BRIEF REPORT / RAPPORT BREF

Adrenal 11-beta hydroxysteroid dehydrogenaseactivity in response to stress

Marisa Zallocchi, Laura Matkovi�, and María C. Damasco

Abstract: This work studied the effect of stresses produced by simulated gavage or gavage with 200 mmol/L HCl twohours before adrenal extraction, on the activities of the 11β-hydroxysteroid dehydrogenase 1 and 11β-hydroxysteroiddehydrogenase 2 isoforms present in the rat adrenal gland. These activities were determined on immediately preparedadrenal microsomes following incubations with 3H-corticosterone and NAD+ or NADP+. 11-dehydrocorticosterone wasmeasured as an end-product by TLC, and controls were adrenal microsomes from rats kept under basal (unstressed)conditions. 11β-hydroxysteroid dehydrogenase 1 activity, but not 11β-hydroxysteroid dehydrogenase 2 activity, was in-creased under both stress-conditions. Homeostatically, the stimulation of 11β-hydroxysteroid dehydrogenase 1 activitywould increase the supply of glucocorticoids. These, in turn, would activate the enzyme phenylethanolamine N-methyltransferase, thereby improving the synthesis of epinephrine as part of the stress-response.

Key words: acidosis, adrenal, HSD, stress.

Résumé : Le présent travail a examiné l’effet du stress produit par un gavage simulé ou un gavage avec 200 mmol/Lde HCl, deux heures avant l’ablation de la surrénale, sur les activités des isoformes 11β-hydroxystéroïde déshydrogé-nase 1 et 11β-hydroxystéroïde déshydrogénase 2 contenues dans la glande surrénale de rat. Ces activités ont été déter-minées sur des microsomes des glandes préparées immédiatement après des incubations avec de la 3H-corticostérone etNAD+ ou NADP+. La 11-déshydrocorticostérone a été mesurée en tant que produit terminal par CCM; des microsomesde surrénale de rats maintenus en conditions basales (sans stress) ont servi de témoins. Seule l’activité de la 11β-hy-droxystéroïde déshydrogénase 1 a augmenté dans les deux conditions de stress. Du point de vue homéostasique, la sti-mulation de l’activité de la 11β-hydroxystéroïde déshydrogénase 1 augmenterait l’apport de glucocorticoïdes. Ceux-ci, àleur tour, activeraient l’enzyme phényléthanolamine N-méthyl transférase, augmentant ainsi la synthèse de l’adrénalineen réponse au stress.

Mots clés : acidose, surrénale, HSD, stress.

[Traduit par la Rédaction] Zallocchi et al. 425

In the mid-1950s, Selye observed that in situations of fearor displeasure, which he called stress, animals respond witha number of characteristic changes, including an increase inthe size of the adrenal gland, a decrease in the mass oflymph tissue, and the involution of the thymus. Today, weknow that these changes are principally a reaction to gluco-corticoids secreted in response to increased activity of thehypothalamic–pituitary–adrenal axis (Meaney et al. 1996).We also know that these responses take place 30 min afterthe onset of stress, and that glucocorticoids govern processes

that are fundamental for the normal functioning of mostcells.

The concentration of intracellular glucocorticoids is largelyregulated by the enzyme 11β-hydroxysteroid dehydrogenase(HSD), which has two known isoforms: 11β-hydroxysteroiddehydrogenase 1 (HSD1), which is bidirectional and acts invivo by reducing inactive steroids into their active forms(cortisone into corisol and 11-dehydrocorticosterone intocorticosterone); and 11β-hydroxysteroid dehydrogenase 2(HSD2), which is unidirectional and catalyses the oxidationof active into inactive glucocorticoids. Because the gluco-corticoids are unable to bind to the intracellular receptor,they have no activity (Rusvai and Náray-Fejes-Tóth 1993;Hirasawa et al. 1997).

Both isoforms are found in the adrenal cortex (Shimojo etal. 1996) and, in situations of stress, are involved in regulat-ing the production and secretion of active glucocorticoids inplasma and in increasing the formation of epinephrine by ex-erting their action on the chromaffin cells of the adrenal me-dulla (Zuckerman-Levin et al. 2001).

Can. J. Physiol. Pharmacol. 82: 422–425 (2004) doi: 10.1139/Y04-035 © 2004 NRC Canada

422

Received 30 September 2003. Published on the NRCResearch Press Web site at http://cjpp.nrc.ca on 22 July 2004.

M. Zallocchi, L. Matkovi�,1 and M.C. Damasco. Dpto.Química Biológica, 4to. Piso, Pabellón 2, Facultad deCiencias Exactas y Naturales (UBA), Ciudad Universitaria,Ciudad Autónoma de (1428) Buenos Aires, Argentina.

1Corresponding author (e-mail: mzallo@qb.fcen.uba.ar).

The hypothesis of this study is that stress regulates the ac-tivity of both isoforms of adrenal hydroxysteroiddehydrogenase (HSD).

Materials and methods

ChemicalsTritiated corticosterone ([3H1,2,6,7]corticosterone; specific

activity, 70 µCi/µmol) was purchased from New EnglandNuclear, Life Sciences Products (Boston, Mass.). Silica gel60 F254 plates and ethyl acetate were from Merck-Darmstadt (Federal Republic of Germany). The other re-agents were obtained from Sigma (St. Louis, Mo.) orAldrich (Milwaukee, Wis.).

AnimalsThe animals used for these experiments were treated in

accordance with the principles and guidelines of the Guidefor the Care and Use of Laboratory Animals. Male SpragueDawley rats, 300–350 g, were maintained on rat chow andwater ad libitum. All in vivo experiments were conductedbetween 0900 and 1100.

Food was withdrawn 12 h before the experiments. Intactanimals were divided into three groups: basal (unstressed),HCl, and cannula.

Stressed animals

HClIn the HCl group, acidosis was achieved by gavage

(oropharyngeal gastric intubation), with an overload of10 mL of 200 mmol/L HCl administered two hours beforethe adrenal glands were removed. This is a standard proce-dure used by the National Toxicology Program and the Envi-ronmental Protection Agency for the chronic treatment oftest animals for carcinogens, and by other groups to testascorbate (Chen et al. 2000), chlorine, and chloramines(Carlton et al. 1986). This treatment resulted in metabolicacidosis within two hours (acute acidosis), which was con-firmed by blood parameters (Igarreta et al. 1999).

CannulaIn the cannula group, the acid overload was simulated

(placement of rubber catheter only) two hours before re-moval of the adrenal glands.

Preparation of adrenal microsomesWhole adrenal glands were excised, perfused, resus-

pended in a 50-mmol Tris–HCl/L buffer (pH, 8) containing1 mmol MgCl2/L, and homogenyzed with a potter teflonhomogenyzer. Homogenates were centrifuged at 10 000g for30 min. The supernatant was then centrifuged at 105 000gfor 60 min. The microsomal fraction obtained was resus-pended in phosphate buffer. Total protein was determinedusing the Bradford method (Bradford 1976).

Single-point HSD activityThe activity of each isoenzyme was determined by mea-

suring the conversion rate of [3H1,2,6,7]corticosterone into[3H1,2,6,7]11-dehydrocorticosterone. Microsomal suspensions,containing 200 µg protein/mL, were incubated with 4 nmol[3H1,2,6,7]corticosterone/L (Monder et al. 1994; Gomez-Sanchez

et al. 2001). The cofactor for HSD1 was 500 µmolNADP+/L, and for HSD2 was 500 µmol NAD+/L. For eachflask, a 500 µL Tris–HCl buffer containing 1 mmol MgCl2/L(pH, 8) was used as incubation media. The reaction wasstopped after 15 min at 37 °C with 3 mL ethyl acetate, andsteroids were extracted. Steroids were separated using thin-layer chromatography with chloroform–ethanol 96% (92:8).[3H1,2,6,7]corticosterone and [3H1,2,6,7]11-dehydrocorticoster-one were eluted, and radioactivity was counted in a liquidscintillation counter (Wallace 1409 DSA). HSD activity wasexpressed as the total pmols of 11-dehydrocorticosteroneper mg of protein.

Plasma levels of corticosteroneTwo hours after the stress and before the adrenal glands

were removed, blood was withdrawn from the aorta. Plasmacorticosterone levels were determined using radioimmuno-assay, in accordance with the method described by Gomez-Sanchez et al. (1975).

Statistical analysisAll values are presented as mean ± SE. Differences be-

tween groups were evaluated using analysis of variance(ANOVA) and then the Tukey–Kramer test. Statistical signif-icance was set at p < 0.05.

To determine HSD activity, the basal and cannula groupseach had four rats, and the HCl group had six rats.

To determine corticosterone plasma levels, there were fouranimals in each group.

Results

The activity of the HSD1 isoform was significantly higherin animals in the cannula and HCl groups than that in thebasal group. In the stressed groups, HSD1 isoform activitywas significantly higher in the HCl overload group than inthe cannula group (Fig 1).

No significant differences were observed in the activity ofadrenal HSD2 in the different groups (Fig 1).

As expected, stressed animals had levels of corticosterone10 times higher than basal unstressed animals (Table 1).

Discussion

We quantified HSD1 dehydrogenase activity as a measureof active enzyme, because reductase activity is unstable inhomogenates. Our results show that stress produces a signifi-cant increase in adrenal HSD1 activity (Fig. 1). Even thoughit is a bidirectional enzyme, HSD1 acts in vivo by reducing11-dehydrocorticosterone to cortisone and corticosterone tocortisol, thus increasing the supply of active glucocorticoids.

The higher levels of HSD1 correspond to the increase inthe levels of circulating glucocorticoids observed in the ani-mals subjected to stress by either cannula or HCl overload.In both groups, the increase in plasma concentration ofcorticosterone observed was 10-fold (Table 1).

One explanation for the higher HSD1 activity observed inthe HCl group than in the cannula group could be the organ-ism’s need to maintain acid–base homeostasis; the great im-portance of glucocorticoids in the regulation of proton

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elimination at the renal level has been demonstrated (Baylisand Brenner 1978; Malnic et al. 1997).

The presence of HSD2 activity in the adrenal gland pointsto many potential physiological roles for this enzyme.Among these appears to be the protection of mineralocorti-coid receptors against glucocorticoid occupancy. Anothermay include acting as a mechanism to accommodate patternsof glucocorticoid secretion from the adrenal gland (Rolandand Funder 1996). In the stressed groups we studied, we didnot observe any change in HSD2 activity from the basalgroup; this is understandable, because HSD2 deactivatesglucocorticoids and, in stressful situations, the increase incorticosterone would be essential to generate the homeo-static response.

In the adrenal gland, the cortex and the medulla are inti-mately related (Borruel et al. 1968; Ehrhart-Bornstein et al.1998), and glucocorticoids are important for the mainte-nance of chromaffin cells and their production of epineph-rine (Axelrod and Reisine 1984; Doupe et al. 1985).

Recently, Zuckerman-Levin et al. (2001) demonstrated theimportance of glucocorticoids in the adrenal medulla re-sponse to stress. Their intra-adrenal presence is essential forthe secretion of epinephrine; patients with glucocorticoid de-ficiency showed decreased production of this catecholamine.

The increased activity of HSD1 during stress could havetwo functions: it increases the supply of glucocorticoids nec-essary for generating the homeostatic response to stress; and,in a paracrine manner, it contributes to the increased activityof phenylethanolamine N-methyltransferase in the chrom-affin cells, which synthesyze epinephrine from norepine-

phrine (Zuckerman-Levin et al. 2001). Both of these actionsallow the animal to respond better to stressful situations.

Acknowledgements

This work was supported by grants from ConsejoNacional de Investigaciones Científicas y Técnicas(CONICET) and University of Buenos Aires. We thank Dr.Prof. Carlos P. Lantos for his critical review of this manu-script and M.E. Otero for technical assistance.

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Treatment Corticosterone (ng/mL of plasma)

Basal 29±7Cannula 231±26***HCl 353±38***

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Table 1. Plasmatic levels of corticosterone in basaland stressed groups.

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