The mineralocorticoid, aldosterone, and the glucocorticoids, cortisol and corticosterone, are produced uniquely in the adrenal cortex. These steroids act by binding to intracellular receptors which then act to modulate gene transcription in target tissues. The (patho)physiology of corticosteroid action has recently been illuminated by genetic analysis of congenitaldeficiency syndromes and by producing 'knockout' mice with gene deficiencies for glucocorticoid or mineralocorticoid receptors.
The mineralocorticoid steroid, aldosterone, is produced in the (outer) zona glomerulosa of the adrenal cortex by a series of enzymatic modifications of cholesterol, including the unique oxidation of the methyl group at carbon 18 to the aldehyde (CH3 to CHO). Aldosterone is secreted in response to elevated concentrations of angiotensin II or plasma potassium, in turn reflecting relative or absolute sodium deficiency. Normal secretion rates and circulating concentrations of aldosterone are much lower than those of glucocorticoids.
The physiological glucocorticoid in most species is cortisol (hydrocortisone), and is corticosterone in rats and mice. Glucocorticoids are produced in the mid-zone of the adrenal cortex, the zona fasciculata, in response to adrenocorticotrophic hormone (ACTH) from the anterior pituitary. ACTH secretion is stimulated by a variety of physical or psychological stressors (hypoglycaemia, fear, pain, heavy exercise, infection); there is also a circadian rise and fall in basal ACTH and cortisol concentrations.
Cortisol and aldosterone differ not only in their secretion rates and plasma concentrations, but also in the extent to which they are protein-bound in plasma. Approximately half of circulating aldosterone is bound with low affinity to albumin, and half is free. For cortisol, although both relative and absolute values vary with steroid concentration, only about 4% is free. The majority of the remainder is bound with high affinity to transcortin (corticosteroid binding globulin, CBG). In contrast with the physiological glucocorticoids, synthetic glucocorticoids (prednisone, prednisolone, dexamethasone, fludrocortisone) have relatively low affinity for CBG.
In tissues with a rapid circulatory transit time, the intracellular steroid concentrations available to activate receptors reflect free concentrations in plasma. In other tissues, most notably the liver and spleen, the transit time is sufficient to allow the dissociation of steroid from albumin, so that steroid concentrations available for receptor binding or metabolism reflect free plus albumin bound. Basal levels of cortisol are approximately 80% CBG-bound, with the free and albumin -bound proportions rising relatively slowly until peak cortisol secretion rates are achieved.
In contrast with albumin binding, CBG binding may have a number of physiological roles. The first is to act as a reservoir of steroid, in that CBG-bound glucocorticoid is not metabolised. Secondly, the binding of steroid by CBG is very temperature -dependent, so that the higher the temperature the lower the affinity; free steroid levels in skin capillaries (approximately 28oC) are thus one half to one third of those in viscera (37oC), and high levels are also found at sites of inflammation. Third, at such sites neutrophil elastase can specifically cleave CBG, releasing bound steroid to modulate the inflammatory response. Whether this represents the totality of the physiology of CBG is unknown; there are no known cases of its congenital absence, suggesting as yet unrecognised possible developmental roles.
Corticosteroid receptors (Fig. 1)
In contrast with receptors for neurotransmitters, cytokines and peptide/protein hormones, classical steroid receptors are intracellular rather than on the cell membrane. Receptors for mineralocorticoids (MR) and glucocorticoids (GR), together with those for androgens (AR) and progestins (PR), form a subfamily within the steroid/thyroid/retinoid/orphan receptor super family. MR and GR, in the absence of hormone, are primarily in the cell cytoplasm, invested by a series of associated proteins including members of the heat shock protein and immunophilin families. These proteins maintain the receptor in a form with high affinity for steroid, and prevent the receptor from interacting with DNA in the absence of hormone. On binding the steroid, the receptor sheds its associated proteins, translocates to the nucleus, and binds as a dimer (commonly MR:MR or GR:GR, but with increasing evidence for MR:GR) to particular nucleotide sequences on target genes, known as response (or regulatory) elements. The receptors then initiate (or on occasion, repress) the transcription of mRNA encoding the proteins which are corticosteroid -responsive in the particular target tissue.
Model of aldosterone action in a physiological mineralocorticoid target cell. Aldosterone (A) enters the cell and binds to mineralocorticoid receptors (MR). The aldosterone-MR complex then moves into the nucleus and binds to sequences on DNA (hormone response elements: HRE), which in turn alter the transcription of messenger RNA and protein synthesis, ultimately leading to sodium retention.
In such tissues, cortisol (F) is normally excluded, by metabolism to receptor-inactive cortisone by the enzyme 11b hydroxysteroid dehydrogenase. If the enzyme is blocked or deficient, cortisol can bind as well as aldosterone to MR and, in addition, to glucocorticoid receptors (GR), and under both circumstances can activate HRE like aldosterone.
The best studied response element, often termed the glucocorticoid response element or the hormone response element, is the 15-nucleotide GGTACAnnnTGTTCT 'consensus' sequence. The first ambiguity is that such glucocorticoid response elements appear more or less equally responsive to activated MR, GR, PR and AR, posing the question of how cells which contain multiple receptors discriminate between signals. This is particularly the case for glucocorticoids, in that GR appear to be expressed in all nucleated cells in the body.
One way in which some specificity between MR and GR responses is achieved is by their differential action at sites other than 15-nucleotide response elements. Specifically, longer 'composite response elements' have been identified on DNA that bind not only MR or GR, but other proteins which influence gene transcription e.g. fos/jun heterodimers of the early immediate response gene series. At such composite response elements - or indeed, in the cytoplasm, as a result of direct protein-protein interactions - GR, but not MR, can block the effect of the fos/jun transcription factor. This builds some specificity into the system at the level of the nuclear response.1
This is particularly an issue in that the receptors known as MR are physiologically mineralocorticoid receptors in epithelial tissues such as kidney and colon, where aldosterone acts to increase transepithelial sodium transport. However, in other tissues MR are occupied and activated by physiological glucocorticoids. In addition, it has now been clear for more than a decade that MR have identical, and very high, affinity for aldosterone and cortisol, a counterintuitive finding given the clear physiological actions of aldosterone despite its very low plasma concentration.2 This ambiguity provokes two questions. The first is how cortisol is excluded from epithelial MR, allowing aldosterone to occupy the non-discriminating MR; the second is the physiological roles of non-epithelial MR, for which cortisol has approximately 10 times higher affinity than for GR, which we commonly think of as 'its' receptor.
11b hydroxysteroid dehydrogenase
Just as the 'signature' of aldosterone is the unique aldehyde group at C18, the signature of a glucocorticoid is a hydroxyl group at C11. Although on occasion we give patients cortisone acetate, the cortisone (with a keto group at C11) has to be reduced to hydrocortisone (cortisol) in the liver before it can bind to GR and act as a glucocorticoid. This hepatic conversion reflects the activity of an enzyme called 11b hydroxysteroid dehydrogenase Type 1, which, although bidirectional, acts predominantly in most tissues as a reductase converting cortisone to cortisol.
In epithelial tissues in which aldosterone acts as a physiological mineralocorticoid - renal collecting tubules, colon, salivary glands, sweat glands - a second enzyme, 11b hydroxysteroid dehydrogenase Type 2 (11-HSD2), converts cortisol to receptor-inactive cortisone. The enzyme is essentially unidirectional, has a very low Km (i.e. is very efficient) and its cloning in 19943 has allowed the demonstration of its crucial role in MR selectivity in vivo, and thus in salt and blood pressure homeostasis.
The syndrome of apparent mineralocorticoid excess, described almost two decades ago, is characterised by severe juvenile hypertension, salt sensitivity and very high ratios of urinary cortisol to cortisone metabolites. In 1995, the first report of a point mutation in the gene coding for 11-HSD2 was published in a family with 3 members affected by the syndrome. Since then, approximately 15 different mutations/deletions have been found in individuals and families with the syndrome.4 This 'experiment of nature' confirms the crucial role of 11 -HSD2 in excluding glucocorticoids from occupying (and activating) epithelial MR, thus conferring aldosterone specificity on the inherently non-discriminating MR. While clearly 11-HSD2 is necessary for in vivo epithelial MR selectivity, it may or may not act alone. On kinetic and other grounds, it would come as no surprise if the conversion of cortisol to cortisone were only one of a number of mechanisms which are necessary for the physiological integrity of the mineralocorticoid response.
These MR are presumably high affinity glucocorticoid receptors, although their physiological roles are as yet quite unclear. They are abundant in the hippocampus, and in more modest amounts in other tissues (e.g. heart, liver). In animal studies, 'inappropriate' occupancy of such unprotected MR by aldosterone produces hypertension (after intracerebroventricular infusion of aldosterone) and cardiac fibrosis (after peripheral infusion of aldosterone to salt-loaded rats). The extent to which these animal studies can be extrapolated to the clinical situation, and the true physiological roles of such receptors, in rat and man, remain to be explored.
Receptor deficiency syndromes
Pseudohypoaldosteronism (PHA) was first described5 almost 40 years ago at the Royal Children's Hospital in Melbourne as a syndrome of hyponatraemia, hyperkalaemia, hyperreninaemia and markedly elevated plasma and/or urinary aldosterone levels. Since the patient was insensitive to administered mineralocorticoid (deoxycorticosterone acetate), this suggested that the syndrome may represent a defect in the normal aldosterone effect or mechanisms. A decade ago, monocytes from patients with PHA were shown not to bind tritiated aldosterone, in contrast with control monocytes, suggesting that the syndrome reflected mutation(s) in the MR rendering them unable to bind and respond to aldosterone.
Very recently, two lines of evidence have suggested that this may not be the case. First, the nucleotide sequence coding for MR has been obtained in unrelated patients in Australia, France and the U.S.A. and has been shown to be normal. Secondly, in a study of a dozen families with the syndrome, no linkage was found with a series of markers on chromosome 4 near the site of the gene coding for human MR. This constitutes indirect but strong evidence for some other explanation for the lack of steroid binding, and the clinical defect in aldosterone action. In March 1996, the autosomal recessive form of PHA was shown to reflect defects in the a or b subunits of the amiloride-sensitive epithelial sodium channel.6
A series of studies has detailed receptor mutations, commonly producing lower affinity receptors. These patients have increased levels of ACTH, producing higher cortisol levels to compensate for the lower receptor affinity and, as an adverse effect, higher production rates of adrenal androgens. Although the elevated cortisol levels may be 'appropriate' for the lower affinity GR, they appear to spill over into MR, resulting in salt retention and raised blood pressure. Recently, an incidence of allelic variation of GR has been reported of the order of 9%, by correlating the extent of suppression with the GR sequence in over 200 normal people given a dexamethasone suppression test.
Gene deficiencies leading to absence of receptors (receptor knockouts)
The successful 'knocking out' (KO) of the gene coding for GR in embryonic stem cells, and the breeding of heterozygous GR +/- and homozygous GR -/- mutant mice, was reported last year.7 Most -/- GRKO mice go blue and die from pulmonary atelectasis within hours of birth, although a small proportion (5-10%) of them survive, and when mated, produce uniquely GR -/- mice. At birth, the -/- GRKO mice have essentially no adrenal medullae, and the enzyme which converts noradrenaline to adrenaline is absent. They also have very low or absent levels of glucocorticoid-induced enzymes in liver and elsewhere, despite very high concentrations of ACTH and corticosterone (the physiological glucocorticoid in mice). Heterozygous GRKO +/- mice have no increased incidence of postpartum mortality or obviously distinctive phenotype; they do, however, have concentrations of ACTH and corticosterone elevated to levels between those of control and GRKO -/- mice. This is evidence for the importance of having normal cellular concentrations of GR for homeostasis.
The same laboratory has now succeeded in producing MRKO mice. MRKO -/- mice start to lose weight approximately 4-6 days postpartum, and without sodium supplementation, die some days later. Not surprisingly, renin and aldosterone levels are elevated, even at birth. Other studies, probing epithelial and non-epithelial roles for MR, are currently under way.
In terms of mineralocorticoid action, there are at least 4 outstanding remaining challenges:
- establishing the aldosterone-induced proteins responsible for transepithelial sodium transport
- documenting the central nervous system pathways involved in mineralocorticoid hypertension
- exploring how salt-loading is necessary for the cardiovascular effects of aldosterone
- weighing the importance of the recently documented rapid non-genomic effects of aldosterone
For glucocorticoids, the remaining questions are much less focused, understandably given the protean roles of these steroids in development, differentiation and homeostasis. Two broad areas that might be addressed are the relative roles of glucocorticoids acting via GR in modulating versus mediating the stress response, and secondly, the physiological implications of an always-occupied receptor, which would appear to be the case for MR in non-epithelial, non-11-HSD2 protected tissues.
The following statements are either true or false.
1. Falling plasma concentrations of sodium stimulate the secretion of aldosterone from the adrenal medulla.
2. Mineralocorticoid and glucocorticoid receptors are found inside cells rather than on the cell membrane.
Answers to self-test questions
- Pearce D, Yamamoto KR. Mineralocorticoid and glucocorticoid receptor activities distinguished by nonreceptor factors at a composite response element. Science 1993;259:1161-5.
- Krozowski ZS, Funder JW. Renal mineralocorticoid receptors and hippocampal corticosterone-binding species have identical intrinsic steroid specificity. Proc Natl Acad Sci USA 1983;80:6056-60.
- Albiston AL, Obeyesekere VR, Smith RE, Krozowski ZS. Cloning and tissue distribution of the human 11b hydroxysteroid dehydrogenase type 2 enzyme. Mol Cell Endocrinol 1994;105:R11-R17.
- Funder JW. Apparent mineralocorticoid excess, 11 b hydroxysteroid dehydrogenase and aldosterone action: closing one loop, opening another. Trends Endocrinol Metab 1995;6:248-51.
- Cheek DB, Perry JW. A salt-wasting syndrome in infancy. Arch Dis Child 1958;33:252-6.
- Chang SS, Grunder S, Hanukoglu A, Rosler A, Mathew PM, Hanukoglu I, et al. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nature Genetics 1996;12:248-53.
- Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, Aguzzi A, et al. Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev 1995;9:1608-21.