Neuronal adenosine A2A receptors signal ergogenic effects of caffeine

The natural plant alkaloid caffeine (1,3,7-trimethylxantine) is one of the most common ergogenic substances for physical activity practitioners and athletes^{1,2,3,4,5,6,7,8,9,10}. Caffeine increases endurance^{1,8,9,10,11,12}, intermittent^7,13,14 and resistance^4,15 exercise in humans. In rodents, its ergogenic effects are conserved because caffeine increases running time on the treadmill at constant^16,17 and accelerated speeds^18,19. Sports sciences promote nonselective phosphodiesterase (PDE) inhibition^7,8 and increased calcium mobilization^2,7,8 as mechanisms for these ergogenic effects. However, the primary pharmacological effect of caffeine is the nonselective antagonism of adenosine A₁ and A_2A receptors (A₁R, A_2AR)^20,21,22,23.

Adenosine can act as an inhibitory modulator of the Central Nervous System (CNS) associated with tiredness and drowsiness^{24,25,26,27,28,29}. During exercise, circulating ADP/AMP/adenosine levels increase due to ATP hydrolysis^30,31. However, there is still no substantial evidence on the role of adenosine in exercise-induced fatigue. It is just known that the nonselective A₁R and A_2AR agonist 5′-(N-ethylcarboxamido)adenosine (NECA), injected into the rat brain, abolishes the ergogenic effects of caffeine¹⁶.

Since there is increasing evidence that the adenosine modulation system critically controls allostasis²⁹ and A_2AR have a crucial role in the ability of caffeine to normalize brain function^30, we hypothesized that caffeine decreases fatigue during exercise through antagonism of A_2AR in the CNS. We combined the use of pharmacology (SCH 58261 and caffeine) and transgenic mice with tissue-selective deletion of A_2AR, to test this hypothesis in an incremental running test with indirect calorimetry (or ergospirometry). A_2AR knockout (KO) mice allow assessing if the ergogenic effect of caffeine persists in the absence of A_2AR; the use of SCH 58261, the current reference for A_2AR antagonists^32,33, allows directly assessing the ergogenic role of A_2AR. SCH 58261 has excellent selectivity and affinity for A_2AR^32,33, and affords motor benefits in animal models of Parkinson’s disease as does caffeine, supporting the recent FDA approval of the A_2AR antagonist Istradefylline for PD treatment³³. Our goal is to assess the ergogenicity of A_2AR using the pharmacological and genetic tools described above.

SCH 58261: pharmacological inactivation of A_2AR is ergogenic

SCH 58261 was psychostimulant for males, but not for females, since SCH 58261 only increased male locomotion in the open field (F_1,39 = 4.5, η² = 0.1, β = 0.54, 95% CI 58.8–72.1, P < 0.05, Fig. 1A).

The running power of females (F_7,77 = 221, P < 0.05, Fig. 1B) and males (F_7,84 = 183, P < 0.05, Fig. 1B’) increased at each stage of the exercise test. Submaximal V̇O₂ also increased to the maximum (V̇O₂max, dotted line) of females (F_8,77 = 168, P < 0.05, Fig. 1B) and males (F_7,84 = 14.3, P < 0.05, Fig. 1B’). Female (F_8,70 = 180, P < 0.05, Fig.S2A) and male (F_8,70 = 164, P < 0.05, Fig.S2B) submaximal V̇CO₂ kinetics was similar to V̇O₂. SCH 58261 had no effect on these submaximal values.

We demonstrated for the first time that SCH 58261 is ergogenic since SCH 58261 increased V̇O₂max (F_1,36 = 27.7, η² = 0.44, β = 0.99, 95% CI 0.16–0.2, P < 0.5, Fig. 1C) and running power (F_1,35 = 55, η² = 0.61, β = 1.0, 95% CI 1.0–1.3, P < 0.05, Fig. 1D) in both sexes.

SCH 58261 had no effect on increasing RER of females (F_7,70 = 6.9, P < 0.5, η² = 0.43, β = 0.99, Fig. 1E) and males (F_7,84 = 9.4, η² = 0.57, β = 0.99, P < 0.5, Fig. 1E’). Exercise test raised the animals’ core (F_1,26 = 5.5, η² = 0.17, β = 0.62, 95% CI 28.7–29.39, P < 0.05, Fig. 1F) and tail temperature (F_1,22 = 81, η² = 0.78, β = 0.99, 95% CI 24.2–25.6, P < 0.05, Fig. 1G), with no effect of SCH 58261. Figure 1 shows the heating of the mouse’s tail in post-exercise recovery (rec) in relation to rest. Three females at estrus (Fig.S3C) were excluded from temperature experiments due to large exercise-induced tail hyperthermia⁴⁰. The previous results refer to females in diestrus (Fig.S3A), proestrus (Fig.S3B), and metestrus (Fig.S3D).

Neuronal A_2AR antagonism is ergogenic

Caffeine increases exercise performance in rodents^{16,17,19,26,40} and humans^{1,4,8,9,10,11,12,13,14,15,24,28,51,52}. Our results show the key role of A_2AR in the ergogenic effects of caffeine using pharmacological and genetic tools. Thus, the potent and selective A_2AR antagonist SCH 58261 displayed an ergogenic effect similar to that of caffeine, and the ergogenic effect of caffeine was abrogated in A_2AR KO mice.

SCH 58261 and caffeine improved V̇O₂max, running and critical power of wild type mice. These results are in line with the improved running time observed in caffeine-treated rats^16,26,53 and mice¹⁹. Further evidence for the ergogenic effect of caffeine is based on its ability to increase muscle power and endurance output in rodents^{54,55,56,57,58}. For the first time, we demonstrated that the selective antagonism of A_2AR is ergogenic. Also, for the first time, we demonstrated that the genetic inactivation of A_2AR impaired the ergogenic effects of caffeine. Tissue-specific A_2AR KO selectively in forebrain neurons further allowed showing that these ergogenic effects of caffeine are due to the antagonism of A_2AR in forebrain neurons. Thus, we suggest that caffeine decreases central fatigue during exercise. Moreover, caffeine decreased RER in the submaximal stages of the exercise test, an effect also abrogated in A_2AR KO mice. However, exercise-induced core and tail hyperthermia were similar among animals treated with SCH 58261 or caffeine, except for A_2AR KO mice, suggesting possible A₁R-A_2AR-mediated interactions^56,57 in the temperature control⁵¹.