THE NEED FOR THE PRESENCE OF CALCIUM FOR THE STIMULATIONIN VITROOF RAT ADRENAL GLANDS BY ADRENOCORTICOTROPHIC HORMONE

Abstract

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.

Lipolytic Action of Corticotropin on Rat Adipose Tissue In Vitro12

“…It would seem reasonable to suggest that the fatty acids from cholesterol esters and neutral fat in the adrenal cortex might serve as a source of fatty acyl-CoA and acetyl-CoA from which the steroids may be synthesized and that ACTH may serve to initiate the hydrolysis of fatty acid esters. Further support for the analogy between ACTH action on the adrenal cortex and adipose tissue is found in the fact that when the lipolytic effect on adipose tissues is studied in artificial media instead of plasma, the requirements for maximal activity appear to be very similar to those reported necessary for stimulation of adrenal slices by ACTH in vitro (10,20). These same conditions are not required for the in vitro lipolytic action of epinephrine and norepinephrine (10,21,22).…”

“…per ml. ), serotonin (20 2. Corticotropin A and various fractions in the purification of f8-corticotropin as well as a product of pepsin digestion of fB-corticotropin with high adrenocorticotropic hormone (ACTH) activity all had potent lipolytic activity.…”

Hormonal alterations in experimental diabetes: Role of a primary disturbance in calcium homeostasis

“…22]. Calcium potentiates the release of ACTH by pituitary tissue [23] and stimulates adrenal steroidogenesis [24]. Moreover.…”

The effect of calcium ions on the metabolism of exogenous cholesterol by rat adrenal mitochondria

“…Since the trophic effect of ACTH with relation to increased corticosteroid production is prevented by prior treatment of the animal with cycloheximide the involvement of a putative protein factor in the regulation of the cholesterol desmolase system has been proposed [3]. There have also been reports which implicate calcium ions in the response of steroidogenic tissues to trophic hormone stimulation [4][5][6][7]. In these investigations [6,7] the influences of trophic hormones and calcium ions on the activity of the mitochondrial cholesterol desmolase towards endogenous cholesterol have been reported.…”