Activation of adenosine A2A or A2B receptors causes hypothermia in mice

Abstract

Extracellular adenosine is a danger/injury signal that initiates protective physiology, such as hypothermia. Adenosine has been shown to trigger hypothermia via agonism at A₁ and A₃ adenosine receptors (A₁AR, A₃AR). Here, we find that adenosine continues to elicit hypothermia in mice null for A₁AR and A₃AR and investigated the effect of agonism at A_2AAR or A_2BAR. The poorly brain penetrant A_2AAR agonists CGS-21680 and PSB-0777 caused hypothermia, which was not seen in mice lacking A_2AAR. MRS7352, a likely non-brain penetrant A_2AAR antagonist, inhibited PSB-0777 hypothermia. While vasodilation is probably a contributory mechanism, A_2AAR agonism also caused hypometabolism, indicating that vasodilation is not the sole mechanism. The A_2BAR agonist BAY60-6583 elicited hypothermia, which was lost in mice null for A_2BAR. Low intracerebroventricular doses of BAY60-6583 also caused hypothermia, indicating a brain site of action, with neuronal activation in the preoptic area and paraventricular nucleus of the hypothalamus. Thus, agonism at any one of the canonical adenosine receptors, A₁AR, A_2AAR, A_2BAR, or A₃AR, can cause hypothermia. This four-fold redundancy in adenosine-mediated initiation of hypothermia may reflect the centrality of hypothermia as a protective response.

Keywords: A(2A)AR; A(2B)AR; Adenosine; Hypothermia; Paraventricular hypothalamus; Preoptic area.

Fig. 1. Adenosine-mediated hypothermia remains intact in Adora1^−/−;Adora3^−/− mice

(A,B) Tb and activity response to adenosine (ADO, 100 mg/kg, i.p.) or vehicle (Veh) in C57BL/6J (WT) and Adora1^−/−;Adora3^−/− mice. (C,D) Average Tb (0–60 min) and total physical activity (10–60 min). Data are mean ± SEM, n = 5–6/group in a crossover design; ***p < 0.001 vs vehicle within genotype.

Fig. 2. Systemic CGS-21680 administration causes hypothermia and decreases physical activity

(A) Tb response to the indicated i.p. CGS-21680 doses in C57BL/6J mice. (B,C) Effect of CGS-21680 on average Tb (0–60 min) and physical activity (10–60 min). Data are mean ± SEM, n = 3-7/group; **p<0.01, ***p < 0.001 vs vehicle.

Fig. 3. Systemic CGS-21680 causes hypothermia through A_2AAR, not involving A₁AR, A₃AR, or mast cells

(A-C) Tb and activity response to CGS-21680 (0.5 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Adora2a^−/− mice. (D-F) Tb and activity response to CGS-21680 (0.5 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Adora1^−/−;Adora3^−/− mice. (G-I) Tb and activity response to CGS-21680 (0.5 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Kit^{W−sh/W−sh} mice. Data are mean ± SEM, n = 3–7/group; *p<0.05, **p<0.01, ***p < 0.001 vs vehicle within genotype, *p < 0.05 or ***p =0.001 vs vehicle.

Figure 4. No hypothermic response to intracerebroventricular CGS-21680

(A–C) Tb and activity response to i.c.v. CGS-21680 (0.01 or 0.1 mg/kg) or vehicle in C57BL/6J mice. Data are mean ± SEM, n = 4–6/group in a crossover design.

Fig. 5. Effect of A_2AAR antagonists on PSB-0777-induced hypothermia

(A–D) Tb and activity response to SCH442416 (0.6 mg/kg, i.p.) or vehicle at-15 min and then PSB-0777 (1 mg/kg, i.p.) or saline at 0 min. (E) Structure of SCH442416. (F-I) Tb and activity response to MRS7352 (3 mg/kg, i.p.) or vehicle at-15 min and then PSB-0777 (1 mg/kg, i.p.) or saline at 0 min. (J) Structure of MRS7352. Data are mean ± SEM, n = 5–12/group, *p < 0.05 or ***p <0.001 indicates significance at this level or better vs the other three groups by 1-way ANOVA with Sidak multiple comparison testing.

Fig. 6. CGS-21680 reduces metabolic rate prior to Tb

The effect of CGS-21680 (0.25 mg/kg, i.p.) or vehicle in C57BL/6J (WT) on (A) Tb, (B) total energy expenditure (TEE), (C) respiratory exchange ratio (RER), (D) physical activity, and (E) food intake. Data are mean ± SEM, n = 11/group, *p < 0.05.

Fig. 7. BAY60-6583 causes hypothermia and decreased physical activity via A_2BAR

(A-C) Tb and physical activity response to the indicated i.p. BAY60-6583 doses in C57BL/6J mice. (D-F) Tb and activity response to BAY60-6583 (1 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Adora2b^−/− mice. (G-I) Tb and activity response to BAY60-6583 (1 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Kit^{W−sh/W−sh} mice. Data are mean ± SEM, n = 3–8/group; *p<0.05, **p<0.01, ***p < 0.001 vs vehicle within genotype.

Fig. 8. Intracerebroventricular BAY60-6583 causes hypothermia

(A–C) Tb and physical activity response to BAY60-6583 (0.04 or 0.004 mg/kg, i.c.v.) in Adora1^−/−;Adora3^−/− mice. Data are mean ± SEM, n = 7/group (vehicle, 0.004 mg/kg) or n =2 for 0.04 mg/kg. (D-F) Tb and physical activity response to BAY60-6583 (0.004 mg/kg, i.c.v.) or vehicle in Adora2b^−/− or C57BL/6J (WT) mice. Data are mean ± SEM, n = 5–8/group in a crossover design; *p<0.05, **p<0.01 vs vehicle within genotype.

Fig. 9. BAY60-6583 reduces metabolic rate before Tb

The effect of BAY60-6583 (1 mg/kg, i.p.) or vehicle in C57BL/6J (WT) mice on (A) Tb, (B) total energy expenditure (TEE), (C) respiratory exchange ratio (RER), (D) physical activity, (E) food intake. Data are mean ± SEM, n = 11/group, *p < 0.05.

Fig. 10. Neuronal activation after intracerebroventricular infusion of BAY60-6583

(A) BAY60-6583 (0.004 mg/kg, i.c.v.) or vehicle was infused into in Adora1^−/−;Adora3^−/− mice, with brains removed 1 h later and stained for Fos. (A-D) Representative coronal Fos immunofluorescence images following vehicle (left) or BAY60-6583 (right) treatment. Nuclei are: POA, preoptic area; PVH, paraventricular hypothalamus; Arc, arcuate; VMH, ventromedial hypothalamus; and DMH, dorsomedial hypothalamus. (E) Quantitation of Fos-positive neurons. Means and p values are indicated.