2A receptor (A 2A R)-mediated tonic inhibition of dopamine D 2 receptor (D 2 R) signaling in the striatopallidal pathway. 2A R antagonist; 2A R in complex with either 4-[2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethyl]phenol (ZM241385), a well-known potent inverse agonist, or caffeine revealed the same cardinal structural features, suggesting that caffeine can stabilize the receptor in an inverse agonistic conformation. 2A R-based ligand binding to isolated receptor conformations from the crystal structures predicted caffeine to be a neutral antagonist. 2A R is thus under intense debate as its determination would have critical consequences not only for understanding the mechanism underlying the antiparkinsonian action of this drug but also for designing new PD pharmacotherapies. This is particularly relevant given that constitutive A 2A R signaling is thought to increase in certain pathological conditions including PD. 2A R’s constitutive activity would be promising therapeutic drugs. Here, we found that caffeine acted as an A 2A R inverse agonist in cells and in a tissue model for PD. Caffeine is considered a putative drug for the treatment of Parkinson’s disease (PD). (1, 2) Its antiparkinsonian effect is thought to be mediated by blocking the well-known adenosine Areceptor (AR)-mediated tonic inhibition of dopamine Dreceptor (DR) signaling in the striatopallidal pathway. (1) Caffeine is proposed to act as a weak (μM) AR antagonist; (3) however, recent structural studies suggest that caffeine may behave as an inverse agonist. (4) Indeed, the comparison of crystal structures of an engineered AR in complex with either 4-[2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-][1,3,5]triazin-5-yl-amino]ethyl]phenol (ZM241385), a well-known potent inverse agonist, or caffeine revealed the same cardinal structural features, suggesting that caffeine can stabilize the receptor in an inverse agonistic conformation. (4) Nonetheless, a parallel study predicting AR-based ligand binding to isolated receptor conformations from the crystal structures predicted caffeine to be a neutral antagonist. (5) The intrinsic efficacy of caffeine, antagonist vs inverse agonist, on the AR is thus under intense debate as its determination would have critical consequences not only for understanding the mechanism underlying the antiparkinsonian action of this drug but also for designing new PD pharmacotherapies. This is particularly relevant given that constitutive AR signaling is thought to increase in certain pathological conditions including PD. (6, 7) Therefore, inverse agonists (8) able to block AR’s constitutive activity would be promising therapeutic drugs. Here, we found that caffeine acted as an AR inverse agonist in cells and in a tissue model for PD.

2A R. We found that 2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680), a well-established selective A 2A R agonist, induced cAMP accumulation by ∼3.5-fold over the basal, whereas caffeine reduced the basal level of cAMP by ∼10-fold (Figure 2A R-mediated ERK phosphorylation (Figure 2A R. A limited number of strategies can be applied to measure inverse agonism of ligands acting at G protein-coupled receptors (GPCRs). The most conventional among them consists of determining alterations in basal cell signaling upon receptor overexpression. Accordingly, we evaluated the effects of caffeine on basal levels of both 3′,5′-cyclic adenosine monophosphate (cAMP) and phosphorylation of ERK in cells transiently expressing the AR. We found that 2-[-(2-carboxyethyl)phenyl-ethylamino]-5′--ethylcarboxamidoadenosine (CGS21680), a well-established selective AR agonist, induced cAMP accumulation by ∼3.5-fold over the basal, whereas caffeine reduced the basal level of cAMP by ∼10-fold (Figure 1 a). Interestingly, a dose-dependent effect was found for caffeine, which was comparable, although with lower potency and efficacy, to that obtained for the well-described inverse agonist ZM241385 (Figure S1, Supporting Information ). In addition, an inhibitory effect of caffeine was also observed in basal levels of AR-mediated ERK phosphorylation (Figure 1 b). These signaling data supported the view that caffeine acts as an inverse agonist by reducing the constitutive activity of the AR.

Figure 1 Figure 1. Intrinsic efficacy of caffeine. (a) cAMP measurement in HEK293 cells expressing A 2A R and incubated with saline, 200 nM CGS21680, and 100 μM caffeine. Saline-stimulated cAMP was set as 100%, and bars represent the mean ±SEM of four independent experiments. Letters (a, b, and c) designate a significant difference between treatments (P < 0.05). (b) Extracellular signal regulated kinase 1/2 (ERK1/2) activation in HEK293 cells expressing A 2A R and incubated with saline, 200 nM CGS21680 and 100 μM caffeine. Saline-stimulated cAMP was set as 100%, and bars represent the mean ± SEM of four independent experiments. Letters (a, b, and c) designate a significant difference between treatments (P < 0.05). (c) Design of the predicted FRET-based A 2A R sensor conformations in response to a full agonist (left) or to an inverse agonist (right). (d) Specific MRS5424 binding to the A 2A RFlAsH/CFP in the absence (middle) or presence (right) of caffeine. Scale bar: 10 μm. Images are representative of four independent experiments. (e) FRET changes of the A 2A RFlAsH/CFP in response to a full or an inverse agonist. Shown are the changes of the calculated FRET ratio (F FlAsH /F CFP ), induced by rapid superfusion of adenosine (100 μM) or caffeine (300 μM). Traces are representative of at least five separate experiments. (f) Plot of the effects on cAMP accumulation of adenosine and caffeine versus the changes that they induced in the normalized FRET ratio (F FlAsH /F CFP ). The ΔFRET ratio (A-1) induced by each single A 2A R ligand (adenosine (100 μM) or caffeine (300 μM); n = 5) is represented vs the effects on cAMP accumulation (setting saline-stimulated effects to 0%) achieved for the same ligand (adenosine (100 μM) or caffeine (300 μM); n = 4).

2A R (A 2A RFlAsH/CFP) 2A R biosensor by using MRS5424, 2A R agonist (Figure To further confirm the intrinsic efficacy of caffeine, we used an approach that is independent of downstream biochemical responses by recording its action directly at the level of the receptor. To this end, we used an intramolecular biosensor based on Förster resonance energy transfer (FRET) for the AR (A (9) as depicted in Figure 1 c. This type of GPCR biosensor permits the measurement of changes in receptor conformation upon ligand binding in live cells and the rigorous measurement of the intrinsic efficacy of an agonist (full, partial, or inverse) directly at the level of the receptor and independently from variation in receptor number and/or cell conditions. (9-11) We first ascertained that caffeine bound to and displaced a full agonist from the AR biosensor by using MRS5424, (12) a fluorescent AR agonist (Figure 1 d, left panel). HEK293 cells expressing the receptor biosensor perfused with MRS5424 alone were selectively labeled at the plasma membrane (Figure 1 d, middle panel). Perfusion of caffeine in addition to MRS5424 blocked most of the cell fluorescence (Figure 1 d, right panel), indicating that MRS5424 and caffeine competed for the same binding site on the receptor.

2A RFlAsH/CFP biosensor to compare the action of adenosine and caffeine directly at the level of the receptor. Thus, we evaluated changes on FRET signals from a cyan fluorescent protein (CFP) sequence introduced in the C-terminus of the A 2A R to FlAsH, a selectively reactive fluorescein in the third intracellular loop, upon ligand challenging. We observed that adenosine caused a fast decrease in FRET, whereas caffeine caused FRET to increase with slower kinetics (Figure 2A R to adopt distinct conformations in response to adenosine or caffeine, which correlates with the distinct functional efficacies of these two ligands (Figure 2A -adrenergic receptor. 2A - but also β 1 -adrenergic receptors, in which inverse agonists were also found to trigger a very distinct off mode of the receptor’s activation switch. Next, we performed FRET studies in live cells expressing the Abiosensor to compare the action of adenosine and caffeine directly at the level of the receptor. Thus, we evaluated changes on FRET signals from a cyan fluorescent protein (CFP) sequence introduced in the C-terminus of the AR to FlAsH, a selectively reactive fluorescein in the third intracellular loop, upon ligand challenging. We observed that adenosine caused a fast decrease in FRET, whereas caffeine caused FRET to increase with slower kinetics (Figure 1 e). Both these opposite changes and the distinct kinetics of change in intramolecular FRET (see also Figure S2, Supporting Information ) are evidence of the capacity of the AR to adopt distinct conformations in response to adenosine or caffeine, which correlates with the distinct functional efficacies of these two ligands (Figure 1 f). Of note, the magnitude of caffeine-induced FRET changes was lower than for adenosine-mediated changes. This fact may be attributed to close energy transfer efficiencies in inverse agonism and basal conformational states. Thus, we performed additional photobleaching experiments and observed that caffeine mediated a slight but significant increase of FRET efficiency compared to control conditions (Figure S3, Supporting Information ). These results are in agreement with similar studies done with the α-adrenergic receptor. (10) Overall, an inverse agonism ascribed to caffeine could be postulated, an observation consistent with that obtained not only for α- but also β-adrenergic receptors, in which inverse agonists were also found to trigger a very distinct off mode of the receptor’s activation switch. (10, 13)

2A R inverse agonist under physiological conditions, we generated 6-hydroxydopamine (6-OHDA)-lesioned mice as an animal model for PD, 2A R increased functionality. 2A R since caffeine did not affect locomotion in A 2A R deficient (A 2A R-KO) mice (Figure 2A R-dependent caffeine locomotor effect as previously reported 2A R activity that would be responsible of caffeine-mediated locomotion effects. The inhibitory effect of inverse agonists on constitutive GPCR signaling is usually straightforwardly assessed in heterologous cell systems, but not in native tissues. (14) To test our hypothesis that caffeine acts as an AR inverse agonist under physiological conditions, we generated 6-hydroxydopamine (6-OHDA)-lesioned mice as an animal model for PD, (15) a disease related to AR increased functionality. (6, 7) We examined the effect of caffeine on locomotor activity in both control (sham) and 6-OHDA-lesioned mice. The administration of caffeine (10 mg/kg) produced hyper-motility in both 6-OHDA-lesioned and sham animals. This increased mobility was selectively mediated via the AR since caffeine did not affect locomotion in AR deficient (AR-KO) mice (Figure 2 a). The caffeine-induced motor effect was markedly intensified in the 6-OHDA-lesioned mice (Figure 2 a). These data confirmed the AR-dependent caffeine locomotor effect as previously reported (16, 17) and further showed that the 6-OHDA lesion resulted in enhanced AR activity that would be responsible of caffeine-mediated locomotion effects.

Figure 2 Figure 2. Evaluation of the effects of caffeine in a mouse model of Parkinson’s disease. (a) Locomotor activity assessed in the open-field paradigm. Caffeine-evoked locomotor activity is expressed as percentage increase compared to baseline activity (which is set to 0% after saline injection). 6-OHDA (n = 6); SHAM (n = 6); KO (n = 8); vertical bars indicate mean ± SEM, and letters (a, b, and c) designate a significant difference between groups (P < 0.05). (b) Western blot analysis of striatal synaptosomes showing that TH expression is reduced upon 6-OHDA lesion, while A 2A R is not altered. Load control used for quantitating was α-actinin. A representative blot of four samples is shown. (c) cAMP measurement of striatal synaptosomes and incubated with 10 μM forskolin, 200 nM CGS21680, and 100 μM caffeine. Basal cAMP levels were set as 0%, and bars represent the mean ± SEM of four independent experiments.

2A R and quantified the extent of the 6-OHDA lesion by the loss of tyrosine hydroxylase (TH) (Figure P < 0.05) of TH expression upon 6-OHDA lesion, confirming dopamine denervation. Conversely, we did not find a significant difference (P > 0.05) in levels of A 2A R expression between sham and 6-OHDA-lesioned mice. Various studies have shown either no alteration or an increase of striatal A 2A R expression under dopaminergic denervation, 2A R basal function described in PD We next evaluated the expression of AR and quantified the extent of the 6-OHDA lesion by the loss of tyrosine hydroxylase (TH) (Figure 2 b). We observed a significant reduction (< 0.05) of TH expression upon 6-OHDA lesion, confirming dopamine denervation. Conversely, we did not find a significant difference (> 0.05) in levels of AR expression between sham and 6-OHDA-lesioned mice. Various studies have shown either no alteration or an increase of striatal AR expression under dopaminergic denervation, (18, 19) indicating that the increase in AR basal function described in PD (6, 7) would be mostly explained by mechanisms other than receptor overexpression alone.

2A R agonist CGS21680 did not induce cAMP generation in A 2A R-KO animals (Figure 2A R-KO mice, but decreased cAMP levels (P < 0.05) in 6-OHDA-lesioned mice (Figure 2A R activity, an effect of caffeine could not be detected. However, we observed a clear inverse agonistic action of caffeine under pathological conditions where constitutive A 2A R activity would be increased. It could be then postulated that A 2A R inverse agonists would be more efficient than neutral antagonists in the management of PD. Indeed, several putative A 2A R blockers have recently been in clinical trials, and one of the most promising is preladenant, 2A R activity but also drug-mediated adverse effects. The last set of experiments was designed to further confirm our hypothesis. Thus, cAMP accumulation was determined in striatal synaptosomes from sham and 6-OHDA-lesioned mice in response to forskolin, CGS21680, or caffeine. Two positive controls validated the reliability of the approach used: (1) forskolin, a direct activator of adenylyl cyclases, induced cAMP production in all conditions; and (2) the selective AR agonist CGS21680 did not induce cAMP generation in AR-KO animals (Figure 2 c). Caffeine did not exert any effect on sham or AR-KO mice, but decreased cAMP levels (< 0.05) in 6-OHDA-lesioned mice (Figure 2 c). Interestingly, the prototypic inverse agonist ZM241385 (100 nM) also decreased cAMP basal levels with similar efficacy (∼10%) only in 6-OHDA-lesioned mice. Hence, in normal physiological conditions where basal cAMP accumulation is probably not exclusively dependent on AR activity, an effect of caffeine could not be detected. However, we observed a clear inverse agonistic action of caffeine under pathological conditions where constitutive AR activity would be increased. It could be then postulated that AR inverse agonists would be more efficient than neutral antagonists in the management of PD. Indeed, several putative AR blockers have recently been in clinical trials, and one of the most promising is preladenant, (20, 21) which has been precisely characterized as an inverse agonist. (5) However, we did not observe significant differences in the efficacy of caffeine and a more potent drug, i.e., ZM241385, in the tissue model for PD. However, it would seem likely that depending on the pathological status the choice of a low- or high-potency drug would permit the fine modulation not only of AR activity but also drug-mediated adverse effects.