Guanosine-Mediated Anxiolytic-Like Effect: Interplay with Adenosine A1 and A2A Receptors

Abstract

Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A₁R (CPA, CCPA) or A_2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, whereas when administered alone, each compound was ineffective as compared to the control group. Interestingly, the pretreatment with nonselective antagonist caffeine or selective A₁R (DPCPX) or A_2AR (ZM241385) antagonists did not modify the GUO-induced anxiolytic-like effect. Finally, binding assay performed in hippocampal membranes showed that [³H]GUO binding became saturable at 100-300 nM, suggesting the existence of a putative GUO binding site. In competition experiments, ADO showed a potency order similar to GUO in displacing [³H]GUO binding, whereas AR selective agonists, CPA and CGS21680, partially displaced [³H]GUO binding, but the sum of the two effects was able to displace [³H]GUO binding to the same extent of ADO alone. Overall, our results strengthen previous data supporting GUO-mediated anxiolytic-like effects, add new evidence that these effects are blocked by A₁R and A_2AR agonists and pave, although they do not elucidate the mechanism of GUO and ADO receptor interaction, for a better characterization of GUO binding sites in ARs.

Keywords: A1R; A2AR; adenosine; behavior; caffeine; guanosine.

Figure 1

Dose-effect and time-course of guanosine (GUO)-mediated anxiolytic-like effect evaluated by elevated plus maze (EPM) test during a 5 min session. Dose-effect (GUO 45 min): (A) number of open arm entries [F_(3,44) = 3.929, p < 0.02]; (B) time spent in open arms [F_(3,44) = 4.441, p < 0.01]; (C) ratio time in open arms and total time in arms [F_(3,44) = 6.939, p < 0.001]. (D–F) Time-course (30 mg/kg GUO): (D) number of open arm entries [F_{(4,51) =} 6.710, p < 0.0005]; (E) time spent in open arms [F_(4,51) = 7.672, p < 0.0001]; (F) ratio time in open arms and total time in arms [F_(4,51) = 6.148, p < 0.0005]. Ctrl: Control group. Each bar represents the mean value ± SD. Tukey test: * p < 0.05, ** p < 0.01, *** p < 0.001. Ctrl raw mean values: (A) 6.75; (B) 60.05 s; (C) 0.27; (D) 8.58; (E) 95.84 s; (F) 0.45.

Figure 2

Functional competition between GUO and adenosine (ADO) or selective agonists for A₁R and A_2AR on GUO-mediated anxiolytic-like effect evaluated by EPM test during a 5 min session. Pretreatment with ADO (10 mg/kg or 30 mg/kg) resulted in a complete block of anxiolytic-like effect of GUO (30 mg/kg) with significant decrease in (A) number of open arm entries [F_(5,62) =6.972, p < 0.0001], (B) time spent in open arms [F_(5,62) = 6.566, p < 0.0001] and (C) the ratio of time in open arm and total time in arms [F_(5,62) = 18.33, p < 0.0001], when compared to GUO treated group. Pretreatment with A₁R agonist CPA (0.1 mg/kg) or with A_2AR agonist CGS (0.4 mg/kg) was able to completely block the anxiolytic-like effect of GUO (30 mg/kg) with significant decrease in (D) number of open arm entries [F_(5,60) = 5.450, p < 0.0005], (E) time spent in open arms [F_(5,60) = 22.29, p < 0.0001] and (F) the ratio of time in open arm and total time in arms [F_(5,60) = 15.54, p < 0.0001], when compared to GUO treated group. CGS alone produced a significant reduction in time spent in open arms and ratio of time in open arm and total time in arms as compared to control group (Ctrl): each bar represents the mean value ± SD. Tukey test: * p < 0.05, ** p < 0.01; *** p < 0.001. Ctrl raw mean values: (A) 6.23; (B) 76.23 s; (C) 0.33; (D) 7; (E) 97.88 s; (F) 0.43.

Figure 3

Effects of A₁R and A_2AR antagonists on anxiolytic-like effect of GUO evaluated by EPM test during a 5 min session. (A–C) Functional competition between GUO and nonselective ARs antagonist caffeine (Caff). Caffeine alone (30 mg/kg) induced anxiolytic effects, as shown by significant increase in time spent in open arms and ratio of time in open arms and total time in arms as compared to control group (Ctrl). Pretreatment with caffeine (30 mg/kg) did not block anxiolytic-like effect of GUO and showed significant increase in time in open arms and ratio of time in open arms and total time in arms as compared to control group. (A–C) Functional competition between caffeine (30 mg/kg) and ADO (30 mg/kg) showed preservation of caffeine anxiolytic effect with significant increase in time spent in open arms and ratio of time in open arms and total time in arms as compared to control group. (D–F) Functional competition between GUO and selective AR antagonists. Selective A₁R antagonist DPCPX (1 mg/kg) was not able to block GUO anxiolytic-like effect, as shown by significant increase in time in open arms and ratio of time in open arms and total time in arms as compared to control group. DPCPX alone did not show significant changes of all EPM parameters as compared to control group. Pretreatment with selective A_2AR antagonist ZM241385 (0.4 mg/kg) was not able to block GUO (30 mg/kg) anxiolytic-like effect, as shown by significant increase in time in open arms and ratio of time in open arms and total time in arms as compared to control group, and ZM241385 alone did not show significant changes of all EPM parameters as compared to control group. Caffeine pretreatment: number of open arm entries [F_(4,61) = 4.021, p < 0.01], time spent in open arms [F_(4,61) = 6.074, p < 0.0005], ratio time in open arms and total time in arms [F_(4,61) = 6.267, p < 0.0005]. DPCPX and ZM241385 pretreatment: number of open arm entries [F_(5,76) = 2.558, p < 0.05], time spent in open arms [F_(5,76) = 4.637, p = 0.001], ratio time in open arms and total time in arms [F_(5,76) = 4.303, p < 0.002]. Each bar represents the mean value ± SD. Tukey test: * p < 0.05, ** p < 0.01, *** p < 0.001. Ctrl raw mean values: (A) 6.07; (B) 55.35 s; (C) 0.31; (D) 8.44; (E) 75.79 s; (F) 0.33.

Figure 4

Anxiolytic-like effect of GUO evaluated by light–dark box (LDB) and open field test (OFT) test during a 5 min session. (A,B) The LDB data analysis revealed that GUO treatment (30 mg/kg) significantly increased the time spent in light box, as compared to control (Ctrl) group, whereas the number of entries although increased did not become significant. This anxiolytic-like effect of GUO treatment was completely blocked by pretreatment with nonselective ARs agonist ADO. ADO treatment alone did not affect behavior as compared to control group. (C,D) The OFT data analysis revealed that GUO treatment (30 mg/kg) significantly increased both the number of central transitions and the amount of time spent in the center of the arena compared to control group. This anxiolytic-like effect of GUO was completely blocked by pretreatment with ADO, and ADO treatment alone did not affect behavior as compared to control group. LDB: time in light [F_(3,41) = 6.146, p < 0.002], number of entries [F_(3,41) = 1.843]. OFT: time in center [F_(3,42) = 4.330, p < 0.01], number of central transitions [F_(3,42) = 9.930, p < 0.0001]. Each bar represents the mean value ± SD. Tukey test: * p < 0.05, ** p < 0.005, *** p < 0.0005. Ctrl raw mean values: (A) 26.69 s; (B) 2.29; (C) 24.29 s; (D) 8.86.

Figure 5

[³H]GUO binding to hippocampal membranes. (A) The saturation isotherm studies showed that the binding became saturable at [³H]GUO concentrations ranging between 100 and 300 nM. The pooled data resolved for the presence of a single high affinity binding site with an apparent K_D = 80 ± 34 nM; B_max= 2339 ± 339 fmol/mg /protein. (B) Displacement of [³H]GUO binding by GUO and nonselective ARs agonist ADO in rat hippocampal membranes. Competition binding between GUO and ADO showed for ADO almost the same potency order of GUO to displace [³H]GUO (pIC₅₀ 6.069 ± 0.2074 and pIC₅₀ −6.251 ± 0.1649, respectively), although ADO was able to displace only 70% of [³H]GUO binding. (C) [³H]GUO displacement (70 nM) by 500 µM of GUO, ADO, caffeine and selective agonists CPA or CGS21680 in rat hippocampal membranes.ADO was almost as effective as GUO in displacing [³H]GUO binding. Selective A₁R agonist CPA or selective A_2AR agonist CGS21680 displaced respectively 57% and 11% of [³H]GUO binding. Nonselective ARs antagonist caffeine displaced only 24% of [³H]GUO binding.