Binding of the radioligand [35S]adenosine 5'-O-(2-thiodiphosphate) and intracellular calcium response in rat liver parenchymal cells.
The use of the radioligand [35S]adenosine 5'-O-(2-thiodiphosphate) (ADP beta 35S) for the determination of P2y-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 Ki values of 1.5 +/- 0.47 microM for UTP, 1.8 +/- 0.35 microM for adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), 31 +/- 6.2 microM for ATP and 35 +/- 6.1 microM for GTP. In addition, we showed that ADP beta 35S is highly resistant to degradation by ecto-nucleotidases, with only 14.5 +/- 1.4% of total ADP beta 35S present being degraded after 1 hr, and that the binding of ADP beta 35S to its binding sites was modulated by EDTA. The Ki value of ATP shifted to 8.1 +/- 1.2 microM 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 EC50 values of 3.5 +/- 0.22 microM for UTP, 20.7 +/- 3.1 microM for ATP, 38.3 +/- 6.4 microM for ADP beta S and 73.6 +/- 13.7 microM 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 P2u-purinoceptor and/or "nucleotide receptor", in that UTP is at least as active as ATP.
van Rhee
A M
AM
Center for Bio-Pharmaceutical Sciences, Division of Medicinal Chemistry, Leiden, The Netherlands.
van Winden
E C
EC
Nagelkerke
J F
JF
de Bont
H J
HJ
IJzerman
A P
AP
Soudijn
W
W
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured / Q000187:drug effects; D004492:Edetic Acid; D008099:Liver / Q000378:metabolism*; D008297:Male; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D011983:Receptors, Purinergic / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013873:Thionucleotides / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology
D011983:Receptors, Purinergic; D013462:Sulfur Radioisotopes; D013873:Thionucleotides; C030812:adenosine 5'-O-(2-thiodiphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D002118:Calcium; D014544:Uridine Triphosphate