Effects of ACTH on the last step of aldosterone biosynthesis

J. sferoid Biochem. Vol. 33, No. 6, pp.

0022-4731/89$3.00+ 0.00 Pergamon Press plc

1253-1255,1989

Printed in Great Britain

SHORT COMMUNICATION EFFECTS OF ACTH ON THE LAST STEP OF ALDOSTERONE BIOSYNTHESIS EDUARDO N. COZZA,* NORA R. CEBALLOSand CARLOS P. LANTOS Laboratorio de Esteroides, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires and Programa de Regulation Hormonal y Metabolica, CONICET, Buenos Aires, Argentina (Received 13 December 1988; received for publication 22 August 1989)

Summary-The production of tritiated aldosterone and tritiated SM (a saponifiable 18-hydroxycorticosterone derivative) by rat adrenals were studied at various incubation times in absence or presence of two concentrations of ACTH. Tritiated 18-hydroxycorticosterone or 1Gdeoxyaldosterone served as precursors. The lower ACTH concentration (150 PM) increased the production of tritiated aldosterone. Whereas, the higher ACTH concentration (1.5 PM) stimulated tritiated aldosterone production at shorter incubation time (30min), while after 60 min it inhibited. This time dependency would reflect variations in the levels of endogenous steroids. On the other hand, the effects of ACTH on tritiated SM production were opposite to those on tritiated aldosterone. In effect, while 150pM ACTH inhibited SM production, 1.5 PM ACTH stimulated it. These results suggest that ACTH promotes opposite effects on the productions of aldosterone and SM and therefore both productions would be coordinated under the regulation of ACTH.

INTRODUCTION It is well known that ACTH not only promotes glucocorticoid production by the adrenal but is also a short-term stimulator of aldosterone biosynthesis (l-41 by acting in both early and late pathway [S]. On the other hand, we have reported (61that 18-hydroxycorticosterone, an intermediate in the biosynthetic pathway to aldosterone. can be metabolized to a saponifiable 18-hydroxycorticosterone derivative (SM). After treatment with an alcoholic solution of potassium bicarbonate, SM yields 18-hydroxycorticosterone [6]. However, the chemical structure of SM remains to be determined. In the present work, the time-course of the conversion of [1,2-sH]18-hydroxycorticosterone to aldosterone and SM were each investigated under two different ACTH concentrations. The lower ACTH concentration (150pM) increased the production of tritiated aldosterone at all incubation times, while the higher one (1.5 PM) corresponded to experiments in which tritiated aldosterone production had been found to be decreased [7]. In addition, the same productions were studied from [l ,2-3H]18-deoxyaldo-

*Author to whom all correspondence should be addressed: Eduardo N. Cozza, J. A. Haley VAH-IllM, 13000 Bruce B. Downs Blvd., Tampa, FL 33612, U.S.A. Abbreviations: The following trivial names have been used, 18-hydroxycorticosterone, 1l&18,21-trihydroxy-4pregnene-3,20-dione. ll-deoxyalddsterone, 21:hydroxy1l/3,18-oxido,4,pregnene-3,20-dione. SM is an abbreviation for Saponifiable Material, see Ref. [6].

steome, an anhydride of the former, which has also been shown to be precursor of aldosterone [S, 91. MATERIALS

AND METHODS

Tissue preparation Male rats of the CHBB-Thorn strain weighing 200-250 g were decapitated. Their adrenals were excised, freed from adherent tissue, placed on ice and quartered. In some experiments capsules were separated from the whole adrenal. Adrenal quarts, capsules or cores were pooled according to Lantos et a1.[7]. Mitochondria fractionation Mitochondria were obtained as previously described [6]. Mitoplasts (matrix plus inner membrane), intermembrane and outer membrane were obtained essentially as described elsewhere [lo]. purity of mitochondrial fractions were checked by measuring specific mitochondrial markers, succinate dehydrogenase [ll] (inner membrane), kynureine hydroxylase [12] (outer membrane) and adenylate kinase [13] (intermembrane). Incubations 0.8 @i of [1,2-3H]18-hydroxycorticosterone or [1,23H]18-deoxyaldosterone were incubated with 100 mg of quartered rat adrenals, or capsules or cores or whole mitochondria or mitochondrial fractions from that amount of tissue, at 37°C in 13.5 mM Krebs-Ringer bicarbonate glucose buffer @H 7.4), with or without ACTH at indicated concentrations.

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Analysis of incubates

Incubations were stopped by extracting supematants with 2 vol. of methylene chloride 3 times. Adrenal tissues were homogenized in 20% aqueous ethanol and homogenates were extracted similarly. 0.02 ,uCi[i4C]aldosterone were added to the combined extracts. Then, these extracts were evaporated under N, and the dried residue analyzed by paper chromatography [6] or by ,RP-HPLC [14], as described elsewhere. Drugs

[1,2-‘H]l8-hydroxycorticosterone (SA = 52 Ci/mmol) was from Amersham Radiochemicals. Tritiated 18-deoxyaldosterone was prepared from the former by dehydration with HCl [8]. [i4C]aldosterone (SA = 57 Ci/mmol) was obtained from New England Nuclear. Radioinert 18-deoxyaldosterone-21-acetate was a kind gift from Dr Marcel Harnik (Biotechnology Center, University of Tel-Aviv, Israel). Bovine ACTH was kindly provided by Dr Eduardo Passeron (Laboratorios ELEA, Buenos Aires, Argentina). RESULTS AND DISCUSSION

In order to evaluate the zonal localization of the formation of SM, tritiated 18-hydroxycorticosterone was incubated in the presence of adrenal cores and capsules. When incubates were analyzed by HPLC, tritiated SM was only obtained in the samples proceeding from incubations performed with adrenal capsules, with a retention time of 25.4 min (Table 1). However, non-polar radioactive fractions were also obtained by HPLC in incubations of 18-hydroxycorticosterone with adrenal cores (retention time 19.7 min and 29.7 min). When the material corresponding to those peaks was saponified and chromatographed, no changes were observed in retention times in comparison with the original run. These results suggest that non-saponifiable products were obtained by incubation of tritiated 18-hydroxycorticosterone with rat adrenal cores. Tritiated SM was also converted into 18-hydroxycorticosterone almost exclusively by capsules (Table 1). These results suggest that either SM is not produced in adrenal cores, or that the concentrations of endogenous 18-hydroxycorticosterone and SM were very high, producing a large dilution of the tracer. In order to evaluate whether or not different concentrations of endogenous could be responsible of the difference between cores and capsules in producing and transforming SM, we carried out the following experiments. Tritiated SM, obtained from incubations of capsules with

18-hydroxycorticosterone, was incubated with cores. As before, no 18-hydroxycorticosterone was obtained. With the same purpose in mind, we compared the productions of tritiated 18-hydroxycorticosterone from incubations of adrenal capsules with SM from two different sources. The first SM sample corresponded to the SM fraction isolated from incubations of adrenal capsules with tritiated 18-hydroxycorticosterone. The second SM sample corresponded to the first SM sample combined with the SM-like fraction isolated from incubations of adrenal cores with tritiated 18-hydroxycorticosterone (retention times 24.9-25.9 min). We found that both tritiated 18-hydroxycorticosterone productions were very similar. These findings suggest that endogenous SM in adrenal core, if any, is very low and not responsible for the differences between core and capsule activities shown in Table 1. When adrenal capsule mitochondria were fractioned into mitoplast, intermembrane and outer membrane, and these fractions incubated with tritiated 18-hydroxycorticosterone, we found that most of the activity for the transformation of 18-hydroxycorticosterone into SM was found in the outer membrane fraction (Table 1). We conclude that in the adrenal capsule mitochondria, 18-hydroxycorticosterone may be transformed into aldosterone or into SM. It seems reasonable to speculate that these alternatives have to be under regulation. In order to evaluate the possible role of ACTH in that putative regulation, quartered rat adrenal were incubated with [l,2-3H]18-hydroxycorticosterone in the absence or presence of ACTH. The upper panel of Fig. 1 depicts the time-course of the conversion of tritiated 18-hydroxycorticosterone to aldos0.3

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Table I. Localization of the production of SM

Fraction Quartered rat adrenals Adrenal capsules Adrenal cores Capsule mitochondria Capsule mitochondria fractions: Mitoplasts Intermembrane Outer membrane

% Enzymatic activity From SM From I8-OH-B to 18-OH-B to SM 100 81 21 85

loo 92 18 88

I2 9 88

7 10 93

Tritiated 18-hydroxycorticosterone or SM were incubated with quartered rat adrenals, or rat adrenal cores and capsules, or capsule mitochondria, or capsule mitochondrial mitoplasts or intermembrane or outer membrane (see Materials and Methods). Each fraction corresponded to 100 mg of adrenal tissue. After incubation, tritiated SM produced from l8-hydroxycorticosterone or this last steroid produced from tritiated SM were measured in the supernatants as indicated in the text. Enzymatic activity of quartered rat adrenals was arbitrary as 100%. Results are mean of one of three experiments performed in duplicates.

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Fig. I. Time-course of the conversion of tritiated 18-hydroxycorticosterone to aldosterone (panel A) and SM (panel B) of triplicate incubation samples from three different experiments containing 100 f 5 mg quartered rat adrenals and either OpM (a), 15OpM (A) or l.SpM (m) ACTH. Values are means & SEM. *Significance of differences with controls as calculated by r-test (P c 0.05).

Short communication Table 2. Effect of ACTH on the conversion of IB-deoxvaldosterone to aldosterone at 90 min Treatment Controls ACTH 1.5~M ACTH 150 PM

Tritiated aldosterone (pmol) 0.63 + 0.04 0.38 f 0.02* 1.09 + 0.04’

Tritiated 18-deoxyaldosterone was incubated with 100 + 5 mg quartered rat adrenals at 37°C and pH 7.4 with or without ACTH. For details see the text. lf (0.01.

terone in the absence or presence of 150pM ACTH. As expected, ACTH constantly stimulated this conversion, and reaches plateau above controls. However, by contrast, 1.5 PM ACTH, which is a very high dose, depresses the conversion to aldosterone after 60 min (Fig. 1). This caused the aldosterone plateau to fall below the corresponding control values. This effect of higher ACTH concentration would be reflecting strong changes in the specific radioactivity of the aldosterone precursors caused by increases in the level of endogenous steroids. The lower panel of Fig. 1 depicts the time-course of the conversion of tritiated 18-hydroxycorticosterone to SM. Many of the effects of ACTH on this transformation are reversed from the above case. Both ACTH concentrations displace the initial part of the curve to the right, although 150pM ACTH does so only with marginal significance. From 60 min on, the higher ACTH concentration increases the conversion of tritiated 18-hydroxycorticosterone to SM. Whereas lower ACTH concentration decreases this conversion. Thus, aldosterone would be increased while SM decreased. Under this hypothesis, we would reason that the higher ACTH concentration has to produce a lower conversion of tritiated 18-hydroxycorticosterone into SM due to the increase of endogenous steroids as mentioned above. However, as shown in the lower panel of Fig. 1, 1.5 nM ACTH increased, instead of decreased, the production of tritiated SM. We have to take these results with caution because of the high dose of ACTH. Although the reasons do not seem to be very clear, we could hypothesize that the effects of ACTH on SM productions might be dose-dependent or that, at least for high ACTH doses, two pools of 18-hydroxycorticosterone exist in mitochondria. This last hypothesis is peculiarly interesting because of the different mitochondrial loci for aldosterone and SM productions. Further experiments are necessary to evaluate this point. Table 2 lists the conversions of tritiated 18-deoxyaldosterone to aldosterone. At 90 min, the time interval at which these conversions were measured, the lower ACTH concentration increases and the higher concentration decreases the conversions to tritiated aldosterone. According to these results 18-deoxyaldosterone and its precursor 18-hydroxycorticosterone seem to be similarly affected by ACTH. ACTH is known to regulate several steps of the biosynthesis of aldosterone [l, 51. We are reporting herein a new locus under regulation by ACTH, very close to the formation of aldosterone, that is the formation of SM. It has been recently postulated that 18-hydroxycorticosterone could either metabolize to aldosterone or to SM according to homoestatic requirements [6], the latter as an

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inactivating alternative in case aldosterone became unnecessary or harmful. In line with this postulate the observed symmetry of ACTH effects on both alternative pathways is of particular interest. Acknowfedgemenrs-Authors wish to thank Mrs Maria 0. de Bedners for skilful technical assistance. This work was supported by grants from Consejo National de Investigaciones Cientificas y Tecnicas and Universidad de Buenos Aires, Argentina. REFERENCES

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