The roles of iPLA2, TRPM8 and TRPA1 in chemically induced cold hypersensitivity

Abstract

Background: The cooling agents menthol and icilin act as agonists at TRPM8 and TRPA1. In vitro, activation of TRPM8 by icilin and cold, but not menthol, is dependent on the activity of a sub-type of phospholipase A2, iPLA2. Lysophospholipids (e.g. LPC) produced by PLA2 activity can also activate TRPM8. The role of TRPA1 as a primary cold sensor in vitro is controversial, although there is evidence that TRPA1 plays a role in behavioural responses to noxious cold stimuli. In this study, we have investigated the roles of TRPM8 and TRPA1 and the influence of iPLA2 on noxious cold sensitivities in naïve animals and after local administration of menthol, icilin and LPC. The roles of the channels in cold sensitivity were investigated in mice lacking either TRPM8 (Trpm8-/-) or TRPA1 (Trpa1-/-).

Results: Intraplantar administration of icilin evoked a dose-dependent increase in sensitivity to a 10 degrees C stimulus that was inhibited by iPLA2 inhibition with BEL. In contrast the cold hypersensitivities elicited by intraplantar menthol and LPC were not inhibited by BEL treatment. BEL had no effect on basal cold sensitivity and mechanical hypersensitivities induced by the TRPV1 agonist, capsaicin, and the P2X3 agonist alpha,beta-methylene ATP. Both Trpm8-/- and Trpa1-/- mice showed longer latencies for paw withdrawal from a 10 degrees C stimulus than wild-type littermates. Cold hypersensitivities induced by either icilin or LPC were absent in Trpm8-/- mice but were retained in Trpa1-/- mice. In contrast, cold hypersensitivity evoked by menthol was present in Trpm8-/- mice but was lost in Trpa1-/- mice.

Conclusions: The findings that iPLA2 inhibition blocked the development of cold hypersensitivity after administration of icilin but failed to affect menthol-induced hypersensitivity agree well with our earlier in vitro data showing a differential effect of iPLA2 inhibition on the agonist activities of these agents. The ability of LPC to induce cold hypersensitivity supports a role for iPLA2 in modulating TRPM8 activity in vivo. Studies on genetically modified mice demonstrated that the effects of icilin and LPC were mediated by TRPM8 and not TRPA1. In contrast, menthol-induced cold hypersensitivity was dependent on expression of TRPA1 and not TRPM8.

Figure 1

Temperature dependence of withdrawal latencies. A. Response time for rat hind-limb paw withdrawal (lick or lift) from a cold plate set at various temperatures for unrestrained, freely moving rats. B. Paw withdrawal latencies for left and right hind-limbs in lightly restrained rats. Note the similar results obtained with these two methods and the close correspondence between the values for left and right limbs in restrained animals. Data shows mean ± SEM for 9 rats.*** p < 0.001 vs. 20°C readings.

Figure 2

Icilin-induced cold hypersensitivity is inhibited by the iPLA2 inhibitor, BEL. A. Ipsilateral paw withdrawal latencies in lightly restrained rats at various times after intra-plantar administration of 0.6, 6 or 60 μg icilin or vehicle alone. B. Prior administration of 30-300 μg BEL dose dependently inhibited the cold hypersensitivity induced by 60 μg icilin. Data show mean ± SEM for 6 rats/group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs vehicle. In B, † p < 0.05, †† p < 0.01 when compared with vehicle/icilin treated group.

Figure 3

Menthol-induced cold hypersensitivity is not inhibited by BEL. A. Intraplantar administration of menthol (3-25 mg) induced a time- and dose-dependent reduction in ipsilateral paw withdrawal latency in freely-moving rats. B. Prior administration of BEL (30-300 μg) had no effect on the cold hypersensitivity evoked by 25 mg menthol. Data show mean ± SEM for 6 rats/group. * p < 0.05, *** p < 0.001 vs vehicle.

Figure 4

LPC-induced cold hypersensitivity is not inhibited by BEL. A. Intraplantar administration of LPC evoked a dose-dependent cold hypersensitivity in lightly restrained rats. B. Prior administration of BEL (30-300 μg) did not inhibit the cold hypersensitivity evoked by 60 μg LPC. Data show mean ± SEM for 6 rats/group. ** p < 0.01, *** p < 0.001 vs vehicle.

Figure 5

Cold hypersensitivities evoked by icilin and LPC are absent in Trpm8^-/- mice but retained in Trpa1^-/- mice. Effects of intraplantar administration of either 60 μg LPC (A) or 100 μg icilin (B) on cold withdrawal latencies in lightly restrained Trpm8^-/- and wild-type littermate mice. Neither compound had an effect on withdrawal latency for the injected (ipsilateral) paw in Trpm8^-/- mice. Both compounds reduced the ipsilateral paw withdrawal latencies in wild-type littermates, but had no effect on withdrawal latencies for the uninjected (contralateral) paw. Intraplantar LPC (C) or icilin (D) reduced paw withdrawal thresholds for ipsilateral paws in both Trpa1^-/- and wild-type littermate mice. Note that a greater cold hypersensitivity was seen in Trpa1^-/- than in wild-type mice. Data show mean ± SEM for 6 mice/group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs relevant predose values. † p < 0.05, †† p < 0.01 for contralateral-ipsilateral difference in wild-type mice.

Figure 6

Menthol-evoked cold hypersensitivity is absent in Trpa1^-/- mice but present in Trpm8^-/- mice. A. Intraplantar administration of 25 mg menthol evoked marked cold hypersensitivity in the ipsilateral paw of lightly restrained Trpm8^-/- mice. Note that a greater cold hypersensitivity was seen in Trpm8^-/- than in wild-type mice. B. Menthol evoked cold hypersensitivity in wild-type but not Trpa1^-/- mice. Data show mean ± SEM for 6 mice/group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs relevant predose values. † p < 0.05, †† p < 0.01, ††† p < 0.001 for contralateral-ipsilateral difference in wild-type mice.

Figure 7

TRPA1 agonists AITC and cinnamaldehyde evoke cold hypersensitivity. Intraplantar injection of either AITC (100 μg) or cinnamaldehyde (50 μg) evoked marked cold hypersensitivity in the ipsilateral paw of lightly restrained wild-type mice but had no effect when administered to Trpa1^-/- mice. Data show mean ± SEM for 6 mice/group. *** p < 0.001 vs pre-dose latencies.