Pharmacological analysis of CCK(2) receptor ligands using COS-7 and SK-N-MC cells, expressing the human CCK(2) receptor.
A series of CCK(2) receptor ligands were analysed with respect to their interaction with binding sites in the membranes of COS-7 cells and SK-N-MC cells transiently expressing the human CCK(2) receptor (short isoform). The ligands were YF476, YM022, AG041R, L-740,093, JB93182, PD134308, and PD136450. Their binding was analysed by radioligand competition using [3H]L-365,260 as the labelled ligand. Saturation binding analysis indicated that [3H]L-365,260 interacted with a single class of binding sites. In competition binding experiments using COS-7-cell membranes, all seven ligands were incubated together with 2 nM [3H]L-365,260. The data for four of the compounds fitted a one-site model (pK(i) values: YM022: 9.2+/-0.02; YF476: 9.6+/-0.04; L-740,093: 9.2+/-0.01; and AG041R: 8.3+/-0.06), while the data for the three others fitted a two-site model (pK(i) values: JB93182: 8.8+/-0.04 and 6.0+/-0.15; PD134308: 9.0+/-0.04 and 6.1+/-0.15; and PD136450: 9.0+/-0.02 and 5.4+/-0.41). SK-N-MC cell membranes and 2 nM [3H]L-365,260 were incubated together with YM022, YF476, JB93182, and PD134308. The data for YM022 and YF476 fitted a one-site model (pK(i) values: YM022: 9.3+/-0.06; YF476: 9.4+/-0.02), while the data for JB93182 and PD134308 fitted a two-site model (pK(i) values: JB93182: 8.7+/-0.06 and 6.2+/-0.06; PD134308: 9.1+/-0.06 and 7.0+/-0.17). Competition binding experiments in the presence of the GTP-analogue guanylylimidodiphosphate, using either of the two cell types, produced similar binding data for PD134308 and JB93182 as in the absence of GTP-analogue. The human receptor seems to exist in a low and/or high affinity state. The shift from low to high affinity does not seem to reflect the degree of G protein coupling.
Nilsson
Isabelle
I
Division of Pharmacology, Faculty of Health Sciences, University of Linköping, University Hospital, S-581 85, Linköping, Sweden. isani@imv.liu.se
Monstein
Hans Jürg
HJ
Lindström
Erik
E
Håkanson
Rolf
R
Svensson
Samuel
S
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008962:Models, Theoretical; D009447:Neuroblastoma; D011869:Radioligand Assay; D043887:Receptor, Cholecystokinin B; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000378:metabolism; D011949:Receptors, Cholecystokinin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured
D008024:Ligands; D043887:Receptor, Cholecystokinin B; D011956:Receptors, Cell Surface; D011949:Receptors, Cholecystokinin; D006160:Guanosine Triphosphate