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The Journal of Clinical Endocrinology & Metabolism
Copyright © 2000 by The Endocrine Society
Vol. 85, No. 5
Printed in U.S.A.
COMMENT
Mannheim Classification of Nongenomically Initiated
(Rapid) Steroid Action(s)
ELISABETH FALKENSTEIN, ANTHONY W. NORMAN,
AND
MARTIN WEHLING
Institute of Clinical Pharmacology, Faculty for Clinical Medicine at Mannheim (E.F., M.W.),
University of Heidelberg, Mannheim, Germany; and Department of Biochemistry (A.W.N.),
University of California–Riverside, Riverside, California
ABSTRACT
There is increasing evidence for rapid effects of steroids that are
incompatible with the classical model of genomic steroid action. To
address the diversity of mechanisms for rapid steroid signaling de-
I
N THE CLASSICAL model of steroid action, the effector
mechanism involves the binding of steroids either to
receptors present in the nucleus or in the cytosol, followed
by translocation of the receptor-ligand complex to the nucleus, with subsequent modulation of transcription and protein synthesis. The considerable latency of genomic steroid
effects (⬎30 min) is the consequence of these time-consuming
steps of action.
Over the past years, it has become increasingly clear that
rapid actions of steroids, occurring within a few minutes
after the addition of the agent, exist that are incompatible
with the classical model of action. Such effects have been
described for all classes of steroids and related compounds,
such as 1␣,25-dihydroxyvitamin D3 (the steroid hormone
metabolite of vitamin D3) and triiodothyronine (1). In addition to in vitro effects on intracellular signaling pathways,
research on rapid in vivo actions of steroids in animals and
humans was significantly intensified during recent years.
Investigations in this regard comprise actions on vasoregulation, the central nervous system, and a wide array of other
organs (1). Besides in vivo effects of aldosterone and neurosteroids, which are described below, nongenomic progesterone signaling is believed to play a role in human infertility
(2) and rapid effects of estrogens on myocardial ischemia and
vasoregulation have gained attention due to a potential role
in beneficial effects of postmenopausal hormone replacement therapy (3).
In general, it has become obvious that the mechanism of
rapid steroid signaling is not uniform, and a variety of modes
of rapid action have been described (1). A classification of
rapid steroid effects with regard to the mechanisms involved,
Received September 14, 1999. Revision received December 3, 1999.
Accepted December 29, 1999.
Address correspondence and requests for reprints to: Dr. Martin
Wehling, Institute of Clinical Pharmacology, Faculty of Clinical Medicine at Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer,
Mannheim, Germany 68167.
scribed over the past years, a classification of rapid steroid effects has
been proposed to promote the discussion and understanding of nongenomic steroid action. (J Clin Endocrinol Metab 85: 2072–2075,
2000)
thus, seems to be necessary and may prove helpful for the
discussion and understanding of nongenomic steroid action.
The following classification has been proposed and discussed at the “First International Meeting on Rapid Responses to Steroid Hormones,” held in Mannheim, Germany,
from September 18 –20, 1998 (4). The Mannheim classification scheme proposed for nongenomic responses initiated by
steroid hormones is presented as Fig. 1. According to this
classification, the following categories would be possible: AI,
BI, AIIa, AIIb, BIIa, and BIIb. Examples for AI, AIIa, AIIb, and
BIIb are given below. However, there are no examples for
categories BI and BIIa known, to date.
Classification AI (Direct Action, No Receptor
Involved)
Nongenomic effects of steroids may be induced at high
steroid concentrations without receptor involvement by
modulation of protein function reflecting changes in membrane physicochemical properties. The apparent steroid
specificity of these effects may, thus, reflect variable lipophilicity and polarity.
In 1961, Willmer (5) proposed that steroids could be inserted
into the phospholipid bilayers of membranes, thereby altering
the fluidity of the membrane. These nonspecific, nongenomic
effects on physicochemical membrane properties have been
described in various cells such as breast cancer cells (6), vaginal
epithelial cells (7), and human spermatozoa (8).
Shivaji and Jagannadham (8) investigated interactions of
progesterone, 17-␣-hydroxyprogesterone, testosterone, and
estradiol (E2) with membrane vesicles prepared from phosphatidylserine and from lipid extracts of human and hamster
spermatozoa. The results indicated that progesterone at high
concentrations decreases the fluidity of membranes, aggregates membrane vesicles, induces fusion of membrane vesicles, and renders vesicles permeable to hydrophilic molecules like carboxyfluorescein. In contrast, testosterone and E2
at the same concentrations (␮m) had very little effect on
2072
CLASSIFICATION OF NONGENOMIC STEROID ACTION
2073
FIG. 1. Mannheim classification of
nongenomic steroid actions. Dotted arrows indicate a hypothetical category
with no example yet known. Other arrows indicate examples for categories
with given examples, which are explained in the text.
membrane fluidity, membrane aggregation, fusion, and leakage. Thus, steroid specificity may be apparent even in the
absence of protein. Nonspecific steroid actions can be expected at supramicromolar concentrations, but may also occur at much lower concentrations; the vitamin D3 metabolite
1␣,25(OH)2D3 influences growth zone cell membrane fluidity in rat costochondral chondrocytes at nm concentrations
(9).
Classification AII-a (Direct Action via Classical
Intracellular Receptors)
The possible involvement of the classic steroid receptor
(belonging to the superfamily of steroid hormone receptors)
in mediating some rapid responses has been suggested by
recent studies; for example, the estrogen receptor (ER) ␣ has
been shown to be involved in rapid estrogen signaling. In
isolated early passage, ovine fetal pulmonary artery endothelial cells rapid stimulation of endothelial nitric oxide synthase (NOS) activity by 17␤-E2 (10⫺10–10⫺6 M) has been demonstrated within 5 min (10). In NCI-H441 human bronchiolar
epithelial cells, 17␤-E2 (10⫺8 m) stimulated NOS activity with
a maximum effect of 143% above basal levels within 5 min.
These effects were insensitive to actinomycin D, but completely inhibited by the antagonists of the classical estrogen
receptor tamoxifen and ICI 182,780. In addition, overexpression of ER␣ caused a 45% increase in E2-17␤-mediated acute
augmentation of NOS activity (11).
Membrane receptors for estrogen have also been detected
in GH3/B6 rat pituitary tumor cells by antibodies directed
against epitopes of the classical intracellular receptors (12).
These cells exhibited rapid PRL release (within 5 min) after
treatment with nanomolar levels of estrogen.
Classification AII-b (Direct Steroid Action via
Nonclassical Receptors)
The majority of rapid effects of steroids on cellular signaling and function seem to be transmitted by membrane
receptors unrelated to the classic intracellular steroid receptors. The pharmacological characteristics of these membranebinding sites, which have been described for all classes of
steroids, are clearly distinct from classic intracellular steroid
receptors, thus pointing to the involvement of putative, novel
receptors at the membrane level.
A well-described example of this class of rapid steroid
action are acute aldosterone effects on various ion transport
mechanisms and second messenger systems (1, 13–16).
In human mononuclear leucocytes and vascular smooth
muscle cells (VSMCs), aldosterone or fludrocortisone significantly stimulate the generation of inositol-1,4,5-trisphosphate (IP3) within 30 sec, with an EC50 of approximately 0.1
nm. Cortisol is a very weak agonist, with an EC50 of approximately 1 ␮m. Furthermore, the classic mineralocorticoid
(MR) antagonist canrenone did not block these effects at
100-fold excess concentrations. Similarly, diacylglycerol production was increased in VSMCs within 30 sec by subnanomolar concentrations of aldosterone, but only by supramicromolar concentrations of cortisol (17).
As IP3 releases calcium from intracellular IP3-sensitive
stores, the effects of aldosterone on free intracellular calcium
([Ca2⫹]i) were investigated in VSMCs and pulmonary artery
endothelial cells. An immediate increase of [Ca2⫹]i was seen
after the addition of aldosterone; [Ca2⫹]i, then reached a
plateau within 2–3 min. The EC50 for aldosterone was ⬃0.1
nm, whereas cortisol and other glucocorticoids were active
only at or above ␮m concentrations. Pretreatment with the
aldosterone antagonist spironolactone (10 ␮m) for 5 or 30 min
did not antagonize the effect of aldosterone (14).
In addition, in human mononuclear leucocytes, membrane-binding sites for aldosterone have been found with
binding characteristics that are in agreement with the functional data mentioned above; these sites, therefore, may mediate rapid aldosterone action. Specific saturable binding of
aldosterone to microsomal membranes was demonstrated
with a Kd of ⬃0.1 nm for the radioligand; displacement experiments showed a Kd of ⬃0.1 nm for aldosterone (18, 19).
The MR receptor antagonist canrenone and cortisol were
inactive as ligands up to ␮m concentrations, whereas fludrocortisone and desoxycorticosterone acetate showed intermediate activity.
These data on rapid aldosterone action and potential binding sites are incompatible with an involvement of classic
intracellular type-I-MR receptors, which do not discriminate
between aldosterone and cortisol, and bind the antagonist
canrenone with higher affinity.
In addition to these in vitro effects, convincing in vivo
evidences have been described for rapid aldosterone action.
2074
FALKENSTEIN ET AL.
For example, aldosterone effects on baroreceptor neuron discharge frequency have been described in the dog, which
occur as early as 15 min after application of the steroid (20).
Moreover, aldosterone was found to significantly increase
peripheral vascular resistance and blood pressure in humans, whereas cardiac output was decreased within 5 min
after injection of aldosterone (21). The results of the latter
study have been confirmed with modern invasive techniques
in catheterization studies, recently. It was shown that systemic vascular resistance significantly increased after iv application of 0.5 mg aldosterone within 3 min (22). Further
clinical significance for rapid aldosterone action was found
in a study in which calf phosphocreatine concentrations were
monitored by nuclear magnetic resonance spectroscopy at
rest and under stress (23). Aldosterone (0.5 mg iv) significantly facilitated phosphocreatine recovery after stress, an
effect starting within 8 min after application of the steroid.
Another prominent example of this category of nongenomically initiated steroid action is the seco-steroid hormone 1␣,25(OH)2-vitamin D3 [1␣,25(OH)2D3]. The rapid effects of 1␣,25(OH)2D3 have been demonstrated in a variety
of systems (24) and include rapid stimulation of the intestinal
calcium transport in the perfused chick intestine (termed
transcaltachia) (25), as well as rapid activation of protein
kinase C (26 –28) and mitogen-activated protein (MAP) kinase (29, 30).
In transcaltachia, the application of 1␣,25(OH)2D3 at subnanomolar amounts to the basal lateral surface of the intestinal epithelial cell resulted in a very prompt (within 2 min)
increase in the rate of appearance of 45Ca2⫹ in the perfusate
exiting via the celiac artery, an effect not blocked by actinomycin D (31). Moreover, 1␣,25(OH)2D3 (10⫺8 m) significantly
increased MAP kinase phosphorylation, with the earliest
response detectable at 30 sec (30).
Because 1␣,25(OH)2D3 is a conformationally flexible molecule, a series of analogs locked in the cis and trans conformation
have been used to evaluate the optimal shape for activation. The
cis-locked conformers, but not the trans-locked analogs, can
mimic the rapid membrane effect of 1␣,25(OH)2D3 but are only
weak agonists for the genomic responses. In addition, the cis
analogs bind poorly to the nuclear receptor (30) and a specific
antagonist, 1␤,25(OH)2D3, for 1␣,25(OH)2D3-mediated rapid
responses has been identified (31).
These results suggest that the nuclear hormone D receptor
is not involved in these effects. In addition, membrane-binding
sites potentially transmitting rapid effects of 1␣,25(OH)2D3
have been characterized in chick intestine (32–34). Studies on
hormone D analogs provide convincing structural correlation
between binding to the membrane receptor and the ability to
initiate transcaltachia.
Classification BII-b (Indirect: Steroid Needs Partner
Ligand as Coagonist, Nonclassic Receptor Involved)
Indirect modulation of cell function by a steroid acting as
coagonist has been shown for neuroactive steroids that can
rapidly alter the excitability of neurons via modulation of
GABAergic effects (35). These neuroactive steroids include
naturally occurring steroids, steroids synthesized in oligo-
JCE & M • 2000
Vol 85 • No 5
dendrocytes of the brain (neurosteroids and their sulfate
derivatives) and synthetic steroids (36).
Neuroactive steroids that have been thoroughly studied
are particularly 3␣-hydroxy ring A-reduced pregnane steroids, including allopregnanolone (3␣, 5␣-P) and tetrahydrodeoxy corticosterone (THDOC). These steroids are potent
positive allosteric modulators of GABAA-gated inward Cl⫺ion conductance (100% potentiation at 10 nm) (37), whereas
pregnenolone sulfate and dehydroepiandrosterone sulfate
display antagonistic properties at GABAA receptors (38, 39).
The effects of 5␣-pregnan-3␣,21-diol-20-one (5␣-THDOC)
were tested in pyramidal neurons in in vitro slice preparations of the adult rat frontal neocortex by the use of intracellular microelectrodes (40). This neurosteroid (10 ␮m) increased and prolonged the inhibitory postsynaptic potential.
The mean maximal synaptic conductance of the early,
GABAA receptor-mediated inhibitory postsynaptic potential
was enhanced to more than 700% of control and the mean
synaptic conductance at the maximum of the late, partially
GABAB receptor-mediated, inhibitory postsynaptic potential
to approximately 400%; the progesterone/glucocorticoid receptor antagonist RU 38486 does not prevent this increase.
Responses to the iontophoretically applied specific GABAA
receptor agonist muscimol, but not to the specific GABAB
receptor agonist 1-baclofen, were enhanced by 5␣-THDOC.
In a recent study, the effect of 3␣,5␣-P on [3H]-noradrenalin (NA) release from superfused hippocampal synaptosomes was examined (41). Release of [3H]-NA was elicited by
5 ␮m GABA; it was further augmented by 3␣,5␣-P at concentrations from 0.1– 0.3 ␮m. With no GABA added to the
superfusion medium, 3␣,5␣-P (0.01–10 ␮m) did not modify
the basal release of [3H]-NA.
Progesterone action through the GABAA-receptor complex has also been linked to analgesic properties of this hormone (42). These neurosteroid effects apparently involve a
binding site on the GABA receptor that has not yet been
localized on the known primary structure of the GABAAreceptor complex and are seen only if GABA is present.
The stimulatory properties of 3a-reduced pregnane neuroactive steroids or their precursor, progesterone, at GABA
receptors are thought to exert several psychopharmacological effects in animals and humans. For example, anesthetic
effects have been used clinically in humans and are still used
in veterinary medicine (43), and anticonvulsant properties
have led to the development of synthetic derivatives of
3␣-reduced pregnane neuroactive steroids, which are under clinical investigation for the treatment of epilepsy disorders (44).
In summary, the Mannheim classification of nongenomically initiated (rapid) steroid action(s) may help to describe
the existing variety of mechanisms involved in rapid steroid
action. Only if this diversity of potential mechanisms is recognized can there be a full appreciation of the scope of this
emerging field. Future development of this proposed classification must address the issue of different signaling pathways as a major discriminant. However, given the paucity of
data in this regard, to date, this dimension has not been
incorporated into the present version.
CLASSIFICATION OF NONGENOMIC STEROID ACTION
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