Characterization of oxytocin-binding sites in primary rat brain cell cultures

Abstract

Detection and characterization of oxytocin-binding sites in dissociated brain cell cultures were performed, using a highly selective iodinated oxytoxin antagonist ([ 125I]OTA). Dissociated cells derived from hypothalamus and extrahypothalamic forebrain of 16-day-old fetal rats were maintained in chemically defined medium in order to enrich the cultures in neuronal cells. Specific binding of [ 125I]OTA, demonstrated in both hypothalamic and forebrain cell cultures, was temperature- and time-dependent; maximal binding was obtained by incubating the iodinated ligand for 60 min at 37 °C. The binding parameters were shown to be identical in both cell type cultures. The Scatchard plot analysis suggested the presence of a single class of binding sites of high affinity ( K d about 0.06 nM) and low binding capacity ( B max about 4 fmol/dish). The specificity of these binding sites tested in competition experiments revealed that the unlabelled OT antagonist was the most potent in inhibiting specific [ 125I]OTA binding ( K i = 0.1nM). A lower affinity, of the nM range was demonstrated for oxytocin (OT), arginine-vasopressin (AVP) and the V 1 antagonist, whereas the V 2 AVP agonist poorly competed for [ 125I]OTA binding sites ( K i about 250 nM). In conclusion, the [ 125I]OTA binding characteristics, identical in both hypothalamic and forebrain cultures, fulfil the classical conditions required for the existence of receptor sites since the binding was reversible, saturable and specific. As these characteristics were similar to those already described in the adult rat, at the central level in hippocampus, and at the periphery in the mammary gland, it could be postulated that [ 25I]OTA binds to an OT receptor site. Therefore, fetal rat brain cell cultures may provide a relatively simplified in vitro model for studying the OT receptor and its regulation.