RESUMO
The corticotropin releasing factor (CRF) exerts its effects by acting on its receptors and on the binding protein (CRFBP), and has been implicated in alcohol use disorder (AUD). Therefore, identification of the exact contribution of each protein that mediates CRF effects is necessary to design effective therapeutic strategies for AUD. A series of in vitro/in vivo experiments across different species were performed to define the biological discrete role of CRFBP in AUD. First, to establish the CRFBP role in receptor signaling, we developed a novel chimeric cell-based assay and showed that CFRBP full length can stably be expressed on the plasma membrane. We discovered that only CRFBP(10 kD) fragment is able to potentiate CRF-intracellular Ca2+ release. We provide evidence that CRHBP gene loss increased ethanol consumption in mice. Then, we demonstrate that selective reduction of CRHBP expression in the center nucleus of the amygdala (CeA) decreases ethanol consumption in ethanol-dependent rats. CRFBP amygdalar downregulation, however, does not attenuate yohimbine-induced ethanol self-administration. This effect was associated with decreased hemodynamic brain activity in the CRFBP-downregulated CeA and increased hemodynamic activity in the caudate putamen during yohimbine administration. Finally, in alcohol-dependent patients, genetic variants related to the CRFBP(10 kD) fragment were associated with greater risk for alcoholism and anxiety, while other genetic variants were associated with reduced risk for anxiety. Taken together, our data provide evidence that CRFBP may possess both inhibitory and excitatory roles and may represent a novel pharmacological target for the treatment of AUD.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Alcoolismo/genética , Proteínas de Transporte/genética , Consumo de Bebidas Alcoólicas/fisiopatologia , Alcoolismo/fisiopatologia , Tonsila do Cerebelo/fisiopatologia , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Regulação para Baixo/genética , Expressão Gênica/genética , Humanos , Sistema Hipotálamo-Hipofisário/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Sistema Hipófise-Suprarrenal/fisiopatologia , Fluxo Sanguíneo Regional/genética , Especificidade da Espécie , Adulto JovemRESUMO
'Thrombin aptamers' are based on the 15-nucleotide consensus sequence of d(GGTTGGTGTGGTTGG) that binds specifically to thrombin's anion-binding exosite-I. The effect of aptamer-thrombin interactions during inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) and antithrombin (AT) has not been described. Thrombin inhibition by HCII without glycosaminoglycan was decreased approximately two-fold by the aptamer. In contrast, the aptamer dramatically reduced thrombin inhibition by >200-fold and 30-fold for HCII-heparin and HCII-dermatan sulfate, respectively. The aptamer had essentially no effect on thrombin inhibition by AT with or without heparin. These results add to our understanding of thrombin aptamer activity for potential clinical application, and they further demonstrate the importance of thrombin exosite-I during inhibition by HCII-glycosaminoglycans.
Assuntos
Antitrombinas/farmacologia , Cofator II da Heparina/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Trombina/antagonistas & inibidores , Dermatan Sulfato/farmacologia , Interações Medicamentosas , Glicosaminoglicanos/farmacologia , Humanos , Modelos Moleculares , Trombina/química , Trombina/genéticaRESUMO
A Burkholderia strain isolated from soil is capable of inhibiting the growth of bacteria, plant-pathogenic fungi, pathogenic yeasts, and protozoa. Inhibition does not involve cell contact or the presence of living cells, suggesting that at least a substantial portion of the antimicrobial activity is due to the excretion of extracellular compounds.