Effects of mono- and divalent ions on the binding of the adenosine analogue CGS 21680 to adenosine A 2 receptors in rat striatum

Abstract

The effect of monovalent and divalent cations on equilibrium binding of the adenosine A 2-selective agonist ligand CGS 21680 (2-[ p-(2-carbonylethyl)phenylethylamino]-5′- N-ethylcarboxamidoadenosine) to membranes prepared from rat striatum was examined. Competition experiments with cyclohexyladenosine, 2-chloroadenosine, N-ethylcarboxamidoadenosine and CGS 21680 suggest that at 2 nM [ 3H]CGS 21680 binds to a single site with the pharmacology of an A 2a receptor. Magnesium and calcium ions caused a concentration-dependent increase in binding that reached about 10-fold at 100 mM. Manganese ions had a biphasic effect on binding with a maximal increase at 5 mM. Lithium, sodium and potassium ions all caused a concentration-dependent decrease of binding. Sodium was most potent, potassium least. At 200 mM ion concentration, the inhibition of binding was 88% by sodium, 47% by lithium and 29% by potassium ions. The effect of sodium chloride was the same as that of sodium acetate. The effect of sodium ions was essentially similar to that of Gpp(NH)p. However, sodium ions produced a larger effect than even maximally effective concentrations of Gpp(NH)p. The maximal inhibition by Gpp(NH)p was about 55% at 2 nM radioligand concentration irrespective of the magnesium concentration. The maximal effect of sodium ions was reduced by increasing concentrations of magnesium ions. Increasing magnesium ion concentration from 1 to 100 mM increased the half-maximally effective concentration of Gpp(NH)p almost 10-fold and that of sodium ions less than 2-fold. Furthermore, sodium ions and Gpp(NH)p had additive effects. The binding of an agonist to striatal A 2a receptors shows an unusually large dependence on both divalent and monovalent cations that can only partly be explained by a change in the coupling to G s proteins.