Regional differences in the effect of guanine nucleotides on agonist and antagonist binding to adenosine A1-receptors in rat brain, as revealed by autoradiography

Abstract

The effect of increasing doses of GTP on agonist and antagonist binding to adenosine A1-receptors in different regions of rat brain was studied by autoradiography. A high concentration of GTP (100μM) practically eliminated the binding of the agonist [ 3H] n 6-cyclohexyladenosine in all regions. However, there were regional differences in the effects of low concentrations of GTP (0.1–10 μM). In some regions, for example the hippocampus, all concentrations of GTP decreased [ 3H] n 6-cyclohexyladenosine binding, by decreasing the B max. In other structures, e.g. the superior colliculus, there was a biphasic response to GTP. Concentrations of 0.1–3 μ M increased agonist binding, apparently due to a decrease in K D, whereas higher concentrations also decreased binding in these regions. The effects of GTP were mimicked by the stable GTP analogue guanosine-5'- O-(3-thiotriphosphate). GTP (0.5–100μM) increased the binding of the antagonist [ 3H]8-cyclopentyl-1,3-dipropylxanthine in all regions, but most markedly in those where GTP had a biphasic effect on agonist binding. Decreasing the levels of endogenous adenosine by increasing the concentration of adenosine deaminase and adding the 5'-nucleotidase inhibitor α-β-methylene adenosine-5'-diphosphate gave an increase in [ 3H]8-cyclopentyl-1,3-dipropylxanthine binding and diminished the response to GTP. In sections treated with adenosine deaminase and α-β-methylene adenosine-5'-diphosphate, GTP steadily decreased [ 3H] n 6-cyclo-hexyladenosine binding in all regions. Thus, the GTP-induced increase in both agonist and antagonist binding may be due to a displacement of endogenous adenosine. In the presence of 1 mM EDTA, GTP had a monophasic effect on the binding of [ 3H] n 6-cyclohexy-ladenosine in all regions. In the presence of 2 mM MgCl 2 a biphasic response to GTP was seen in all regions. In EDTA washed sections, the effect of MgCl 2 on [ 3H] n 6-cyclohexyladenosine binding was more pronounced in the superior colliculus, where we had observed a biphasic response to GTP. The results suggest that there are regional differences in the effects of GTP on adenosine A1-receptor binding in rat brain, that reflect regional differences in the magnesium-dependent binding of endogenous adenosine, which is bound to the receptor by tight binding, is very difficult to remove, and easily interferes with radioligand binding in in vitro experiments. There may be regional differences in the sensitivity of A1-receptor-G-protein complexes to magnesium, that reflect a heterogeneity of the G-proteins to which the A1-receptors are coupled.