RESUMEN
Alpha-2 adrenergic receptors (alpha2 AR) mediate incorporation of guanosine 5'-O-(gamma-thio)triphosphate ([35S]GTPgammaS) into isolated membranes via receptor-catalyzed exchange of [35S]GTPgammaS for GDP. In the current study, we used [35S]GTPgammaS incorporation to characterize the intrinsic activity and potency of agonists and antagonists at the cloned mouse alpha2a/d and human alpha2a, alpha2b, and alpha2c ARs. Full agonists increased [35S]GTPgammaS binding to membranes by 2- to 3-fold. Antagonists did not increase [35S]GTPgammaS binding but competitively inhibited agonist-stimulated [35S]GTPgammaS binding. Compounds with intrinsic activities less than that of the full agonists norepinephrine (NE) or epinephrine (EPI) were capable of antagonizing agonist-stimulated [35S]GTPgammaS binding. The agonistic properties of a number of alpha2 AR ligands were characterized at each alpha2 AR subtype. The rank order of agonist potency for selected compounds at the human receptors (with intrinsic activity compared with NE, defined as 1.0) was: alpha2a: Dexmedetomidine (0.73) > guanabenz (0.38) > UK-14304 (1.02) > clonidine (0.32) > ST-91 (0.63) > NE (1.00). alpha2b: Dexmedetomidine (1.10) > clonidine (0.18) > guanabenz (0.71) > NE (1.00) > ST-91 (0.44) > UK-14304 (0.59). alpha2c: Dexmedetomidine (1.03) > NE (1.00) > UK-14304 (0.75) > ST-91 (0.32) > or = clonidine (0.23) >> guanabenz (0). This report provides a functional characterization of adrenergic receptor ligands at human and mouse alpha2a/d AR. It also illustrates the utility of [35S]GTPgammaS incorporation as a functional marker of receptor activation.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Receptores Adrenérgicos alfa 2/metabolismo , Adenilil Ciclasas/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Antagonistas Adrenérgicos alfa/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Vectores Genéticos , Humanos , Ligandos , Ratones , Unión Proteica , Quinolizinas/metabolismo , Ensayo de Unión Radioligante , Receptores Adrenérgicos alfa 2/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
We have isolated and characterized from human prostate novel splice variants of the human alpha1A-adrenoceptor, several of which generate truncated products and one isoform, alpha(1A-4), which has the identical splice site as the three previously described isoforms. Long-PCR on human genomic DNA showed that the alpha(1A-4) exon is located between those encoding the alpha(1A-1) and alpha(1A-3) variants. CHO-K1 cells stably expressing alpha(1A-4) showed ligand binding properties similar to those of the other functional isoforms as well as agonist-stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that alpha(1A-4) is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.