Publicação
Characterization of the levels and G-protein coupling of adenosine A2A receptors in hippocampus and striatum of transgenic rats overexpressing adenosine A2A receptors in the brain
| Resumo: | Background: The transmembrane G-protein coupled adenosine A2A Receptors (A2AR) are one of the main brain targets of adenosine, an homeostatic neuromodulator (Fredholm et al., 2007). Evidence was found of upsurge of A2AR expression, accompanied by coupling and affinity alterations associated to cognitive deficits in ageing (Diógenes et al., 2007; Lopes et al., 1999b) and neurodegeneration, namely Alzheimer’s Disease (Eskelinen et al., 2009). However, it is not known whether A2AR are involved in the development of pathology and aging phenotype; and if the overexpression happens as cause or a consequence of disease. Objectives: Test the hypothesis that inducing neuronal A2AR overexpressing in the forebrain (CAMkIIa promoter driven human A2A receptor rats) is sufficient to mimic functional and structural changes observed in aged rats. Methods: Radioligand binding of [3H] ZM241385 (antagonist of A2AR) to perform saturation and competition assays in neuronal membranes from striatum and hippocampus of wild-type (WT) and A2AR over-expression transgenic rats (TG). Results: Saturation binding for A2AR, showed an increase in Bmáx of the TG compared to WT both in the striatum (1249±79 and 931,6±72,9 fmol/mg, n=3) and the hippocampus (731,2±156,1 fmol/mg versus undetectable levels in WT). The affinity constant (KD) was similar in the striatum (0,123±0,045 nM) of TG and WT (0,106±0,05 nM); whereas in the hippocampus it was 0,62±0,39 nM for TG (n=3). Studies of Competition with GppNHp were performed to investigate the coupling of A2AR to G-proteins. In the striatum, the GTP shift1 was 166% (n=4) in WT and it was superior in TG: 194 (n=4). Analytical comparison showed significant difference (p-value of 0,03). In the hippocampus, TG GTP shift was 211% (n=4), showing no significant difference from the striatum of TG. Conclusions: The major conclusion of the present study is that over-expression of A2A receptors in the forebrain induces increased coupling of G-protein to A2AR in striatum and hippocampus. This profile is similar to the one found in aged animals (Lopes et al., 1999b), albeit with lower magnitude. This suggests the contribution of other factors, besides over-expression, to the modification of GTP binding profile. |
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| Autores principais: | Bento, Marta Leal, 1992- |
| Assunto: | Receptor A2A de adenosina Proteínas de ligação ao GTP Cognição Neurociências |
| Ano: | 2016 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Background: The transmembrane G-protein coupled adenosine A2A Receptors (A2AR) are one of the main brain targets of adenosine, an homeostatic neuromodulator (Fredholm et al., 2007). Evidence was found of upsurge of A2AR expression, accompanied by coupling and affinity alterations associated to cognitive deficits in ageing (Diógenes et al., 2007; Lopes et al., 1999b) and neurodegeneration, namely Alzheimer’s Disease (Eskelinen et al., 2009). However, it is not known whether A2AR are involved in the development of pathology and aging phenotype; and if the overexpression happens as cause or a consequence of disease. Objectives: Test the hypothesis that inducing neuronal A2AR overexpressing in the forebrain (CAMkIIa promoter driven human A2A receptor rats) is sufficient to mimic functional and structural changes observed in aged rats. Methods: Radioligand binding of [3H] ZM241385 (antagonist of A2AR) to perform saturation and competition assays in neuronal membranes from striatum and hippocampus of wild-type (WT) and A2AR over-expression transgenic rats (TG). Results: Saturation binding for A2AR, showed an increase in Bmáx of the TG compared to WT both in the striatum (1249±79 and 931,6±72,9 fmol/mg, n=3) and the hippocampus (731,2±156,1 fmol/mg versus undetectable levels in WT). The affinity constant (KD) was similar in the striatum (0,123±0,045 nM) of TG and WT (0,106±0,05 nM); whereas in the hippocampus it was 0,62±0,39 nM for TG (n=3). Studies of Competition with GppNHp were performed to investigate the coupling of A2AR to G-proteins. In the striatum, the GTP shift1 was 166% (n=4) in WT and it was superior in TG: 194 (n=4). Analytical comparison showed significant difference (p-value of 0,03). In the hippocampus, TG GTP shift was 211% (n=4), showing no significant difference from the striatum of TG. Conclusions: The major conclusion of the present study is that over-expression of A2A receptors in the forebrain induces increased coupling of G-protein to A2AR in striatum and hippocampus. This profile is similar to the one found in aged animals (Lopes et al., 1999b), albeit with lower magnitude. This suggests the contribution of other factors, besides over-expression, to the modification of GTP binding profile. |
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