Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense effectors

Abstract

An involvement of the transient receptor potential vanilloid (TRPV) 1 channel in the regulation of body temperature (T(b)) has not been established decisively. To provide decisive evidence for such an involvement and determine its mechanisms were the aims of the present study. We synthesized a new TRPV1 antagonist, AMG0347 [(E)-N-(7-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)acrylamide], and characterized it in vitro. We then found that this drug is the most potent TRPV1 antagonist known to increase T(b) of rats and mice and showed (by using knock-out mice) that the entire hyperthermic effect of AMG0347 is TRPV1 dependent. AMG0347-induced hyperthermia was brought about by one or both of the two major autonomic cold-defense effector mechanisms (tail-skin vasoconstriction and/or thermogenesis), but it did not involve warmth-seeking behavior. The magnitude of the hyperthermic response depended on neither T(b) nor tail-skin temperature at the time of AMG0347 administration, thus indicating that AMG0347-induced hyperthermia results from blockade of tonic TRPV1 activation by nonthermal factors. AMG0347 was no more effective in causing hyperthermia when administered into the brain (intracerebroventricularly) or spinal cord (intrathecally) than when given systemically (intravenously), which indicates a peripheral site of action. We then established that localized intra-abdominal desensitization of TRPV1 channels with intraperitoneal resiniferatoxin blocks the T(b) response to systemic AMG0347; the extent of desensitization was determined by using a comprehensive battery of functional tests. We conclude that tonic activation of TRPV1 channels in the abdominal viscera by yet unidentified nonthermal factors inhibits skin vasoconstriction and thermogenesis, thus having a suppressive effect on T(b).

Figure 1.

Chemical structure and in vitro pharmacology of AMG0347. A, Structural formula of AMG0347 as confirmed by spectral analysis (supplemental Materials and Methods, available at www.jneurosci.org as supplemental material). B, Concentration-dependent effects of AMG0347 on the activation of the TRPV1 channel by heat, protons, and capsaicin. For comparison, the effects of AMG9810 on channel activation by the same stimuli are shown. Channel activation was assessed based on the uptake of ⁴⁵Ca²⁺ by cultured Chinese hamster ovary cells stably transfected with the rat TRPV1 channel. The ⁴⁵Ca²⁺ uptake assay was conducted three times for each AMG compound–stimulus combination.

Figure 2.

AMG0347 produces hyperthermia in wild-type rats and mice but not in TRPV1-deficient mice. A, Effects of intravenous AMG0347 (doses indicated) or its vehicle on the colonic (deep body) temperature of rats at a neutral T_a of 28°C and on the abdominal (deep body) temperature of mice at a neutral T_a of 31°C. B, Effects of AMG0347, abdominal needle prick, or RTX on the abdominal temperature of TRPV1^+/+ (wild-type) and TRPV1^−/− (TRPV1-deficient) mice. When the stimulus was expected to result in hyperthermia (AMG0347 administration and needle prick), the experiment was conducted at a neutral T_a (31°C); when the stimulus was expected to result in hypothermia (RTX), the experiment was performed at a subneutral T_a (26°C). The number of animals in each group (n) is indicated.

Figure 3.

AMG0347 hyperthermia is brought about by activation of autonomic thermoeffectors and is independent of basal T_b and T_sk. A, Effects of AMG0347 or its vehicle on the colonic temperature, heat loss index, and oxygen consumption of rats at T_a of 28°C (upper end of the TNZ), 24°C (lower end of the TNZ), or 17°C (below the lower end of the TNZ). Colonic temperature is an index of deep T_b, the heat loss index is an index of skin vasodilation, and oxygen consumption is an index of thermogenesis. B, Results of a linear correlation analysis between the magnitude of AMG0347-induced hyperthermia and the values of either T_b or T_sk immediately before AMG0347 administration. Based on the data presented in A.

Figure 4.

AMG0347 hyperthermia is absent in rats with localized intra-abdominal TRPV1 desensitization. The colonic temperature response of rats pretreated with RTX (20 μg/kg, i.p.) or its vehicle to administration of AMG0347 at thermoneutrality (T_a of 24°C) is shown.

Figure 5.

Sites of TRPV1 desensitization in RTX-pretreated rats. The sensitivity of TRPV1 channels in different bodily compartments was determined in vehicle-pretreated rats, in rats pretreated with a low desensitizing dose (20 μg/kg, i.p.) of RTX, and in rats pretreated with a high dose (200 μg/kg, i.p.) of RTX. Five tests were performed: the eye-wiping test (determines the sensitivity of TRPV1 channels in the eye); the centrally induced tail-vasodilation test (determines the sensitivity of TRPV1 channels in the brain); the Bezold–Jarisch reflex test (determines the sensitivity of TRPV1 channels in the heart and lungs); the hot-plate test (assesses the sensitivity of TRPV1 channels in the skin, primarily of the paws); and the writhing test (determines the sensitivity of TRPV1 channels in the peritoneal cavity). For methodological details, see Materials and Methods. The results of all tests are summarized in the schematics at the bottom.