Pharmacological blockade of the cold receptor TRPM8 attenuates autonomic and behavioral cold defenses and decreases deep body temperature

Abstract

We studied N-(2-aminoethyl)-N-(4-(benzyloxy)-3-methoxybenzyl)thiophene-2-carboxamide hydrochloride (M8-B), a selective and potent antagonist of the transient receptor potential melastatin-8 (TRPM8) channel. In vitro, M8-B blocked cold-induced and TRPM8-agonist-induced activation of rat, human, and murine TRPM8 channels, including those on primary sensory neurons. In vivo, M8-B decreased deep body temperature (T(b)) in Trpm8(+/+) mice and rats, but not in Trpm8(-/-) mice, thus suggesting an on-target action. Intravenous administration of M8-B was more effective in decreasing T(b) in rats than intrathecal or intracerebroventricular administration, indicating a peripheral action. M8-B attenuated cold-induced c-Fos expression in the lateral parabrachial nucleus, thus indicating a site of action within the cutaneous cooling neural pathway to thermoeffectors, presumably on sensory neurons. A low intravenous dose of M8-B did not affect T(b) at either a constantly high or a constantly low ambient temperature (T(a)), but the same dose readily decreased T(b) if rats were kept at a high T(a) during the M8-B infusion and transferred to a low T(a) immediately thereafter. These data suggest that both a successful delivery of M8-B to the skin (high cutaneous perfusion) and the activation of cutaneous TRPM8 channels (by cold) are required for the hypothermic action of M8-B. At tail-skin temperatures <23°C, the magnitude of the M8-B-induced decrease in T(b) was inversely related to skin temperature, thus suggesting that M8-B blocks thermal (cold) activation of TRPM8. M8-B affected all thermoeffectors studied (thermopreferendum, tail-skin vasoconstriction, and brown fat thermogenesis), thus suggesting that TRPM8 is a universal cold receptor in the thermoregulation system.

Figure 1.

Chemical structure and in vitro pharmacology of M8-B. A, Structural formula of M8-B as confirmed by spectral analysis. B, Concentration-dependent effects of M8-B on the activation of rat TRPM8 channels (stably expressed in Chinese hamster ovary cells) by cold (10°C), icilin (1 μm), and menthol (100 μm). C, Concentration-dependent effects of M8-B on the activation of native TRPM8 channels in murine primary sensory neurons by cold (15°C) and menthol (250 μm). Here and in other figures, the number of experiments is shown in parentheses at each curve.

Figure 2.

M8-B decreases deep T_b in Trpm8^+/+ rats and mice, but not in Trpm8^−/− mice. A–C, The figure shows the effect of intravenous infusion of M8-B (dose indicated) or its vehicle on colonic T_b in Trpm8^+/+ rats (A) and the effects of intraperitoneal infusion of M8-B or its vehicle on deep (abdominal) T_b in Trpm8^+/+ mice (B) and Trpm8^−/− mice (C). Experiments were conducted at a subneutral T_a (19°C for rats, 26°C for mice). Here and in other figures, the time of M8-B infusion is shown with a gray bar.

Figure 3.

RTX readily produces hypothermia in both Trpm8^+/+ and Trpm8^−/− mice. A, B, The figure shows the effects of the intraperitoneal infusion of RTX (dose indicated) or its vehicle on the deep (abdominal) T_b in Trpm8^+/+ (A) and Trpm8^−/− (B) mice. Experiments were conducted at the subneutral T_a of 26°C. The time of RTX infusion is shown with a gray bar.

Figure 4.

M8-B decreases T_b when administered peripherally but not centrally. A–C, The figure shows the effects of intravenous (A), intrathecal (B), and intracerebroventricular (C) infusion of M8-B at the same dose (indicated) or of the vehicle on colonic T_b in rats. Experiments were conducted at a subneutral T_a of 19°C.

Figure 5.

M8-B suppresses cold-induced c-Fos expression in the LPB. A, How borders of the LPBC and LPBE were determined for cell counting. Based on the schematic drawing of the −9.12 mm coronal plane of rat brain (Paxinos and Watson, 2007), a template was made that encompassed two targeted areas (the LPBC and LPBE; dashed green line) and two landmarks [the border of the superior cerebellar peduncle (scp) and the dorsal border of the pons; solid green line]. From each animal, a pontine tissue section most representative of the −9.12 mm coronal plane was selected. The template was appropriately scaled and overlapped with the tissue section to best fit the landmarks, and the number of c-Fos-positive cells within the targeted areas was counted. B, Representative photomicrographs of four pontine sections containing c-Fos-positive neurons. The sections were obtained from rats treated with M8-B or its vehicle and exposed to either a warm (32°C) or cold (4°C) environment. C, Quantitative analyses of c-Fos-immunoreactive cells in the LPBC and LPBE, separately (left, middle) and together (right).

Figure 6.

The thermoregulatory effect of M8-B strongly depends on T_a. The figure shows the effect of intravenous infusion of M8-B or its vehicle on colonic T_b in rats. The rats were kept in an environmental chamber before and during the infusion and transferred to another environmental chamber immediately after the infusion. A–H, The first chamber had an ambient temperature set to either a deep subneutral level of 15°C (A) or a supraneutral level of 32°C (B–H). The second chamber had T_a set to 15°C (A), 32°C (B), 28°C (C), 24°C (D), 21°C (E), 19°C (F), 14°C (G), or 11°C (H). The mean T_a to which the animals were exposed in each experiment is shown with a red line. Note that small SE values for T_a (<0.1°C) make SE bars invisible.

Figure 7.

Relationship between the hypothermic response to M8-B infusion and mean T_sk during the response. T_sk varied across experiments, because rats were exposed to different ambient temperatures. Data were generated in the experiments shown in Figure 6B–H and fitted into a two-line model. The best-fit set of two lines was determined by the minimum sum of square roots of deviations from the lines. The best-fit lines intersect at a “threshold” T_sk of 23.2°C. Below this threshold, the hypothermic response index depends on T_sk (r² = 0.85).

Figure 8.

M8-B delay onset of the tail-skin vasoconstrictor response to a cold environment. A, Changes in colonic T_b and HLI in rats transferred to a cool environment (21°C) after intravenous infusion of M8-B or its vehicle in a warm environment (32°C). The transfer took place immediately after the end of infusion (at 45 min). B, Relationships between HLI and T_b in M8-B-treated and vehicle-treated rats at onset of the hypothermic response (45–65 min). Here and in Figures 9B, 10B, and 11B, the direction of time is shown with arrows, and the times corresponding to the earliest and latest data points are indicated.

Figure 9.

Thermogenesis is inhibited at onset of the T_b response to a high dose of M8-B at a constant subneutral T_a. A, Changes in colonic T_b and VO₂ in response to intravenous infusion of M8-B or its vehicle in a cool environment (19°C). B, Relationships between VO₂ and T_b at onset of the hypothermic response (10–60 min) in M8-B-treated and vehicle-treated rats.

Figure 10.

Nonshivering thermogenesis in BAT is suppressed at onset of the T_b response to M8-B infusion and the subsequent cold exposure. Rats were infused with M8-B or its vehicle in a warm environment (32°C) and transferred to a cold environment (11°C) immediately thereafter (at 45 min). A, Changes in colonic T_b and BAT thermogenesis. B, Relationships between BAT thermogenesis index and T_b in M8-B-treated and vehicle-treated rats.

Figure 11.

M8-B blocks the hyperthermic response and warmth-seeking behavior induced by menthol. After rats were infused intravenously with M8-B or its vehicle in a warm environment (32°C), they were treated with menthol epidermally and released into a thermogradient apparatus, where they could select their preferred T_a. A, The effects of M8-B or its vehicle on deep (abdominal) T_b and preferred T_a. B, Relationships between preferred T_a and T_b in M8-B-treated and vehicle-treated rats during the first 60 min in the thermogradient.

Figure 12.

Cutaneous TRPM8 controls autonomic and behavioral thermoeffectors.