Chapter 5 Nucleotide radiolabels as tools for studying P2Y receptors in membranes from brain and lung tissue

Abstract

Publisher Summary This chapter reveals that nucleotide radiolabels have been used successfully to localize and to quantify P2Y receptors in brain tissue. Radiolabelled nucleotides are a valuable tool for analysing the density of certain P2Y receptors in membranes or after solubilization and purification of P2Y receptors. Extracellular nucleotides have a wide range of physiological effects in many different tissues through activation of specific P2 receptors, which comprise transmitter gated ion channels (P2X receptors) and C-protein linked metabotropic P2Y receptors. The chapter presents the radioligand binding analysis by preparing the membranes from rat lung, and plasma membranes or synaptosomal membranes from rat brain cortex. [ 3 H]UTP is used to label uridine nucleotide selective P2Y receptor types in lung membranes. Similarly, in rat synaptosomal membranes, binding of [ 35 S]ATPαS that is used to label preferentially P2Y receptors is enhanced by the additional presence of diadenosine polyphosphates. In both tissues, lung and brain, binding density for UTP is considerably lower than for [ 3 H] α,β,P-MeATP, a P2X specific ligand, for [ 35 S]ATPαS and for [ 3 H]Ap 4 A. In brain membranes, ligand specificity of ATPαS binding is consistent with P2Y receptor pharmacology, whereas in lung membranes ATPαS seemed to label various types of receptors.