TRPV1 stimulation triggers apoptotic cell death of rat cortical neurons

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Abstract

Transient receptor potential vanilloid 1 (TRPV1) functions as a polymodal nociceptor and is activated by several vanilloids, including capsaicin, protons and heat. Although TRPV1 channels are widely distributed in the brain, their roles remain unclear. Here, we investigated the roles of TRPV1 in cytotoxic processes using TRPV1-expressing cultured rat cortical neurons. Capsaicin induced severe neuronal death with apoptotic features, which was completely inhibited by the TRPV1 antagonist capsazepine and was dependent on extracellular Ca2+ influx. Interestingly, nifedipine, a specific L-type Ca2+ channel blocker, attenuated capsaicin cytotoxicity, even when applied 2–4 h after the capsaicin. ERK inhibitor PD98059 and several antioxidants, but not the JNK and p38 inhibitors, attenuated capsaicin cytotoxicity. Together, these data indicate that TRPV1 activation triggers apoptotic cell death of rat cortical cultures via L-type Ca2+ channel opening, Ca2+ influx, ERK phosphorylation, and reactive oxygen species production.

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Materials and methods

Preparation of neuron-rich cultures from rat cerebral cortex. All animals used in the present study were treated in accordance with the guidelines of the Kyoto University Animal Experimentation Committee and the Japanese Pharmacological Society. Primary cultures of dissociated cortical neurons were prepared from the cerebral cortex of fetal Wistar rats (18 days of gestation), according to procedures described previously [13], [14]. Briefly, single cells mechanically dissociated from the

Functional expression of TRPV1 in rat cortical cultures

We examined whether cultured rat cortical cells expressed functional TRPV1. RT-PCR and Western blot analyses revealed that TRPV1 is expressed at the mRNA and protein level after 11-13 days in culture (Fig. 1A and B). Furthermore, calcium imaging with fura-2 AM demonstrated that a TRPV1-specific agonist, capsaicin (10 μM), can induce an increase in ([Ca2+]i) that was markedly attenuated by a TRPV1-specific antagonist, capsazepine (10 μM). This suggests that TRPV1 is activated in response to

Discussion

Here, we found that overstimulation of TRPV1 in rat cortical neurons led to apoptotic cell death via ERK phosphorylation, L-type VDCC opening, ROS production and caspase-3 activation, which agrees with previous studies showing that capsaicin can induce apoptotic cell death in rat mesencephalic dopamine neurons [11], [12].

We found that NADA, which is an endogenous TRPV1 agonist, concentration-dependently exert a neurotoxic effect on cortical neurons. An antagonist of TRPV1 largely inhibited

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