In human and rat lung membranes [ 35s]GTPγS binding is a tool for pharmacological characterization of G protein-coupled devucleotide receptors

Abstract

The P2Y receptor family is activated by extracellular nucleotides such as ATP and UTP. P2Y receptors regulate physiological functions in numerous cell types. In lung, the P2Y 2 receptor subtype plays a role in controlling Cl − and fluid transport Besides ATP or UTP, also diadenosine tetraphosphate (Ap 4A), a stable nucleotide, seems to be of physiological importance. In membrane preparations from human and rat lung we applied several diadenosine polyphosphates to investigate whether they act as agonists for G protein-coupled receptors. We assessed this by determining the stimulation of [ 35S]GTPγS binding. Stimulation of [ 35S]GTPγS binding to G proteins has already been successfully applied to elucidate agonist binding to various G proteincoupled receptors. Ap nA ( n = 2 to 6) enhanced [ 35S]GTPγS binding similarly in human and rat lung membranes, an indication of the existence of G protein-coupled receptor binding sites specific for diadenosine polyphosphates Moreover, in both human and rat lung membranes comparable pharmacological properties were found for a diadenosine polyphosphate ([ 3H]Ap 4A) binding site. The affinity for Ap 2A, Ap 3A, Ap 4A, Ap 5A, and Ap 6A was also comparable. 8-Diazido-Ap 4A and ATP were less potent, whereas the pyrimidine nucleotide UTP showed hardly any affinity. Thus, we present evidence that different diadenosine polyphosphates bind to a common G protein-coupled receptor binding site in membranes derived either from human or rat lung.