Characterization of [ 35 S]‐ATPαS and [ 3 H]‐α,β‐MeATP binding sites in rat brain cortical synaptosomes: regulation of ligand binding by divalent cations

We made a comparative analysis of the binding characteristics of the radioligands [ 35 S]‐ATPαS and [ 3 H]‐α,β‐MeATP in order to test whether these ligands can be used to analyse P 2 ‐purinoceptors in synaptosomal membranes from rat brain cortex. Synaptosomes possess sites with high affinity for [ 35 S]‐ATPαS ( K d =22.2±9.1 n M , B max =14.8 pmol mg −1 protein). The rank order of the competition potency of the different compounds (ATPαS, ATP, ATPγS>ADPβS, 2‐MeSATP>deoxyATP, ADP>>UTP, α,β‐MeATP, AMP, Reactive Blue‐2, suramin, isoPPADS) is consistent with pharmacological properties of P 2Y ‐purinoceptors. Under identical conditions [ 35 S]‐ATPαS and [ 3 H]‐α,β‐MeATP bind to different binding sites at synaptosomal membranes from rat brain cortex. The affinity of the [ 3 H]‐α,β‐MeATP binding sites ( K d =13.7±1.8 n M , B max =6.34±0.28 pmol mg −1 protein) was 38 fold higher than the potency of α,β‐MeATP to displace [ 35 S]‐ATPαS binding ( K i =0.52 μ M ). ATP and ADPβS competed at both binding sites with different affinities, 60 fold and 175 fold, respectively. The other agonists tested (2‐MeSATP, UTP, GTP) did not affect specific [ 35 H]‐α,β‐MeATP binding at concentrations up to 100 μ M . The antagonists (suramin, isoPPADS, Evan's Blue) showed completely different affinities for both binding sites. Binding of [ 35 S]‐ATPαS on synaptosomes was regulated by GTP, which is indicative for G‐protein coupled receptors. The K d value for the high affinity binding site was reduced in the presence of GTP about 5 fold (from 1.8 n M to 8.6 n M ). In the presence of Mg 2+ the affinity was increased ( K d 1.8 n M versus 22 n M in the absence of Mg 2+ ). The binding of both radioligands was regulated in an opposite manner by physiological concentrations of Ca 2+ and Mg 2+ . Binding of [ 3 H]‐α,β‐MeATP to synaptosomal membranes was increased 3 fold by raising the Ca 2+ concentration from 10 μ M to 1 m M , whereas the addition of Mg 2+ in the same concentration range resulted in an 80% reduction of the binding. In contrast, [ 35 S]‐ATPαS binding was not influenced at the same range of Ca 2+ or Mg 2+ concentrations (10 μ M to 1 m M ). The addition of Mg 2+ (5 m M ) increased the affinity of [ 35 S]‐ATPαS for the high affinity site 10 fold. Diadenosine polyphosphates had a bimodal effect on [ 35 S]‐ATPαS binding to synaptosomal membranes. AP 5 A and Ap 6 A enhanced binding of [ 35 S]‐ATPαS 1.6 fold in a concentration range between 0.1 and 50 μ M . Ap 3 A was a weak inhibitor with a K i value of 7.2 μ M . Ap 4 A, AP 5 A and Ap 6 A inhibited with K i values>100 μ M . These data support the concept that diadenosine polyphosphates do not directly interact with ATPαS binding sites. In conclusion, on the basis of present knowledge of the interaction of P 2 ‐purinoceptor active compounds with P 2X ‐ and/or P 2Y ‐purinoceptors, our data strongly suggest that [ 35 S]‐ATPαS is a useful tool to study P 2Y ‐purinoceptors. Thus, the [ 35 S]‐ATPαS binding site might to a large extent represent P 2Y ‐purinoceptors in synaptosomes from rat brain cortex. The nucleotide binding is regulated by G proteins, indicated by the effects of GTP/Mg 2+ on binding.