Methylglyoxal evokes pain by stimulating TRPA1

Abstract

Diabetic neuropathy is a severe complication of long-standing diabetes and one of the major etiologies of neuropathic pain. Diabetes is associated with an increased formation of reactive oxygen species and the electrophilic dicarbonyl compound methylglyoxal (MG). Here we show that MG stimulates heterologously expressed TRPA1 in CHO cells and natively expressed TRPA1 in MDCK cells and DRG neurons. MG evokes [Ca(2+)]i-responses in TRPA1 expressing DRG neurons but is without effect in neurons cultured from Trpa1(-/-) mice. Consistent with a direct, intracellular action, we show that methylglyoxal is significantly more potent as a TRPA1 agonist when applied to the intracellular face of excised membrane patches than to intact cells. Local intraplantar administration of MG evokes a pain response in Trpa1(+/+) but not in Trpa1(-/-) mice. Furthermore, persistently increased MG levels achieved by two weeks pharmacological inhibition of glyoxalase-1 (GLO-1), the rate-limiting enzyme responsible for detoxification of MG, evokes a progressive and marked thermal (cold and heat) and mechanical hypersensitivity in wildtype but not in Trpa1(-/-) mice. Our results thus demonstrate that TRPA1 is required both for the acute pain response evoked by topical MG and for the long-lasting pronociceptive effects associated with elevated MG in vivo. In contrast to our observations in DRG neurons, MG evokes indistinguishable [Ca(2+)]i-responses in pancreatic β-cells cultured from Trpa1(+/+) and Trpa1(-/-) mice. In vivo, the TRPA1 antagonist HC030031 impairs glucose clearance in the glucose tolerance test both in Trpa1(+/+) and Trpa1(-/-) mice, indicating a non-TRPA1 mediated effect and suggesting that results obtained with this compound should be interpreted with caution. Our results show that TRPA1 is the principal target for MG in sensory neurons but not in pancreatic β-cells and that activation of TRPA1 by MG produces a painful neuropathy with the behavioral hallmarks of diabetic neuropathy.

Figure 1. Methylglyoxal is an intracellular TRPA1 agonist.

(A) Application of MG evokes concentration-dependent [Ca²⁺]_i-responses in CHO cells expressing mouse or human TRPA1. (B) MG (0.5 mM) elicits inward currents with the characteristic rapid onset and inactivation in the presence of 2 mM Ca²⁺ (top panel). In Ca²⁺-free solutions (1 mM EGTA) the inward current grew more slowly and addition of Ca²⁺ produced the typical current surge followed by a rapid inactivation of the current (bottom panel). (C) MG rapidly activates TRPA1 in excised inside-out patches, with a markedly higher potency than when applied extracellularly (D, compare with A). (E) The current voltage relationship in membrane patches containing several channels demonstrated a reversal potential close to 0 mV and channel block or inactivation at positive membrane potential. (F) Current-voltage relationship for a single channel in an inside-out patch stimulated with MG.

Figure 2. TRPA1 is expressed in MDCK cells.

(A) The TRPA1 agonists AITC, H₂O₂, MG and cinnamaldehyde stimulate [Ca²⁺]_i-responses in MDCK cells. (B, C) The selective TRPA1 antagonist AP18 (10 µM) produces a rightward shift of AITC and MG evoked [Ca²⁺]_i-responses in MDCK cells.

Figure 3. Methylglyoxal stimulates DRG neurons expressing TRPA1.

(A) Pseudocoloured images illustrating [Ca²⁺]_i-concentrations in Fura-2 loaded cultured DRG neurons. MG evokes [Ca²⁺]_i-responses in TRPA1 containing neurons. (B) Typical examples of [Ca²⁺]_i-responses in capsaicin sensitive DRG neurons from Trpa1^+/+ and Trpa1^−/− mice. (C) MG activates characteristic TRPA1 currents in DRG neurons (holding potential -60 mV). Before addition of 2 mM Ca²⁺ this neuron was superfused with a solution containing 15 µM Ca²⁺, which prevents the Ca²⁺-induced current surge.

Figure 4. Methylglyoxal is a reversible TRPA1 agonist.

The cysteine substitutions C665S, C641S and C621S (in human TRPA1) do not significantly affect responses evoked by MG (A), but dramatically reduce the sensitivity to AITC (B). (C) The lysine substitution K712Q (mouse TRPA1) reduced [Ca²⁺]_i-responses evoked by AITC (50 µM). (D) The K712Q mutation reduced the amplitude of [Ca²⁺]_i-responses evoked by MG (2 mM) and Δ⁹-tetrahydrocannabiorcol (20 µM; C16) in individual CHO cells (n = 22–64). (E) Outward TRPA1 current responses (+60 mV) evoked by MG (1 mM) in CHO cells decay relatively slowly when MG is removed (recorded under Ca²⁺-free conditions). Application of DTT (2 mM) produces a rapid, partial current inactivation. (F) Currents evoked by AITC (50 µM) under the same conditions inactivate relatively slowly, but are resistant to DTT and leave TRPA1 refractory to stimulation. (G) Currents elicited by the oxidant H₂O₂ (5 mM) remain at a stable level after removal of H₂O₂, but are rapidly and reversibly inactivated by DTT. Data were analyzed by ANOVA followed by Dunnett’s post-hoc test (panel B) or by t-test (*P<0.05, **P<0.01, ***P<0.001 compared to the wildtype channel).

Figure 5. Methylglyoxal produce persistent sensory neuropathy.

(A) Intraplantar injections of MG (250 nmoles in 25 µl) evokes nociceptive behaviors in wildtype mice but is without effect in Trpa1^−/− mice (n = 6). Injections of the GLO-1 inhibitor Sr-p-Bromobenzylglutathione cyclopentyl diester (GLOI, 50 mg/kg, 1h before test) did not acutely affect the withdrawal threshold in the paw pressure (B) or von Frey (C) tests compared to mice treated with vehicle (n = 6). (D-G) Effect of GLOI or vehicle injections administered every 2 days during a 2 week treatment period on the paw withdrawal latency from a 50°C hotplate (D), 10°C coldplate (E), the paw withdrawal threshold in the paw pressure test (F) and in response to stimulation with von Frey filaments (G). The data points are mean ± SEM from n = 6 mice, except for the last time point (15 days after the final injection) where n = 3. Data were analyzed by t-test (panel A) or ANOVA followed by Tukey’s HSD test (**P<0.01, ***P<0.001 compared to vehicle treated group).

Figure 6. Intra-epidermal nerve fiber density is unaffected by 2 weeks GLO-1 inhibition.

(A) PGP9.5 positive nerve fibers crossing the basal membrane into the epidermis (the white arrow highlights an example) in 8 µm sections of plantar skin (scale bar = 20 µm). (B) Following two weeks treatment with the GLO-1 inhibitor (GLO-I, 50 mg/kg every other day), the number of intra-epidermal nerve fibers crossing into the epidermis/mm skin was unchanged in both Trpa1^+/+ mice and in Trpa1^−/− littermates. Data were analyzed by ANOVA followed by Tukey’s HSD test (n = 6 mice, *P<0.05 compared to vehicle treated Trpa1^+/+ mice).

Figure 7. TRPA1 does not influence insulin release.

(A) MG (0.5 mM) evokes indistinguishable [Ca²⁺]_i-responses in β-cells from Trpa1^+/+ (black) and Trpa1^−/− (red) mice (n = 15-28). (B) Illustration of the time course of MG and tolbutamide (200 µM) evoked [Ca²⁺]_i-responses in β-cells. (C, D) Effect of the TRPA1 antagonist HC030031 (HC, 100 mg/kg) or vehicle (Veh) administered 30 min before glucose or vehicle in the glucose tolerance test (2 g/kg, i.p.) performed in Trpa1^+/+ (C) and Trpa1^−/− mice (D). Data points are mean ± s.e.m of n = 6 mice and were analyzed by ANOVA followed by Tukey’s HSD test (*P<0.05, **P<0.01, ***P<0.001 compared to the group treated with vehicle and glucose, †P<0.05 compared to the group treated with vehicle and vehicle).