Mechanism of the antimineralocorticoid effects of spirolactones

Kidney International, Vol. 20 (1981), pp. 1—6

EDITORIAL REVIEW

Mechanism of the antimineralocorticoid effects of spirolactones P. CORVOL, M. CLAIRE, M. E. OBLIN, K. GEERING, and B. RossIER

Spirolactones are antimineralocorticoid compounds, which were introduced into therapeutics in 1960. They are useful in the treatment of hyperaldosteronism and essential hypertension, although their side effects (gynecomastia, impotence, menstrual cycle abnormalities) limit their use. They are known to antagonize the effect of aldosterone at the level of the target

cells, and until recently this mechanism was the only one generally implicated. In 1972, Erbier showed that spironolactone inhibits aldosterone biosynthesis in vitro [1]. Since then, several authors have confirmed this effect, both in animals and man, and have therefore challenged the relative importance of peripheral aldosterone antagonism in favor of an inhibitory effect of these drugs on aidosterone biosynthesis. This editorial review attempts to clarify the findings and to determine whether antialdosterones act mainly at the target cell, or through inhibition of aldosterone production, or alternatively by a combination of each effect. Action of spirolactones on mineralocorticoid receptors

Aldosterone is thought to induce a three step cascade of events [2]: (1) stereospecific binding of aldosterone to a cytoplasmic receptor followed by translocation of the steroid receptor complex into the nucleus, where it interacts with specific sites of the genome, (2) induction of the synthesis of mRNA or rRNA, and (3) translation of the induced mRNA into specific proteins, which, in turn, directly or indirectly increase sodium

reabsorption (and/or potassium or hydrogen ion secretion)

specific competitive antagonists and to consider the various molecular mechanisms by which spirolactones could act. (1) Spirolactones: specific competitive antagonists? Specific competitive antagonists should fulfill the following criteria: (1) dose-dependent antagonistic activity inducing a decrease in the potency of the antagonist, (2) specificity, and (3) competitive interaction at the receptor level. For studying the action of aldosterone and spirolactones, two

experimental models have been used most extensively: the adrenalectomized rat and the urinary bladder of the toad. (a) Antagonistic activity. It is well established that, in the adrenalectomized rat, aldosterone induces a sodium reabsorp-

tion and a potassium excretion [10, 16]. A direct effect on hydrogen ion excretion has also been suggested [17]. For practical reasons, the urinary sodium/potassium concentration ratio in 4-hour urine samples from adrenalectomized rats has generally been used as a screening test for mineralocorticoid and antimineralocorticoid activity (Kagawa's test [18]). In this model, spirolactones enhance the urinary sodium-potassium ratio previously depressed by the administration of mineralocorticoids. The expected dose-response curve is obtained for competitive inhibition. In the toad bladder, aldosterone enhances sodium reabsorption without effect on potassium ion movement, but two studies have shown increased hydrogen ion secretion in toad and turtle bladders [19, 20]. Spirolactones inhibit, in a dose-dependent manner, the aldosterone-induced sodium reabsorption as mea-

across the epithelial cells of target organs such as mammalian sured by the short-circuit current method [12, 19]. Using renal tubules or amphibian urinary bladder. General reviews of Lineweaver-Burk plots of changes in short-circuit current, this mechanism have recently been published [3, 4] and articles Porter and Kinsey showed that SC 19886 (Fig. 1) was able to dealing specifically with the mineralocorticoid receptor [5], the competitively inhibit the aldosterone-stimulated sodium transrole of RNA [6], or the aldosterone-induced proteins [7] are also port [21]. One striking finding that emerges from various studies is that available. The possibility that a steroid hormone could directly compete the spirolactones display no or very little agonistic activity on with aldosterone at the level of the target cells was first urinary sodium-potassium ratios in the adrenalectomized rat. suggested by Sala and Luetscher, who found that cortisone At very high doses, some spirolactones such as SC 8109 show a inhibited the antinatriuretic effect of injected aldosterone in rats small but significant agonistic activity and, thus, are "partial" [8]. Landau et al [9] described in 1955 that progesterone blocked antagonists [22]. In the toad, which cannot be adrenalectothe sodium-retaining action of aldosterone in a patient with mized, the secretion of endogenous aldosterone can only be Addison's disease. In adrenalectomized rats, Celia and Kagawa partially suppressed by soaking the animals in saline solution (1957) subsequently demonstrated that steroidal derivatives, [23]. Thus, the urinary bladder used in vitro for testing the the spirolactones, act by a similar mechanism, that is, by direct agonistic and antagonistic activity is exposed to variable competition with aldosterone at the target cell level [10]. Since amounts of aldosterone before removal. This fact might explain that time, a great number of clinical or experimental studies

have provided evidence that the spirolactones are specific competitive antagonists of aldosterone at the level of the mineralocorticoid receptor (for review, see Refs. 11—14). Before

Received for publication May 6, 1980 and in revised form December 19, 1980

accepting these conclusions, it is necessary to examine careful-

0085-2538/81/0020-0001 $01.20

ly the pharmacologic data indicating that spirolactones are

© 1981 by the International Society of Nephrology

2

Corvol et a! HO

___-x___ Steroids

sc 19886

SC 9420 Spironolactone

11 Activity in vivo

Binding

in vitro

-O (5 mg/kg]

sc 8109

SC 14226 Canrenoate

SC 26304

SC 9376 Canrenone

96%

24%

Spironolactone

-coo SC 23133

[10 mg/kg]

Prorenone

63%

0.06%

Fig. 1. Structures of aldosterone antagonists described in the text.

K-canrente

the observation that spirolactones (up to 25 M) either had no agonistic action or depressed to some extent the sodium transport baseline [24]. In one study [25], two spirolactones SC 26304 and prorenone (SC 23133) were found to have a small but significant agonistic effect. This effect could not be reproduced,

[20 mg/kg] 0%

0%

however, in another study concerning prorenone [14], and the general conclusion is that most spirolactones, including spiron17-0-Methyl 5,6 olactone, lack agonistic activity. This feature is in general not dihydrocanrenoic acid shared by other antisteroid drugs; for example, the antiestrogen Fig. 2. Comparison of in vivo and in Vitro activity of three spirolactones: nafoxidin [26] or the antiglucocorticoid cortexolone (see review (a) Percentage of increase in sodium-potassium ratio compared with the aldosterone-treated reference group; (b) Percentage of the competi[27]) have significant agonistic activity.

potency of aldosterone taken as reference. The 100% corresponds In both experimental systems the antagonistic action of tive to the concentration ratio of unlabeled to 3H-aldosterone needed to terone, indicating that the antagonist does not bind to the from Peterfalvi et at [30].)

spirolactones is fully reversible upon further addition of aldos- obtain 50% inhibition of binding to mineralocorticoid sites. (Data is receptor in an irreversible manner [11, 14].

(b) Specificity. Spirolactones block the effect of various mineralocorticoids such as deoxycorticosterone [18] and aldos- from receptor studies because spirolactones generally exhibit a terone [18, 28]. This property permits a convenient and repro- high affinity for the mineralocorticoid receptor [28, 40]. Drug

ductible bioassay in man: spirolactones reverse the sodium- metabolism to less active or inactive metabolites partly acretaining and potassium-excreting effect of 9a-fluorohydrocorti- counts for this apparent discrepancy [41]. Spironolactone is sone [29]. Specificity for mineralocorticoid effects is also shown dethioacetylated into canrenone (SC 9376), which exists in by the fact that spironolactone acts differently from amiloride in equilibrium with canrenoate (SC 14266), the acid carboxylic toad bladder [30]. Moreover, in this model the effect of vaso- form [42]. In receptor studies [28, 39], canrenone has 10% of the pressin, which enhances sodium reabsorption to the same affinity of spironolactone for the aldosterone receptor, whereas extent as aldosterone but by a different mechanism involving canrenoate does not bind to the receptors. The critical importance of the presence of the lactone ring for the binding of cyclic AMP, is not inhibited by spironolactone [31]. (c) Action at the receptor level and structure-activity rela- spirolactones to receptors has been demonstrated using methyltionship. Mineralocorticoid binding sites have been identified ated canrenoate (Fig. 2), a canrenoate analog that is inactive and characterized in a number of target organs, both in mam- both in the rat kidney [28] and in the toad bladder (Rossier et al, mals and amphibians. Two types of binding sites have been unpublished results). described: type I displays affinity constants, which make them (2) Mechanism of action of spirolactones at the receptor good candidates for mineralocorticoid receptors, whereas type level. Our understanding of the molecular mechanism of action II could represent glucocorticoid receptors [32—35]. Several of spirolactones has recently progressed by the use of labeled studies show that spirolactones can compete for the type I antagonists with high specific activity. Marver et al [43] first demonstrated that in the rat kidney a 3H-labeled spirolactone binding sites [36—39]. In vivo, spirolactones are not very potent drugs because high (SC 26304) bound to cytoplasmic proteins, which are probably molar antagonist/agonist ratios are required for producing an mineralocorticoid receptors. These investigators did not detect effect. Thus, in the adrenalectomized rat, a dose of S mg of any translocation of the antagonist-receptor complexes into the spironolactone per kilogram of body wt must be given to nucleus under three different experimental conditions. Claire et antagonize the effect of I p.g of aldosterone acetate per kilogram a! [40] obtained similar results with another labeled spirolacof body wt [28]. This dose is much higher than that expected tone, 1,2,-3H-prorenone, which bound with high affinity to

Extracellular

/

Cytoplasm

space

1[lIj' /

Agonist• Antagonist LI-

03+

Spirolactones

3

mechanism B which has been postulated for cortexolone [27]. Mechanism C implies a high affinity binding of the antagonistreceptor complex to the chromatin and can thus be ruled out.

Nucleus

0

a

Mechanism D which has been proposed for nafoxidin, an antiestrogen [26], is not likely for the action of spirolactones because no cytosolic receptor depletion was observed in the study of Claire et al [40].

C

= =

z

©

Fig. 3. Models for steroid agonist and antagonist mechanism of action

at the subcellular level: interactions with receptors and chromatin acceptor sites. One asterisk (*) denotes the active form of the receptor; two asterisks (**), the inactive form,

Inhibition of aldosterone biosynthesis by spirolactones Spironolactone has first been shown to inhibit aldosterone biosynthesis in incubated adrenal quarters in vitro by Erbler in 1972 [1]. Aldosterone synthesis stimulated by ACTH, potassium, or angiotensin II was inhibited by l0- to l0- M spironolactone. A dose-response curve was obtained from these studies, and Erbler concluded that spironolactone could block the conversion of corticosterone into aldosterone. Since 1972, several authors have confirmed the inhibition of aldosterone biosynthesis by spirolactones in adrenal preparations of human, bovine [44], duck [45], and guinea pig [46]. The kinetics of the inhibition of aldosterone biosynthesis have been investigated by Delarue et al [47] in an in vitro frog interrenal perfusion model: the lag period between the infusion of the drug

mineralocorticoid receptors (Kd = 1.6 X i0 M) [40]. Again, and the beginning of the response was less than 10 mm. the cytosolic 3H-prorenone receptor complex did not yield any Aldosterone output rose back to its initial value approximately 100 mm after stopping drug administration. Spirolactones and nuclear binding. These results have been interpreted within the context of a their y carboxylic acid analogs share these effects. model proposed by Feldman, Funder, and Edelman [33] for the (1) Site of action of spirolactones on steroid biosynthesis in interaction of aldosterone with its receptors (see Fig. 3). This vitro. Spirolactones have been reported to act at different steps model can be useful for understanding the mechanism of action of aldosterone biosynthesis (Fig. 4). Spironolactone, canrenof spirolactone. It implies the following assumptions: (1) pas- one, and K canrenoate inhibit mitochondrial ll3 and 18sive diffusion of ligand across the outer plasma and the nuclear hydroxylase [44, 45, 48]. Canrenone appears to be the most membranes allowing complete equilibration between the extra- active compound and was found to be much more effective in cellular and the intracellular compartment, (2) presence of inhibiting 1 8-hydroxylases than were 11 p-hydroxylases in the receptors in two allosteric states in equilibrium: one active and adrenals from patients with primary hyperaldosteronism [44]. one inactive (in the presence of an agonist, the equilibrium is But, the action of spirolactones is not limited to these hydroxyshifted towards the "active" form; in the presence of an lases; pretreatment of guinea pig with spironolactone induces antagonist, towards the "inactive" form), (3) free receptors are an inhibition of adrenal 21-hydroxylase [46], and administration unable to translocate into the nuclear compartment, (4) the of spironolactone to rats at high doses (200 mg/kg of body wt receptor-agonist complex translocating into the nucleus and per day) impaires the testicular l7a-hydroxylase [49]. Unlike then (5) binding to specific acceptor sites on the chromatin. All the structural requirement for receptor antagonism, antistesteps 1, 2, 4, 5 as well as recycling of the receptors in the roidogenesis activity does not seem to be dependent on the cytoplasm are assumed to be reversible. presence of a lactonic ring, but high concentrations (10 to Spirolactones could theoretically inhibit the action of aldos- i0 M) of the inhibitors are always required to inhibit biosynterone by four different mechanisms (Fig. 3). In the first one thesis. Several molecular mechanisms have been proposed for the (A), the antagonist receptor complex is supposed to be unable to translocate into the nucleus. In mechanism B, translocation effects of spirolactones on steroidogenesis. Erbler [50] suggestoccurs but the complex has little or no affinity for the chromatin ed that canrenone might act as a false substrate for 11 3- and 18acceptor. In mechanism C, the antagonist receptor complex hydroxylases and, indeed, adrenal tissue hydroxylates canrenalso translocates into the nucleus, then binds to chromatin but one and other spirolactones [44]. A competitive type of inhibifails to trigger the transcription mechanism. Mechanism D tion has been suggested by Cheng for 18-hydroxylation [44] but implies an impairment of receptor recycling, leading to cytoso- spirolactones may also inhibit hydroxylation by binding to lic receptor depletion. cytochrome P450 [44, 46] or even "destroy" the cytochrome The data obtained by Marver et al [43] and Claire et al [40] P450 of adrenal and testicular tissue when animals are pretreatfavor the mechanism A, because there was no radioactive 3H- ed with high doses [46, 49]. This latter effect, however, is spirolactone receptor complex found in the nuclei under differ- probably nonspecific because it is shared by other steroids that ent conditions. But, as suggested by Claire et al, methodologic do not affect steroidogenic tissue. artifacts such as redistribution of receptors during homogeniza(2) Effect of spirolactones on aldosterone secretion in vivo. tion and high nonspecific binding of spirolactones could explain Although spirolactones inhibit aldosterone biosynthesis in vithe absence of binding to chromatin. If not due to artifacts, this tro, their action on plasma aldosterone level is much more absence of binding to chromatin could also be in favor of controversial. That spirolactones might indeed block aldoster-

4

Corvol et a! Cholesterol

for inhibiting receptor-binding of aldosterone. If spirolactones were to act by inhibiting the biosynthesis of aldosterone, they

DS-Pregnenolone

would, therefore, have to be concentrated at the sites of

aldosterone synthesis in the adrenal cortex, This possibility has not been excluded to date. 21 -Hydroxylase (2) In vitro, spirolactones can affect nonspecifically several hydroxylases that are involved not only in aldosterone but also Deoxycorticosterone in glucocorticoid biosynthesis. 1 113-Hydroxylase (3) Depression of aldosterone secretion has not always been Corticosterone observed under short-term spirolactone treatment and, when 1 8-Hydroxylase present, is transient and of small magnitude. 18-OH-Corticosterone (4) During long-term spirolactone treatment in animals and 1 8-Deshydrogenase man, aldosterone levels are increased to the same extent as with [Aldosterone nonantialdosterone diuretic therapy. Fig. 4. Effects of spirolactones at various steps of the biosynthesis of These conclusions concerning the antialdosterone effect of aldosterone. The solid line (—) denotes the major inhibiting property; spirolactones probably also apply to the mechanisms of their the broken line (---), the other possible sites of action. side effects. Spirolactones are potent antiandrogenic drugs at the level of the dihydrotestosterone receptor [58, 59]. But here again, spirolactones appeared to induce either a slight decrease one biosynthesis in vivo came first from a study on a patient in plasma androgen levels [60] or even no alteration at all [61]. with primary aldosteronism. In this study, SundQjord et al [51] 'I!

Progesterone

showed

a decrease in aldosterone secretion rate during 4

P. CORVOL M. CLAIRE

month's treatment with doses up to 100 to 200 mgldaily. But, a carefully controlled study performed on normal volunteers with diuretic-induced secondary hyperaldosteronism demonstrated

M. E. OBLIN

only a transient effect of canrenone on plasma alclosterone levels [52]. Similarly, Conn and Hinerman [53] found only an early and transient decrease of urinary aldosterone during

Paris and Lausanne

spironolactone treatment of primary aldosteronism. In fact, the authors noted that, in some cases, the observed decrease was followed by an important increase (50 to 100%) in the aldosterone excretion. The same observation was made by Abshagen et a! [54] for plasma aldosterone levels in normal subjects under continuous spironolactone treatment: after 3 days, the aldosterone levels increased to the same extent as that in triamterene-

K. GEERING B. ROSsIER

Acknowledgments This work was supported by an ATP from INSERM (44.76.76) and a grant from FNS (3.274.0.78). Dr. 0. Peters helped in the preparation of the manuscript.

Reprint requests to Dr. P. Corvo!, JNSERM U 36, 17, rue du Fer a Moulin, 75005 Paris, France

treated patients. Gaillard et al [55] also failed to observe a depression of aldosterone secretion rate after the administration of 400mg of spironolactone to normal young men and, instead,

obtained an increase of aldosterone secretion without any change in aldosterone metabolism, cortisol secretion rate, or

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