ROS production. Compared to EGTA, which enhanced the bupivacaineinduced ROS production at 16 h after treatment, CaCl2 significantly inhibited such change.  In SH-SY5Y cells, bupivacaine reduced MMP and increased intracellular ROS production, resulting in activation of caspase-3. Both NNC55-0396 and EGTA greatly enhanced bupivacaine-induced MMP depolarization, intracellular ROS accumulation and caspase-3 activation. However, CaCl2 significantly suppressed these changes induced by bupivacaine.
Conclusion(s): Mitochondrial dysfunction is critically involved in bupivacaine-induced apoptosis. Bupivacaine may induce cell death via blocking the T-type Ca2+ channel and resulting activation of mitochondrial apoptotic pathway.

10 / Bupivacaine induces apoptosis via blocking T-type Ca2+ channels and resulting activation of mitochondrial apoptotic pathway in SH-SY5Y cells