Characterization of corticotropin releasing factor (CRF) and arginine vasopressin in median eminence extracts on sephadex gel-filtration

Abstract

Hypothalamic median eminence extracts (MEE) were subjected to gel filtration on Sephadex G-25 and G-75 columns to characterize the corticotropin releasing factor (CRF) in relation to arginine vasopressin (AVP). CRF activity was measured using monolayer cultured anterior pituitary cells, and AVP was measured by radioimmunoassay. Sephadex G-25 chromatography of AVP immunoreactivity of MEE (NIAMDD-Rat HE-RP-1) showed three peaks on elution with 0.1 N HCI and four peaks on elution with 0.2M acetic acid. But freshly prepared MEE showed only one peak on elution with 0.1 N HCI. These results suggest that AVP in NIAMDD-Rat HE-RP-1 is polymerized or aggregated on the Sephadex G-25 column, especially when eluted in 0.2M acetic acid. Two main peaks of CRF activity appeared consistently on both Sephadex G-25 and G-75 chromatography. One was near the void volume and the other was retarded. The small molecular CRF (small-CRF) peak was coeluted with immunoreactive AVP and 125I-AVP, on both chromatographies on elution with 0.1 N HCI. The large molecular CRF (big-CRF) appeared between the void volume and I-39 ACTH on Sephadex G-75 chromatography. Big-CRF from freshly prepared MEE had no AVP immunoreactivity. AVP showed CRF activity in pituitary cell cultures, but its CRF activity accounted for no more than 20% of the CRF activity of NIAMDD-Rat HE-RP-1. The log dose-response characteristics of the CRF activities of small-CRF and AVP differed. These results suggest that the median eminence has at least two substances with CRF activity: one is large molecular CRF, and the other is small molecular CRF which may have a vasopressin-like molecular weight. AVP may account for a part of the CRF activity of small molecular CRF but is not identical with genuine CRF.