LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels

Abstract

Background and purpose: Retinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ , is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels.

Experimental approach: We performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours.

Key results: LE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies.

Conclusions and implications: This blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels.

Keywords: LE135; TRPA1; TRPV1; mechanical allodynia; nociception; thermal hyperalgesia.

Figure 1

LE135 activates both recombinant and native TRPV1 channels. Representative images (A) and traces (B) illustrate that LE135-elicited intracellular Ca²⁺ responses are decreased in Trpv1^−/− DRG neurons compared with that from Trpv1^+/+ mice. AITC evoked similar intracellular Ca²⁺ responses in Trpv1^+/+ and Trpv1^−/− DRG neurons. Each trace corresponds to the change of fluorescence ratio in a single neuron in response to 20 μM LE135, 300 nM capsaicin (CAP), 100 μM AITC and 100 mM KCl at indicated times. (C) Percentage of DRG neurons responding to LE135, capsaicin, AITC and KCl in neurons isolated from Trpv1^+/+ or Trpv1^−/− mice (n ≥ 350 per genotype). Representative current–voltage relationships (D) and the time course (E) of LE135 (1 and 10 μM)-activated outward (at +60 mV) and inward (at −60 mV) currents in a TRPV1-expressing HEK293T cell. (F) Concentration–response curve of LE135-activated inward currents at −60 mV in WT TRPV1 is fitted with the logistic equation: Y = Y_min + (Y_max − Y_min)/(1 + 10∧[(log EC₅₀ − X)*Hill slope)], where Y is the response at a given concentration, Y_max and Y_min are the maximum and minimum response, X is the logarithmic value of the concentration and Hill slope is the slope factor of the curve. EC₅₀ is the concentration that gives a response halfway between Y_max and Y_min. The graph inset illustrates the maximal responses (current densities) evoked by saturating concentrations of LE135 (100 μM) and capsaicin (10 μM).

Figure 2

Structural basis of LE135 activation of TRPV1 channels. (A) Concentration–response curves of LE135-activated inward currents at −60 mV in WT and TRPV1 channel mutants with disrupted protein phosphorylation, proton or heat activation domains. (B) Concentration–response curves of LE135-activated inward currents at −60 mV in WT and TRPV1 mutants carrying single or double point mutations in the vanilloid-binding pocket. (C) Schematic diagram illustrates structural elements required for activation/modulation of TRPV1 channels by capsaicin (red filled circle), protein phosphorylation (green triangle), protons (orange diamond) and heat (purple star). (D) EC₅₀ values and Hill coefficients of LE135-activated response in WT and TRPV1 mutants derived from concentration–response curves in (A) and (B). **P < 0.01 and ***P < 0.001 versus WT. Concentration–response curves are fitted with the logistic equation as described in Figure 1.

Figure 3

TRPV1 channels mediate LE135-induced nocifensive responses. Bar chart illustrates that in contrast to vehicle, intraplantar injection of LE135 (100 nmol/20 μL) produced robust flinching and licking responses that were significantly reduced in Trpv1^−/− mice. LE135-evoked nocifensive responses were not significantly affected in Trpa1^−/− mice compared with WT mice. ***, ⁺⁺⁺P < 0.001 versus vehicle and Trpv1^−/− respectively; NS, not significant versus Trpa1^−/−. n = 6–10 animals per condition.

Figure 4

Pharmacological or genetic blockade of TRPV1 function abolishes LE135-induced thermal hyperalgesia. Time course of thermal hypersensitivity in animals treated with LE135. Intraplantar injection of LE135 (30 nmol/10 μL, red trace) induced thermal hyperalgesia in Trpv1^+/+ mice as reflected by a decrease in paw withdrawal latency. Administration of AMG9810 (10 mg kg⁻¹, i.p.,) removed the effect of LE135. LE135-elicited thermal hypersensitivity was also abolished in Trpv1^−/− mice. *** P < 0.001 versus vehicle; ⁺⁺⁺ P < 0.001 versus AMG9810; and ^### P < 0.001 versus Trpv1^−/−. Please note that no effect was observed upon injection of vehicle alone. n = 5–10 animals per condition.

Figure 5

Both TRPV1 and TRPA1 channels contribute to LE135-induced mechanical allodynia. Time course of mechanical allodynia in animals treated with LE135. Intraplantar injection of LE135 (30 nmol/10 μL) produced mechanical allodynia as reflected by a decrease in paw withdrawal threshold in Trpv1^+/+ mice, which was not significantly reduced by i.p. injection of AMG9810 (10 mg·kg⁻¹), or genetic deletion of TRPA1 or TRPV1 channels. However, LE135-elicited mechanical hypersensitivity was completely abolished in the Trpa1^−/− mice pre-treated with AMG9810 (10 mg·kg⁻¹). ***P < 0.001 versus LE135; ⁺⁺⁺P < 0.001 versus AMG9810; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus Trpv1^−/−; and ^$$P < 0.01, ^$$$P < 0.001 versus Trpa1^−/−.

Figure 6

LE135 directly activates TRPA1 channels. LE135 (1 and 10 μM) activated an outward current at +60 mV and an inward current at −60 mV in a TRPA1-expressing HEK293T cell. Current traces in (A) show representative current–voltage relationships of LE135-activated currents. The time course of the current is shown in (B). (C) Concentration–response curve of LE135-activated inward currents taken at −60 mV in TRPA1-expressing HEK293T cells is fitted with the logistic equation as described in Figure 1. The graph inset illustrates the maximal responses (current densities) evoked by saturating concentrations of LE135 (100 μM) and AITC (100 μM). Representative pictures (D) and traces (E) illustrate that LE135-elicited intracellular Ca²⁺ responses are abolished in Trpv1^−/− DRG neurons pre-treated with TRPA1 antagonist HC030031 (upper panel) or Trpa1^−/− DRG neurons pre-treated with TRPV1 antagonist AMG9810 (lower panel). Each trace corresponds to the change of fluorescence ratio in a single neuron. (F) Percentage of DRG neurons responding to LE135, capsaicin (CAP), AITC and KCl in neurons isolated from Trpv1^−/− or Trpa1^−/− mice (n ≥ 350 per genotype).

Figure 7

A single lysine residue is required for LE135 activation of TRPA1 Channels. Concentration–response curves of LE135-activated currents in WT and TRPA1 mutants carrying a single point mutation K710R or triple cysteine mutations (C621S, C641S, and C665S). EC₅₀ values of LE135-activated response in WT and TRPA1 mutants derived from concentration–response curves in (A) are indicated. Concentration–response curves are fitted with the logistic equation, as described in Figure 1. ND, not determined. The graph inset illustrates that the hTRPA1-K mutant channel was activated by a TRPA1 agonist, flufenamic acid (FFA; 300 μM).