Differential characterization of binding sites for adenine and uridine nucleotides in membranes from rat lung as possible tools for studying P2 receptors in lung

Nucleotide receptors (P2 receptors) are involved in stimulating Cl(-)secretion in airway epithelia. These receptors may play a key role in development of new therapeutic strategies in the treatment of cystic fibrosis. However, the diversity of nucleotide binding sites in lung tissue has not yet been clarified. Here we studied the characteristics of various nucleotide binding sires in rat lung membranes by equilibrium binding analysis of several P2 receptor specific ligands. Displacement studies revealed a recognition site for adenosine 5'-O-( 1-thiotriphosphate) ([S-35]ATP alpha S; K-d 243 nM). From this site the ligand is readily displaced by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), a typical agonist for P2Y(1) receptors and also by a,p-methylene adenosine 5'-triphosphate (alpha,beta-MeATP), a typical agonist for P2X receptors. [H-3]alpha,beta-MeATP labelled specific binding sites (K-d 56 nM) in rat lung membranes. Analysis of binding of [H-3]UTP to lung membranes revealed a high-affinity binding site (K-d 44 nM). Membrane-bound [H-3]UTP was not displaced even by high concentrations of ATP, indicating no common binding site for UTP and ATP. Furthermore, specific binding of P-1,P-4-di(adenosine 5')tetraphosphate ([3H]Ap(4)A; K-d 91 nM) was found in lung membranes. Thus, we demonstrate at least four distinct types of nucleotide binding sites in lung membranes: Two have characteristics comparable to P2X and P2Y(1) receptors, while two further sites still have to be identified, one binding Ap(4)A and the other binding UTP very specifically. (C) 1998 Elsevier Science Inc.