Adenosine A1 and A2A receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices

Abstract

6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson's disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 μM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A₁ (A₁R) and/or A_2A receptors (A_2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A₁R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A₁R agonist, did not alter GUO effects. Regarding A_2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A_2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A₁R antagonist DPCPX, and the A_2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A₁R-A_2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

Keywords: 6-Hydroxydopamine, Striatal slices; Adenosine A1 and A2A receptors; Guanosine.

Fig. 1

Effects of A₁R modulation on 6-OHDA-induced toxicity in striatal slices. Experimental design is describe in a. Striatal slices were pre-incubated with A₁R agonist CCPA (100 nM; b, c) or A₁R antagonist DPCPX (250 nM; d, e). Slices were incubated with 6-OHDA (100 μM) and/or co-incubated with GUO (100 μM). 6-OHDA-induced mitochondrial membrane potential (ΔΨ) (b, d) and ROS levels (c, e). Data are expressed as percentage of controls normalized among individual experiments and represent means with SEM (n = 6). (*) when p < 0.05 compared with control or (#) compared to 6-OHDA group (one-way ANOVA followed by Tukey’s test)

Fig. 2

Effects of A_2AR modulation on 6-OHDA-induced toxicity in striatal slices. Experimental design is describe in a. Striatal slices were pre-incubated with of A_2AR agonist CGS 21680 (CGS, 30 nM; b, c) or A_2AR antagonist SCH 58261 (SCH, 50 nM; d, e). Slices were incubated with 6-OHDA (100 μM) and/or co-incubated with GUO (100 μM). 6-OHDA-induced mitochondrial membrane depolarization (b, d) and ROS levels increase (c, e). Data are expressed as percentage of controls normalized among individual experiments and represent means with SEM (n = 6). (*) when p < 0.05 compared with control or (#) compared to 6-OHDA group (one-way ANOVA followed by Tukey’s test)

Fig. 3

A₁R and A_2AR modulation on ATP levels in striatal slices. Experimental design is describe in a. Effect of pre-incubation of A₁R antagonist, DPCPX (250 nM) (b) or A_2AR agonist, CGS21680 (CGS, 30 nM) (c) on 6-OHDA-induced ATP depletion. Slices were incubated with 6-OHDA (100 μM) and/or co-incubated with GUO (100 μM). Data are expressed as μmol ATP/μg of protein of each sample and represent means with SEM (n = 3). (*) when p < 0.05 compared with control or (#) compared to 6-OHDA group (one-way ANOVA followed by Tukey’s test)