RESUMEN
Antagonists of the B1 bradykinin receptor (B1R), encoded by the BDKRB1 gene, offer the promise of novel therapeutic agents for inflammatory and neuropathic pain. However, the in vivo characterization of the pharmacodynamics of B1R antagonists is hindered by the low level of B1R expression in healthy tissue and the profound species selectivity exhibited by many compounds for the B1R. To circumvent these issues we generated two genetically engineered rodent models. The first is a transgenic rat over-expressing the human B1R under the control of the neuronal-specific enolase promoter; we previously reported the utility of this model in assessing human B1R receptor occupancy in the central nervous system of the rat. The second model, reported here, utilized gene-targeting by homologous recombination to replace the genomic coding sequence for the endogenous mouse B1R with that of the human B1R. The mRNA expression profile of the humanized Bdkrb1 (hBkdrb1) allele is similar to that of the mouse Bdkrb1 (mBkdrb1) in the wild-type animal. Furthermore, in vitro assays indicate that tissues isolated from the humanized mouse possess pharmacological properties characteristic of the human B1R. Therefore, we have generated a humanized B1R mouse model that is suitable for testing the efficacy of human B1R-selective compounds.
Asunto(s)
Acetamidas/farmacología , Receptor de Bradiquinina B1 , Sulfonas/farmacología , Acetamidas/química , Animales , Antagonistas del Receptor de Bradiquinina B1 , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Estructura Molecular , ARN Mensajero/genética , Receptor de Bradiquinina B1/genética , Receptor de Bradiquinina B1/fisiología , Sulfonas/química , Transcripción GenéticaRESUMEN
We have developed an efficient and selective radioligand, the [35S]-radiolabeled dihydroquinoxalinone derivative, 4, for an ex vivo receptor occupancy assay in transgenic rats over-expressing the human bradykinin B1 receptor.
Asunto(s)
Antagonistas del Receptor de Bradiquinina B1 , Quinoxalinas/síntesis química , Quinoxalinas/farmacología , Animales , Animales Modificados Genéticamente , Unión Competitiva/efectos de los fármacos , Humanos , Marcaje Isotópico , Ligandos , Ensayo de Unión Radioligante , Ratas , Receptor de Bradiquinina B1/química , Relación Estructura-Actividad , Radioisótopos de AzufreRESUMEN
The pharmacological properties of the kinin B1 receptor in binding the endogenous kinin peptides are known to differ across species. Molecular cloning has revealed that these pharmacological differences arise from the diversity within the BDKRB gene. In this report, the molecular diversity of the human BDKRB1 gene is expanded by the identification of eight single nucleotide polymorphisms (SNPs) in the coding sequence of the receptor, three of which change the amino acid sequence of the receptor. The molecular cloning and pharmacological characterization of two primate B1 receptors, rhesus and African Green monkey, reveals that they exhibit the same high degree of selectivity for des-Arg10 kallidin (Lys-bradykinin) relative to des-Arg9 bradykinin that is observed with the human kinin B1 receptor. Previous mutagenesis studies of the human B1 receptor have implicated extracellular domain (EC) IV in conferring this selectivity for des-Arg10 kallidin, by interacting with the N-terminal Lys residue of the peptide. The pharmacological analysis of chimeric B1 receptors, in which EC-IV of the human B1 receptor is replaced with the corresponding domain of either rat or dog, supports the proposal that EC-IV is an important determinant in conferring ligand selectivity.
Asunto(s)
Bradiquinina/análogos & derivados , Polimorfismo de Nucleótido Simple , Receptores de Bradiquinina/genética , Receptores de Bradiquinina/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Bradiquinina/metabolismo , Bradiquinina/farmacología , Chlorocebus aethiops , Clonación Molecular , Perros , Frecuencia de los Genes , Humanos , Calidina/análogos & derivados , Calidina/metabolismo , Calidina/farmacología , Macaca mulatta , Datos de Secuencia Molecular , Ratas , Receptor de Bradiquinina B1 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
By using a combination of genetic, pharmacological, and anatomical approaches, we show that the melanocortin 4 receptor (MC4R), implicated in the control of food intake and energy expenditure, also modulates erectile function and sexual behavior. Evidence supporting this notion is based on several findings: (i) a highly selective non-peptide MC4R agonist augments erectile activity initiated by electrical stimulation of the cavernous nerve in wild-type but not Mc4r-null mice; (ii) copulatory behavior is enhanced by administration of a selective MC4R agonist and is diminished in mice lacking Mc4r; (iii) reverse transcription (RT)-PCR and non-PCR based methods demonstrate MC4R expression in rat and human penis, and rat spinal cord, hypothalamus, brainstem, pelvic ganglion (major autonomic relay center to the penis), but not in rat primary corpus smooth muscle cavernosum cells; and (iv) in situ hybridization of glans tissue from the human and rat penis reveal MC4R expression in nerve fibers and mechanoreceptors in the glans of the penis. Collectively, these data implicate the MC4R in the modulation of penile erectile function and provide evidence that MC4R-mediated proerectile responses may be activated through neuronal circuitry in spinal cord erectile centers and somatosensory afferent nerve terminals of the penis. Our results provide a basis for the existence of MC4R-controlled neuronal pathways that control sexual function.