G Protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of adenosine A1 and striatal A2A receptors

Abstract

The effect of guanine nucleotide-binding protein (G protein) modifiers on the binding of the adenosine A2A receptor agonist 2-[4-(2-p-carboxyethyl[3H])phenyl-amino]-5'-N-ethylcarboxamidoadenosine ([3H]CGS 21680) and of the adenosine A1 receptor agonist [3H]R-phenylisopropyladenosine ([3H]R-PIA) to rat cortical and striatal membranes was studied. Guanosine 5'-(beta,gamma-imido)triphosphate (1-300 microM), which uncouples all G proteins, more effectively inhibited [3H]CGS 21680 (30 nM) binding in the cortex than [3H]R-PIA (2 nM) binding to cortical or striatal membranes or [3H]CGS 21680 (30 nM) binding in the striatum. N-Ethylmaleimide (1-300 microM) or pertussis toxin (1-100 microg/ml), which uncouple G(i)/G(o) protein-coupled receptors, more effectively inhibited [3H]R-PIA binding to cortical or striatal membranes and [3H]CGS 21680 binding in the cortex than [3H]CGS 21680 binding in the striatum. Cholera toxin (2.5-250 microg/ml), which uncouples G(S) protein-coupled receptors, more effectively inhibited [3H]CGS 21680 binding in the striatum than [3H]CGS 21680 binding in the cortex and less effectively inhibited [3H]R-PIA binding to cortical or striatal membranes. Treatment of solubilised cortical membranes with pertussis toxin (50 microg/ml) decreased [3H]CGS 21680 (30-100 nM) binding which almost fully recovered after reconstitution with G(i)/G(o) proteins. The K(i) for displacement of [2-3H]-(4{2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin+ ++-5-ylamino]ethyl}phenol) ([3H]ZM 241385, 1nM) by CGS 21680 was 110 nM (95%CI: 98-122 nM) in non-treated, 230 (167-292) nM in pertussis toxin (25 microg/ml)-treated and 222 (150-295) nM in cholera toxin (50 microg/ml)-treated cortical membranes; in contrast, the K(i) for displacement of [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazol o(1,5-c)pyrimidine ([3H]SCH 58261, 1 nM) by CGS 21680 was 74 (57-91) nM in non-treated, 71 (44-100) nM in pertussis toxin-treated and 147 (100-193) nM in cholera toxin-treated cortical membranes. Finally, CGS 21680 displaced monophasically the binding of the A1 antagonist, [3H]8-cyclopentyl-1,3-dipropylxanthine (2 nM), and the A1 agonist, [3H]R-PIA (2 nM), in 2 or 10 mM Mg(2+)-medium, either at 25 degrees C or 37 degrees C, in cortical or striatal membranes. These results indicate that CGS 21680 does not bind to A1 receptors and that limbic CGS 21680 binding sites differ from striatal-like A2A receptors since they couple to G(i)/G(o) proteins, as well as to G(s) proteins.

Transducing system operated by adenosine A2A receptors to facilitate acetylcholine release in the rat hippocampus

“…The present results are particularly important in light of the several reports showing that the prototypical adenosine A 2A receptor agonist, CGS 21680, essentially binds to extrastriatal regions of the central nervous system to sites apparently different from adenosine A 2A receptors (Cunha et al, 1996(Cunha et al, , 1999Johansson and Fredholm, 1995;Johansson et al, 1993;Lindström et al, 1996). The functional demonstration of pharmacologically well-defined adenosine A 2A receptors in hippocampal neurons extends previous demonstrations that adenosine A 2A receptors mRNA is present in the hippocampus (Cunha et al, 1994a;Dixon et al, 1996;Peterfreund et al, 1996), namely in neurons (Lopes et al, 2001), and constitutes a clear demonstration that adenosine A 2A receptors are not confined to the striatum.…”

“…However, some studies have cast doubts on the real selectivity of CSC and of ZM 241385 (Cunha et al, 1996;de Mendonc ßa and Ribeiro, 1994;Lindström et al, 1996;Lopes et al, 1999a) towards adenosine A 1 receptormediated responses. Thus, we decided to test the ability of SCH 58261 to antagonize the CGS 21680-and HENECAinduced facilitation of acetylcholine release, since this compound has repeatedly been shown to behave as a selective antagonist of adenosine A 2A receptors (Cunha et al, 1999;Fredholm et al, 1998;Lindström et al, 1996). As illustrated in Fig.…”

“…As occurs for the modulation of glutamate (Ambrósio et al, 1997;Lopes et al, 2002) and serotonin release (Okada et al, 2001), the modulation of acetylcholine release by adenosine in the hippocampus is also controlled by both adenosine A 1 and A 2A receptors (Cunha et al, 1994a;Jin and Fredholm, 1997). The presence of adenosine A 2A receptors in the hippocampus has been confirmed using molecular biology studies, like polymerase chain reaction (PCR), in situ hybridization (Cunha et al, 1994b) and Northern blot analysis (Peterfreund et al, 1996), but binding studies have revealed an atypical binding pattern (Cunha et al, 1996) and G protein coupling (Cunha et al, 1999) of these putative adenosine A 2A receptors. Some functional peculiarities of the facilitatory effects of the prototypical adenosine A 2A receptor agonist, CGS 21680 (Fredholm et al, 1994), include the strict dependency on co-activation of adenosine A 1 receptors in the control of glutamate release (Lopes et al, 2002), the atypical pharmacology in the control of GABA release (Cunha and Ribeiro, 2000a) and the coupling to protein kinase C in the control of excitatory synaptic transmission (Cunha and Ribeiro, 2000b;Lopes et al, 2002).…”

Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes

“…8 The apparent discrepancy between these findings and a role of adenosine A 2A receptors can be explained considering the possible coupling of (MEK1) or pertussis toxin-inhibitable G i proteins. [26][27][28] It is noteworthy that stimulation of adenosine A 2A -receptor protects liver sinusoidal endothelial cells against reperfusion injury 9 and reduces TNF-␣ release by lipopolysaccharide-stimulated Kupffer cells. 29 However, in both these cell types a cyclic-AMP dependent pathway appears to be coupled with the stimulation of adenosine A 2A -receptors.…”

Caffeine and Adenosine

“…Besides the high affinity A1 and A2A receptors, the cloned adenosine receptors also include the high affinity A3 receptor, and the low affinity A2B receptor. Other entities have been proposed based on functional and/or binding studies (e.g., an atypical A2A receptor in the rat hippocampus [4]; the A3 receptor in the frog motor nerve endings [5]). The first proposal for the existence of an A3 AR was based upon pharmacological characteristics, namely high affinity for agonists and xanthine sensitivity [5].…”