Biological and immunological characterization of corticotropin-releasing activity in the bovine adrenal medulla

Abstract

Corticotropin-releasing factor (CRF)-like activity in bovine adrenal medulla extracts were characterized by measurement of adrenocorticotropin (ACTH) release from rat anterior pituitary cells in vitro, and by a sensitive heterologous radioimmunoassay (RIA) for bovine hypothalamic CRF. Bovine adrenal medulla was boiled in 2 M acetic acid, homogenized, and submitted to acetone precipitation, followed by batch-wise treatment with C-18 resin. The partially purified adrenal medulla extract showed significant stimulation of ACTH release in vitro and CRF-like immunoreactivity (CRF-IR). After subsequent ion exchange chromatography on a SP-Sephadex column, most CRF bioactivity (CRF-BA) and CRF-IR were eluted with weakly basic materials in the SP-II fraction in which synthetic CRF is eluted. Minor CRF-BA and CRF-IR were also eluted in the SP-III fraction which contained basic peptides. Upon Sephadex G-50 gel filtration of the SP-II fraction, CRF-BA and CRF-IR coeluted, but slightly later than synthetic bovine CRF. However, rechromatography of the major CRF activity on a Sephadex G-50 column and reverse phase and ion exchange high performance liquid chromatographies (HPLC) indicated that CRF-BA and CRF-IR were eluted in the identical fraction as synthetic bovine CRF. Gel filtration in the SP-III fraction on a Sephadex G-50 column showed a few low CRF-BA peaks which lacked CRF-IR. This CRF-BA, however, contributed to less than 5% of the total CRF-BA. These results indicate that the majority of CRF-BA and CRF-IR in the bovine adrenal medulla is chromatographically indistinguishable from bovine hypothalamic CRF. The CRF concentration in the bovine adrenal medulla was 0.53 ng/g wet wt. as measured by two different RIAs, for CRF, and was comparable to that measured by bioassay (0.60 ng/g) in terms of synthetic CRF.