Transient receptor potential A1 is a sensory receptor for multiple products of oxidative stress

Abstract

Transient receptor potential A1 (TRPA1) is expressed in a subset of nociceptive sensory neurons where it acts as a sensor for environmental irritants, including acrolein, and some pungent plant ingredients such as allyl isothiocyanate and cinnamaldehyde. These exogenous compounds activate TRPA1 by covalent modification of cysteine residues. We have used electrophysiological methods and measurements of intracellular calcium concentration ([Ca(2+)](i)) to show that TRPA1 is activated by several classes of endogenous thiol-reactive molecules. TRPA1 was activated by hydrogen peroxide (H(2)O(2); EC(50), 230 microM), by endogenously occurring alkenyl aldehydes (EC(50): 4-hydroxynonenal 19.9 microM, 4-oxo-nonenal 1.9 microM, 4-hydroxyhexenal 38.9 microM) and by the cyclopentenone prostaglandin, 15-deoxy-delta(12,14)-prostaglandin J(2) (15d-PGJ(2), EC(50): 5.6 microM). The effect of H(2)O(2) was reversed by treatment with dithiothreitol indicating that H(2)O(2) acts by promoting the formation of disulfide bonds whereas the actions of the alkenyl aldehydes and 15d-PGJ(2) were not reversed, suggesting that these agents form Michael adducts. H(2)O(2) and the naturally occurring alkenyl aldehydes and 15d-PGJ(2) acted on a subset of isolated rat and mouse sensory neurons [approximately 25% of rat dorsal root ganglion (DRG) and approximately 50% of nodose ganglion neurons] to evoke a depolarizing inward current and an increase in [Ca(2+)](i) in TRPA1 expressing neurons. The abilities of H(2)O(2), alkenyl aldehydes and 15d-PGJ(2) to raise [Ca(2+)](i) in mouse DRG neurons were greatly reduced in neurons from trpa1(-/-) mice. Furthermore, intraplantar injection of either H(2)O(2) or 15d-PGJ2 evoked a nocifensive/pain response in wild-type mice, but not in trpa1(-/-) mice. These data demonstrate that multiple agents produced during episodes of oxidative stress can activate TRPA1 expressed in sensory neurons.

Figure 1.

H₂O₂ activates TRPA1 expressed in CHO cells. A, H₂O₂ activates TRPA1 with a concentration-dependent latency in Ca²⁺-containing solutions. Currents recorded in calcium- containing solution showing characteristic “threshold” with a sudden increase in membrane current. B, Ca²⁺ potentiates H₂O₂-induced TRPA1 currents. Current response to 10 mm H₂O₂ in a TRPA1 CHO cell, −60 mV. Note the slow increase in current in calcium-free solution followed by a rapid current increase when Ca²⁺ (2 mm) is added. C, Current–voltage relationship of H₂O₂-evoked current with 2 s voltage ramp in a TRPA1 CHO cell in calcium-free solution revealed a reduced current at positive potentials. D, Kinetics of H₂O₂-evoked TRPA1 current in calcium free solution. Note the time and voltage-dependent inactivation at more positive potentials that accounts for reduced conductance seen with voltage ramp protocols (C) (Figs. 2C, 4C). Holding potential −60 mV with 20 mV interval steps to from −80 to +180 mV.

Figure 2.

Single-channel activity evoked by H₂O₂. Cell attached TRPA1 single-channel currents. A, Few brief openings seen in the absence of H₂O₂ (top trace), but robust channel activity elicited by 1 mm H₂O₂ (lower trace) at −100 mV. B, Voltage-dependent single-channel current activity showing inactivation at positive membrane potentials. Holding potential, −60 mV; traces are offset for clarity. C, Voltage ramp illustrating single-channel inactivation at positive membrane potentials (the trace shown is the average of 5 sweeps).

Figure 3.

Concentration-dependent effect of H₂O₂ on TRPA1. A, Concentration-dependent time course of [Ca²⁺]_i-responses to stimulation with H₂O₂ in CHO cells expressing TRPA1. Traces are mean ratios from quadruplicate wells. B, Concentration–response curves constructed from the experiment shown in (A) 90 and 600 s after addition of H₂O₂ (mean ± SEM). C, The time required for half-maximal activation (T_1/2) is concentration dependent (data points are mean ± SEM of 4 measurements). D, Fe²⁺ potentiates the effect of H₂O₂, suggesting that H₂O₂ acts via intracellular production of hydroxyl radicals. Concentration–response curves for H₂O₂-evoked increase in [Ca²⁺]_i in normal and Fe²⁺-loaded TRPA1 cells are shown. Fe²⁺-loaded cells were incubated with 100 μm FeSO₄ for 1 h and then washed so that no extracellular FeSO₄ was present during the experiment (mean ± SEM; n = 4).

Figure 4.

H₂O₂ activates TRPA1-containing DRG and nodose neurons. A, Pseudocolored images illustrating [Ca²⁺]_i responses evoked by H₂O₂ (5 mm) measured with Fura-2 in rat DRG neurons. B, Currents evoked by H₂O₂ (1 mm) in DRG neurons in the presence (left) and absence (right) of extracellular calcium (at a holding potential of −60 mV). The current rapidly inactivated when Ca²⁺ was applied after an initial current has developed in Ca²⁺-free conditions. C, Current–voltage plot for the H₂O₂ response in a DRG neuron generated by a slow 2 s voltage ramp in Ca²⁺-free solution. Note the characteristic reduced conductance at positive membrane potential. D, Change in [Ca²⁺]_i (340/380 ratio) of typical DRG neurons in response to sequential applications of H₂O₂, AITC, and capsaicin (Caps) showing H₂O₂-sensitive and H₂O₂-insensitive TRPV1 expressing neurons. E, [Ca²⁺]_i responses in DRG (top sequence) and nodose neurons (bottom sequence) to sequential application of H₂O₂ (5 mm), AITC (50 μm), and capsaicin (1 μm). All neurons in the culture were identified by the [Ca²⁺]_i increase elicited by application of 50 mm K⁺. H₂O₂, AITC and capsaicin stimulated a larger proportion of neurons dissociated from nodose than dorsal root ganglia. The number of neurons tested in each group was between 363 and 744.

Figure 5.

Lipid peroxidation products and 15d-PGJ₂ are TRPA1 agonists. A, [Ca²⁺]_i increases evoked by 4-ONE (3 μm), 4-HNE (30 μm), and 15d-PGJ₂ (20 μm) in DRG neurons. Sequential applications of AITC and capsaicin (Caps) show that 4-ONE, 4-HNE, and 15d-PGJ₂ activate the same subset of TRPV1-expressing, capsaicin-sensitive neurons as AITC. B, Left, 4-HNE-evoked current in a TRPA1 CHO cell initially in calcium-free solution. Admission of calcium led to rapid inactivation. Right, Current–voltage relationship of 4-HNE-evoked current. C, Left, 4-HNE-evoked current in a DRG neuron (external solution containing 15 μm Ca²⁺, −60 mV). Right, Current–voltage relationship of the 4-HNE-evoked current in the same neuron. D, Time course (left) and voltage-dependent kinetics (right) for 15d-PGJ₂-evoked TRPA1 current in a CHO cell. Right, Current–voltage relationship of 15d-PGJ₂-evoked current.

Figure 6.

Concentration dependence of lipid mediators and dithiothreitol sensitivity. A, Concentration–response curves for 4-ONE, 4-HNE, 4-HHE, and 15d-PGJ₂ in TRPA1 CHO cells (mean ± SEM, n = 4, representative of at least 3 experiments). B, Application of DTT reverse [Ca²⁺]_i-responses induced by application of H₂O₂, but not 4-HNE or 15d-PGJ₂.

Figure 7.

H₂O₂, 4-ONE and 15d-PGJ₂ activate TRPA1 in a membrane-delimited manner. A–C, Application of 5 mm H₂O₂ (A), 5 μm 4-ONE (B) and 20 μm 15d-PGJ₂ (C) evoke single-channel activity in excised inside-out patches (−100 mV, each trace is representative of at least 3 experiments).

Figure 8.

H₂O₂ and 15d-PGJ₂ induce pain-related behavior in vivo by activating TRPA1. A, B, Duration of nocifensive (licking/flinching) behavior in wild-type mice evoked by intraplantar hindpaw injections of H₂O₂ in saline (A) or 15d-PGJ₂ in saline containing 10% DMSO (B). The injection volume was 25 μl. Pain-related behavior was recorded over 5 min (mean ± SEM, n = 6 for each group). C, D, Wild-type (+/+) and TRPA1-deficient mice (−/−) were injected in the hind paw with H₂O₂ (C) (2.2 μmol/25 μl) or 15d-PGJ₂ (D) (32 nmol/25 μl). Pain-related behavior (licking, biting, flinching, or shaking of the injected paw) was recorded for 3 min after injection. The nocifensive responses induced by H₂O₂ and 15d-PGJ₂ were dramatically reduced or absent in mice lacking TRPA1 (mean ± SEM; n = 6 in each group; **p < 0.01).