Paclitaxel alters the evoked release of calcitonin gene-related peptide from rat sensory neurons in culture

Abstract

Peripheral neuropathy (PN) is a debilitating and dose-limiting side effect of treatment with the chemotherapeutic agent, paclitaxel. Understanding the effects of paclitaxel on sensory neuronal function and the signaling pathways which mediate these paclitaxel-induced changes in function are critical for the development of therapies to prevent or alleviate the PN. The effects of long-term administration of paclitaxel on the function of sensory neurons grown in culture, using the release of the neuropeptide calcitonin gene-related peptide (CGRP) as an endpoint of sensory neuronal function, were examined. Dorsal root ganglion cultures were treated with low (10 nM) and high (300 nM) concentrations of paclitaxel for 1, 3, or 5 days. Following paclitaxel treatment, the release of CGRP was determined using capsaicin, a TRPV1 agonist; allyl isothiocyanate (AITC), a TRPA1 agonist; or high extracellular potassium. The effects of paclitaxel on the release of CGRP were stimulant-, concentration-, and time-dependent. When neurons were stimulated with capsaicin or AITC, a low concentration of paclitaxel (10nM) augmented transmitter release, whereas a high concentration (300 nM) reduced transmitter release in a time-dependent manner; however, when high extracellular potassium was used as the evoking stimulus, all concentrations of paclitaxel augmented CGRP release from sensory neurons. These results suggest that paclitaxel alters the function of sensory neurons in vitro, and suggest that the mechanisms by which paclitaxel alters neuronal function may include functional changes in TRP channel activity. The described in vitro model will facilitate future studies to identify the signaling pathways by which paclitaxel alters neuronal sensitivity.

Keywords: CGRP; Chemotherapy-induced peripheral neuropathy; Dorsal root ganglion culture; Neuropeptide release; Neurotoxicity; Paclitaxel; Peripheral sensory neuron; TRPA1; TRPV1.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

Figure 1

Paclitaxel alters capsaicin-evoked release of CGRP from sensory neurons in culture. A) Each column represents the mean ± SEM of CGRP released in fmol/well/10 min from wells treated with 10 nM or 300 nM paclitaxel for 5 days. The first open column of each group represents basal release (B), the shaded column represents release in the presence of 30 nM capsaicin (C), and the second open column of each group represents the recovery of basal release following stimulation (B). An asterisk indicates a significant difference in capsaicin-evoked release compared to release from the vehicle-treated neurons (p

Figure 2

Paclitaxel alters AITC-evoked release of CGRP from sensory neurons in culture. A) Each column represents the mean ± SEM of CGRP released in fmol/well/10 min from wells treated with 10 nM or 300 nM paclitaxel for 5 days. The first open column of each group represents basal release (B), the shaded column represents release in the presence of 30 nM AITC (A), and the second open column of each group represents the recovery of basal release following stimulation (B). An asterisk indicates a significant difference in AITC-evoked release compared to release from the vehicle-treated neurons (p–12) using a two-way ANOVA with Bonferroni’s post-hoc test. B) Each column is the mean ± SEM of total CGRP content in fmol/well (N=10–12) from vehicle treated cultures (Veh) or paclitaxel treated cultures (Pac) as indicated.

Figure 3

Paclitaxel augments potassium-evoked release of CGRP from sensory neurons in culture. A) Each column represents the mean ± SEM of CGRP released in fmol/well/10 min from wells treated with 10 nM or 300 nM paclitaxel for 5 days. The first open column of each group represents basal release (B), the shaded column represents release in the presence of 50 mM KCl (HK), and the second open column of each group represents the recovery of basal release following stimulation (B). An asterisk indicates a significant difference in HK-evoked release compared to release from the vehicle-treated neurons (p

Figure 4

Paclitaxel does not decrease the survival of sensory neurons in culture. Sensory neuronal cultures were treated with vehicle (open columns, Veh) or with 10 nM or 300 nM paclitaxel for 5 days (Pac; shaded columns) and then cell viability was measured. As a positive control, vehicle-treated cultures were exposed to H2O2 (300 μM) for 1 hour 24 hours prior to analysis of cell survival (shaded columns). The number of annexin V positive cells (A) and propidium iodine (PI) positive cells (B) were counted in a minimum of 5 fields from 3 different harvests and normalized to the total number of neurons in the field. Each column is the mean ± SEM of % of positively stained neurons. An asterisk indicates a significant difference from untreated controls (p<0.05, N=3) using a one way-ANOVA and Bonferroni’s post-hoc test.

Figure 5

Paclitaxel (10 nM) increases the release of CGRP from sensory neurons in culture in a time-dependent manner. A) Each column represents the mean ± SEM of CGRP released in fmol/well/10 min from wells treated with vehicle or 10 nM paclitaxel for 1, 3, or 5 days as indicated. The first open column of each group represents basal release (B), the shaded column represents release in the presence of 30 nM capsaicin (C), and the second open column of each group represents the recovery of basal release following stimulation (B). An asterisk indicates a significant difference in capsaicin-evoked release compared to release from the vehicle-treated neurons (p–12) using a two-way ANOVA with Bonferroni’s post-hoc test. B) Each column is the mean ± SEM of total CGRP content in fmol/well (N=10–12) from cultures treated with vehicle (Veh) or paclitaxel (Pac) for the duration of time as indicated.

Figure 6

Time-dependent effects of 300 nM paclitaxel on the release of CGRP from sensory neurons in culture. A) Each column represents the mean ± SEM of CGRP released in fmol/well/10 min from wells treated with vehicle or 300 nM paclitaxel for 1, 3, or 5 days as indicated. The first open column of each group represents basal release (B), the shaded column represents release in the presence of 30 nM capsaicin (C), and the second open column of each group represents the recovery of basal release following stimulation (B). An asterisk indicates a significant difference in capsaicin-evoked release compared to release from the vehicle-treated neurons (p–12) from cultures treated with vehicle (Veh) or paclitaxel (Pac) for the duration of time as indicated. An asterisk indicates a significant difference in the total content of CGRP compared to the vehicle-treated cultures (p<0.05, N=10–12) using a one way-ANOVA and Bonferroni’s post-hoc test.