Activation and desensitization of TRPV1 channels in sensory neurons by the PPARα agonist palmitoylethanolamide

Abstract

Background and purpose: Palmitoylethanolamide (PEA) is an endogenous fatty acid amide displaying anti-inflammatory and analgesic actions. To investigate the molecular mechanism responsible for these effects, the ability of PEA and of pain-inducing stimuli such as capsaicin (CAP) or bradykinin (BK) to influence intracellular calcium concentrations ([Ca²⁺](i)) in peripheral sensory neurons, has been assessed in the present study. The potential involvement of the transcription factor PPARα and of TRPV1 channels in PEA-induced effects was also studied.

Experimental approach: [Ca²⁺](i) was evaluated by single-cell microfluorimetry in differentiated F11 cells. Activation of TRPV1 channels was assessed by imaging and patch-clamp techniques in CHO cells transiently-transfected with rat TRPV1 cDNA.

Key results: In F11 cells, PEA (1-30 μM) dose-dependently increased [Ca²⁺](i). The TRPV1 antagonists capsazepine (1 μM) and SB-366791 (1 μM), as well as the PPARα antagonist GW-6471 (10 μM), inhibited PEA-induced [Ca²⁺](i) increase; blockers of cannabinoid receptors were ineffective. PEA activated TRPV1 channels heterologously expressed in CHO cells; this effect appeared to be mediated at least in part by PPARα. When compared with CAP, PEA showed similar potency and lower efficacy, and caused stronger TRPV1 currents desensitization. Sub-effective PEA concentrations, closer to those found in vivo, counteracted CAP- and BK-induced [Ca²⁺](i) transients, as well as CAP-induced TRPV1 activation.

Conclusions and implications: Activation of PPARα and TRPV1 channels, rather than of cannabinoid receptors, largely mediate PEA-induced [Ca²⁺](i) transients in sensory neurons. Differential TRPV1 activation and desensitization by CAP and PEA might contribute to their distinct pharmacological profile, possibly translating into potentially relevant clinical differences.

Figure 1

Concentration-dependent increase in [Ca²⁺]_i elicited by PEA in differentiated F11 cells. (A) Representative traces showing the effect of PEA (0.5–10 μM) on [Ca²⁺]_i in differentiated F11 cells. Each trace is from a single cell, representative of the entire population (n = 12–133 cells in at least three separate experimental sessions). In this and following panels and figures, the bar on the top or at the bottom of each trace corresponds to the duration of drug exposure. (B) Concentration-dependent effect of PEA. Peak [Ca²⁺]_i values recorded in different cells after exposure to 0.5–30 μM PEA were expressed as percent of increase versus basal levels; the solid line represents the fit of the normalized data to a standard binding equation of the following form: y = max/(1 + x/EC₅₀)ⁿ, where x is the drug concentration and n the Hill coefficient. Fitted values for n were 1.8 ± 0.1. (C) A representative trace showing the effect on [Ca²⁺]_i of two successive 30 s exposures to PEA (10 μM), separated by a 15 min interval. (D) Quantification of the effects of a Ca²⁺-free extracellular solution, of the exposure to a pharmacological cocktail containing 10 μM NIM, 1 μM ω-CTX and 150 nM ω-AGA, or of 10 μM NIM, 1 μM ω-CTX, or 150 nM ω-AGA, applied separately on PEA-induced [Ca²⁺]_i. Data are expressed as percent of [Ca²⁺]_i increase induced by 10 μM PEA in standard Ca²⁺-containing solution. Each point is the mean±SEM of 20–32 separate determinations performed in at least three experimental sessions. In this and following figures, asterisks denote values statistically different from controls (P < 0.05).

Figure 2

Effect of CB₁R and CB₂R antagonists on WIN- and PEA-induced [Ca²⁺]_i increases in differentiated F11 cells. (A) Quantification of the effect of the CB₁R-antagonist SR1 (500 nM), or of the CB₂R-antagonist SR2 (500 nM), as well as of a Ca²⁺-free extracellular solution, on [Ca²⁺]_i changes induced by the non-selective CBR agonist WIN 55 212-2 (WIN, 500 nM), or by PEA (10 μM). Data are expressed as percent of [Ca²⁺]_i increase relative to respective controls (500 nM WIN or 10 μM PEA). Each point is the mean±SEM of 10–19 separate determinations performed in at least three experimental sessions. (B) pERK1/2 immunofluorescence in F11 cells in control conditions (left panel), or exposed for 10 min to PEA (10 μM; middle panel) or to WIN55,212 (500 nM; right panel). The scale bar is 10 μm. The rightmost panel shows the quantification of the data, expressed as arbitrary units (A. U.) of fluorescence intensity. Each bar is the mean of at least 4 fields from cells analysed in at least three different experimental sessions.

Figure 3

Effect of CAP on [Ca²⁺]_i in differentiated F11 cells. (A) Superimposed representative traces showing the effect on [Ca²⁺]_i of a single exposure to 50 μM CAP or to 50 μM CAP + 1 μM CPZ. (B) Concentration-dependent effect of CAP. Peak [Ca²⁺]_i values recorded after exposure to 0.1–50 μM CAP were expressed as percent of increase versus basal levels; the solid line represents the fit of the normalized data to a standard binding equation of the following form: y = max/(1 + (x/EC₅₀)ⁿ, where x is the drug concentration and n the Hill coefficient. Fitted values for n were 1.1 ± 0.4. Each point is the mean±SEM of 7–42 separate determinations performed in at least three experimental sessions. (C) Quantification of the effects of TRPV1 antagonists (CPZ and SB-366791; each at 1 μM) on CAP- or PEA-induced [Ca²⁺]_i responses; data are expressed as percent of [Ca²⁺]_i increase relative to respective controls (50 μM CAP or 10 μM PEA). Each point is the mean±SEM of 19–21 separate determinations performed in at least 3 experimental sessions. (D) Superimposed representative traces showing the effect on [Ca²⁺]_i of a single exposure to 50 μM CAP in control conditions, or after 10 min pre-incubation with 0.5 μM PEA. Each trace is from a single cell, representative of the entire population (n = 35 for each experimental condition).

Figure 4

Regulation of [Ca²⁺]_i by PPARα ligands. Representative traces showing the effect of 10 μM GW-6417 on CLO (1 mM)- (A) and PEA (10 μM)-induced (B) [Ca²⁺]_i changes in differentiated F11 cells. The duration of GW-6417 exposure is indicated by the bar on top of the traces. (C) Quantification of the effects of 10 μM GW-6471, of a Ca²⁺-free extracellular solution, and of 1 μM CPZ on CLO-, GW-7647, CAP, and PEA-induced [Ca²⁺]_i increase. Data are expressed as percent of [Ca²⁺]_i increase relative to their respective controls (1 mM CLO; 10 μM GW-7647; 50 μM CAP; 10 μM PEA;). Each point is the mean±S.E.M. of 10–34 separate determinations performed in at least 3 experimental sessions.

Figure 5

Effect of BK on [Ca²⁺]_i in differentiated F11 cells. Representative traces showing the effect of three subsequent exposures to BK (250 nM) on [Ca²⁺]_i in differentiated F11 cells obtained in control conditions (A) or after exposure to 0.5 μM PEA 10 min before and during the second BK exposure (B). (C) Quantification of the effects of 1 μM CPZ, of a Ca²⁺-free extracellular solution, and of 0.5 μM PEA on BK-induced [Ca²⁺]_i increase (second BK exposure), and of drug washout during the third BK exposure. Data are expressed as percent of [Ca²⁺]_i increase relative to controls (250 BK in the first pulse). Each point is the mean±SEM of 20–34 separate determinations performed in at least three experimental sessions.

Figure 6

Activation of TRPV1 channels by PEA in TRPV1-transfected CHO cells. (A) Representative images showing PEA- (10 μM) and CAP- (5 μM) induced [Ca²⁺]_i changes in CHO cells transiently-transfected with TRPV1 cDNA. To identify transfected cells, EGFP fluorescence was also monitored in the same microscopic field. Note that significant drug-induced [Ca²⁺]_i changes only occurred in transfected (EGFP-positive) and not in non-transfeced (EGFP-negative) CHO cells. (B) Representative whole-cell inward currents from a TRPV1-transfected cell held at −60 mV and exposed to 0.1–5 μM CAP, as indicated. (C) Representative whole-cell inward currents from a TRPV1-transfected cell held at −60 mV and exposed to 0.1–30 μM PEA, as indicated. (D) Quantitative comparison of TRPV1 activation by CAP and PEA. Peak current data were expressed as pA/pF (to facilitate comparison among cells of different sizes), and expressed as a function of agonist concentrations. The solid lines represent fits of the experimental data to the following binding isotherm: y = max/(1 + x/EC₅₀)ⁿ, where x is the drug concentration and n the Hill coefficient. The fitted values for n were 1.6 ± 0.6 for CAP, and 0.9 ± 0.1 for PEA. Each point is the mean±SEM. of 6–26 (for CAP) or 9–23 (for PEA) determinations. (E) Effect of the removal of Ca²⁺_e on 1 μM CAP- or 10 μM PEA-induced peak inward current at −60 mV. Each point is the mean±SEM of 12–15 determinations.

Figure 7

PPARα-mediated activation of TRPV1 channels by CLO and PEA, but not by CAP. Representative whole-cell current traces evoked in the same TRPV1-expressing CHO cell showing the effect of the PPARα antagonist GW-6471 (10 μM) on TRPV1 currents elicited by CAP (5 μM) (A), CLO (100 μM) (B), and PEA (10 μM) (C). CHO cells were held at −60 mV; the bars on top of each panel represent the duration of GW-6471 exposure; in each panel, the arrow indicates the addition of the agonist (exposure time: about 3 s). (D) Quantification of the effects of GW-6471 (10 μM) and of CPZ (3 μM) on CAP-, CLO-, and PEA-evoked peak TRPV1 currents. Each point is the mean±S.E.M. of 8–16 determinations for each agonist.

Figure 8

Desensitization of TRPV1-mediated currents by CAP and PEA. (A, B) Representative whole-cell current traces evoked in the same TRPV1-expressing CHO cell upon 30 s exposure to 1 μM CAP (A) or to 10 μM PEA (B), followed by drug washout; currents were recorded using a voltage ramp protocol (from −80 to +80 mV; 1.6 mV/ms). Plotted traces are: control (no agonist), peak of agonist-evoked currents, plateau of agonist-evoked currents (after 30 s of agonists exposure) and washout, as indicated. (C) Representative whole-cell inward currents evoked in TRPV1-expressing CHO cells held at −60 mV during 30 s exposures to 1 μM CAP or to 10 μM PEA, as indicated. The percentage of the current desensitized during exposure to each agonist is indicated. The bars on the top of each trace correspond to the duration of agonist exposure.

Figure 9

Exposure to sub-effective PEA concentrations reduce CAP-induced TRPV1 currents. Representative whole-cell current traces recorded in TRPV1-expressing CHO cells held at −60 mV in response to two subsequent 3 s exposures to 0.5 μM CAP, separated by a 1 min interval. The second CAP exposure was performed in control condition (n = 4; A), or upon pre-incubation with 0.01 μM PEA for 1 min (n = 12; B). The bars on the top of each trace correspond to the duration of drug exposures. Each trace is from a single cell, representative of the entire population. The percentage of current decrease induced by PEA exposure is indicated.