Transient receptor potential channel ankyrin-1 is not a cold sensor for autonomic thermoregulation in rodents

Abstract

The rodent transient receptor potential ankyrin-1 (TRPA1) channel has been hypothesized to serve as a temperature sensor for thermoregulation in the cold. We tested this hypothesis by using deletion of the Trpa1 gene in mice and pharmacological blockade of the TRPA1 channel in rats. In both Trpa1(-/-) and Trpa1(+/+) mice, severe cold exposure (8°C) resulted in decreases of skin and deep body temperatures to ∼8°C and 13°C, respectively, both temperatures being below the reported 17°C threshold temperature for TRPA1 activation. Under these conditions, Trpa1(-/-) mice had the same dynamics of body temperature as Trpa1(+/+) mice and showed no weakness in the tail skin vasoconstriction response or thermogenic response to cold. In rats, the effects of pharmacological blockade were studied by using two chemically unrelated TRPA1 antagonists: the highly potent and selective compound A967079, which had been characterized earlier, and the relatively new compound 43 ((4R)-1,2,3,4-tetrahydro-4-[3-(3-methoxypropoxy)phenyl]-2-thioxo-5H-indeno[1,2-d]pyrimidin-5-one), which we further characterized in the present study and found to be highly potent (IC50 against cold of ∼8 nm) and selective. Intragastric administration of either antagonist at 30 mg/kg before severe (3°C) cold exposure did not affect the thermoregulatory responses (deep body and tail skin temperatures) of rats, even though plasma concentrations of both antagonists well exceeded their IC50 value at the end of the experiment. In the same experimental setup, blocking the melastatin-8 (TRPM8) channel with AMG2850 (30 mg/kg) attenuated cold-defense mechanisms and led to hypothermia. We conclude that TRPA1 channels do not drive autonomic thermoregulatory responses to cold in rodents.

Keywords: TRPA1; TRPM8; cold exposure; hypothermia; thermoregulation.

Figure 1.

Cold exposure reveals no autonomic thermoregulatory deficiency in Trpa1^−/− mice. A, During exposure to 8°C, there was no weakness in the defense of deep (colonic) T_b by Trpa1^−/− mice (n = 8), compared with Trpa1^+/+ mice (n = 6). The T_b dynamics did not differ between the genotypes. Both the tail skin vasoconstriction response (a decrease in the tail T_sk) and the thermogenic response (an increase in oxygen consumption) in Trpa1^−/− mice were no less robust than the responses in Trpa1^+/+ mice. B, Functional TRPA1 deficiency of Trpa1^−/− mice (n = 6) compared with Trpa1^+/+ mice (n = 5) was confirmed by a decreased pain response to intraplantar mustard oil (0.75%, 20 μl).

Figure 2.

Compound 43 is a potent antagonist of the rat TRPA1 channel in vitro and in vivo. A, In Chinese hamster ovary cells expressing rat TRPA1 channel, enantiopure (right) compound 43 (but not its left enantiomer compound 44) potently blocked the increase in ⁴⁵Ca²⁺ uptake induced by cold (4°C) or AITC (80 μm). For each compound–stimulus combination, the ⁴⁵Ca²⁺ uptake was measured twice. B, Compound 43 (but not compound 44) attenuated the nocifensive response of rats to intraplantar administration of AITC (0.01%, 25 μl). For each group, n = 8.

Figure 3.

Pharmacological blockade of TRPA1 does not affect autonomic cold defenses in rats. A, Compared with their vehicle (n = 6), neither the TRPA1 antagonists compound 43 (n = 13) and A967079 (n = 5) nor the TRPM8 antagonist AMG2850 (n = 13) affected the dynamics of deep T_b or tail T_sk in rats in the warmth. At time 0, the rats were transferred from one environmental chamber set to 30°C to a second chamber, which was also set to 30°C (see Materials and Methods). All compounds were administered at the same dose (30 mg/kg, i.g.) 30 min before switching the chambers. B, Compared with their vehicle (n = 11), the TRPA1 antagonists compound 43 (n = 6) and A967079 (n = 8), but not the TRPM8 antagonist AMG2850 (n = 6), had no effect on either the deep T_b response to cold or cold-induced vasoconstriction (a decrease in the tail T_sk). At time 0, the rats were transferred from 30°C to 3°C. All compounds were administered at the same dose (30 mg/kg, i.g.) 30 min before the transfer. C, Rats treated with compound 43 or A967079, but not with their vehicle, had a high concentration of the respective compound in their blood plasma at 3.5 h after compound administration. B, C, Results were obtained from the same rats.