Characterization of [35S]-ATP alpha S and [3H]-alpha, beta-MeATP binding sites in rat brain cortical synaptosomes: regulation of ligand binding by divalent cations.
1. We made a comparative analysis of the binding characteristics of the radioligands [35S]-ATP alpha S and [3H]-alpha, beta-MeATP in order to test whether these ligands can be used to analyse P2-purinoceptors in synaptosomal membranes from rat brain cortex. 2. Synaptosomes possess sites with high affinity for [35S]-ATP alpha S (Kd = 22.2 +/- 9.1 nM, Bmax = 14.8 pmol mg-1 protein). The rank order of the competition potency of the different compounds (ATP alpha S, ATP, ATP gamma S > ADP beta S, 2-MeSATP > deoxyATP, ADP > > UTP, alpha, beta-MeATP, AMP, Reactive Blue-2, suramin, isoPPADS) is consistent with pharmacological properties of P2Y-purinoceptors. 3. Under identical conditions [35S]-ATP alpha S and [3H]-alpha, beta-MeATP bind to different binding sites at synaptosomal membranes from rat brain cortex. The affinity of the [3H]-alpha, beta-MeATP binding sites (Kd = 13.7 +/- 1.8 nM, Bmax = 6.34 +/- 0.28 pmol mg-1 protein) was 38 fold higher than the potency of alpha, beta-MeATP to displace [35S]-ATP alpha S binding (Ki = 0.52 microM). ATP and ADP beta S 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 [35H]-alpha, beta-MeATP binding at concentrations up to 100 microM. The antagonists (suramin, isoPPADS, Evan's Blue) showed completely different affinities for both binding sites. 4. Binding of [35S]-ATP alpha S on synaptosomes was regulated by GTP, which is indicative for G-protein coupled receptors. The Kd value for the high affinity binding site was reduced in the presence of GTP about 5 fold (from 1.8 nM to 8.6 nM). In the presence of Mg2+ the affinity was increased (Kd 1.8 nM versus 22 nM in the absence of Mg2+). 5. The binding of both radioligands was regulated in an opposite manner by physiological concentrations of Ca2+ and Mg2+. Binding of [3H]-alpha, beta-MeATP to synaptosomal membranes was increased 3 fold by raising the Ca2+ concentration from 10 microM to 1 mM, whereas the addition of Mg2+ in the same concentration range resulted in an 80% reduction of the binding. In contrast, [35S]-ATP alpha S binding was not influenced at the same range of Ca2+ or Mg2+ concentrations (10 microM to 1 mM). The addition of Mg2+ (5 mM) increased the affinity of [35S]-ATP alpha S for the high affinity site 10 fold. 6. Diadenosine polyphosphates had a bimodal effect on [35S]-ATP alpha S binding to synaptosomal membranes. AP5A and Ap6A enhanced binding of [35S]-ATP alpha S 1.6 fold in a concentration range between 0.1 and 50 microM. Ap3A was a weak inhibitor with a Ki value of 7.2 microM. Ap4A, AP5A and Ap6A inhibited with Ki values > 100 microM. These data support the concept that diadenosine polyphosphates do not directly interact with ATP alpha S binding sites. 7. In conclusion, on the basis of present knowledge of the interaction of P2-purinoceptor active compounds with P2x- and/or P2Y-purinoceptors, our data strongly suggest that [35S]-ATP alpha S is a useful tool to study P2Y-purinoceptors. Thus, the [35S]-ATP alpha S binding site might to a large extent represent P2Y-purinoceptors in synaptosomes from rat brain cortex. The nucleotide binding is regulated by G proteins, indicated by the effects of GTP/Mg2+ on binding.
Schäfer
R
R
Institut für Neurobiochemie, Otto-von-Guericke-Universität Magdeburg, Germany.
Reiser
G
G
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism*
D002413:Cations, Divalent; D008024:Ligands; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium