ACTHの分泌機序に関する研究特に視床下部ACTH放出因子 (CRF) の生検定法について

抄録

The author has tried to establish a convenient and accurate bioassay method for the determination of C.R.F. (corticotrophin relasing factor) activity of the hypothalamic extract. The principle of the method lies in the combination of two in vitro techniques. In the first step three pituitary halves taken from the freshly sacrified female rats were incubated in vitro for the liberation of ACTH into the incubation medium. In the second step the expected ACTH secretion into the medium was determined by the in vitro assay technique of ACTH-like activity following the description of Tsuji and Yasui, which is characterised through its specificity, high sensitivity and reproductbility (Folia Endo-crinologica Japonica. 41 : 643, 1965).
The method and the results obtained are summarized as follows :
1) As the first test, tissue for the liberation of ACTH three pituitary halves taken from three different rats were placed in two flasks and used in the form of pooled samples. One flask served as test and the other flask as the control. Preincubation of pituitary halves for 60 minutes brought about high sensitivity and well standardized reactivity of the pooled pituitary tissues. After preincubation for sixty minutes three halves of the above-described pituitary halves were placed in the fresh incubation medium after tho-rough washing. A standardized portion of the rat hypothalamus encircling the median eminence was cut out with a razor blade and crude extract was placed with cold galcial acetic acid following the description of Vernikos-Danellis. This extract (henceforth abr. M.M.E.) was added to the incubation medium, in which 3 halves of rat pituitary halves are placed. After final incubation for 60 minutes the expected ACTH secretion in the medium was determined after Tsuji-Yasui's bioassay method of ACTH. The characteris-tic of this last step lies in the use of pooled samples of four beef adrenocortical slices weigh-ing about 100 mg in total as test material. The two-step procedures made it possible to determine the C.R.F. activity of M.E.E. qualitatively and even semi-quantitatively. The sensitivity of the method for C.R.F. activity was in the level of M.E.E. prepared from one animal.
2) At first the author has tried to verify the specifictiy of this assay technique through the exclusion of possible sources of error originating from this M.E.E.
The following points were checked : i) No ACTH-like activity was observed in the M.E.E. ii) No ACTH destroying or potentiating effect was observed in the M.E.E. iii) C.R.F. contained in the M.E.E. was stable for heating up to 100°C for 30 minutes, henceforth all experiments were carried out using M.E.E. heated to 100°C for 30 minutes. This procedure excludes various enzymic actions. iv) No C.R.F. activity was shown in the crude extract of brain cortex. v) It was proved that the M.E.E. acts directly upon the incubated pituitary tissues and induces the release of ACTH. The simultaneous examination of ACTH content of the incubated pituitary tissues revealed the decrease of the ACTH content in the tissue.
3) To verify the presence of ACTH itself in the incubation medium, in which pituitary tissues were placed, a 4 point assay was carried out with the incubation medium and ACTH (Third international standard preparation of ACTH). There was always a 'exact parallelism. In the following experiments for the assay of expected ACTH secretion in the incubation medium two control flasks with two concentrations of ACTH standard preparation, i.e, 10^-2 and 10^-3 mU/flask were incubated simultaneously for the guarantee of the ACTH assay.
4) The specificity of this assay system for the determination of C.R.F. activity was clearly demonstrated in the comparative studies, in which crude extract from brain cortex, serotonin, pitressin, histamine, acetylcholin and bradykinin were used as the test material.