Peripheral Adenosine A3 Receptor Activation Causes Regulated Hypothermia in Mice That Is Dependent on Central Histamine H1 Receptors

Abstract

Adenosine can induce hypothermia, as previously demonstrated for adenosine A1 receptor (A1AR) agonists. Here we use the potent, specific A3AR agonists MRS5698, MRS5841, and MRS5980 to show that adenosine also induces hypothermia via the A3AR. The hypothermic effect of A3AR agonists is independent of A1AR activation, as the effect was fully intact in mice lacking A1AR but abolished in mice lacking A3AR. A3AR agonist-induced hypothermia was attenuated by mast cell granule depletion, demonstrating that the A3AR hypothermia is mediated, at least in part, via mast cells. Central agonist dosing had no clear hypothermic effect, whereas peripheral dosing of a non-brain-penetrant agonist caused hypothermia, suggesting that peripheral A3AR-expressing cells drive the hypothermia. Mast cells release histamine, and blocking central histamine H1 (but not H2 or H4) receptors prevented the hypothermia. The hypothermia was preceded by hypometabolism and mice with hypothermia preferred a cooler environmental temperature, demonstrating that the hypothermic state is a coordinated physiologic response with a reduced body temperature set point. Importantly, hypothermia is not required for the analgesic effects of A3AR agonists, which occur with lower agonist doses. These results support a mechanistic model for hypothermia in which A3AR agonists act on peripheral mast cells, causing histamine release, which stimulates central histamine H1 receptors to induce hypothermia. This mechanism suggests that A3AR agonists will probably not be useful for clinical induction of hypothermia.

Graphical abstract

Fig. 1.

MRS5698 causes dose-dependent hypothermia and decreased physical activity. (A) Tb response to the indicated MRS5698 dose injected intraperitoneally into C57BL/6J mice. (B) Physical activity response to indicated MRS5698 dose in arbitrary units each minute. The effect of MRS5698 on (C) mean Tb (of 1–60 minutes), (D) duration of hypothermia, and (E) physical activity calculated from the data in (A) and (B). Data are mean ± S.E.M., n = 6–13/group; *P < 0.05, **P < 0.01, ***P < 0.001. Every tenth S.E.M. is shown in (A). In (B) data are graphed as splines and S.E.M.s were omitted for visual clarity.

Fig. 2.

MRS5698-induced hypothermia is lost in Adora3^−/− mice. Tb response to MRS5698 (10 mg/kg i.p.) or vehicle in (A, B) Adora1^−/− (KO) and C57BL/6J (WT) mice and (C, D) Adora3^−/− (KO) and C57BL/6J (WT) mice. Data are mean ± S.E.M., n = 3–5/group in a crossover design, *P < 0.05, ***P < 0.001. Every tenth S.E.M. is shown.

Fig. 3.

Pretreatment with compound 48/80 attenuates MRS5698 hypothermia. (A) C57BL/6J mice were pretreated with compound 48/80 (48/80) or saline (sal) for 5 days (see Materials and Methods). On the next day, at time 0, mice were treated with MRS5698 (10 mg/kg i.p.) or vehicle. Data are mean ± S.E.M., n = 6/group, every tenth S.E.M. is shown. (B) Mean Tb (of 1–60 minutes) calculated from the data in (A), *P < 0.05, ***P < 0.001.

Fig. 4.

Comparison of the hypothermic response to peripheral versus central dosing with A₃AR agonist MRS5841. (A) Tb and (B) mean Tb response to MRS5841 (10 mg/kg) or vehicle injected intraperitoneally into C57BL/6J (WT) or Adora3^−/− (A₃AR KO) mice. MRS5841 causes hypothermia in wild-type but not Adora3^−/− mice. (C) Tb and (D) mean Tb response to MRS5841 (0.5 mg/kg = 25 nmol) or vehicle injected intracerebroventricularly into C57BL/6J mice. Data are mean ± S.E.M., n = 4–7/group in (A), n = 15/group in (C); every tenth S.E.M. is shown, **P < 0.01.

Fig. 5.

A₃AR agonist–induced hypothermia is mediated by central histamine H₁ receptors. (A) Tb and (B) mean Tb (of 1–60 minutes) values in response to pretreatment with H₁R antagonist pyrilamine (10 mg/kg i.p.) or saline followed 15 minutes later by MRS5698 (10 mg/kg i.p., at time 0) or vehicle in C57BL/6J mice. (C) Tb and (D) mean Tb in response to pretreatment with H₂R antagonist zolantidine (10 mg/kg i.p.) or saline followed 15 minutes later by MRS5698 (10 mg/kg i.p.) or vehicle. (E) Tb and (F) mean Tb in response to pretreatment with H₄R antagonist JNJ7777120 (10 mg/kg i.p.) or vehicle. (G) Tb and (H) mean Tb in response to pretreatment with intracerebroventricular pyrilamine (0.5 mg/kg) or saline followed 15 minutes later by MRS5698 (10 mg/kg i.p.) or vehicle. Data are mean ± S.E.M., n = 4/group; every tenth S.E.M. is shown in (A), (C), (E), and (G); *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

MRS5698 reduces metabolic rate prior to Tb. Effect of MRS5698 (10 mg/kg i.p.) or vehicle dosed to C57BL/6J mice (A) Tb, (B) mean Tb (of 1–60 minutes), (C) total energy expenditure (TEE), and (D) mean TEE (of 1–60 minutes). The TEE falls before Tb (nadir at ∼30 minutes versus ∼60 minutes for Tb). Parameters were measured every 13 minutes; data are mean ± S.E.M., n = 6/group; *P < 0.05, **P < 0.01.

Fig. 7.

Mice treated with MRS5698 seek cooler temperatures. C57BL/6J mice were treated with MRS5698 (3 mg/kg i.p.) or vehicle and placed in a thermal gradient. The mice were monitored by video, and the (A) position within the gradient and the (C) velocity were calculated. The mean (B) position and (D) velocity for each mouse from 6 to 35 minutes after dosing was calculated. Mean ± S.E.M., n = 6/group, crossover design; *P < 0.05.

Fig. 8.

Dose-dependent reversal of neuropathic pain by MRS5698. PWT measured in the ipsilateral (A) and contralateral (B) paws before chronic constriction injury (CCI; day 0), after 7 days of constriction and before drug treatment (day 7), and at the indicated times after administration of MRS5698 (●, 0.1; ▪, 0.3; ▴, 1 mg/kg i.p.) or vehicle (○, 0.1% dimethyl sulfoxide in saline). Mean ± S.D., n = 5/group; *P < 0.05 day 7 versus day 0, ^†P < 0.05 indicated time versus day 7.

Fig. 9.

Pyrilamine does not reverse the analgesic effect of MRS5698. PWT measured in the ipsilateral (A) and contralateral (B) paws before chronic constriction injury (CCI; day 0), after 7 days of constriction and before drug treatment (day 7), and at the indicated times after administration of saline then vehicle (○), saline then MRS5698 (▴), pyrilamine then vehicle (□), or pyrilamine then MRS5698 (▪). The pyrilamine (10 mg/kg i.p.) or its vehicle (saline) was given 15 minutes before MRS5698 (3 mg/kg i.p.) or its vehicle (0.1% dimethyl sulfoxide in saline). Mean ± S.D., n = 5/group; *P < 0.05 day 7 versus day 0, ^†P < 0.05 indicated time versus day 7.