DHEA, PREG and Their Sulphate Derivatives on Plasma and Brain After CRH and ACTH Administration

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DHEA, PREG and Their Sulphate Derivatives on Plasma and Brain after CRH and ACTH Administration

J. M. Torres1 and E. Ortega1,2,3

(Accepted January 29, 2003)

The term neurosteroids applies to steroids that are synthesized in the nervous system, either de novo from cholesterol or from steroid hormone precursors. RIA was used to determine plasma and brain levels of the neurosteroids pregnenolone (PREG), dehydroepiandrosterone (DHEA), and their sulfate derivatives (PREG-S and DHEA-S) in male and female rats after administration of two typical stress hormones: corticotropin-releasing hormone (CRH) and adrenocorti- cotropin hormone (ACTH). In all cases, the parameters measured were detectable in plasma and brain. PREG, PREG-S, and DHEA increased significantly in plasma and brain after CRH and ACTH administration in males and females. Because neurosteroids play an important role in mammalian physiology, including that of humans, stress situations may alter the physiological functions regulated by these neurosteroids.

KEY WORDS: Neurosteroids; CRH; ACTH; brain.

INTRODUCTION trated and adrenalectomized rats (3). Subsequent in vitro studies confirmed neurosteroidogenenic activity The term neurosteroids applies to steroids that by demonstrating the gene expression of several are synthesized in the nervous system, either de novo steroidigenic enzymes in the brain, in particular P450 from cholesterol or from steroid hormone precursors. side-chain-cleavage (P450scc); 17hydroxylase/C17– Evidence accumulated over the last decade indicates 20lyase (P450c17); 3 hydroxiesteroid dehydrogenase that the brain is capable of the de novo biosynthesis (3BHSD); steroid sulfokinase; steroid sulfatase; and the of neurosteroids independent of gonads, adrenals, or enzymes steroid 5 reductases and 3 oxidoreductase other peripheral steroidogenic organs (1,2). The first (3–5). It has further been demonstrated that neuro- proposal of steroidogenesis in the brain was based steroidogenesis is accomplished by a tripartite contri- on the observation that pregnenolone (3hydroxy- bution of astrocytes, oligodendrocytes, and neurons, 5pregnene-20-one) (PREG), dehydroepiandrosterone the three types of brain cell (4). (3hydroxy-5androstene-17-one) (DHEA) and their Various biological effects of neurosteroids have sulfate (S) derivatives accumulate in the brain of cas- been observed, such as electrical stimulation of neurons, involvement in behavioral activities, and growth/ 1 Department of Biochemistry and Molecular Biology, Faculty of differentiation of glial cells in vitro (6). The neuros- Medicine, University of Granada, 18012 Granada, Spain. teroids PREG, DHEA, and their sulfates, as well as re- 2 Institute of Neurosciences, University of Granada, 18012 Granada, duced metabolites of progesterone such as 35THP, Spain. can act as allosteric modulators of neurotransmitter re- 3 Address reprint requests to: E. Ortega, Department of Biochem- istry and Molecular Biology, Faculty of Medicine, University of ceptors, such as GABA-A, NMDA, and sigma recep- Granada, Avda. de Madrid s/n, 18012, Granada, Spain. Tel: 34- tors (1). Thus, the concentration of neurosteroids at 958-243519; Fax: 34-958-249015; E-mail: [email protected] different sites in the brain varies according to environ- 1187 0364-3190/03/0800–1187/0 © 2003 Plenum Publishing Corporation NR2808_465537.qxd 05/23/2003 2:49 AM Page 1188

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mental and behavioral circumstances, such as stress, steroids fractions were evaporated under nitrogen in a water bath sex recognition, or aggression (1). and the residue dissolved in isooctane until chromatographic sepa- Earlier studies published by our group demon- ration by CELITE 535. Plasma samples (2 ml) mixed with radioactive steroids to evaluate the recoverability were extracted with ethyl strated that stress modulates the reproductive function acetate and the residues were dissolved in isooctane until chromato- in rats, at least in part by decreasing LH secretion graphic separation by CELITE 535. Pregnanolone was eluted with through an increase in corticotropin releasing hormone Benzene:Isooctane (30%) and DHEA was eluted with Bezene: (CRH) and corticotropin hormone (ACTH) (7–10). Isooctane (50%). The different fractions collected were evaporated More recently, we reported that CRH and ACTH ad- to dryness under stream of nitrogen and keep until the RIA were performed. On the day of the RIA the residues were dissolved in 1 ml ministration increases levels of the neurosteroid allo- of PBS. Aliquots of 200 l per tube were used in each respective pregnanolone (35THP) in plasma and brain of rats RIA. The RIA were performed as previously described (3,16,17) (11). PREG, DHEA, and their sulfates play an impor- using reagents kindly provided by Roussel-Uclaf. tant role in the psychology of mammals, including hu- Statistical Analysis. Statistical calculation of the results was mans (1). It may therefore be of value to study the effect done with the Wilcoxon signed ranks test. The data are expressed as mean SD. of abnormal concentrations of these neurosteroids, with a view to the possible treatment of functional and trophic disturbances of the nervous system (1,12). RESULTS The present study was designed to test the effects of the peripheral administration of ACTH and CRH on Brain and plasma of male and female rats showed the plasma and brain levels of PREG, DHEA, and their measured concentrations of PREG, PREG-S, DHEA, sulfate derivatives in male and female rats. and DHEA-S. Plasma and brain concentrations of free PREG and DHEA increased significantly after ACTH and EXPERIMENTAL PROCEDURE CRH administration in both males and females. How- ever, the increase of PREG in brain and plasma of both Animals. Adult male and female Wistar rats of over 150 g body sexes was greater than that of DHEA (Figs. 1 and 2). weight were used in this study. The animals were divided in three After ACTH and CRH administration, PREG-S groups, each comprising both males and females: one group was in- increased significantly in plasma and brain of males jected with 0.75 nM of CRH (CRH 1–41, Sigma-Aldrich) (13), the second group was injected with 0.5 g of ACTH (ACTH 1–24, Syn- and females (Fig. 3), although the increase was smaller acthen, Novartis Pharmaceuticals), and a third group received no than that of PREG. In the other hand, DHEA-S did not treatment and served as controls, according to our previous reports significantly increase in either sex, either in plasma or (11). All experiments were carried out in accordance with the Na- brain after CRH and ACTH administration (Fig. 4). tional Institutes of Health Guidelines on the care and use of Labo- ratory animals (NIH Publications No. 8023). Brain and Blood Collection. After 30 min of CRH or ACTH administration, the rats were killed by decapitation, following guidelines of the American Association for the Accreditation of Laboratory Animal Care. The brains were rapidly removed, frozen on dry ice, and stored at 80°C until analysis. Plasma samples obtained by centrifugation at 2000 rpm were kept at 80°C until analysis. Extraction Procedure and RIA. The entire brain (2 g) was ho- mogenated in phosphate buffer saline (PBS) pH 7.4 with Teflon glass homogenized, mixed with radioactive steroids to evaluate the recoverability and vortexed with ethyl acetate: isooctane 1:1. The homogenate was centrifuged at 4000 rpm for 5 min. The aqueous phase contained the sulfated steroids, and extracts contained the free steroids and steroids conjugated to fatty acids. Sulfated steroid frac- tion was subjected to solvolysis in ethyl acetate to generate free steroids (14). After evaporation of the organic solvent of the extracts (containing the free steroids and steroids conjugated to fatty acids), the pellet was extracted with methanol (MeOH) aqueous, thereafter sonicated and centrifuged. A clean-up step was performed by solid- phase extraction on AMPREP silica minicolumns C18 (15). Samples were deposited on columns which had been previously activated Fig. 1. Plasma (PL) ng/ml and brain (B) ng/g pregnenolone (PREG) successively with MeOH, ddH20, and aqueous MeOH. measured in male and female rats treated with ACTH and CRH, and 2 ml of the extracts were transferred to the cartridge AMPREP controls. Each bar represents the mean SD for 5–7 animals. Sta- C18 and free steroids were eluted with aqueous MeOH. The free tistical differences versus control animals were at less P .01 (#). NR2808_465537.qxd 05/23/2003 2:50 AM Page 1189

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Fig. 2. Plasma (PL) ng/ml and brain (B) ng/g dehydroepiandros- Fig. 4. Plasma (PL) ng/ml and brain (B) ng/g dehydroepiandrosterone (DHEA) measured in male and female rats treated with terone sulfate (DHEA-S) measured in male and female rats treated ACTH and CRH, and controls. Each bar represents the mean SD with ACTH and CRH, and controls. Each bar represents the mean for 5–7 animals. Statistical differences versus control animals were SD for 5–7 animals. There is no statistical differences between at less P .05 (*). ACTH and CRH treated animals versus control animals.

and N-methyl-D-aspartate (NMDA) receptor functions, DISCUSSION modulation of sigma receptor function, regulation of myelinization (19), neuroprotection, and growth of Over the past decade, it has become clear that the axons and dendrites. Neurosteroids have also been brain is a steroidogenic organ that synthesizes a num- shown to modulate the expression of particular sub- ber of steroids, denominated neurosteroids. In general, units of GABA-A and NMDA receptors, providing ad- they mediate their actions, not through classic steroid ditional sites at which these compounds can regulate hormone nuclear receptors, but through ion-gated neu- neural function (1,18). rotransmitter receptors (18). Functions attributed to Because the neurosteroid concept applies to all specific neurosteroids include modulation of GABA-A mammalian species, including humans, it may be important to study the effect of abnormal concentrations of these neurosteroids, with a view to the possible treatment of functional and trophic disturbances of the nervous system (1,12). Our results clearly demonstrated that the administration to an intact animal of CRH and ACTH, two hormones hypersecreted in stress situations, significantly increased plasma and brain levels of PREG, PREG-S, and DEHA in both genders studied. Never- theless, there is not significant increase in DHEA-S levels in either plasma and brain after ACTH and CRH administration. Our results are in accordance with those previously reported by Baulieu et al. (20) demonstrating a lack of significant increase in DHEA- S levels in anterior and posterior brain after ACTH administration. However, our results are in contrast with previous studies (20) that reported that ACTH admin- Fig. 3. Plasma (PL) ng/ml and brain (B) ng/g pregnenolone sulfate istration increased DHEA-S levels in rat plasma. The (PREG-S) measured in male and female rats treated with ACTH discrepancies between the previous reports and our and CRH, and controls. Each bar represents the mean SD for 5–7 animals. Statistical differences versus control animals were at less own results could be due to differences in the experi- P .05 (*). mental protocol. In previous paper ACTH was injected NR2808_465537.qxd 05/23/2003 2:50 AM Page 1190

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SC on three consecutive morning and rats were killed mice against lactating female intruders (15). PREG-S 2 h after the last injection (3,20). In our study, ACTH induces deficits in a passive avoidance memory task was IP injected once, and rats were killed 30 min after (27), improves acquisition and retention of a food injection In our opinion the lack of a significant in- search task (28), and enhances ethanol tolerance (29). crease in DHEA-S could be due to the long half-life of DHEA produces a marked and significant decrease of these compounds, precluding the observation of sig- PREG-S concentrations in the brain, which in turn in- nificant variations in the concentrations of this steroid creases the GABAergic tone. DHEA and PREG down- during the time period of our experiment. regulate reactive astroglia in the male rat brain after a With respect to DHEA values, we have found in- penetrating brain injury (30). Finally, PREG and other creased levels of DHEA in both brain and plasma after neurosteroids present in the olfactory bulb of rats ap- administration of two typical stress hormones. In con- pear to be involved in the chain of events linked to a trast, Barbaccia et al. (21) found no increase of DHEA heterosexual exposure of animals (24). In our experi- in the cerebral cortex of rats accustomed to being han- ments, PREG, PREG-S, and DHEA increased after the

dled after stress situation such as CO2 inhalation for administration of two stress hormones. Stress situa- 1 min or 1 min after a 5-min foot shock. It has been tions may therefore alter physiological functions regu- suggested that a stress situation could modify plasma lated by these neurosteroids. and brain neurosteroids levels (20,22), although con- Previous reports indicated that rodent adrenals do tradictory data exist to the respect, perhaps because of not produce sizable amounts of DHEA, unlike pri- differences in experimental protocols; for example, a mates (23). In this line, earlier studies indicated that stress situation such as the heavy stress following the concentration of DHEA was not affected by ACTH anesthesia and surgery (20) increases DHEA-S in administration (3,23,24). These data are not consistent plasma and anterior brain but not in posterior brain 2 with the results of the present study. Differences be- days after sham adrenalectomy and orchidectomy. The tween the experiments may explain these discrepan- same heavy stress (anesthesia and surgery) increases cies. Our results clearly demonstrate that DHEA and DHEA-S in anterior brain but not in plasma or poste- DHEA-S are present in plasma in sizable amounts and rior brain 15 days after sham adrenalectomy and or- that these steroids are, at least in part, of adrenal ori- chidectomy. Both experiments were made for the same gin. Three observations support this hypothesis. First, investigators and using the same technique to measure DHEA increase in a similar manner after the adminis- DHEA-S, suggesting that differences in the experi- tration of ACTH and CRH. ACTH increases the mental protocol could be reflected in the final results. biosynthesis of adrenal steroids, while CRH appears to Brain contains various regions that respond to modify the biosynthesis of both adrenal and testicular acute stress, modifying neurosteroids levels (3,20); steroids (13). Second, we found (unpublished observa- therefore a measure of neurosteroid levels in different tions) comparable amounts of DHEA-S and corticos- brain areas could be more adequate to study the effects terone in rat plasma under different experimental of both CRH and ACTH. Unfortunately, in this paper conditions. Third, the response of neurosteroids to as in other previously reported studies (23,24), we ACTH and CRH appears to be greater in females than have used in our experimental procedure the whole in males, in line with previous reports by our group on brain. The aim of our paper is to offer a general view the effects of acute alcohol intoxication on adrenal about the short time effects (30 min) of two typical hormones in humans (31,32). stress hormones, ACTH and CRH on neurosteroids Although it seems unlikely, we cannot rule out pregnanolone and DHEA and their respective sulfate the possibility that CRH and ACTH penetrated areas derivatives. of the brain involved in the biosynthesis of neuros- The increased levels of neurosteroids after CRH teroids. In fact, diazepan binding inhibitor (DBI), a and ACTH administration may have important behav- protein essential for ACTH-induced steroidogenesis in ioral and biochemical consequences. In fact, PREG-S the adrenal cortex, has been reported in the brain (33). affects cognitive processes in the rat when injected To summarize, our results demonstrated an in- into the nucleus basalis magnocellularis (25), and both crease in neurosteroid levels in brain and plasma after DHEA-S and PREG-S show memory-enhancing ef- the administration of ACTH and CRH, two typical fects in foot-shock avoidance training (26). DHEA has stress hormones. Because neurosteroids play an im- a trophic effect on mouse neurons, reinforces long- portant role in the physiology of mammals, including term memory of an active avoidance behavior (24), humans, stress situations may alter physiological func- and inhibits the aggressive behavior of castrated male tions regulated by these neurosteroids. NR2808_465537.qxd 05/23/2003 2:50 AM Page 1191

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