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.
G protein-coupled corticotropin-releasing hormone receptors in rat retina
OLIANAS, MARIA CONCETTA;ONALI, PIER LUIGI
1995-01-01
Abstract
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.
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