In Vitro Assay for ACTH-Releasing Activity Using ACTH Radioimmunoassay

Abstract

Several procedures have been reported for the assay of corticotrophine-releasing factor (CRF), each having its advantages and disadvantages. This report deals with an in vitro assay of ACTH releasing activity utilizing pituitary incubation combined with ACTH radioimmunoassay.Rat half pituitary was preincubated in 2 ml Krebs Ringer bicarbonate buffer containing 0.2 % glucose and 0.25 % BSA (KRBG-BSA) for 1.5 hr (45 min × 2). The medium was replaced by 1 ml KRBG-BSA and incubated for 30 min. Then the medium was again replaced by 1 ml KRBG-BSA or KRBG-BSA containing test materials and incubated for another 30 min. The amount of ACTH assayed by radioimmunoassay in the 2nd 30 min incubation was compared with in the 1st 30 min incubation and expressed as percentage.In ACTH radioimmunoassay, anti-ACTH serum was diluted to 1 : 1,500-3,000. The 125I-α 1-24ACTH-antibody system was not affected by lysine-vasopressin (LVP), argininevasopressin (AVP), rat's pituitary LH, GH and prolactin. Human 1-39ACTH was used as ACTH standard, and the dilution curve of incubation medium was paralleled with the standard curve. Repeatability of immunoassayable ACTH within-assay was 174 ± 5.0 pg/tube (CV = 2.9 %).A log dose-relationship was observed between the amounts of stalk median eminence extracts (SME ; NIAMDD) added to the incubation medium and its ACTH releasing activities. The sensitivity of this assay method was at least 0.1 SME or 10 mU of LVP and AVP.Using this method, it found that LVP, AVP, norepinephrine (100 ng/m1-200 ng/ml) and 5-hydroxytryptophane (1 μg/ml) had ACTH releasing activities but LH-RH, TRH, glucagon, dopamine, phentolamine, propranolol, haloperidol, prostaglandin Ei and indomethacin did not affect the release of ACTH.