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Keywords:

  • aldosterone synthase;
  • brain;
  • glucocorticoids;
  • heart;
  • 11β-hydroxylase;
  • mineralocorticoids

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. The adrenal corticosteroids
  5. Non-epithelial actions of corticosteroids
  6. Corticosteroid biosynthesis in the cardiovascular system
  7. Corticosteroid biosynthesis in the central nervous system
  8. Conclusions
  9. Acknowledgements
  10. References

1. The major corticosteroids aldosterone and cortisol (corticosterone in rodents) are secreted from the adrenal cortex under the regulation of the renin–angiotensin system and the hypothalamic–pituitary–adrenal axis.

2. In addition to their accepted roles in such processes as blood pressure regulation, glycogenesis, hepatic glyconeogenesis and immunosuppression, the corticosteroids have been implicated in the development of cardiac fibrosis, modulation of hippocampal neuron excitability, memory formation and neurodegeneration.

3. The advent of sensitive molecular biological techniques has produced a wealth of evidence to support the existence of extra-adrenal corticosteroidogenic systems. Most attention has been paid to the cardiovascular system and the central nervous system, where the full array of enzymes required for the de novo synthesis of corticosteroids from cholesterol has been identified.

4. Although the evidence for local corticosteroid production is strong, the quantities of steroid would be small compared with adrenal production. Therefore, it is still a matter of debate as to whether extra-adrenal corticosteroids are of any physiological significance. This will depend on factors such as local concentration, proximity to target cells and, possibly, to tissue-specific control mechanisms.


List of abbreviations:
-HSD

3β-Hydroxysteroid dehydrogenase

11β-HSD

11β-Hydroxysteroid dehydrogenase

AngII

Angiotensin II

CYP11B1

Gene encoding 11β-hydroxylase

CYP11B2

Gene encoding aldosterone synthase

CYP11A1

Gene encoding side-chain cleavage enzyme

DOC

Deoxycorticosterone

GR

Glucocorticoid receptor

HPA axis

Hypothalamic–pituitary–adrenal axis

MR

Mineralocorticoid receptor

P450c21

21-Hydroxylase enzyme

P450scc

Side-chain cleavage enzyme

RAS

Renin–angiotensin system

RT-PCR

Reverse transcriptase polymerase chain reaction

SCO

Subcommissural organ

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. The adrenal corticosteroids
  5. Non-epithelial actions of corticosteroids
  6. Corticosteroid biosynthesis in the cardiovascular system
  7. Corticosteroid biosynthesis in the central nervous system
  8. Conclusions
  9. Acknowledgements
  10. References

With the development of more sensitive molecular biological techniques, it has become apparent that the adrenal cortex is not the only tissue capable of aldosterone and cortisol production. Low-level secretion from extra-adrenal tissues, together with the discovery that aldosterone and cortisol act on ‘non-classical’ (i.e. non-electrolyte transporting) target tissues, raises the possibility of a novel paracrine or autocrine role for these hormones. To sustain this hypothesis, it is necessary to establish that the biosynthetic apparatus is present and active in these tissues and that the tissues also possess the receptors necessary to transduce their effects. Research into extra-adrenal corticosteroid production has focused particularly on two areas that fulfil these criteria: the cardiovascular system and the central nervous system.

In the present review, we will give a brief introduction to the adrenal corticosteroids, summarise some of the non-epithelial actions of corticosteroids on the cardiovascular system and the brain and then present the current evidence for the production of adrenal corticosteroids within these tissues, paying particular attention to the expression of the enzymes 11β-hydroxylase and aldosterone synthase.

The adrenal corticosteroids

  1. Top of page
  2. Summary
  3. Introduction
  4. The adrenal corticosteroids
  5. Non-epithelial actions of corticosteroids
  6. Corticosteroid biosynthesis in the cardiovascular system
  7. Corticosteroid biosynthesis in the central nervous system
  8. Conclusions
  9. Acknowledgements
  10. References

Adrenal corticosteroid synthesis and its regulation have been reviewed extensively elsewhere,1,2 so here we shall confine ourselves to a brief summary. The corticosteroids are synthesized from cholesterol within the adrenal cortex. Although androgens are also secreted by the adrenal cortex, the term ‘corticosteroids’ as used in the present review is intended to include only the mineralocorticoids and glucocorticoids. The main mineralocorticoid effect is to increase extracellular volume and blood pressure by stimulating sodium reabsorption in exchange for K+ and H+ at electrolyte-transporting epithelia. The glucocorticoids affect a wide range of physiological processes, including glycogenesis, hepatic glyconeogenesis and protein catabolism, as well as having immunosuppressive and hypertensive effects. These actions are effected through the mineralocorticoid receptor (MR) or glucocorticoid receptor (GR) in target tissues.

In humans, the major mineralocorticoid is aldosterone and the major glucocorticoid is cortisol (corticosterone is the major glucocorticoid in the rat). Most steroidogenic reactions are catalysed by enzymes of the cytochrome P450 family, which are located within the mitochondria and require adrenodoxin as a cofactor. 21-Hydroxylase and 17α-hydroxylase are microsomal and do not require this cofactor. The rate-limiting step of corticosteroid synthesis is the delivery of cholesterol to the side-chain cleavage enzyme on the inner mitochondrial membrane, which is catalysed by the steroidogenic acute regulatory (StAR) protein.

A major difference in cortisol and aldosterone synthesis occurs at the terminal stages mediated by the CYP11B1-encoded 11β-hydroxylase and the CYP11B2-encoded aldosterone synthase (see Fig. 1). These enzymes have 93% identity and this is reflected in their shared 11β-hydroxylation and 18-hydroxylation functions. However, aldosterone synthase is also capable of an additional 18-oxidation. The two enzymes are not coexpressed within the adrenal cortex: 11β-hydroxylase is present in the zona fasciculata/reticularis, whereas aldosterone synthase is found only within the thin zona glomerulosa at the outer edge of the cortex.

image

Figure 1. Simplified diagram of the major corticosteroid biosynthetic pathways in the rat and human adrenal cortex.

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Adrenal glucocorticoid synthesis is regulated almost exclusively by the hypothalamic–pituitary–adrenal (HPA) axis. The main regulator of cortisol production is adrenocorticotrophic hormone (ACTH), which is secreted by the anterior pituitary and stimulates CYP11B1 transcription. Adrenocorticotrophic hormone secretion follows a circadian rhythm and can also be increased by physical and emotional stresses. Aldosterone secretion in the adrenal cortex is mainly under the control of the renin–angiotensin system (RAS). Two principal stimuli activate renin secretion from the juxtaglomerular cells of the kidney: a fall in blood volume and pressure, which is sensed by the renal baroreceptor system, and a decrease in sodium arriving at the macula densa. The signalling pathway between the macula densa and the juxtaglomerular cells remains unclear. Angiotensin (Ang) II is the active component of the RAS. In addition to other direct pressor effects, AngII stimulates CYP11B2 transcription. K+ is also a key agonist. Doses of K+ too small to affect plasma K+ concentration can significantly stimulate synthesis directly by depolarizing the zona glomerulosa cell membrane. Adrenocorticotrophic hormone is less important, except acutely and in high doses. Aldosterone stimulates Na+ reabsorption and, therefore, is a key regulator of sodium homeostasis and consequently of extracellular volume. Aldosterone also promotes excretion of K+ and H+.

The ligand-binding domains of MR and GR are 57% identical and bind common steroids with varying degrees of affinity. One important consequence of this is that MR activation can occur when mineralocorticoids are absent due to the ability of the MR to also bind cortisol. In vivo, however, certain tissues demonstrate selectivity, with MR binding aldosterone even when circulating cortisol is high. This is due to the presence of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which converts cortisol in those tissues to cortisone. Another isoform, 11β-HSD1, has a higher Km for glucocorticoids, but generally acts as a reductase. This is important for maintaining high tissue glucocorticoid levels, but renders 11β-HSD1 a poor protector of MR.

At its most rapid, the classical steroid hormone receptor action through GR or MR, the so-called genomic mode of action, has a latent period of approximately 30 min. Over the past decade, corticosteroid effects have been described that occur too rapidly to be explained by genomic action. For example, the addition of aldosterone to cultures of human mononuclear leucocytes results in a raised intracellular free calcium concentration within 2 min.3 Furthermore, these rapid non-genomic effects do not require protein synthesis and, while an extremely elusive membrane-bound mineralocorticoid receptor cannot be completely ruled out,3 non-genomic MR interactions are a distinct possibility.

Non-epithelial actions of corticosteroids

  1. Top of page
  2. Summary
  3. Introduction
  4. The adrenal corticosteroids
  5. Non-epithelial actions of corticosteroids
  6. Corticosteroid biosynthesis in the cardiovascular system
  7. Corticosteroid biosynthesis in the central nervous system
  8. Conclusions
  9. Acknowledgements
  10. References

The cardiovascular system

Deoxycorticosterone (DOC) and aldosterone have long been known to promote collagen deposition in vessels, thus enhancing vascular repair at the expense of compliance. This effect, termed ‘remodelling’, was originally regarded as an adaptation to the arterial hypertension caused by aldosterone excess. However, chronic aldosterone administration is now known to stimulate collagen deposition by cardiac fibroblasts independently of blood pressure elevation in the rat myocardium, thus increasing myocardial stiffness.4,5 The prevention of aldosterone binding through administration of the MR antagonist spironolactone reduces significantly the risk of both morbidity and mortality among patients with severe heart failure.6 The time-course and the efficacy of spironolactone in mediating these protective effects suggest a genomic mode of aldosterone action, but there is also evidence of a rapid non-genomic cardiovascular effect in vivo. Clinical studies have shown a small but significant increase in systemic vascular resistance (SVR) within 3 min of aldosterone administration when compared with a placebo group.7 This rapid aldosterone effect could cause compensatory adjustments in cardiac output during changes in posture.

In the rat, continuous low intracerebroventricular (i.c.v.) doses of aldosterone increase systemic blood pressure, apparently acting through MR. These doses have no effect when administered subcutaneously and are too low to alter electrolyte balance.8,9 Glucocorticoids and mineralocorticoids may interact to mediate these pressor/depressor responses;10 GR and MR have opposing actions on blood pressure and different latent periods and durations of action when given by the i.c.v. route.11 The prevention of hypertension through i.c.v. infusion of MR antagonists does not prevent the development of cardiac hypertrophy and fibrosis caused by systemic mineralocorticoid excess, so these central pressor effects of aldosterone are distinct from those on fluid and electrolyte balance, salt appetite and the trophic effects on the vasculature and the heart.

The brain

Corticosteroids also exert profound effects on brain metabolism and, consequently, on behaviour. Their lipophilic properties should ensure that access of exogenous steroid to the many MR and GR within the brain is not impeded by the blood–brain barrier. This is the case for corticosterone and progesterone, but the multidrug resistance P-glycoprotein can pump specific steroids, including cortisol and aldosterone, back across the barrier.12,13 In the mouse, P-glycoprotein was found to reduce levels of cerebellar cortisol by approximately two-thirds. This selectivity also applies in humans, where cortisol is the major glucocorticoid. The GR is especially abundant in the hippocampus, lateral septum, cerebral cortex, amygdala and the nucleus tractus solitarius. Most brain regions possess fewer MR, with high concentrations limited to the hippocampus, the septum and the granular cells of the cerebellum.14,15

There is little 11β-HSD2 in the brain after birth and its distribution does not generally coincide with regions of high MR expression.16 High 11β-HSD2 levels in the adult rat brain are restricted to the subcommissural organ (SCO), with lower amounts detected in the hypothalamic ventromedial nucleus and some discrete cells of the amygdala.17 The SCO is involved with the central control of aldosterone secretion and sodium homeostasis, whereas amygdala MR occupation has been implicated in the control of salt appetite.18 However, the majority of brain MR is likely to be occupied by glucocorticoid. The 11β-HSD1 isoform is much more widely distributed in the brain. Its reductase activity amplifies glucocorticoid action in the hippocampus19 and may modulate the neurotoxic effects of excitatory amino acids (see below), although it is uncertain whether this is of significance in vivo.20

If we assume that the adult rat brain corticosteroid receptors are, for the most part, non-selective and that most of the corticosteroid within the brain is derived from the adrenal cortex, then one would expect central corticosteroid receptor occupancy to vary with the circadian rhythm of adrenal corticosterone production. A general trend has emerged from in vitro studies of corticosteroid effects on the electrophysiological properties of CA1 hippocampal neurons. Briefly, intermediate corticosteroid levels, broadly corresponding to predominant MR occupation, result in small transmitter responses and ionic conductances that would tend to have a stabilizing effect. Higher levels of corticosteroid, which result in the occupation of both MR and GR, or adrenalectomy (ADX; where no receptors are occupied at all) evoke larger responses. This results in a U-shaped dose–response curve (see Fig. 2).21 Large responses could help return the brain to a normal state after a stressful experience. However, such suppressive responses can render the cells vulnerable to damage: increases in calcium influx, for example, will lead to calcium build-up in the cell. So, if high levels of corticosteroid are sustained, this suppression will become harmful. Studies in humans have found that raised cortisol is associated with neurodegeneration in the hippocampus,22 although investigations using aged non-human primates did not.23

image

Figure 2. Representation of ionic conductances and transmitter responses of CA1 hippocampal neurons in vitro, as a function of the mineralocorticoid receptor (MR)/glucocorticoid receptor (GR) occupancy ratio (arbitrary scale), which rises with the total corticosteroid concentration ([CORT]). Responses during predominant MR occupation are generally small and have a neuroprotective effect, whereas responses during conditions corresponding to adrenalectomy (ADX) or to simultaneous MR and GR occupation are relatively large and, over sustained periods, can lead to neurodegeneration. (Adapted from Joels and de Kloet.21)

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Stressful experiences and steroid hormone administration also produce short-term deficits in episodic and spatial memory. Corticosteroids may thereby modulate long-term potentiation (LTP), a phenomenon often linked to memory formation.24,25

At the other extreme, adrenalectomy results in degeneration of mature granule cells in the adult rat dentate gyrus.26 This can be prevented by exogenous corticosterone or aldosterone, indicating that the occupation of MR, normally occupied by the basal concentrations of corticosterone, is sufficient to protect the granule cells.

The HPA axis is regulated by corticosteroids through a negative feedback loop. Administration of small doses of receptor agonists and antagonists to the dorsal hippocampus shows that the balance of MR- and GR-mediated effects within the brain can have a critical effect on HPA axis regulation.27,28 The HPA axis of animals subjected to chronic stress can become adapted to these conditions, altering adrenal sensitivity to ACTH. Rats handled during development have more effective glucocorticoid feedback mechanisms and, in old age, have lost fewer neurons, possibly as a result of suffering less from chronically raised corticosteroid levels.29

Corticosteroid biosynthesis in the cardiovascular system

  1. Top of page
  2. Summary
  3. Introduction
  4. The adrenal corticosteroids
  5. Non-epithelial actions of corticosteroids
  6. Corticosteroid biosynthesis in the cardiovascular system
  7. Corticosteroid biosynthesis in the central nervous system
  8. Conclusions
  9. Acknowledgements
  10. References

The ability of the cardiovascular system to produce corticosteroids was first suggested in vivo when several corticosteroidogenic enzymes were found to be expressed in vascular cells. Endothelial and smooth muscle cells isolated from human pulmonary artery transcribed mRNA encoding aldosterone synthase, MR, 3β-HSD (types I and II), 21-hydroxylase and 18-hydroxylase, but not the side-chain cleavage enzyme or 11β-hydroxylase.30–32 Human vascular endothelial cells (HUVEC) derived from umbilical veins transcribed the CYP11B1 and CYP11B2 genes.34 The components of the RAS are expressed in blood vessels and in the heart as a local system, regulated at an autocrine-paracrine level.33 In HUVEC, AngII and K+ increased CYP11B2 but not CYP11B1 mRNA levels, whereas ACTH raised CYP11B1 but not CYP11B2 mRNA.34

In the rat, perfusion of mesenteric arteries isolated from the normotensive Wistar-Kyoto (WKY) rat strain yielded detectable amounts of corticosterone and aldosterone, albeit at much lower levels than those found in the adrenal cortex or plasma.35 The angiotensin-converting enzyme inhibitor quinapril significantly reduced aldosterone production, but left corticosterone production unaffected. Elevating AngII or K+ levels by perfusion significantly increased aldosterone secretion.

In vivo, AngII, ACTH and low-sodium diets all caused increased cardiac aldosterone and corticosterone levels in subsequently analysed tissues.36CYP11B1 and CYP11B2 transcripts were present in the four cardiac chambers of the adult Wistar rat heart. Cardiac CYP11B transcript levels were regulated in a manner similar to the adrenal cortex: ventricular CYP11B1 mRNA was increased by AngII or ACTH, whereas AngII or low-sodium diets upregulated CYP11B2 transcription.36 The changes in cardiac corticosteroid concentrations were not always proportional to those observed in plasma and there were also temporal differences between the two, suggesting that extra-adrenal regulatory systems, although they may be broadly similar to their adrenal counterparts, act independently of them.

Only two CYP11B genes are found in humans,37 but rat studies are complicated by the presence of CYP11B3 and CYP11B4. CYP11B4 seems only to be a pseudogene, but CYP11B3 is expressed in the adrenal zona fasciculata-reticularis for a short period after birth and is regulated by ACTH. The CYP11B3 gene product is an enzyme possessing 18- and 11-hydroxylase activities.38 Following an expression pattern similar to that seen in the adrenal cortex, CYP11B3 mRNA is present in 21-day-old, but not adult, rat hearts.36

The genes for MR, GR, P450scc, P450c21 and type II 3β-HSD are transcribed in all chambers of the normal human adult heart as well as the fetal heart and the aorta.39 Using reverse transcription–polymerase chain reaction (RT-PCR), CYP11B1 transcripts could be detected in all these samples, with the exception of the ventricles. CYP11B2 transcription was limited to the fetal heart and to the adult aorta, with no positive results gained from any of the normal adult cardiac chambers. Quantitative analysis of the transcript levels in heart samples estimated them, where present, to be 100–10 000-fold lower than those in the adrenal gland.39

The development of aldosterone-induced cardiac fibrosis is dependent on a simultaneously increased sodium intake,40 so it is unlikely that a direct action of aldosterone on heart MR is solely responsible. Furthermore, specific actions on MR by aldosterone would presumably require the presence of 11β-HSD2 in rat cardiac fibroblasts. Whereas 11β-HSD2 transcription has been identified in normal human heart tissue,39 neither the enzyme nor the MR can be detected in rat cardiac fibroblasts.41 Instead, it has been proposed that locally produced aldosterone could compensate for the absence of cardiac 11β-HSD2, creating a concentration high enough to occupy MR without the need for glucocorticoid inactivation.42 Rat myocardial aldosterone concentration has been estimated at 16 nmol/L, a level 17-fold higher than in plasma.43 Delcayre et al.43 speculate that this could be due to slower aldosterone degradation in the heart, to intracellular sequestration or to local delivery of aldosterone to extracellular spaces rather than into the bloodstream. Differential splicing of mRNA could alter the translational efficiency or the half-life of a message in particular tissues where high local steroid concentrations are required. Regardless of the mechanism, the relatively high local aldosterone concentration in the heart suggests some independent paracrine or autocrine action. However, under normal conditions it is more likely that cardiac MR are occupied by glucocorticoid. The local corticosterone concentration would be expected to be much higher than that of aldosterone and to be augmented by the reductase function of 11βHSD1 in rat cardiac fibroblasts, which converts circulating levels of 11-dehydrocorticosterone to active corticosterone.41

Cardiac aldosterone expression may only be of significance under pathological conditions, perhaps contributing to cardiac remodelling in hypertension or heart disease. Aldosterone levels are elevated in the failing human heart, exceeding circulating concentrations, whereas plasma aldosterone levels remain unaffected.44 Cardiac levels of CYP11B1 and CYP11B2 transcripts are both raised during heart failure45 and a direct correlation has been identified between myocardial CYP11B2 mRNA expression and collagen volume in the failing human heart.46 After myocardial infarction (MI), Wistar rats had increased cardiac levels of CYP11B2 transcripts and aldosterone in the non-infarcted areas of their left ventricles, although CYP11B1 mRNA and corticosterone levels actually fell in these areas. Myocardial infarction did not affect adrenal CYP11B2 expression or aldosterone secretion.47 Therefore, conditions after MI would favour the occupancy of cardiac MR by mineralocorticoid rather than glucocorticoid when compared with the normal.

Although certain attempts to block the action of angiotensin have had minimal effects on the progression of cardiac fibrosis,48 there is some evidence that postinfarction myocardial remodelling involves the activation of cardiac aldosterone production by AngII. In one study, MI resulted in increased local production of AngII, whereas plasma levels were unaffected.47 The postinfarction rise in cardiac aldosterone levels was blocked by losartan, an antagonist of the angiotensin AT1 receptor. Losartan treatment also prevented the development of myocardial fibrosis, as did treatment with the MR antagonist spironolactone. However, spironolactone was unable to prevent the postinfarction rises in aldosterone and AngII. Angiotensin II has growth-promoting properties and could be responsible for the proliferation of myofibroblasts observed in fibrosis. The density of the AT1 receptors in myofibroblasts can be increased by aldosterone, which would enhance the action of AngII after MI. Spironolactone treatment blocks the increase in AT1, suggesting that aldosterone acts through MR to achieve this effect.49

Cardiac steroid production may also play a more general role in the aetiology of hypertension. The stroke-prone spontaneously hypertensive rat (SHRSP) is a genetic model for human hypertension, although the precise cause of its hypertension is unknown. Aortic CYP11B2 mRNA was elevated in 10-week-old hypertensive rats compared with WKY rats.50 In addition, aldosterone production and CYP11B2 transcript levels are significantly higher in the mesenteric arteries of young (2-week-old) SHRSP compared with WKY rats of the same age,51 although these differences do not persist into later life. However, the transcription of the gene encoding the α1-subunit of Na+/K+-ATPase, an aldosterone-dependent activity, is significantly higher in the mesenteric arteries of adult SHRSP.

Corticosteroid biosynthesis in the central nervous system

  1. Top of page
  2. Summary
  3. Introduction
  4. The adrenal corticosteroids
  5. Non-epithelial actions of corticosteroids
  6. Corticosteroid biosynthesis in the cardiovascular system
  7. Corticosteroid biosynthesis in the central nervous system
  8. Conclusions
  9. Acknowledgements
  10. References

The term ‘neurosteroids’ was devised to describe steroids synthesized within the central nervous system from cholesterol or similar early precursors; pregnenolone and dehydroepiandrosterone synthesized from cholesterol within the rat brain were early examples.52 The discovery of the side-chain cleavage enzyme P-450scc within the white matter of the rat brain was significant in neurosteroid research.53 Immunohistochemical P450scc staining led to the belief that the enzyme was confined to glial cells.54CYP11A1 transcripts, which encode P-450scc, were detected in primary glial cell cultures55,56 and these cells can convert cholesterol to pregnenolone in vitro.57 More recently, CYP11A1 transcripts, or P-450scc itself, have been found in neurons such as the Purkinje cells of the rat cerebellum58,59 or primary cells isolated from fetal rat hippocampus.60

CYP11A1 transcripts have been detected in the rat brain by RT-PCR, always at lower levels than those found in adrenal tissue.55,56,61,62 There appear to be no significant sex differences in the distribution of brain CYP11A1 transcripts. In situ hybridization studies show transcripts are most abundant in the pyramidal cell layers of the hippocampus and dentate gyrus and in the granule and Purkinje cells of the cerebellum.59 In humans, CYP11A1 transcripts are present in the brains of children and adults. Hippocampal and frontal lobe specimens have been analysed, with levels in adults tending to be higher than those in children.63

Transcripts for StAR protein, which delivers cholesterol to P-450scc, have been detected in the adult rat central nervous system by RNase protection assay, RT-PCR and in situ hybridization.59 These transcripts are abundant in the cerebral cortex, the pyramidal cell layers of the hippocampus and dentate gyrus and the olfactory bulb. The cerebellum has the highest levels of all. It has been found that StAR mRNA levels in the brain are approximately two to three orders of magnitude lower than those in the adrenal gland and no sex differences are apparent.

Virtually all rat tissues examined, including the brain, contain adrenodoxin mRNA.61,62,64 These transcripts are more abundant than those for CYP11A1 because they can be detected by RNase protection assay and northern blotting, as well as by RT-PCR. Abundant NADPH-cytochrome P450 reductase transcripts are also in the brain and in all other rat tissues that have been analysed.61

The rat isoenzymes of 3β-HSD (types I–IV) are expressed in a tissue-specific manner. In the brain, transcripts for types I, II and IV can be detected by restriction digestion of RT-PCR products.56 Interestingly, the absent type III has a completely different activity to the other isoenzymes, possessing a 3-ketosteroid reductase activity.65In situ hybridization localized these transcripts to the cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus and olfactory bulb, although the probe did not discriminate between the various types. The highest levels of 3β-HSD expression within the hippocampus are found within the CA1–2 fields and the dentate gyrus, whereas in the cerebellum expression is strongest in the Purkinje cells, granule cells and stellate/basket cells.59,66,67 In the rat central nervous system, 3β-HSD mRNA has only been detected in neurons. However, cultured astrocytes and oligodendrocytes express 3β-HSD, suggesting that glial cells may acquire this ability in vitro.57,65

There have been fewer investigations of brain 21-hydroxylase. Failure to detect CYP21 transcripts in the rat brain by an RNase protection assay led to claims that extra-adrenal 21-hydroxylation was mediated by another enzyme.68 However, as with several other genes involved in the pathway, the sensitivity of an amplification technique such as RT-PCR is probably required to detect transcripts. Indeed, such an approach has since detected 21-hydroxylase transcripts in the brains of mice61 and humans.69

The similarity of 11β-hydroxylase and aldosterone synthase created practical difficulties in early studies and it was understandably assumed that aldosterone and cortisol/corticosterone were produced by the same enzyme. An immunohistochemical study of the rat brain using a polyclonal antibody will, in retrospect, have detected both enzymes, particularly because the antibody bound all three adrenocortical zones.70 With the subsequent cloning of the CYP11B1 and CYP11B2 genes, several groups detected CYP11B1 mRNA in rat brain tissue homogenates using RT-PCR,55,61,62,71 RNase protection assays55 and northern blotting.72 Transcripts can be detected in whole brain samples, as well as cerebellum, cerebral cortex, amygdala, hippocampus and hypothalamus homogenates. According to one RT-PCR study, female rats have consistently higher CYP11B1 transcript levels in the hippocampus than males.55 Brain minces are able to convert DOC to corticosterone, proving that 11β-hydroxylase activity is present in the rat brain.71CYP11B1 mRNA cannot be detected in primary cultures of glial cells55 and immunohistochemical studies with a specific monoclonal antibody localized the 11β-hydroxylase enzyme to the cerebellar Purkinje neuron.62 Neurons cultured from another area of strong 11β-hydroxylase immunostaining, namely the hippocampus, were able to 11β-hydroxylate the steroid precursor DOC.73 However, glial expression in these regions cannot be ruled out.

Aldosterone synthase expression in the brain has also been investigated at the transcript level. One group failed to find CYP11B2 transcripts in brain tissue by RNase protection assay, nor could the transcripts be detected in primary cultures of glial cells.55 However, others have since used RT-PCR to detect CYP11B2 mRNA in whole brain and in cortex, cerebellum, brain stem, hippocampus, hypothalamus and amygdala homogenates.61,74 Brain minces converted DOC to aldosterone, corticosterone, 18-hydroxy-deoxycorticosterone and, in the greatest amounts, to 11-dehydrocorticosterone.

Immunohistochemistry showed aldosterone synthase to localize to precisely the same areas of the brain as 11β-hydroxylase. This cannot be attributed to cross-reaction by the respective antibodies because each of these localized to the correct adrenocortical regions.62 Indeed, 11β-hydroxylase and aldosterone synthase shared an extremely similar distribution pattern to those of CYP11A1, 3β-HSD and StAR, as shown by in situ hybridization,59 and that of MR.15 This is highly suggestive of an autocrine or paracrine model of corticosteroid action in the brain.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. The adrenal corticosteroids
  5. Non-epithelial actions of corticosteroids
  6. Corticosteroid biosynthesis in the cardiovascular system
  7. Corticosteroid biosynthesis in the central nervous system
  8. Conclusions
  9. Acknowledgements
  10. References

So much evidence has accumulated to support the local production of corticosteroids in the heart and in the brain that it must now be regarded as a real phenomenon. What is harder to establish is whether the levels of local steroid are capable of real physiological effects and, if so, what these effects may be. For example, although the importance of aldosterone levels in hypertension and cardiac disease is clear,6 there is no direct evidence that local production (as opposed to adrenal production) is a significant factor.

Moreover, because systemic corticosteroids are lipophilic and, therefore, have unimpeded access to all cells, to be of importance local synthesis must result in significantly higher local concentrations of steroid and be controlled locally, and maybe differently, from adrenal synthesis. Whereas some progress has been made in identifying regulatory factors with influence over local cardiovascular production, much more investigation is required in this area and there are no published data on the regulation of brain corticosteroid production. These gaps in our knowledge must be addressed.

The extra-adrenal corticosteroid production systems appear to differ from the adrenal system in two important ways. First, the predicted low levels of local steroid production and the proximity of these sites of production to the corticosteroid receptors strongly suggest a paracrine or autocrine mode of action. Second, the fact that some cells within these local systems are capable of coexpressing 11β-hydroxylase and aldosterone synthase raises new questions about corticosteroid synthesis, which, due to the strict zonation of adrenocortical production, have never had to be addressed previously. For example, would the two coexpressed enzymes compete for substrate? Wouldn't such coexpression ultimately lead to lower levels of steroid product than zonation?

It is by no means certain that the small amounts of product that could be produced in the central nervous system are capable of any substantial physiological effect, particularly when one realizes that the minuscule local production must act against the background of those systemic corticosteroids that manage to cross the blood–brain barrier. However, such effects may be possible when one takes into account the relative dilution of steroids from different sources. For example, adrenocortical CYP11B2 expression has been estimated to occur at more than 1000-fold the levels found in the hippocampus (E Davies and SM MacKenzie, unpubl. obs., 2001), but any resulting adrenal steroid must be diluted through a much greater volume of tissue to reach its endocrine targets than locally produced steroids acting in a paracrine or autocrine mode. In addition, recent work on the human brain suggests that 11β-HSD1 is highly expressed in the cerebellar granule layer and in hippocampal neurons.75 This correlates well with the areas of corticosteroid expression identified in the rat brain and implies that levels of locally produced corticosteroid could be supplemented by the activation of cortisone by 11β-HSD1 in these same regions.

At present, such suggestions remain purely theoretical, but one piece of evidence shows that local corticosteroid production exerts a real physiological effect. 19-Ethynyldeoxycorticosterone is an irreversible inactivator of aldosterone synthase76 that, when administered to the Dahl SS/jr rat model of hypertension, causes a reduction in salt-induced blood pressure. Administration of 5 ng/h 19-ethynyldeoxycorticosterone via an i.c.v. route is sufficient to significantly reduce systemic blood pressure in the Dahl SS/jr rat but, when given subcutaneously, this dose has no effect.71 The SCO, one of the few adult brain regions to demonstrate MR selectivity due to its expression of 11β-HSD2, has been implicated in this central hypertensive effect. This evidence also suggests that aldosterone in the central nervous system controls blood pressure in an entirely different way to the more familiar systemic mechanism. There may be other, as yet undiscovered, instances of local steroids using different mechanisms to exert the same, or even entirely different, effects as their adrenally derived counterparts.

It remains to be seen whether regulation of local corticosteroid production differs significantly from that in the adrenal cortex. The possibility remains that the transcription of corticosteroidogenic genes relies on different tissue-specific factors in the brain than in the adrenal gland or that post-transcriptional differences may apply, perhaps influencing transcript half-life. Even if local and adrenal transcriptional regulation of the CYP11B1 and CYP11B2 genes is found to be identical, differences could apply further up the regulatory cascade, in differential regulation of local RAS, for example.

Our own interests lie in the effects of corticosteroids on blood pressure homeostasis and, specifically, how mutations at the CYP11B1 and B2 loci may contribute to hypertension. We have identified polymorphisms that are significantly associated with essential hypertension through their effects on corticosteroid metabolism.77 At the local level, where expression is so much lower and control of synthesis presumably much finer, could such polymorphisms be of even greater significance?

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. The adrenal corticosteroids
  5. Non-epithelial actions of corticosteroids
  6. Corticosteroid biosynthesis in the cardiovascular system
  7. Corticosteroid biosynthesis in the central nervous system
  8. Conclusions
  9. Acknowledgements
  10. References

The authors thank Professor Robert Fraser (Division of Cardiovascular and Medical Sciences, Western Infirmary) for his invaluable contributions and suggestions during the preparation of this review. ED is funded by Medical Research Council Programme Grant G9317119. SMM is funded by Wellcome Trust Project Grant 060362.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. The adrenal corticosteroids
  5. Non-epithelial actions of corticosteroids
  6. Corticosteroid biosynthesis in the cardiovascular system
  7. Corticosteroid biosynthesis in the central nervous system
  8. Conclusions
  9. Acknowledgements
  10. References
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