Ap4A and ADP‐β‐S binding to P2 purinoceptors present on rat brain synaptic terminals

Abstract

1 Diadenosine tetraphosphate (Ap₄A) a dinucleotide stored and released from rat brain synaptic terminals presents two types of affinity binding sites in synaptosomes. When [³H]-Ap₄A was used for binding studies a K_d> value of 0.10 ± 0.014 nm and a B_max value of 16.6 ± 1.2 fmol mg⁻¹ protein were obtained for the high affinity binding site from the Scatchard analysis. The second binding site, obtained by displacement studies, showed a K_i value of 0.57 ± 0.09 μm. 2 Displacement of [³H]-Ap₄A by non-labelled Ap₄A and P₂-purinoceptor ligands showed a displacement order of Ap₄A > adenosine 5′-O-(2-thiodiphosphate) (ADP-β-S) > 5′-adenylyl-imidodiphosphate (AMP-PNP) > α,β-methylene adenosine 5′-triphosphate (α,β-MeATP) in both sites revealed by the K_i values of 0.017 nm, 0.030 nm, 0.058 nm and 0.147 nm respectively for the high affinity binding site and values of 0.57 μm, 0.87 μm, 2.20 μm and 4.28 μm respectively for the second binding site. 3 Studies of the P₂-purinoceptors present in synaptosomes were also performed with [³⁵S]-ADP-β-S. This radioligand showed two binding sites the first with K_d and B_max values of 0.11 ± 0.022 nm and 3.9 ± 2.1 fmol mg⁻¹ of protein respectively for the high affinity binding site obtained from the Scatchard plot. The second binding site showed a K_i of 0.018 ± 0.0035 μm obtained from displacement curves. 4 Competition studies with diadenosine polyphosphates of [³⁵S]-ADP-β-S binding showed a displacement order of Ap₄A > Ap₅A > Ap₆A in the high affinity binding site and K_i values of 0.023 nm, 0.081 nm and 5.72 nm respectively. The second binding site potency order was Ap₅A > Ap₄A > Ap₆A, with K_i values of 0.28 μm, 0.53 μm and 5.32 μm respectively. 5 Displacement studies of [³⁵S]-ADP-β-S with P₂-purinoceptor agonists showed the following potency pattern: ADP-β-S > AMP-PNP > α,β-MeATP with K_i values of 0.021 nm, 0.029 nm 0.215 nm respectively in the high affinity binding site. 2-Methylthio-adenosine 5′-triphosphate (2MeSATP) was unable to displace [³⁵S]-ADP-β-S in this binding site. The second binding site showed a profile of ADP-β-S > α,β-MeATP> AMP-PNP > 2MeSATP and K_i values of 0.018 μm, 0.212 μm, 0.481 μm and 18.04 μm respectively. 6 These studies suggest the presence of a new P₂-purinoceptor in rat brain synaptosomes with high affinity for diadenosine polyphosphates which we tentatively designate as P_2d.