Downregulation of the oxytocin receptor on cultured astroglial cells.

Cultured astroglial cells obtained from rat fetal hypothalamus express oxytocin (OT) receptors, which have been previously characterized (Di Scala-Guenot and Strosser. Biochem. J. 284: 491-497, 1992), with a radioiodinated OT antagonist. In these cells, at steady-state binding at 37 degrees C, ice-cold acidic treatment released 10% of the bound ligand; with pronase treatment, 52% of the tracer was released. Because the binding was performed with an antagonist, one could assume that the radiolabeled ligand remains locked into the membrane in a state insensitive to the stripping agents rather than being internalized. Receptor downregulation induced by OT was concentration- and time-dependent, leading to a 72% loss of maximal binding capacity without changing the affinity of the receptor. On removal of OT the binding capacity recovered partially and the restoration process was blocked by monensin (20 microM) but not by cycloheximide (20 micrograms/ml), suggesting involvement of receptor recycling. Concerning the early mechanisms involved in the downregulation processes, uncoupling of the receptor from the G protein and the receptor phosphorylation by protein kinase C could be demonstrated. Treatment of the cells with the OT antagonist d(CH2)5OVT was shown to facilitate radioligand binding and to protect the receptor against OT-induced downregulation.