New tools for human fat cell alpha-2A adrenoceptor characterization. Identification on membranes and on intact cells using the new antagonist [3H]RX821002.
The pharmacology of the alpha-2 adrenoceptor of the human adipocyte was improved by using some new alpha-2 antagonists from different chemical families (imidazolines, benzazepines and benzofuroquinolizines) in biological and binding assays. Moreover, investigations were also carried out to define the binding properties of a new imidazolinic antagonist, RX821002 [2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline], which could be a potential radioligand. [3H]RX821002 binding was very rapid and reversible. Saturation isotherms indicated that [3H]RX821002 labeled, with high affinity, a homogeneous population of noninteracting binding sites with a mean Kd of 0.98 +/- 0.05 nM (n = 6). The binding of [3H]RX821002 on the human fat cell alpha-2 adrenoceptor displayed a specificity which is strictly similar to that obtained with [3H]rauwolscine and which is classical for an alpha-2 A adrenoceptor. The binding parameters of [3H]RX821002 were compared with those obtained with the classical alpha-2 antagonist [3H]yohimbine. Analysis of the data indicate: 1) that [3H]RX821002 exhibited higher affinity; 2) that the nonspecific binding of [3H]RX821002 was very low; 3) that the total number of sites (maximum binding values) defined with [3H]RX821002 was significantly higher than that defined with [3H]yohimbine. This difference was not due to a specific preferential labeling of one of the two affinity states of the receptor, but suggested that [3H]yohimbine does not label the whole receptor population; 4) that [3H]RX821002 specific binding was less sensitive to magnesium chloride and GTP than [3H]yohimbine binding; and 5) that [3H]RX821002 can be used suitably for identification of alpha-2 adrenoceptors on the intact adipocyte.(ABSTRACT TRUNCATED AT 250 WORDS)
Galitzky
J
J
Institut National de la Santé et de la Recherche Médicale (INSERM, U.317), Université Paul Sabatier, Toulouse, France.
Larrouy
D
D
Berlan
M
M
Lafontan
M
M
D000273:Adipose Tissue / Q000032:analysis*; D000317:Adrenergic alpha-Antagonists / Q000378:metabolism* / Q000494:pharmacology; D000328:Adult; D004146:Dioxanes / Q000378:metabolism*; D004147:Dioxins / Q000378:metabolism*; D005260:Female; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D019329:Idazoxan / Q000031:analogs & derivatives; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008875:Middle Aged; D011942:Receptors, Adrenergic, alpha / Q000032:analysis*; D014316:Tritium; D015016:Yohimbine / Q000378:metabolism
D000317:Adrenergic alpha-Antagonists; D004146:Dioxanes; D004147:Dioxins; D011942:Receptors, Adrenergic, alpha; D014316:Tritium; D015016:Yohimbine; D006160:Guanosine Triphosphate; C058525:2-methoxyidazoxan; D008274:Magnesium; D019329:Idazoxan