1952) have shown that adrenocorticotrophic hormone (ACTH) increases the steroid production of rat adrenals in vitro. The same has been found with pig and beef adrenal cortex slices by Haynes, Dorfman (1952, 1953). We have found that the presence of calcium in the incubation medium is essential for the stimulation by ACTH. METHODSTwo rat adrenals were bisected and incubated at 37°C in 0.75 ml. of a Krebs-Ringer glucose medium in semi-micro Warburg vessels for one hour. This medium was discarded and the adrenals were incubated for a further two hours in fresh medium with or without added ACTH. The methods used by Saffran and Bayliss (1953) of extracting and measuring the steroids by the ultra violet absorption of the chloroform extractable lipid material have been modified so that 2-5 /ig. steroid may be measured (Elliott el al.). In some of the experiments the steroids have been measured also by the use of 2,5 diphenyl-3-(4-styryl phenyl) tetrazolium chloride, M & B 1767 (Elliott et dl.). RESULTSIn Table 1 are given the results of experiments in which the effect of omitting calcium from the medium was tested. When Krebs-Ringer 0.01 M phosphate was used for the incubation of the glands (20 to 50 mg. tissue/ml, medium), the pH of the medium at the end of the incubation was 6.7 to 6.9, although the medium with and without ACTH added had been adjusted to pH 7.4. The buffer in the medium was therefore increased to 0.03M and calcium was omitted since it precipitates to a variable extent at this phosphate concentration. With this latter medium no stimulation occurred. This is shown in the first values of Columns 5 and 6. The next data in Columns 5 and 6 show that with 0.01 M phosphate also, ACTH has no effect on steroid production in the absence of calcium. To ensure that the low steroid values and lack of ACTH stimulation were not due simply to refractory glands instead of to absence of calcium, four pairs of experiments were carried out, the results of which are given in Columns 1 and 2. The steroid production was two to four times as great for the left adrenal pair to which calcium had been added along with ACTH as for the right pair that had received ACTH but no calcium. In fact these results are similar to the data in Columns 3 and 4 which illustrate the stimulation of steroid production by ACTH in the presence of calcium.These experiments were repeated in Krebs-Ringer 0.025M bicarbonate medium, pH 7.4, because in this medium the pH can be maintained during the incubation and at the same time calcium can remain in solution. The same calcium effects as in the phosphate media were obtained. The results in Columns 7 and 8 show that calcium alone has no effect on the steroid production of the adrenal without added ACTH.The steroid values obtained by use of the tetrazolium compound, although lower than the values obtained by the ultra violet method also showed the same calcium effects.

No abstract

As further evidence that rat adrenals in vitro produce corticoids, the chloroform-soluble substances formed by rat adrenals have been chromatographed on paper in two different solvent systems to compare their mobilities with those of known corticoids. The chromatography was carried out for the shortest possible time compatible with some resolution in order to prevent losses by excessive exposure or by transfer. Eluates of sections of the chromatograms were analyzed for α, β-unsaturated ketone groups and for groups that reduce a tetrazolium derivative (M. and B. 1767). The data may be interpreted as follows: 1. Rat adrenals in vitro produce ultraviolet-absorbing substances with chromatographic mobilities resembling hydrocortisone and corticosterone. Of these substances only 40–70% can be typical corticoids as judged by the reducing: ultraviolet-absorbing ratio. Ultraviolet-absorbing material without reducing properties is also present, most of which has a polarity greater than corticosterone. 2. ACTH, added to rat adrenal glands in vitro, stimulates primarily the production of substances with a polarity similar to that of corticosterone; the production of the other components is also somewhat increased.