Enhanced stability of wild-type and constitutively active alpha(2A)-adrenoceptors by ligands with agonist, silent and inverse agonist properties.
The hypothesis that prolonged treatment of a constitutively active receptor with inverse agonists may lead to increased receptor density was tested for the alpha(2)-adrenoceptor (AR) inverse agonist (+)-RX 811059 at both the wild-type (WT) and Thr(373)Lys alpha(2A) ARs in CHO-K1 cells by monitoring [(3)H]RX 821002 and [(35)S]GTPgammaS binding responses. One-hundred micromolar KCl instead of NaCl in the [(35)S]GTPgammaS membrane binding assay favoured the detection of a high-magnitude constitutive alpha(2A) AR activity. Under this condition, (+)-RX 811059 was an inverse agonist [ E(max) (% vs. basal): Thr(373)Lys alpha(2A) AR (-52+/-2) > WT alpha(2A) AR (-31+/-6)] while atipamezole was a silent neutral antagonist for both WT and Thr(373)Lys alpha(2A) ARs. The B(max) value of [(3)H]RX 821002 binding sites to membranes of transfected CHO-K1 cells was <90% for the Thr(373)Lys alpha(2A) AR compared with the WT alpha(2A) AR (9.1+/-1.4 pmol/mg protein); K(d) values were similar (1.16+/-0.19 nM and 1.51+/-0.15 nM, respectively). Forty-eight-hours' pre-treatment of cells with either 0.1 microM (+)-RX 811059, 1 microM atipamezole or 1 microM of the efficacious agonist d-medetomidine increased the amount of [(3)H]RX 821002 binding sites of both WT (52%-59%) and mutant (306%-447%) Thr(373)Lys alpha(2A) ARs. The same alpha(2) AR ligands also prevented the loss of [(3)H]RX 821002 binding sites as induced by incubation of transfected CHO-K1 cellular membranes at 37 degrees C for 4 h (WT alpha(2A) AR) and 2 h (Thr(373)Lys alpha(2A) AR); 0.1 microM (+)-RX 811059 and 1 microM atipamezole caused an increase compared with the control amount of [(3)H]RX 821002 binding sites to the Thr(373)Lys alpha(2A) AR by 73% and 50%, respectively. In conclusion, no relationship was found between inverse agonism and alpha(2A) AR up-regulation. It is suggested that this is due to structural stabilisation of the alpha(2A) AR, irrespective of the nature of the ligand.
Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 17, avenue Jean Moulin, 81106 Castres Cédex, France. peter.pauwels@pierre-fabre.com
Petrus J
PJ
Pauwels
Stéphanie
S
Tardif
D000317:Adrenergic alpha-Antagonists / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004347:Drug Interactions; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019329:Idazoxan / Q000031:analogs & derivatives* / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D011485:Protein Binding; D011869:Radioligand Assay; D018341:Receptors, Adrenergic, alpha-2 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects
D000317:Adrenergic alpha-Antagonists; D007093:Imidazoles; D018341:Receptors, Adrenergic, alpha-2; C050701:atipamezole; D006160:Guanosine Triphosphate; C065483:ethoxyidazoxan; C058525:2-methoxyidazoxan; D019204:GTP-Binding Proteins; D019329:Idazoxan