2--3--o-(2-4-6-trinitrophenyl)adenosine-5--triphosphate and Astrocytoma

The purinergic receptor signaling system plays an important role in communication between cells in the nervous system and opens new opportunities for screening of potential drugs. Our objective was to explore the pharmacological properties and establish a new methodology for ligand screening for the P2X2 receptor, which has been developed by the combinatorial library approach Systematic Evolution of Ligands by Exponential enrichment (SELEX). To this end, membranes of 1321N1 cells stably transfected with rat P2X2 receptors were resuspended in 2% cholate detergent and subsequently coupled onto an immobilized artificial membrane (IAM). The IAM-cholate-P2X2 mixture was then dialyzed, centrifuged and packed into a FPLC column. Equilibrium binding to the receptor and competition between ATP and the purinergic antagonists suramin and 2'3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) were analyzed by a chromatographic assay using 32P alpha ATP as a radioligand. Our data indicate that suramin does not compete with ATP for the ligand binding site and TNP-ATP is a competitive antagonist, confirming previous studies [C.A. Trujillo, A.A. Nery, A.H. Martins, P. Majumder, F.A. Gonzalez, H. Ulrich, Biochemistry 45 (2006) 224-233]. In addition, we demonstrate that this assay can be used in in vitro selection procedures for RNA aptamers binding to P2X2 receptors. The results demonstrate that the receptor can be immobilized in a stable format and reused over an extended period of time, facilitating the exploration of ligand-receptor interactions and screening of combinatorial pools for possible ligands.

Topics: Adenosine Triphosphate; Animals; Astrocytoma; Binding, Competitive; Cell Culture Techniques; Cell Line, Tumor; Chromatography, Affinity; Drug Evaluation, Preclinical; Fluorescent Dyes; Humans; Ligands; Models, Biological; Neurons; Patch-Clamp Techniques; Purinergic P2 Receptor Antagonists; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Recombinant Proteins; SELEX Aptamer Technique; Suramin; Transfection

ATP is released in a vesicular manner from nerve terminals mainly at higher stimulation frequencies. There is a robust expression of ATP (P2) receptors in the brain, but their role is primarily unknown. We report that ATP analogs biphasically modulate the evoked release of glutamate from purified nerve terminals of the rat hippocampus, the facilitation being mediated by P2X1, P2X2/3, and P2X3 [antagonized by 8-(benzamido)naphthalene-1,3,5-trisulfonate and 2',3'-O-(2,4,6-trinitrophenyl)-ATP] and the inhibition by P2Y1, P2Y2, and/or P2Y4 [antagonized by reactive blue 2 and 2'deoxy-N6-methyladenosine-3',5'-bisphosphate and mimicked by P1-(urinine 5'-),P4-(inosine 5'-) tetraphosphate and 2-methylthio-ADP] receptors. The combination of single-cell PCR analysis of rat hippocampal pyramidal neurons, Western blot analysis of purified presynaptic active zone fraction, and immunocytochemical analysis of hippocampal glutamatergic terminals revealed that the P2 receptors expressed in glutamatergic neurons, located in the active zone and in glutamatergic terminals, were precisely P2X1, P2X2, and P2X3 subunits and P2Y1, P2Y2, and P2Y4 receptors. This provides coincident functional and molecular evidence that P2 receptors are present and act presynaptically as a modulatory system controlling hippocampal glutamate release.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Astrocytoma; Calcium; Cell Line; Cell Line, Tumor; Glutamic Acid; Hippocampus; Kidney; Male; Potassium; Pyramidal Cells; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Presynaptic; Receptors, Purinergic P2; Receptors, Purinergic P2X; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y2; Recombinant Fusion Proteins; RNA, Messenger; Subcellular Fractions; Suramin; Synaptosomes; Transfection; Triazines; Triazoles; Xanthines