5′- N-ethylcarboxamido[8- 3H]adenosine binds to two different adenosine receptors in membranes from the cerebral cortex of the rat

Abstract

In the present study it is reported that [ 3H]NECA binds in a specific and saturable manner to membranes from the cerebral cortex of the rat. Scatchard analysis revealed two binding sites. The high affinity binding site ( K d 10.66 ± 5 nM, B max 0.305 ± 0.05 pmol/mg prot) was characterized by the following features: maximum binding at 25°C, sensitivity to pretreatment with NEM and regulation by Gpp[NH]p, enhancing of binding in the presence of 1.0 mM MgCl 2 and 1.5mM CaCl 2; the rank order of potency of several analogues of adenosine in competing with [ 3H]NECA for binding, was CHA > l-pia > NECA > CADO . The low affinity binding site ( K d 261.8 ± 50 nM, B max 4.19 ± 0.33 pmol/mg prot) showed maximum binding at 0°C, insensitivity to pretreatment with NEM up to 1 mM and to regulation by Gpp[NH]p, and inhibition of binding in the presence of MgCl 12 and CaCl 2 The low affinity site was also present in membranes not pretreated with adenosine deaminase and, even in this condition, an IC 50 of 188.5 ± 36 nM for NECA and an IC 50 of 4.35 ± 0.20 μM for adenosine were found. It is concluded that the high affinity binding site is similar to the A 1 adenosine receptors. The low affinity binding site is not classifiable among the A-type adenosine receptors, although it shows peculiar features shared both with the human platelet A 2 receptor and the adenosine receptor formerly studied with [ 3H]adenosine in membranes from the brain of the rat; these results could reflect heterogeneity of adenosine receptors in central nervous system.