Binding of the radioligand [ 3Sadenosine 5'- O-(2-thiodiphosphate) and intracellular calcium response in rat liver parenchymal cells

Abstract

The use of the radioligand [ 35S]adenosine 5'- O-(2-thiodiphosphate) (ADP β 35S) for the determination of P 2y-purinoceptors on turkey erythrocyte membranes has recently been described. In the present study, we were able to demonstrate specific binding of this radioligand in intact rat liver parenchymal cells. Within 10 min a thermodynamic equilibrium was obtained which lasted for 25 min with a subsequent decline. Displacement studies with several nucleotides were performed yielding K i values of 1.5 ± 0.47 μM for UTP, 1.8 ± 0.35 μM for adenosine 5'- O-(2-thiodiphosphate) (ADPβS), 31 ± 6.2 μM for ATP and 35 ± 6.1 μM for GTP. In addition, we showed that ADP β 35S is highly resistant to degradation by ecto-nucleotidases, with only 14.5 ± 1.4% of total ADP β 35S present being degraded after 1 hr, and that the binding of ADP β 35S to its binding sites was modulated by EDTA. The K i value of ATP shifted to 8.1 ± 1.2 μM upon the addition of 1 mM EDTA to the incubation medium. In these rat liver parenchymal cells all nucleotides promoted calcium entry in a dose-dependent manner with ec 50 values of 3.5 ± 0.22 μM for UTP, 20.7 ± 3.1 μM for ATP, 38.3 ± 6.4 jμM for ADPβS and 73.6 ± 13.7 μM for GTP, with GTP being a partial agonist. Based on the data derived from the present study we discuss the possible correlation between binding and functional experiments and conclude that the described receptor resembles most closely the P 2u-purinoceptor and/or “nucleotide receptor”, in that UTP is at least as active as ATP.