1 The antagonist activity of a series of diinosine polyphosphates (Ip n I, where n=3, 4, 5) was assessed against ATP-activated inward currents at rat P2X 1 ± 4 receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2 Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap 5 A into Ip 5 I) using 5'-adenylic deaminase, and puri®ed using reverse-phase chromatography. 3 Against ATP-responses at rP2X 1 receptors, the potency order for antagonism was (pIC 50 ): Ip 5 I (8.5)4Ip 4 I (6.3)4Ip 3 I (44.5). Ip 5 I (10 ± 100 nM) caused a concentration-dependent rightwards displacement of the ATP concentration-response curve without reducing the maximum ATP e ect. However, the Schild plot was non-linear which indicated Ip 5 I is not a competitive antagonist. Blockade by micromolar concentrations of Ip 5 I was not surmountable. Ip 4 I also behaved as a nonsurmountable antagonist. 4 Against ATP-responses at rP2X 3 receptors, the potency order for antagonism was (pIC 50 ): Ip 4 I (6.0)4Ip 5 I (5.6)4Ip 3 I (44.5). Blockade by Ip 4 I (pA 2 , 6.75) and Ip 5 I (pA 2 , 6.27) was surmountable at micromolar concentrations. 5 Diinosine polyphosphates failed to inhibit ATP-responses at rP2X 2 receptors, whereas agonist responses at rP2X 4 were reversibly potentiated by Ip 4 I and Ip 5 I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X 1 ± 4 receptors. 6 Thus, Ip 5 I acted as a potent and relatively-selective antagonist at the rP2X 1 receptor. This dinucleotide pentaphosphate represents a high-a nity antagonist for the P2X 1 receptor, at which it acts in a competitive manner at low (4100 nM) concentrations but has more complex actions at higher (4100 nM) concentrations.
Diadenosine polyphosphates present at the cytosol can be transported to secretory granules allowing their exocytotic release. Extracellularly, they can act through specific metabotropic or ionotropic receptors, or as analogues of P2X and P2Y nucleotide receptors. The specific ionotropic receptor P4 is present in synaptic terminals, and modulated by protein kinases (PK) A and C and protein phosphatases. Activation of PKA or PKC, directly or through membrane receptors, results in a decrease of affinity or in reduction of the Ca 2+ transient respectively. Adenosine and ATP, both products of the extracellular destruction of diadenosine polyphosphates, acting through A I or P2Y receptors respectively, are important physiological modulators at the P4 receptor.z 1998 Federation of European Biochemical Societies.Key words: Adenine dinucleotide; Ap n A; Nucleotide receptor; Protein kinase; Protein phosphatase; Purinergic receptor Diadenosine polyphosphates: intracellular rolesK,g-adenine dinucleotides have emerged as extracellular signalling molecules together with adenosine and ATP in neural and non-neural tissues. Adenine dinucleotides, also termed diadenosine polyphosphates, are made up of two adenosine moieties bridged by a phosphate chain whose length varies between three and six phosphates. It is generally accepted that some aminoacyl-tRNA synthetases and enzymes with adenylyl reaction intermediates produce these compounds as secondary products [1^3].Diadenosine polyphosphates are present in prokaryotic and eukaryotic cells, their levels, which are below the WM range, increase to WM levels after oxidative or heat shock treatment [1,4,5]. In eukaryotic cells, their concentration also depends on the cell cycle and Ap R A can activate DNA replication and DNA repair [6,7].In cell cytoplasm, diadenosine polyphosphates are able to regulate enzymes, ion channels and transporters. They behave as enzyme inhibitors involved in the nucleotide phosphate transfer mediated by adenosine kinase and adenylate kinase, as they are transition state analogues of these enzymes [8,9]. Nevertheless, they behave as activators of the cytosolic 5P-nucleotidase [10]. Plasma membrane proteins can be modulated on the intracellular side by diadenosine polyphosphates. This e¡ect has been demonstrated in the K e channel present in cardiac cells, where they mimic the e¡ect of ATP [11]. In addition, in pancreatic L-cells, an increase in the extracellular glucose levels produces an enhancement of up to 40 times of Ap Q A and Ap R A, reaching intracellular concentrations of 11.2 WM an 13.6 WM respectively, activating the K e channel involved in insulin secretion [12]. Moreover, ATP as well as Ap R A positively modulate the nucleoside transporter, which is a crucial step in the recovery of extracellular adenosine, on the intracellular side.Cytosolic hydrolases and phosphorylases, speci¢c for the phosphate chain length, cleave adenine dinucleotides to form adenine mononucleotides in both pro-and eukaryotic cells [13,14]. One of these enzymes, d...
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