Olianas M C, Onali P
Department of Neurosciences, University of Cagliari, Italy.
Regul Pept. 1995 Mar 7;56(1):61-70. doi: 10.1016/0167-0115(95)00007-x.
The presence of corticotropin-releasing hormone (CRH) receptors in rat retinal membranes was investigated by using [125I-Tyro]-ovine CRH ([125I]oCRH) as radioligand. The receptor binding was rapid, reversible, saturable and specific. The [125I]oCRH binding was completely displaced by different CRH-related peptides with a rank order of potency similar to that displayed in stimulating rat retinal adenylyl cyclase activity. Two populations of binding sites were detected: one with high affinity (Kd = 1.7 nM) and the other with low-affinity (Kd = 130 nM). The GTP analogue guanosine 5'-O-(3'-thiotriphosphate) reduced the high-affinity binding and increased the relative proportion of sites with low-affinity. Incubation of rat retinal membranes with the RM/1 antibody, which recognizes the carboxyl-terminus of the alpha subunit of the G protein Gs, prevented the CRH stimulation of adenylyl cyclase. In immunoblots, the RM/1 antibody recognized an immunoreactive protein band of 45 kDa and a protein with a similar electrophoretic mobility was ADP-ribosylated by cholera toxin. These data provide evidence for the presence of specific CRH receptors in rat retina and contribute to define the CRH signalling system in this tissue.
采用[125I-酪氨酸]-羊促肾上腺皮质激素释放激素（[125I]oCRH）作为放射性配体，研究大鼠视网膜膜中促肾上腺皮质激素释放激素（CRH）受体的存在情况。受体结合迅速、可逆、可饱和且具有特异性。[125I]oCRH结合完全被不同的CRH相关肽取代，其效力顺序与刺激大鼠视网膜腺苷酸环化酶活性时显示的顺序相似。检测到两类结合位点：一类具有高亲和力（Kd = 1.7 nM），另一类具有低亲和力（Kd = 130 nM）。GTP类似物鸟苷5'-O-(3'-硫代三磷酸)降低了高亲和力结合，并增加了低亲和力位点的相对比例。用识别G蛋白Gsα亚基羧基末端的RM/1抗体孵育大鼠视网膜膜，可阻止CRH对腺苷酸环化酶的刺激。在免疫印迹中，RM/1抗体识别出一条45 kDa的免疫反应性蛋白带，且具有相似电泳迁移率的一种蛋白被霍乱毒素ADP核糖基化。这些数据为大鼠视网膜中存在特异性CRH受体提供了证据，并有助于确定该组织中的CRH信号系统。
