adp-beta-s and diadenosine-tetraphosphate

Our aim was to investigate the neuromodulatory role of diadenosine tetraphosphate (Ap(4)A). Ap(4)A-binding sites were detected in striatum and hippocampus membranes using [(35)S]-ADP beta S as radioligand and Ap(4)A and epsilon-(Ap(4)A), di-ethenoadenosine tetraphosphate, as displacers. Effects of epsilon-(Ap(4)A) on extracellular glutamate levels were studied using intracerebral perfusion. Both areas contain high-affinity binding sites for [(35)S]-ADP beta S with K(d) values in the low nM range. [(35)S]-ADP beta S binding was displaced by Ap(4)A and epsilon-(Ap(4)A). At 1 and 10 microM doses, epsilon-(Ap(4)A) markedly decreased glutamate levels in the striatum. The possibility of Ap(4)A acting as an endogenous modulator of excitatory neurotransmission is discussed.

Topics: Adenosine Diphosphate; Animals; Corpus Striatum; Dinucleoside Phosphates; Dose-Response Relationship, Drug; Extracellular Space; Glutamic Acid; Hippocampus; Male; Neurotransmitter Agents; Rats; Rats, Wistar; Receptors, Purinergic P2; Thionucleotides

Adenosine transport in cultured chromaffin cells was inhibited by purinergic P2y-receptor agonists without significant changes in the affinity constant, the values being between 1 +/- 0.4 and 1.6 +/- 0.6 microM. The Vmax parameter was modified significantly, being 40 +/- 1.0, 26 +/- 5.0, 32 +/- 3.0, and 22 +/- 4.7 pmol/10(6) cells/min for control, adenosine-5'-O-(2-thiodiphosphate), 5'-adenylylimidodiphosphate, and P1,P4-di(adenosine-5'-) tetraphosphate (Ap4A) (100 microM for every effector), respectively. Ap4A, a physiological ligand for P2y receptors in chromaffin cells, showed the highest inhibitory effect (45%). This transport inhibition is explained by an increase in the cytosolic Ca2+ concentration ([Ca2+]i) and the activation of protein kinase C (PKC). Experiments of [Ca2+]i measurement with the fura-2 technique showed that P2y agonists, as well as bradykinin, were able to increase [Ca2+]i, this effect being independent of the presence of extracellular Ca2+. The peptide bradykinin, determined to be coupled to phosphatidylinositol hydrolysis and internal Ca2+ mobilization in chromaffin cells, exhibited a behavior similar to that of P2y agonists in adenosine transport inhibition (39%). P2y agonists and bradykinin increased PKC activity associated with the membrane fraction (about 50% increase in particulate PKC activity with respect to controls). The present studies suggest that adenosine transport is regulated by P2y-purinergic receptors mediated via Ca2+ mobilization and PKC activation.

Topics: Adenosine; Adenosine Diphosphate; Adenylyl Imidodiphosphate; Adrenal Medulla; Animals; Biological Transport; Bradykinin; Carbachol; Cattle; Cells, Cultured; Dinucleoside Phosphates; Dose-Response Relationship, Drug; Kinetics; Phorbol 12,13-Dibutyrate; Protein Kinase C; Receptors, Purinergic; Thionucleotides

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 Kd value of 0.10 +/- 0.014 nM and a Bmax 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 Ki value of 0.57 +/- 0.09 microM. 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-beta-S) > 5'-adenylyl-imidodiphosphate (AMP-PNP) > alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) in both sites revealed by the Ki 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 microM, 0.87 microM, 2.20 microM and 4.28 microM respectively for the second binding site. 3. Studies of the P2-purinoceptors present in synaptosomes were also performed with [35S]-ADP-beta-S. This radioligand showed two binding sites the first with Kd and Bmax 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 Ki of 0.018 +/- 0.0035 microM obtained from displacement curves. 4. Competition studies with diadenosine polyphosphates of [35S]-ADP-beta-S binding showed a displacement order of Ap4A > Ap5A > Ap6A in the high affinity binding site and Ki values of 0.023 nM, 0.081 nM and 5.72 nM respectively. The second binding site potency order was Ap5A> Ap4A > Ap6A,with Ki values of 0.28 microM, 0.53 microM and 5.32 microM respectively.5. Displacement studies of [35S]-ADP-beta-S with P2-purinoceptor agonists showed the following potency pattern: ADP-beta-S > AMP-PNP >alpha,beta-MeATP with Ki 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-beta-S in this binding site. The second binding site showed a profile of ADP-beta-S> a,beta-MeATP> AMP-PNP > 2MeSATP and Ki values of 0.0 18 microM, 0.212 microM, 0.481 microM and 18.04 microM 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 P2d.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Binding, Competitive; Brain Chemistry; Cerebral Cortex; Chromatography, High Pressure Liquid; Dinucleoside Phosphates; In Vitro Techniques; Kinetics; Male; Nerve Endings; Rats; Rats, Wistar; Receptors, Purinergic; Synapses; Synaptosomes; Thionucleotides