Nuclear localization of tritiated aldosterone in the CNS was studied in rats by numerical evaluation of silver grains, deposited over neuronal cell nuclei in thaw-mounted autoradiograms, and compared with the localization obtained after prior administration of a 100-fold excess of radioinert aldosterone, corticosterone or 18-hydroxy-11-deoxycorticosterone (18-OH-DOC). Corticosterone and 18-OH-DOC completely prevented nuclear localization in most regions examined. However, in contrast to pretreatment with aldosterone, pretreatment with corticosterone and 18-OH-DOC did not completely prevent the concentration of radioactivity in the cell nuclei of the indusium griseum. Traces of radioactivity were, furthermore, retained in areas CA1 and CA2 and the dentate gyrus in rats exposed to corticosterone, but not to 18-OH-DOC, prior to [3H]aldosterone. A similar profile of silver grain distribution to that noted with aldosterone was found for corticosterone except that with tritiated corticosterone the most intense concentration of radioactivity occurred in hippocampal areas CA1 and CA2 and not in the indusium griseum. Prior administration of excess deoxycorticosterone acetate abolished nuclear accumulation of tritiated corticosterone. Dihydrotestosterone, on the other hand, failed to compete with tritiated corticosterone at a dose 200-fold in excess of the tritiated steroid. We conclude that (1) a receptor readily shared by aldosterone, corticosterone, 18-OH-DOC and DOC, but not by dihydrotestosterone, is widely distributed throughout the CNS, (2) a receptor shared by aldosterone and 18-OH-DOC, but not by corticosterone may be present in hippocampal areas CA1 and CA2, (3) that both these as well as the receptor accepting dihydrotestosterone can be located within the same cell.

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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.

The uptake of [3H]-aldosterone by the brain and anterior pituitary (AP) was studied after i.v. injection of the isotope into adrenalectomized rats. The AP showed the higher uptake ratio (i.e., radioactivity in tissue/radioactivity in blood), while the brain regions examined (hippocampus, hypothalamus, amygdala, cerebellum and cortex) contained lower levels of radioactivity, although they concentrated the hormone from blood. Neither [3H]-corticosterone nor [3H]-18-hydroxydeoxycorticosterone accumulated in the AP as much as [3H]-aldosterone, while [3H]-corticosterone's uptake was greatest in the hippocampus. Competition experiments demonstrated that [3H]-aldosterone uptake in the AP was inhibited by pretreatment of animals with excess aldosterone, corticosterone and dexamethasone, whereas aldosterone and corticosterone but not dexamethasone competed in the brain regions. Binding sites were demonstrated in vitro in cytosol fractions from AP and several brain regions. Scatchard plot analysis demonstrated high affinity, low capacity binding sites in cytosol from AP and hippocampus. These results suggest the AP and brain areas may be considered as targets for aldosterone, although the functions of the mineralocorticoid in these tissues are a matter of speculation.

Autoradiographic studies with 3H aldosterone demonstrate nuclear concentration of hormone in neurons of the hippocampus, septum, allocortical regions and brain stem reticular formation and motor nuclei of cranial nerves and in the meninges. The results suggest that mineralocorticoids have wide ranging effects on different parts of the central nervous system.