Co‐localization and functional interaction between adenosine A_2A and metabotropic group 5 receptors in glutamatergic nerve terminals of the rat striatum

Abstract

The anti-Parkinsonian effect of glutamate metabotropic group 5 (mGluR5) and adenosine A 2A receptor antagonists is believed to result from their ability to postsynaptically control the responsiveness of the indirect pathway that is hyperfunctioning in Parkinson's disease. mGluR5 and A 2A antagonists are also neuroprotective in brain injury models involving glutamate excitotoxicity. Thus, we hypothesized that the antiParkinsonian and neuroprotective effects of A 2A and mGluR5 receptors might be related to their control of striatal glutamate release that actually triggers the indirect pathway. The A 2A agonist, CGS21680 (1-30 nM) facilitated glutamate release from striatal nerve terminals up to 57%, an effect prevented by the A 2A antagonist, SCH58261 (50 nM). The mGluR5 agonist, CHPG (300-600 lM) also facilitated glutamate release up to 29%, an effect prevented by the mGluR5 antagonist, MPEP (10 lM). Both mGluR5 and A 2A receptors were located in the active zone and 57 ± 6% of striatal glutamatergic nerve terminals possessed both A 2A and mGluR5 receptors, suggesting a presynaptic functional interaction. Indeed, submaximal concentrations of CGS21680 (1 nM) and CHPG (100 lM) synergistically facilitated glutamate release and the facilitation of glutamate release by 10 nM CGS21680 was prevented by 10 lM MPEP, whereas facilitation by 300 lM CHPG was prevented by 10 nM SCH58261. These results provide the first direct evidence that A 2A and mGluR5 receptors are co-located in more than half of the striatal glutamatergic terminals where they facilitate glutamate release in a synergistic manner. This emphasizes the role of the modulation of glutamate release as a likely mechanism of action of these receptors both in striatal neuroprotection and in Parkinson's disease.

Modification upon aging of the density of presynaptic modulation systems in the hippocampus

“…After blocking for 2 h at room temperature with 5% milk in Tris-buffered saline, pH 7.6 containing 0.1% Tween 20 (TBS-T), the membranes were incubated overnight at 4 • C with the different antibodies, namely: widely used mouse anti-␣-tubulin (1:10,000 dilution, from Sigma-Portugal) and goat anti-glyceraldeheyde-3-phosphate dehydrogenase (GAPDH, 1:1000 dilution, from Santa Cruz Biotechnology, Alfagene, Portugal); previously validated (see Pinheiro et al, 2003) mouse anti-synaptophysin (1:1000, from Sigma) and mouse anti-SNAP-25 (1:10,000, from Sigma); previously used (e.g. Köfalvi et al, 2005) guinea-pig anti-vesicular GABA transporter (vGAT, 1:1000, from Calbiochem, PGHitec, Portugal), guinea-pig anti-vesicular glutamate transporters types 1 and 2 (vGluT1 and vGluT2, 1:5000, from Chemicon) and guinea-pig anti-vesicular acetylcholine transporter (vAChT, 1:500, from Chemicon); previously validated rabbit anti-adenosine A 1 receptor (1:1000, from Affinity Bioreagents, Golden, USA; see Rebola et al, 2003b), goat anti-adenosine A 2A receptor (1:500 dilution, from Santa Cruz Biotechnology; see Rebola et al, 2005), rabbit L-15 Cterminus anti-CB 1 receptor (1:500, generously supplied by Dr. Ken Mackie, Indiana University, Bloomington, USA; see Köfalvi et al, 2005), rabbit anti-mGluR5 receptor (1:3000, from Upstate Biotechnology; see Rodrigues et al, 2005b) and goat anti-P2Y1 receptor (1:200, from Santa Cruz Biotechnology; see Rodrigues et al, 2005a). After four washing periods for 10 min with TBS-T containing 0.5% milk, the membranes were incubated with the alkaline phosphatase-conjugated anti-goat, anti-rabbit, anti-mouse or anti-guinea-pig secondary antibody (1:2000, from Amersham) in TBS-T containing 1% milk during 90 min at room temperature.…”

Adenosine A2A receptors in ventral striatum, hypothalamus and nociceptive circuitry

“…In the glutamatergic terminals they are found intrasynaptically, forming heteromers with adenosine A 1 receptors (Ciruela et al, 2006) (Fig. 1) and possibly with presynaptic D 2 , mGlu 5 and CB 1 receptors (Tanganelli et al, 2004;Rodrigues et al, 2005;Carriba et al, 2007). The different A 2A receptor-containing heteromers provide distinct molecular entities with unique functional and pharmacological properties (as reviewed in Ferré et al, 2007).…”

An Update on Adenosine A2A-Dopamine D2 Receptor Interactions: Implications for the Function of G Protein-Coupled Receptors

“…Again, this has the biochemical characteristics of an antagonistic A 2A -D 2 receptor interaction at the second messenger level, which might not depend on A 2A -D 2 receptor heteromerization or which might depend on A 2A -mGlu 5 -D 2 receptor interactions (see above). In fact, A 2A and mGlu 5 receptors have been found to be co-localized in a large proportion of striatal glutamatergic terminals, where they facilitate glutamate release in a synergistic manner [76]. This presynaptic antagonistic A 2A -D 2 receptor interaction can also have implications for the treatment of Parkinson's disease with the co-administration of A 2A antagonists and L-DOPA or D 2 receptor agonists.…”