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Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Turin, 10126 Turin, Italy
Address all correspondence and requests for reprints to: Ezio Ghigo, M.D., Divisione di Endocrinologia, Ospedale Molinette, C.so Dogliotti 14, 10126 Torino, Italy. E-mail: ezio.ghigo{at}unito.it
Abstract
Animal studies indicate that mineralocorticoid receptors (MR) in the hippocampus play a major role in the glucocorticoid feedback control of the hypothalamo-pituitary-adrenal (HPA) axis. Specifically, MR mediate the proactive feedback of glucocorticoids in the maintenance of basal HPA activity. The stimulatory effect of intracerebroventricular and intrahippocampal MR blockade on the HPA axis in animals has been clearly shown, whereas the effect of systemic administration of mineralocorticoid antagonists in humans is still contradictory. To clarify this point, in seven normal young women (aged 25–32 yr; body mass index, 19.0–23.0 kg/m2) we studied the effects of canrenoate (CAN; 200 mg as iv bolus at 2000 h, followed by 200 mg infused in 500 mL saline over 4 h up to 2400 h) or placebo (saline, 1.0 mL as iv bolus at 2000 h, followed by 500 mL over 4 h up to 2400 h) on the spontaneous ACTH, cortisol, dehydroepiandrosterone (DHEA) and aldosterone secretion as well as on the ACTH, cortisol, and DHEA responses to human CRH (2.0 µg/kg as iv bolus at 2200 h) or arginine vasopressin (AVP; 0.17 U/kg as im bolus at 2200 h). Blood samples were taken every 15 min from 2000–2400 h. During placebo, spontaneous ACTH and cortisol levels showed progressive decreases (P < 0.05) from 2000–2400 h (baseline vs. nadir, mean ± SEM, 2.0 ± 0.3 vs. 1.4 ± 0.2 pmol/L and 115.1 ± 23.7 vs. 63.5 ± 24.3 nmol/L), whereas DHEA and aldosterone levels did not change. CRH induced clear increases in ACTH, cortisol, and DHEA levels (peaks, mean ± SEM, 7.1 ± 1.1 vs. 1.6 ± 0.2 pmol/L, 322.9 ± 19.5 vs. 92.8 ± 24.5 nmol/L, and 44.2 ± 2.7 vs. 20.0 ± 3.0 nmol/L; P < 0.05). Similarly, AVP elicited significant increases in ACTH, cortisol, and DHEA levels (3.8 ± 0.3 vs. 1.5 ± 0.1 pmol/L, 211.9 ± 27.2 vs. 67.7 ± 9.7 nmol/L, and 51.6 ± 4.0 vs. 16.3 ± 2.0 nmol/L; P < 0.05). During CAN treatment, ACTH, cortisol, and DHEA levels showed progressive rises, which begun at approximately 60 min and peaked between 2300 and 2400 h (ACTH, 3.4 ± 0.4 vs. 1.1 ± 0.3 pmol/L; cortisol, 314.5 ± 49.6 vs. 123.3 ± 13.2 nmol/L; DHEA, 52.0 ± 8.8 vs. 21.0 ± 2.3 nmol/L; P < 0.05 vs. baseline as well as vs. the same time points during placebo). Aldosterone secretion was not modified by CAN. The ACTH, cortisol, and DHEA responses to human CRH were enhanced by CAN (10.0 ± 1.7 pmol/L, 462.2 ± 36.9 nmol/L, and 66.3 ± 8.8 nmol/L), although statistical significance (P < 0.05) was obtained for cortisol and DHEA only. Also the ACTH, cortisol and DHEA responses to AVP were amplified by CAN (8.0 ± 2.6 pmol/L, 324.0 ± 34.8 nmol/L, and 77.8 ± 4.0 nmol/L); again, statistical significance (P < 0.05) was obtained for cortisol and DHEA only. In conclusion, our study shows that the blockade of MR by CAN significantly enhances the activity of the HPA axis in humans, indicating a physiological role for MR in its control. These results also suggest that the stimulatory effect of CAN on HPA axis is mediated by concomitant modulation of CRH and AVP release.
THE ACTIVITY OF the hypothalamus-pituitary-adrenal (HPA) axis is mainly regulated by the hypothalamic neurohormones CRH and arginine vasopressin (AVP), which are, in turn, under the influence of several neurotransmitters and neuropeptides (1, 2). On the other hand, the glucocorticoid-mediated feedback mechanism, which takes place mainly at pituitary, hypothalamic, and hippocampal levels, is the most important influence integrating the circadian activity of HPA axis activity and adapting the HPA response to stress (3, 4).
Brain glucocorticoid actions are mediated by two receptor types: type 1, or mineralocorticoid receptor (MR), and type 2, or glucocorticoid receptor (GR), both of which have also been cloned in humans (3, 4, 5). Animal studies showed that GRs are distributed everywhere in the brain, but mostly in hypothalamic neurons and corticotroph cells in the pituitary (3). On the contrary, MRs are mainly located in the limbic structures, in particular in the hippocampus, although they are also present in the hypothalamus (3) At the hippocampal level, MRs have lost aldosterone selectivity and bind glucocorticoids 10-fold more than GRs; thus, differently from GRs, MRs are mostly occupied at low glucocorticoid concentrations (3, 4, 5).
Animal data indicate that hippocampal MR activity plays a major role in the glucocorticoid-mediated feedback control of the HPA axis. Specifically, MRs are hypothesized to mainly mediate the proactive feedback of glucocorticoids in the maintenance of basal HPA activity (3), through tonic inhibitory projections to the paraventricular nucleus (6, 7, 8, 9). On the other hand, the progressive occupancy of GRs following the increase of glucocorticoid concentrations seems to mediate, in a coordinated manner with MRs, the reactive feedback that is aimed at controlling the HPA response to stress, in particular at terminating the stress-induced HPA hyperactivation (3).
Although the stimulatory effect of icv and intrahippocampal MR blockade on the HPA axis in animals has clearly been shown, the effects of systemic administration of mineralocorticoid antagonists in humans are still contradictory. In fact, a positive effect of spironolactone on the spontaneous activity of the HPA axis has been shown by some researchers (10, 11), but only a slight and time-restricted stimulatory effect of spironolactone or canrenoate (CAN) on circadian cortisol secretion, without any influence on ACTH release or on ACTH and cortisol responses to human CRH (hCRH) has been reported by others (12, 13).
Based on these premises, to clarify the effect, if any, of mineralocorticoid blockade on HPA axis in humans, we studied the effect of CAN, a well known mineralocorticoid antagonist, although its activity is not fully specific, on the spontaneous ACTH, cortisol, dehydroepiandrosterone (DHEA), and aldosterone secretion as well as on the ACTH, cortisol, and DHEA responses to hCRH or AVP during late evening. In fact, a progressive decrease in HPA activity in late evening and early night hours is well known (2), and there is evidence that during the nadir of the circadian rhythm of ACTH and cortisol secretion MRs are extensively occupied, suggesting a major role of MRs during trough periods (3). Thus, our working hypothesis was that HPA activity during the evening could be the best point to definitely clarify the stimulatory effect, if any, of mineralocorticoid antagonism by CAN.
Subjects and Methods
Drugs
Vials containing 200 mg potassium CAN were purchased from Knoll Pharmaceutical Co. (Milan, Italy). Vials containing 100 µg lyophilized hCRH were purchased from Ferring Pharmaceuticals Ltd. (Copenhagen, Denmark). Vials containing 10 IU AVP were purchased from Parke-Davis (New York, NY).
Study protocol
Seven normal young women (aged 25–32 yr; body mass index, 19.0–23.0 kg/m2) were studied in the early follicular phase.
The study was approved by the ethical committee of the University of Turin, and informed consent to participate in it was obtained from all subjects.
All subjects received the following treatments in random order and at least 3 days apart: 1) placebo plus placebo (saline, 1.0 mL as an iv bolus at 2000 h, followed by 500 mL infused over 4 h up to 2400 h and 1.0 mL saline as an iv bolus at 2200 h), 2) CAN (200 mg as an iv bolus at 2000 h, followed by 200 mg infused in 500 mL saline over 4 h up to 2400 h) plus placebo, 3) placebo plus hCRH (2.0 µg/kg as an iv bolus at 2200 h), 4) placebo plus AVP (0.17 U/kg as an im bolus at 2200 h), 5) CAN plus hCRH, and 6) CAN plus AVP. The tests started at 2000 h after at least 6 h of fasting and 30 min of venous cannulation, kept patent by slow infusion of isotonic saline. Blood samples were taken every 15 min from 2000–2400 h. At each time point in each testing session ACTH, cortisol, DHEA, and aldosterone levels were assayed. Samples from individual subjects were analyzed together.
Plasma ACTH levels (pmol/L) were measured in duplicate by immunoradiometric assay (Allegro HS-ACTH, Nichols Institute Diagnostics, San Juan Capistrano, CA) The sensitivity of the assay was 0.2 pmol/L. The inter- and intraassay coefficients of variation ranged from 6.9–8.9% and from 1.1–3.0%, respectively.
Serum cortisol levels (nmol/L) were measured in duplicate by RIA (CORT-CTK 125 immunoradiometric assay, Sorin, Saluggia, Italy). The sensitivity of the assay was 11.0 nmol/L. The inter- and intraassay coefficients of variation ranged from 6.6–7.5% and from 3.8–6.6%, respectively.
Serum DHEA (nmol/L) was measured in duplicate by RIA (DSL-9000 Active DHEA, Diagostics Systems Laboratories, Inc., Webster, TX). The sensitivity of the assay was 0.068 nmol/L. The inter- and intraassay coefficients of variation ranged from 5.6–10.6% and from 7.0–10.2%, respectively.
Serum aldosterone (pmol/L) was measured in duplicate by RIA (ALDO-MAIA, Biochem Diagnostics, Guidonia, Italy). The sensitivity of the assay was 16.2 pmol/L. The inter- and intraassay coefficients of variation ranged from 11.96–14.06% and from 4.21–9.57%, respectively.
The hormone levels (mean ± SEM) are expressed as absolute values.
Statistical analysis was carried out using ANOVA (Friedman test) and analysis of covariance (multiple ANOVA test).
Results
Basal hormonal levels at 2000 h in different testing sessions
were not significantly different. During placebo administration,
spontaneous ACTH and cortisol secretion showed progressive decreases
from 2000–2400 h (baseline vs. nadir, mean ±
SEM, 2.0 ± 0.3 vs. 1.4 ±
0.2 pmol/L and 115.1 ± 23.7 vs. 63.5 ± 24.3
nmol/L, respectively; P < 0.05), whereas
DHEA and aldosterone levels did not show any significant
variation (24.4 ± 4.0 vs. 22.1 ± 2.7 nmol/L and
254.8 ± 21.8 vs. 220.8 ± 44.8 nmol/L; Figs. 1
and 4).
|
|
and 4
).
|
and 4).
|
and 4).
Aldosterone levels were not modified by CAN administration (279.1
± 11.8 vs. 362.4 vs. 43.1 pmol/L). They were
similar to those recorded during placebo administration (Figs. 1 and 4).
CAN administration also enhanced hCRH-induced ACTH, cortisol, and
DHEA responses (10.0 ± 1.7 pmol/L, 462.2 ±
36.9 nmol/L, and 66.3 ± 8.8 nmol/L, respectively), although
statistical significance (P < 0.05) was obtained for
cortisol and DHEA only (Figs. 2 and 4).
ACTH, cortisol, and DHEA responses to AVP were
enhanced by CAN (8.0 ± 2.6 pmol/L, 324.0 ± 34.8 nmol/L, and
77.8 ± 4.0 nmol/L), although statistical significance
(P < 0.05) was obtained for cortisol and
DHEA only (Figs. 3 and 4).
Side effects
hCRH induced transient facial flushing, whereas AVP induced nausea and vasoconstriction in all subjects; two subjects also reported contraction of diuresis after AVP administration.
CAN administration did not induce significant side effects, but slightly increased the hCRH- or AVP-induced side effects. Stopping the testing or the medication was not required.
Discussion
The results of our present study show that mineralocorticoid antagonism by CAN markedly enhances the activity of HPA axis in the late evening in humans. In fact, CAN elicited an increase in spontaneous ACTH, cortisol, and DHEA secretion and enhanced the adrenal response to both hCRH and AVP. On the contrary, aldosterone secretion was not modified by CAN administration.
MRs have been widely demonstrated to play a major role at the hippocampal level in mediation of the glucocorticoid feedback control of the HPA axis (3, 4, 5). Specifically, MR are hypothesized to mainly mediate the proactive feedback of glucocorticoids in the control of basal HPA activity (3). The stimulatory effect of intracerebroventricular and intrahippocampal MR blockade on the HPA axis in rats has been clearly shown. There is evidence that intraventricular administration of MR antagonists enhances basal trough levels of plasma corticosterone, whereas exposure of rats treated with antimineralocorticoids to another stimulus potentiates the adrenocortical response (14, 15). The same stimulatory effect was recorded with low mineralocorticoid antagonist doses injected into the hippocampus (16), whereas corticosterone, but not dexamethasone, implanted in the same area was able to suppress the adrenalectomy-induced increase in ACTH levels (17). It is noteworthy that intrahippocampal antiglucocorticoids suppress ACTH levels under conditions in which antimineralocorticoids enhance ACTH release (18, 19), suggesting some opposite actions of MRs and GRs in the hippocampus in the balance of the activity of the HPA axis.
Previous studies about the effects of systemic administration of mineralocorticoid antagonists in humans showed contradictory results. In fact, a stimulatory effect of spironolactone and CAN on the HPA axis has been recorded by some researchers (10, 11), whereas others did not find any significant effect of mineralocorticoid antagonism on basal or stimulated HPA axis (12, 13). These conflicting results may be due to the different study protocols, using different timing, doses, and/or routes of administration.
We decided to verify the effects of CAN at the lowest point of corticotroph and adrenal secretion, i.e. late evening, to better understand the activity of MRs during the nadir of the activity of HPA axis. Indeed, cortisol has a higher affinity for hippocampal MR than GR (3). Moreover, GRs and MRs are differently occupied during the peak and the nadir of the circadian rhythm of HPA axis. MRs are extensively occupied under trough periods when GRs are only slightly occupied; this evidence suggested the major role of MRs in the tonic inhibitory effect of glucocorticoid during nadir (3). During circadian peaks, a higher amount of cortisol progressively saturates GRs, suggesting that the suppression of HPA activity at peak levels mainly occurs through GR in a coordinated manner with MR (3).
Our present findings showing that CAN administration markedly increases late evening cortisol and DHEA levels as well as ACTH secretion, although to a lesser extent, clearly demonstrate that brain MRs are deeply involved in the control of the basal activity of the HPA axis. In fact, ACTH, cortisol, and DHEA levels at 2400 h after CAN treatment were clearly higher than those at 2000 h and rose 200% above the nadir after placebo treatment. Moreover, cortisol levels at 2400 h after CAN administration were far higher than 50 nmol/L, which is the limit below which Cushing’s syndrome is ruled out (20).
The clear influence of MRs in the control of the HPA axis is also
demonstrated by showing for the first time that mineralocorticoid
blockade by CAN enhances the ACTH and adrenal responses to either hCRH
or AVP. The stimulatory effect of mineralocorticoid antagonism on the
stimulated activity of the HPA axis has implications concerning the
mechanisms downstream of MRs. There is evidence that modulation of
hypothalamic CRH activity plays a major role in the
hippocampal-mediated glucocorticoid feedback (3, 4), which
could involve neurotransmitter mechanisms including 
-aminobutyric
acidergic pathways (21). Our data suggest that the
mechanisms underlying the stimulatory effect of mineralocorticoid
antagonism on the CRH-induced corticotroph response are likely to at
least partially include actions other than those mediated by CRH. In
fact, there is evidence indicating that the activity of AVP-secreting
neurons or other neurotransmitter pathways is also involved in the
negative glucocorticoid feedback (2, 3, 4). Our findings
showing that CAN enhances the ACTH and adrenal responses to AVP
indicates that the effects of mineralocorticoid antagonism involve
mechanisms partially different even from modulation of AVP. As CRH and
AVP truly synergize to stimulate ACTH and cortisol secretion in humans
(1, 22), it could be hypothesized that the stimulatory
effect of CAN on the HPA axis is mediated by concomitant modulation of
both CRH and AVP.
In agreement with previous data, we also found that the marked stimulatory effect of CAN on adrenal function was not coupled with the same strong effect on ACTH release. This discrepancy is not easy to explain, considering that the central stimuli of the HPA axis usually release more ACTH than cortisol (1). A direct stimulatory effect of CAN on adrenal function cannot be ruled out, although some in vitro data showed an inhibitory, rather than a stimulatory, effect of spironolactone on adrenal steroidogenesis (23). A decrease in the clearance rate of cortisol could also be hypothesized, although this hypothesis seems unlikely taking into account evidence that cortisol metabolism is not modified by potassium CAN in normal subjects (12). On the other hand, the diuretic action of CAN could have increased endogenous vasopressin, which can directly influence adrenal function, independently of ACTH release (24, 25); however, this hypothesis also seems unlikely taking into account that iv administration of potassium CAN did not show any effect on plasma vasopressin concentrations (12). We hypothesize that the cortisol increase during CAN infusion may exert its feedback action mainly through GRs, partially counteracting the MRs blockade by CAN; this could blunt the progressive increase in corticotroph secretion, which, however, is sufficient to clearly increase adrenal secretion.
In the present study we did not find any modification of aldosterone secretion during CAN administration. To our knowledge this point has never previously been addressed. Although aldosterone secretion is mainly dependent on the renin-angiotensin system (26), it is also under the influence of ACTH secretion, as indicated by evidence that even minimal ACTH-(1–24) doses are able to markedly enhance its secretion in normal subjects (27). Thus, in the presence of blocked MR receptors and concomitant increase in corticotroph secretion, an increase in circulating aldosterone levels could have been foreseen. Data showing a direct inhibitory effect of spironolactone on mineralocorticoid secretion at the adrenal level (23) could explain the lack of any increase in aldosterone levels during CAN infusion, which, however, could have been too short to disclose an increase.
In conclusion, our study emphasizes the major role of MRs in the control of the HPA axis in humans, demonstrating that mineralocorticoid antagonism by CAN clearly enhances both spontaneous and stimulated HPA activities.
Acknowledgments
We thank Dr. A. Bertagna and Mrs. A. Barberis for their skillful technical assistance.
Footnotes
1 This work was supported by the Ministero dell’Università e
della Ricerca Scientifica e Tecnologica (no. 9906153187) and the
Foundation for the Study of Endocrinological and Metabolic
Diseases. 
Received October 26, 2000.
Revised March 2, 2001.
Accepted March 20, 2001.
References
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