Characterization of corticotropin-releasing factor receptors in dissociated brain cell cultures

Abstract

Although corticotropin-releasing factor (CRF) receptors have been identified throughout the brain, relatively little is known about the regulation of CRF receptors. Recent investigations aimed at developing an in vitro model studying the regulation of CRF receptors demonstrated CRF binding in brain cell cultures. To test hypothesis that dissociated brain cell cultures contain CRF receptors and may provide a model for studying their regulation, studies characterizing binding of labeled CRF were performed. Dissociated cells derived from hypothalamus and extrahypothalamic forebrain (predominantly cortex) of day 17 fetal rats were maintained in chemically defined medium. We used a stable 125I-labeled analog of ovine CRF, 125I-Tyr 0-ovine CRF ( 125I-oCRF), to identify and characterize CRF receptors. Although specific binding of 125I-oCRF was demonstrated in both hypothalamic and extrahypothalamic cell cultures, the concentration of CRF receptors was much greater (3–5 fold) in extrahypothalamic cells. Binding 125I-oCRF in extrahypothalamic cells was satturable and was composed of high affinity ( K d = 0.51nM) and low affinity ( K d = 17.25nM) sites. Pharmacological displacement of labeled CRF from cells with a variety of CRF fragments and analogs was similar to that in studies of pituitary and brain homogenates. Extrahypothalamic cells studied at several times between 4 and 13 days in culture revealed an increase in the number of CRF receptors; the concentration of CRF receptors at 13 days was 3.5 times that observed at 4 days. Studies directed toward determining whether CRF receptor concentration could be modulated by CRF, adrenocorticotropic hormone 1–12, atropine or a CRF antagonist showed a change (36% decrease) only in response to chronic exposure with CRF. Conclusions: (1) dissociated fetal rat brain cell cultures derived from extrahypothalamic forebrain and hypothalamus contain CRF receptors; (2) CRF receptors in brain cells exhibit a differential distribution and characteristics similar to those previously reported in brain and pituitary; (3) dissociated fetal rat brain cell cultures may provide a relatively simplified in vitro model for studying the regulation of CRF receptors; and (4) CRF down-regulates its own receptor in extrahypothalamic forebrain cells.