Physiol Rev
83: 965–1016, 2003; 10.1152/physrev.00003.2003.
Nongenomic Steroid Action:
Controversies, Questions, and Answers
RALF M. LÖSEL, ELISABETH FALKENSTEIN, MARTIN FEURING, ARMIN SCHULTZ,
HANNS-CHRISTIAN TILLMANN, KARIN ROSSOL-HASEROTH, AND MARTIN WEHLING
Institute of Clinical Pharmacology, Faculty of Clinical Medicine Mannheim,
University of Heidelberg, Heidelberg, Germany
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I. Introduction
A. Early work
B. Genomic versus nongenomic action
II. Receptors
A. The steroid nuclear receptor superfamily: classic receptors
B. Which receptors mediate nongenomic action: the controversy
C. Evidence for the existence of receptors distinct from classic receptors: some examples
D. Additional evidence for and against distinct receptors
III. Effects, Signaling Pathways, and Clinical Implications
A. Glucocorticoids
B. Progesterone
C. Estrogens
D. Androgens
E. Neurosteroids
F. Mineralocorticoids
G. Vitamin D3
H. T3 and T4
I. Does receptor type matter for clinical impact?
IV. Interaction of Nongenomic and Genomic Pathways
V. Synopsis: What Do We Know About Nonclassic Receptors?
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Lösel, Ralf M., Elisabeth Falkenstein, Martin Feuring, Armin Schultz, Hanns-Christian Tillmann, Karin
Rossol-Haseroth, and Martin Wehling. Nongenomic Steroid Action: Controversies, Questions, and Answers.
Physiol Rev 83: 965–1016, 2003; 10.1152/physrev.00003.2003.—Steroids may exert their action in living cells by
several ways: 1) the well-known genomic pathway, involving hormone binding to cytosolic (classic) receptors and
subsequent modulation of gene expression followed by protein synthesis. 2) Alternatively, pathways are operating
that do not act on the genome, therefore indicating nongenomic action. Although it is comparatively easy to confirm
the nongenomic nature of a particular phenomenon observed, e.g., by using inhibitors of transcription or translation,
considerable controversy exists about the identity of receptors that mediate these responses. Many different
approaches have been employed to answer this question, including pharmacology, knock-out animals, and numerous
biochemical studies. Evidence is presented for and against both the participation of classic receptors, or proteins
closely related to them, as well as for the involvement of yet poorly understood, novel membrane steroid receptors.
In addition, clinical implications for a wide array of nongenomic steroid actions are outlined.
I. INTRODUCTION
A. Early Work
In 1942, Hans Selye published a study on the correlation between chemical structure of steroids and their
pharmacological action (437). In this study, rats were
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administered a number of different steroids intraperitoneally, and very rapid anesthetic effects were noticed. In
addition, the main hormone effects known at this time,
namely corticoid, folliculoid, luteoid, and testoid action,
were investigated. Whereas anesthesia occurred within
minutes, the other hormone effects required several hours
or days to become visible. In addition to the drastically
0031-9333/03 $15.00 Copyright © 2003 the American Physiological Society
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B. Genomic Versus Nongenomic Action
According to the generally accepted theory of steroid
action, steroid molecules enter the cell, either passively
by diffussion through the membrane or assisted by any
transporter; subsequently, intracellular receptors, located
in the cytosol or in the nucleus, bind the steroid, thereby
undergoing a conformational change. This leads to the
dissociation of accessory proteins, which have previously
maintained the receptor in an active form, and to an
increase in DNA affinity of the DNA binding domain. The
steroid-receptor complex migrates to the nucleus, where
it influences the transcription of genes into mRNA. Finally, mRNA is translated into protein molecules, which
ultimately exert biological functions. There are several
reviews describing these events comprehensively (36, 37).
This pathway is called “genomic,” because it involves
action on the genome, and from the sequence of events
several properties become obvious.
1) The time required to fully activate this pathway is
comparatively long. For aldosterone, early genes are differentially expressed 1 h after steroid addition (501). Furthermore, protein synthesis requires additional time, and
to exert a given biological function, significant amounts of
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the respective protein must be generated. Clinically, i.e.,
at the level of the whole organism, effects are usually seen
after hours or even days.
2) This pathway is sensitive to inhibitors of transcription (such as actinomycin D) as well as inhibitors of
translation (cycloheximide).
In contrast to the genomic pathway outlined above,
nongenomically mediated phenomena often occur with
very short lag time. As examples, progesterone induces a
significant increase in intracellular calcium in spermatozoa within seconds (58), and aldosterone activates the
Na⫹/H⫹ antiporter within 1 min in colonic crypts (526).
Even more striking are the examples of steroid effects that occur in cells without a functional nucleus, such
as erythrocytes, platelets, or spermatozoa. All these cells
rapidly respond to stimulation by various steroids; details
will be described in the following sections.
As the number of reports on nongenomic steroid
effects has grown tremendously in the past two decades,
it has become likely that the action of steroids in living
cells is mediated by various pathways rather than a single
uniform mechanism. The Mannheim classification scheme
(142) is helpful in describing and separating potential
mechanisms and understanding the distinction between
classic and nongenomic steroid action.
The scheme (Fig. 1) divides pathways of steroid action by several criteria. The main groups A and B differ for
the requirement of a partner agonist to elicit a steroid
response. Within these groups, steroid action can be further classified as nonspecific (I) or displaying ligand specificity (II). According to the identity of the receptors
mediating the specificity in the latter category, a distinction is made between the classic steroid nuclear receptor
(a) and other, nonclassic steroid receptors (b). On the
basis of current findings, the classic steroid receptor itself
is able to drive signaling cascades.
Examples for every class contained in the scheme are
not yet known, and within the classes the specific phenomena vary between species, tissue, cell type, and, obviously, the steroid involved.
II. RECEPTORS
A. The Steroid Nuclear Receptor Superfamily:
Classic Receptors
Steroid molecules all share the minimum skeleton of
four fused rings, which is not only true for the steroids
known from mammals, but also for the structurally related hormonally active substances from insects and
plants (e.g., ecdysone and the brassinosteroids). This
structural relation coincides with a structural similarity in
their animal receptor proteins, which form a protein superfamily together with the receptors for thyroid hor-
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differing time scale, the other striking finding in this work
was the variation of the relative magnitude between the
rapid anesthetic and the delayed “main” hormone action
among the compounds tested. In these days, very little
was known about the mechanisms involved in steroid
hormone action, but it has been concluded that “any one
of these activities may be exhibited . . . irrespective of any
other hormonal properties” which in contemporary perception would be interpreted as the involvement of different receptors and pathways of action. The main hormone action is now known to be mediated by “classic”
steroid receptors, mainly located in the cytosol and the
nucleus, acting on the genome.
Two decades later, in 1963, Klein and Henk (233)
demonstrated acute cardiovascular effects of aldosterone
in men. Within 5 min after administration of the steroid,
peripheral vascular resistance and blood pressure increased whereas cardiac output decreased. The short
time frame suggests a nongenomic mechanism, as explained below. In vitro effects of aldosterone at physiological concentration on Na⫹ exchange in dog erythrocytes were reported almost simultaneously (462). This
experiment very clearly demonstrates the existence of
nongenomic pathways for steroid action, as mammalian
erythrocytes lack a nucleus. Other rapid, presumably nongenomic, steroid effects have been demonstrated later
(125), and when the genomic steroid model was just
emerging, Pietras and Szego (375, 376) already underlined
the diversity of steroid action.
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FIG. 1. The Mannheim classification
of nongenomic steroid action. This
scheme aims to put possible mechanisms
into a system, but there are not examples
known for all classes yet. [From Falkenstein et al. (142), copyright 2000 The Endocrine Society.]
B. Which Receptors Mediate Nongenomic Action:
The Controversy
The commonly accepted mechanism for steroid hormone action, as found in all related textbooks, comprises
the following steps: steroid molecules enter the cell and
bind to their (classic) receptors described in the previous
section. The receptor-steroid complex then translocates
to the nucleus, if not already there, and modifies gene
transcription. As detailed in the previous sections, there
are numerous physiological effects that cannot be mediated by action on the genome, i.e., transcription and translation, as judged from their insensitivity toward appropriate inhibitors and/or the short time frame in which the
responses occur.
Such nongenomic effects obviously involve receptors
as well, if not simply indicating nonspecific steroid efPhysiol Rev • VOL
fects, e.g., on the membrane. A controversy has started,
whether this task is fulfilled by classic receptors as well,
thus representing additional, hitherto unknown functionality, or by different receptors unrelated to them. The
claim of distinction has often been made on the basis of
pharmacological properties, as a specific ligand binding
domain is believed to have a characteristic ligand selectivity pattern, and should, therefore, be at least very similar for genomic and nongenomic action if both were
mediated by the same receptor. Unfortunately, physiological responses to steroids involve living cells capable of
and using additional mechanisms that may interfere with
the determination of a pharmacological profile and its
correlation to ligand selectivity of the isolated receptor. A
very instructive example is the hydroxysteroid dehydrogenase reaction in conjunction with the mineralocorticoid
receptor. Thus some distinctions and classifications
merely based on pharmacology deserve reevaluation. It
must be noted that, unlike the classic steroid receptors,
only few other, novel receptors linked to a defined steroid
effect have been rigorously identified yet, such as receptors for neurotransmitters that are modulated by steroids
(see sect. IID5), the maxi-K channels (493), the Na⫹-K⫹ATPase (which binds cardiotonic steroids), and the
brassinosteroid receptors in plants.
Both the discovery of additional phenomena interfering with cellular steroid action and the yet moderately
successful identification of alternative receptors have
brought new life to the controversy of how nongenomic
steroid action is mediated. In the following sections, evidence for either part is presented and critically discussed.
C. Evidence for the Existence of Receptors
Distinct From Classic Receptors:
Some Examples
Some evidence has been compiled from experimental systems that indicate the participation of receptor
molecules that are not identical or closely related to the
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mones, retinoic acid, and vitamin D (37, 134). Generally, a
classic steroid receptor comprises a NH2-terminal domain
of considerably variable length with possible modulator
function, a highly conserved DNA binding domain, and a
ligand binding domain of ⬃220 –250 amino acids length at
the COOH terminus (35). Isoforms originating from differing promoter usage and other variants have been described for many steroid receptors, but their functional
role is not always known. More than 65 genes coding for
nuclear receptors have been identified in animals (104),
although their ligands are not known in all cases (orphan
receptors). Some of these orphan receptors are likely to
bind steroids as well, such as the farnesoid X receptor
(266), which binds bile acids, a fact that may indicate an
even broader scope of steroid action than previously
known. Other orphan nuclear receptors comprise the peroxisome proliferator-activated receptors (PPAR), which
are involved in regulating glucose and lipid homeostasis.
In their unliganded state, nuclear receptor molecules
are associated with chaperone proteins that are thought
to keep them functional.
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cytosolic, classic steroid receptors. This does not necessarily mean that rapid, nongenomic phenomena are always mediated by nonclassic receptors, e.g., rapid estradiol action is likely to be transduced by its classic receptor (estrogen receptor, ER; see sect. IID3B) in many cases,
and the classic progesterone receptor (PR) was shown
recently to drive rapid signaling cascades. However, there
are numerous physiological steroid responses that are
incompatible with exclusive classic receptor signaling for
reasons which shall be detailed here.
1. Knock-out mice
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2. Pharmacology
A) VITAMIN D3. Vitamin D3 itself is biologically inert, and
it needs to be metabolized to 1,25(OH)2D3 and other
metabolites to achieve its biological activity.
It is well established that 1,25(OH)2D3 can stimulate
biological responses via signal transduction pathways
that utilize the classic nuclear receptor for 1,25(OH)2D3
(vitamin D receptor, VDR) to regulate gene transcription.
It belongs to the superfamily of steroid receptors (see
sect. IIA) and has been cloned, sequenced, and functionally expressed (290). In addition, there is considerable
evidence that 1,25(OH)2D3 can generate rapid, nongenomic biological responses via different signaling pathways (for details, see sect. IIIG).
The naturally occurring isomer of 1,25(OH)2D3 is the
␣-isomer [1␣,25(OH)2D3]. Compared with steroid molecules in the narrow sense, which have a rigid skeleton, it
is unusually flexible in its conformation due to the missing
9 –10 carbon-carbon bond and easily undergoes a cis/
trans-isomerization at the 6 –7 carbon-carbon bond in the
seco B-ring.
In the course of examination of 1,25(OH)2D3 binding
to VDR and a putative membrane receptor, through which
it might induce genomic and nongenomic responses, respectively, a series of analogs locked in either the cis- or
the trans-conformation have been generated. The cislocked conformers activate the rapid, nongenomic pathways but bind poorly to the nuclear receptor and are only
weak agonists for genomic responses (145, 340). In addi-
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The most straightforward way to investigate the involvement of a protein in a physiological process is the
generation of an organism that has the respective gene
disrupted or knocked out. The mineralocorticoid receptor
(MR) knock-out mice MR⫺/⫺, obtained by gene targeting
by Berger et al. (48), die between day 8 and 13 after birth,
with a markedly reduced weight and a severe dehydration
due to failure of sodium reabsorption. They show all signs
of pseudohypoaldosteronism, such as hyperkalemia, hyponatremia, and a strongly activated renin-angiotensinaldosterone system.
Measurements of intracellular calcium were performed
in wild-type mice skin fibroblasts and in fibroblasts from
MR-knock-out (KO) mice after stimulation with aldosterone (207). The basal values of intracellular Ca2⫹ concentration ([Ca2⫹]i) were not significantly different in wildtype and KO fibroblasts, respectively. After addition of 10
nM aldosterone, the [Ca2⫹]i increases in wild-type and
mutant cells were both significant versus baseline levels,
with a trend to even larger and prolonged signals in cells
from KO mice. The response was almost complete within
1 min. In contrast, neither stimulation with 10 nM nor with
1 ␮M cortisol showed a significant increase of intracellular calcium in wild-type cells.
In addition to intracellular calcium measurements,
cAMP levels were determined and basal levels found to be
similar in wild-type and KO mice (207). Incubation of the
cells with 10 nM aldosterone increased intracellular
cAMP levels 2.2-fold within 1 min in wild-type fibroblasts
and interestingly even more in KO mice cells (10.6-fold
increase).
Preincubation of the wild-type mice cells with 10 ␮M
spironolactone, an antagonist at the classic mineralocorticoid receptor, did not significantly decrease aldosterone-induced effects on intracellular cAMP levels (207).
Because aldosterone was used at physiological concentration, unspecific membrane interaction is unlikely.
However, in vitamin D receptor KO cells, nongenomic
effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] such as
opening of ion channels were not detectable (A. W. Norman, personal communication).
Further experiments involving classic receptor KO
animals include ER␣ KO mice, which still exhibit estradiol-induced extracellular regulated kinase (ERK) phosphorylation (483). This observation, however, awaits confirmation in a double (ER␣/ER␤) KO animal. Other experiments conducted with neurons from ER␣ KO mice
demonstrated that the estradiol-induced potentiation of
kainate-induced currents is still present (193). Preincubation with ICI 182780, which is supposed to block both
ER␣ and ER␤, did not blunt the estrogen effect. The
sensitivity toward the cAMP thio analog adenosine 3⬘,5⬘cyclic monophosphothioate, Rp isomer (Rp-cAMPS), suggests a cAMP-dependent pathway.
In ovariectomized and estradiol-primed PR KO mice,
intravenous infusion of progesterone increased lordosis
within 10 min, a phenomenon identical to that seen in
wild-type mice (160).
The rat analog of the porcine progesterone binding
membrane protein (mPR), 25-Dx (436), has been shown to
be expressed to a higher level in female PR KO mice than
in their wild-type cognates (238). Together with other
data on the regulation of its expression, a mechanism has
been suggested by which 25-Dx expression is repressed
through activated PR, which shares some similarities with
regulation in the case of aldosterone.
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tion, the diastereomer 1␤,25(OH)2D3, while unable to
block the genomic effects of 1␣,25(OH)2D3, was found to
be a potent inhibitor of transcaltachia (the rapid, nongenomic stimulation of calcium transport) (339) and
other effects (343).
Extensive studies on structural features of vitamin D
analogs with regard to their differential activities in the
genomic and nongenomic pathways have been elaborated
in chick intestine and osteoblast/ROS 17/2.8 cells (20, 122,
341 and others). Aside other analogs, the 6-s-cis-isomer of
1,25(OH)2D3 has been found to specifically stimulate nongenomic effects such as transcaltachia, but not genomic
biological responses. EC50 values for those effects are in
the subnanomolar, thus physiological, range.
A series of vitamin D3 analogs have been identified
that activate only subsets of the biological responses of
the hormone (342, 343) (see Fig. 2). So far, this feature is
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unique among all other steroid hormones. Several studies
clearly show that the structural characteristics of the
analogs that activate membrane-initiated, rapid pathways
(cis-conformation) are distinct from those that bind to the
nuclear receptor (338) and are only weak agonists for the
genomic responses (63, 542). In contrast, trans-locked
analogs did not promote transcaltachia even at concentrations up to 6.5 nM, and a further analog, the 1␤-epimer
of 1,25(OH)2D3 [1␤,25(OH)2D3], has been found to antagonize the 1␣,25(OH)2D3-mediated response (339). The addition of hybrid analogs of 1␣,25(OH)2D3 modified at the
A-ring and the C,D-ring side chain were described to have
an influence on proliferation rate, proteoglycan production, and protein kinase C (PKC) activity of rat chrondrocytes (66). However, effective binding of these analogs to
the classic VDR was only 0.1% relative to genuine
1␣,25(OH)2D3.
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FIG. 2. Structures of 1␣,25-dihydroxyvitamin D3
[1␣,25(OH)2D3] and some of its analogs. Top: limits of
the conformers generated by rotation around the 6,7
carbon bond. While the 6-s-cis-form resembles a “real”
steroid, the 6-s-trans-form has much less structural similarity. Middle and bottom: analogs of 1␣,25(OH)2D3 that
exhibit certain subsets of its properties. 1␣,25(OH)2lumisterol3 and 1␣,25(OH)2-tachysterol3 are cis- and
trans-locked conformers, i.e., they are unable to undergo rotation. 1␤,25(OH)2D3 is a diastereomer that is an
antagonist of certain rapid responses to 1␣,25(OH)2D3.
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been interpreted as different specificity. This hypothesis
has been challenged, however, by a recent study which
demonstrated that the previously inactive cortisol becomes equally active as aldosterone in rapidly elevating
intracellular pH after addition of carbenoxolone, a 11␤hydroxysteroid dehydrogenase inhibitor (5). This finding
is a striking argument for the involvement of classic MR.
The rise in pH could not be blunted, however, by spironolactone at very high excess, nor by the spirolactone RU
26752. The open-ring congener of the latter, RU 28318,
was able to block the aldosterone-induced effects (see
Fig. 4). From binding studies it is known that RU 28318 is
only a very weak ligand at the MR, as is canrenoate which
has almost two orders of magnitude lower affinity than its
counterpart canrenone (369). Both are, however, almost
equally effective in vivo, as they are converted into each
other and an equilibrium is rapidly established, and the
same is true for RU 28318. The phenomena described by
Alzamora et al. (5) thus share some of the properties of
the classic MR, which is sometimes taken as evidence for
its participation (163), but the sensitivity toward inhibitors is entirely reversed. As ligand selectivity is governed
by molecular attraction and repulsion, it is a function of
receptor (or its ligand binding domain’s) structure. Thus it
is hard to imagine that the same or a very similar ligand
binding domain preserves only part of its selectivity, but
reverses the other (inhibitor) part.
Evidence based on pharmacology generally is not
regarded as compulsory, due to the many cellular events
that are blurring the observation. However, to explain the
data from the study mentioned above by involvement of
classic MR, a cellular mechanism needs to be hypothesized that selectively transforms a (in vitro) weak antagonist (RU 28318) into a strong one (possibly the closedring RU 26752), but at the same time prevents the highaffinity antagonists spironolactone and RU 26752 (if
added directly) from acting at the MR. Given the ligand
(and antagonist) selectivity pattern seen here, a distinct
nonclassic receptor with different selectivity is the more
convincing explanation.
3. Systems where the classic receptor has not
been demonstrated
Several systems have been thoroughly investigated to
demonstrate one or the other classic receptors. The methods employed mainly comprise immunochemistry with
antibodies raised against the respective receptor, and RTPCR. Obviously, a negative proof in such cases is more
difficult, as the failure to detect the protein in question or
its mRNA could also be a matter of sensitivity, or very low
levels that are (falsely) detected may be inferred by impurities in samples. A convincing example is the testosterone action on [Ca2⫹]i in IC-21 macrophages (46). In
these cells, the classic androgen receptor (AR) was not
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The interconversion between the 6-s-trans- and 6-scis-forms has a low energy barrier and therefore occurs
rapidly in solution at room temperature, generating multiple intermediate conformers. As yet, the true equilibrium
ratio of the 6-s-trans- and 6-s-cis-conformers of any vitamin D seco steroid, including 1,25(OH)2D3, has not been
rigorously determined. It has been estimated by computational methods that 88 –99% of 1,25(OH)2D3 exists as the
6-s-trans-conformation, with only 1–12% existing in the
6-s-cis-form. Obviously, not all 1,25(OH)2D3 diastereomers are as active as 1␣,25(OH)2D3. With regard to the
potency for transcaltachia and for 45Ca2⫹ influx in ROS
17/2.8 cells, the diastereomers are different (342). Due to
the facile interconversion of the 6-s-trans- and 6-s-cisconformers of 1,25(OH)2D3, there exist kinetically competent amounts of all conformers available to interact
with any receptors which may, in turn, be linked to the
generation of biological responses.
B) ALDOSTERONE. The pharmacology of the classic MR
has been studied in great detail. Agonists include the
natural and most potent ligand aldosterone, but also deoxycorticosterone, corticosterone (physiologically important in rodents), and cortisol. While aldosterone is bound
by the isolated MR with a dissociation constant (KD) ⬃1
nM, cortisol binds to the same receptor with almost equal
affinity (11, 38, 239, 324). However, under physiological
conditions, the MR is thought to be protected by several
mechansims that metabolize “undesired” ligands to compensate for its low intrinsic specificity (346, 389).
The inhibition of MR activation is beneficial in a
number of diseases, and therefore numerous drugs with
MR antagonist activity have been synthesized and studied,
with spironolactone being the most popular. It has been
shown that under in vitro conditions, the presence of a
lactone ring spiro to the D ring is a prerequisite for
high-affinity binding (369), which is present in spironolactone as well as in its major metabolite canrenone. In vivo,
the open-ring compound canrenoate is rapidly equilibrated with its conversion product canrenone, an active
MR antagonist, while canrenoate is only a very weak MR
ligand in vitro (369) (see Fig. 3).
The relative agonistic and antagonistic activities of
the drug compounds described above have often been
used to distinguish between mechanisms involving the
classic MR and other pathways on a pharmacological
basis. Whereas aldosterone induced phenomena that can
be blunted by the addition of spironolactone (or other
lactone antagonists) point to the involvement of the classic MR, spironolactone insensitivity has reasonably been
interpreted as evidence for the presence of a different
receptor.
Numerous studies have described aldosterone effects that could not be blocked by spironolactone. In
addition, many of these effects were not elicited by cortisol despite its high affinity to the MR, which has often
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detectable by several experimental approaches: immunostaining, Western blotting, as well as RT-PCR using several primer pairs. However, testosterone and testosterone-BSA-fluorescein isothiocyanate (FITC) conjugate
both induced a [Ca2⫹]i response, and confocal techniques
demonstrated exclusive membrane localization of the fluorescent steroid conjugate (for a critical discussion of
steroid conjugates, see sect. V). The human prostatic cell
line PC3 also was reported to be devoid of AR (270). In
these cells, testosterone still induces rapid transient
[Ca2⫹]i increases.
In neuronal cells lacking a functional ER, 17␤-estradiol still activated mitogen-activated protein kinase
(MAPK) signaling and augmented the release of amyloid
␤-precursor protein (279).
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Some other examples are discussed in the sections
on individual steroids.
A different, yet striking example is the rapid effect of
aldosterone on calcium influx and intracellular cAMP in
human proximal tubular cells (unpublished results).
These cells apparently do not have a functional MR, and
these effects thus strongly suggest involvement of a nonclassic receptor.
D. Additional Evidence For and Against
Distinct Receptors
This section serves to summarize evidence for the
involvement of either classic or nonclassic receptor mol-
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FIG. 3. Molecular structures of aldosterone, the physiological ligand of the mineralocorticoid receptor (MR), and some of its antagonists. The closely related cortisol binds to isolated MR with roughly equal affinity but is
converted to an inactive form in vivo. The
closed-ring compounds spironolactone, canrenone, and RU 26752 effectively displace aldosterone from MR, thus explaining their antagonistic action, whereas the open-ring compounds
canrenoate and RU 28318 are known to be very
weak competitors. In vivo, however, an equilibrium forms between open-ring and closed-ring
structures (indicated by arrows). Interestingly,
the open-ring compound RU 28318 inhibits nongenomic aldosterone action, whereas spironolactone is inactive (cf. Fig. 4).
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ecules, or even different mechanisms such as allosteric
action.
Several enzymes have been shown to be susceptible
to activity modulation by steroids, e.g., mitochondrial
ATPase (541). However, the concentrations required often exceed the physiological range by far. Such mechanisms shall not be discussed in detail here.
1. Glucocorticoids
Specific nongenomic glucocorticoid effects have
been suggested for a long time to be mediated via membrane-bound receptors, whereas nonspecific nongenomic
effects are thought to occur due to physicochemical membrane interactions.
Membrane-binding sites for different glucocorticoids
have been described in many tissues and cells. In plasma
membranes from chicken liver, mouse liver, and rat liver,
specific binding sites for cortisol could be demonstrated
(218, 485, 489). In rat liver plasma membranes, two types
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FIG. 4. Effect of aldosterone antagonist on hormone-mediated
increment in Na⫹/H⫹ exchanger activity. Spironolactone or RU 28318
was added to the perfusion medium 10 min before aldosterone and was
maintained throughout the experiment. Parallel samples were run in the
presence of aldosterone alone. Determinations of intracellular pH were
made in chorionic arteries (n ⫽ 4 experiments for each antagonist).
[From Alzamora et al. (5), copyright Lippincott Williams & Wilkins.]
of binding sites for cortisol have been detected, a highaffinity site with low binding capacity and a low-affinity
site with high binding capacity. Further analysis of the
high-affinity binding site by SDS-PAGE showed two protein subunits of 52 and 57 kDa, respectively, and binding
affinities distinct from those of the glucocortocoid receptor (GR) (218). In addition, the binding of corticosterone
to murine and rat liver plasma membranes, to rat kidney
and rat brain plasma membranes, and to calf adrenal
cortex plasma membranes has been shown (219, 486, 487
and others).
Another glucocorticoid responsive site has been
shown in a highly purified rat liver plasma membrane
fraction. This site mediates active transmembrane transport of corticosterone, and it does not seem to be a
member of the ABC transporter or multidrug resistance
transporter superfamily (4, 242).
In 1991, Orchinik et al. (352) identified a corticosteroid receptor in synaptic membranes prepared from
taricha granulosa brain that showed high-affinity binding
of 3H-labeled corticosterone (352). Furthermore, the affinities of corticoids for this binding site were linearly
related to their potencies in rapidly suppressing male
reproductive behavior, thus suggesting its participation in
the regulation of behavior. Evidence for the rapid modulation of Taricha behavior by binding of steroid hormones
to specific membrane-associated receptors is reviewed in
detail by Moore and Orchinik (312) and Moore et al. (313).
In equilibrium saturation binding studies and in titration
studies, nonhydrolyzable guanyl nucleotides were able to
inhibit the binding of 3H-labeled corticosterone to neuronal membranes. The addition of Mg2⫹ enhanced both the
equilibrium binding of [3H]corticosterone and the sensitivity of the receptor to modulation by guanyl nucleotides,
properties that are typical for G protein-coupled receptors (351). Partial purification and further biochemical
characterization of the roughskin newt membrane glucocorticoid receptor revealed a glycoprotein with an apparent molecular mass of 63 kDa and a pI of ⬃5.0. These
characteristics differ from classical glucocorticoid receptor and give strong evidence that these two receptor
proteins are distinct (135). Ligand-binding competition
studies on neuronal membranes of the roughskin newt
demonstrated the potencies of a subset of kappa opioid
ligands to displace [3H]corticosterone binding to the neuronal membranes (136). Displacement was achieved by
dynorphin 1–13 amide, U 50488, naloxone, bremazocine,
and ethylketocyclazocine. Kinetic analysis suggested a
direct rather than an allosteric interaction with the
[3H]corticosterone binding site. The authors conclude
that their results support the hypothesis that the highaffinity membrane binding site for [3H]corticosterone is
located on a kappa opioid-like receptor.
A very compelling comparison of the steroid binding
characteristics of corticosteroid binding sites in plasma,
NONGENOMIC STEROID ACTION
Physiol Rev • VOL
the regulation of the expression of GR and mGR. In the
brain of wild-caught house sparrows it could be shown
that the levels of membrane corticosteroid receptors varied seasonally, being significantly lower during the nesting season than during molting or wintering stages. Affinity of the membrane receptor was not influenced by the
seasonal changes. Furthermore, the cytosolic and membrane receptor numbers were regulated in opposite directions, and thus suggesting not only different functions for
membrane and intracellular receptors but also different
mechanisms that regulate their expression (71).
From the findings discussed above, there is evidence
for both nonclassic receptors and a membrane form of
classic GR, probably originating from alternate transcription, that may mediate nongenomic responses to glucocorticoids.
2. Progesterone
As discussed in section IIIB, it has been shown that
progesterone nongenomically acts on oocyte maturation
in Xenopus. In the past, it has been assumed that the
actinomycin D-insensitive progesterone-induced maturation (456) is mediated by cell surface rather than nuclear
receptors. Thus progesterone covalently attached to either BSA or polymers can induce maturation when applied to the outside of an oocyte (181, 277). However, this
approach is controversial (see sect. IV). Moreover, free
progesterone can induce maturation when applied to the
outside of an oocyte but not when injected directly into
oocytes (277, 284). With regard to this assumption, several membrane progesterone binding sites have been described (reviewed in Refs. 276, 320).
On the other hand, two recent papers reported the
cloning and characterization of cDNAs for Xenopus homologs of the classic PR, termed XPR and XPR-1 (34,
482). In both papers indexes for the involvement of this
homolog in progesterone-induced maturation are given.
XPR-1 antisense oligonucleotides were found to inhibit
progesterone-induced maturation (482), and the injection
of a truncated XPR-mRNA was found to accelerate oocyte
maturation (34). In both cases, the ability of XPR to
promote maturation was not affected by actinomycin D
(34, 482). In a further work, the role of XPR in nongenomic signaling in Xenopus oocytes was examined
(14). The authors found that a fraction of one form of XPR
was attached to the plasma membrane. In addition, it has
been shown that progesterone causes XPR to become
associated with phosphatidylinositol (PI) 3-kinase activity, and an interaction of XPR with p42 MAPK was observed (14).
Recently, a progestin maturation inducing steroid receptor has been characterized on spotted seatrout oocytes (481). The deduced amino acid sequence of this
protein shows seven putative transmembrane domains.
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brain cytosol, and neuronal membranes was reported by
Orchinik et al. (350). Most interestingly, the neuronal
membrane preparations were very selective for corticosterone with affinities more than three orders of magnitude lower for the GR agonist dexamethasone and the
antagonist RU 486. The plasma binding sites, presumably
corticosteroid binding globulins, were less selective. In
brain cytosol, RU 486 bound with highest affinity, followed by dexamethasone ⬃ corticosterone. The authors
suggested that the various types of binding sites can be
unambiguously identified using selectivity profiles and
that rapid responses to corticosteroids in amphibian
membranes are unlikely to be mediated by receptors that
are either related to classic receptors or corticosteroid
binding globulin.
On the other hand, and apart from the results described above, there is evidence that a specialized form of
the GR is localized in the plasma membrane (165). As
already mentioned above, the lysis response of S-49
mouse T-lymphoma cells to glucocorticoids has been, by
means of an anti-GR antibody, attributed to a glucocorticoid receptor-like antigen that is located in the lymphoma
cell membranes (164). Subsequently, membrane glucocorticoid receptors have been reported in rat liver cells (192),
human leukemic cell lines, and cells from human leukemic patients (168). With the use of multiple segregation
techniques, it was possible to separate different sublines
of murine S-49 lymphoma and human CCRF-CEM T-leukemia lines and thus achieve populations enriched and
depleted for glucocorticoid membrane receptors (167,
415). Immunoaffinity purification and Western blot analysis of membrane extracts from these membrane receptor-enriched cell lines showed a membrane GR (mGR)
pattern in a molecular mass range from 42 to 150 kDa
(381). The differences in size between GR (94 kDa) and
mGR (150 kDa) have been suggested to be due to posttranslational modifications of the membrane receptor
(166). Together with mGR, the heat shock proteins hsp70
and hsp90 were coprecipitated as demonstrated by monoclonal antibodies, thus suggesting an interaction of mGR
with these molecules under physiological conditions, although its role remains unclear at the moment (381).
The expression of mouse membrane GR is highly
correlated with the expression of a distinct GR transcript
denoted transcript 1A. The gene encoding the murine GR
spans ⬃110 kb, and the transcripts are assembled from
nine exons. There are five glucocorticoid receptor transcripts known so far (1A-1E), all differing only at their
5⬘-ends and generated by alternative splice mechanisms
in exon 2 (92, 93, 170, 467).
Interestingly, within the 5⬘-untranslated region of GR
transcript 1A there are five small open reading frames
(ORFs), one of them encoding a peptide of ⬃8.5 kDa that
seems to be associated with the translational regulation of
GR (117). Indeed, there is some evidence for a relation in
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in several tissues or cells. For instance, in brain plasma
membrane fractions, progesterone conjugated to radiolabeled BSA has been frequently used to identify putative
steroid receptors. In the medial preoptic area-anterior
hypothalamus of ovariectomized rats, high- and low-affinity progesterone binding sites have been described that
are likely to be associated with G proteins (83, 84, 392,
540). Moreover, photoaffinity labeling done with a progesterone analog in mouse brain membranes identified four
protein bands with molecular masses ranging from 29 to
64 kDa (76).
Progesterone and related compounds were also
found to bind to GABAA receptors (322, 550). Interestingly, in appropriate KO mice, the absence of the ␥-subunit of GABAA receptor leads to a decrease in sensitivity
to neuroactive steroids such as pregnanolone (306).
Consistent with reports on rapid actions of progesterone in the ovary, specific membrane progesterone
binding sites have been discovered in luteal membranes
from various species. However, the binding of the steroid
could only be demonstrated in the presence of digitonin
(68, 297, 390).
In granulosa cells of immature rats, progesterone
was found to inhibit apoptosis (364). It is not likely that
this action is mediated through the classic nuclear PR,
since this protein does not seem to be expressed in these
cells (363). Alternatively, a 60-kDa membrane protein has
been detected in granulosa cells by the use of an antibody
directed against the steroid binding domain of nuclear PR
(c-262) (365). In ligand blot and ligand binding studies, it
has been shown that this protein specifically binds progesterone (363).
The progesterone metabolites 5␣-pregnane-3,20-dione and 3␣-hydroxy-4-pregnen-20-one, which are assumed to modulate breast cell proliferation, were found
to bind specifically to the membrane fraction of MCF-7
breast cancer cells. In saturation analyses, the respective
bindings sites showed dissociation constants of 4.5 and
484 nM, respectively (522).
In search of specific steroid membrane binding sites
and with regard to the reports on rapid progesterone
effects on liver cell conductance (504) and progesterone
membrane binding sites in hepatocytes (217, 488), our
group was able to characterize membrane progesterone
binding sites (mPR) from porcine liver microsomes. After
purification, the binding capacity corresponds to the enrichment of polypeptides of 28 and 56 kDa with the latter
probably representing a dimer of the 28-kDa protein (139,
301). However, the native progesterone membrane receptor is likely to be an oligomeric protein complex of ⬃200
kDa composed at least in part of the 28- and 56-kDa
proteins (300). Interestingly, mPR was localized to intracellular membranes (143). Because the hydrophobicity of
steroids allows for rapid entrance into cells, this finding is
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The protein may therefore represent a G protein-coupled
membrane receptor.
In addition to the well-studied nongenomic steroid
effects in sperm, the existence of corresponding membrane receptors has been examined. The first evidence
supporting the presence of steroid binding sites on human
spermatozoa came from a work by Hyne and Boettcher
(216) where specific 17␤-estradiol receptors were detected for which progesterone could compete. The hypothesis of progesterone receptors on the surface of human sperm was further substantiated by experiments
done with membrane-impermeable agents like BSA-conjugated progesterone (60, 292) and progesterone-BSAFITC (480). Many features of the sperm response to progesterone and contradictory findings with regard to the
presence and the state of the protein involved suggest the
presence of different types of progesterone receptors in
sperm (reviewed in detail in Ref. 400).
The presence of the classic intracellular PR in spermatozoa is discussed controversially. Antibodies directed
against the DNA binding domain of PR did not detect any
specific band (267, 413), confirming earlier experiments
that demonstrated PR not to be present in human sperm
(87). In different studies where an antibody raised against
the steroid binding domain of PR (c-262) was used, protein bands with molecular masses of 50 –52 kDa (413) or
54 and 57 kDa were labeled (267). The application of this
antibody leads to an inhibition of progesterone-induced
Ca2⫹ influx and the acrosome reaction (413). However,
since human PR consists of a ⬃94 kDa A form and a ⬃120
kDa B form (132), the marked proteins are noticeably
smaller in size. Nevertheless, several exon-deleted or
truncated PR variant mRNAs have been observed, at least
in breast cancer cells (253). Recently, a PR transcript
comprising the DNA binding domain as well as the hormone binding domain, has been detected in RT-PCR experiments (414).
In human sperm extracts, analogous to our findings
in porcine liver microsomes (see below), proteins with
molecular masses of 25–30 kDa and 50 – 60 kDa could be
labeled by antibodies against the rat homolog of mPR
(25Dx) (57). Therefore, the existence of an, at least related, progesterone membrane binding protein is very
likely in sperm. For this reason, our group investigated
the influence of a mPR-specific antiserum on rapid progesterone-induced Ca2⫹ fluxes in human sperm. In the
presence of the IgG fraction of a specific antibody (1:10
dilution), a significantly reduced progesterone-induced
Ca2⫹ increase was found (140). These data are in accordance with recent experiments done with the same mPRspecific antibody. Thereby, and by using the same antibody, an inhibition of the progesterone-initiated acrosome reaction by 62% was found (75).
Besides the reproductive system, the presence of
membrane progesterone binding sites has been examined
NONGENOMIC STEROID ACTION
3. Estrogens
Estrogens classically exert their biological effects by
activation of ERs modifying gene expression. Again, ERs,
which regulate the transcriptional activity of target genes
Physiol Rev • VOL
by interacting with different DNA response elements, exist at least in two subtypes, ER␣ and ER␤, deriving from
different genes. In vitro studies suggest that both receptors may play redundant roles in estrogen signaling; however, tissue localization studies indicate different expression patterns for each receptor (198). In addition to their
effects on gene transcription, ERs may be involved not
only in genomic steroid action, but also in rapid nontranscriptional, nongenomic effects. Such nongenomic effects
are initiated at the plasma membrane and are postulated
to be mediated by a membrane-bound ER that seems to be
very similar, if not identical, to the classical intracellular
ERs.
One of the fundamental studies was done in rat pituitary tumor cell lines. Antibodies raised against epitopes
of ER stained membrane proteins of immuno-selected
GH3/B6 cells (354). Moreover, with the use of several
antibodies to ER␣, the membrane version of ER␣ could
be detected making it likely that the membrane and nuclear proteins are highly related. Estrogen at very low
concentrations induces prolactin release from GH3/B6
cells within minutes of application, and the prolactin
release is also elicited or inhibited by ER␣-specific antibodies (512). Similar results could be obtained in CHO
cells transfected with both of the receptors subtypes:
small amounts of both ER␣ and ER␤ were expressed at
the plasma membrane (394).
The evidence of a membrane localization of the subtype ER␣ could be confirmed in cultured hippocampal
neurons. Antibodies directed against ER␣ showed positive membrane staining in nonpermeabilized neurons. On
the other hand, in permeabilized hippocampal neurons,
the labeling for ER␣ could be detected in the perinuclear
area, but abundant staining for ER␣ was found throughout the cell including the neurites. Moreover, the immunoreactivity of ER␣ was reduced throughout the neurons
in the presence of antisense oligonucleotide directed
against the translation start site of ER␣. With the use of
conventional and confocal microscopy, most of the antigen could be localized in the extranuclear compartment
(103).
Activation of the endothelial nitric oxide synthase
(eNOS) leading to increased levels of nitric oxide (NO) is
a fundamental determinant of cardiovascular homeostasis. As previously shown, 17␤-estradiol rapidly stimulates
the eNOS of cultured endothelial cells, which can be
completely blocked by the classical ER antagonists tamoxifen and ICI 182780 (88, 245). Moreover, the stimulation of eNOS by 17␤-estradiol was further enhanced by
overexpression of ER␣ (443). The involvement of ER␣ in
the nongenomic actions of estrogens was confirmed by
the demonstration that the rapid response of eNOS to
17␤-estradiol could be detected in COS-7 cells transfected
with the wild-type ER␣ and eNOS, but not by transfection
with eNOS alone. Again, inhibitors of Ca2⫹ influx, tyrosine
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still compatible with the assumption that mPR may be
involved in nongenomic progesterone action.
Sequence analyses demonstrated that mPR has no
significant identity to any protein with currently known
function, thus nor to the intracellular PR or other steroid
receptors (141, 174). Expression of the mPR-cDNA in
Chinese hamster ovary (CHO) cells leads to an increase in
microsomal progesterone binding compared with microsomes from mock-transfected CHO cells (140). In this
regard, high-affinity binding of progesterone to microsomes from intact bovine lens epithelium has been reported. Using a mPR-specific antibody, a microsomal 28kDa protein with high homology to mPR was detected
(90, 546).
Interestingly, the rat equivalent of mPR, termed
25-Dx (436), has been associated with behavioral processes. In the ventromedial hypothalamus of rats, 25-Dx
expression has been shown to be repressed by progesterone after estradiol priming of ovariectomized animals.
Moreover, compared with wild-type littermates, higher
expression levels of 25-Dx were seen in female PR knockout mice, suggesting a scenario in which the activation of
the intracellular PR represses the expression of 25-Dx
(238).
A very recent addition to the candidate receptors for
rapid progesterone action comes from Zhu et al. (547). A
40-kDa membrane protein was identified after screening a
cDNA library from spotted seatrout ovary, possessing
seven predicted transmembrane domains, which is characteristic for G proteins. Protein recombinantly expressed in Escherichia coli bound progesterone with high
affinity (KD ⫽ 30 nM) and had significant preference for
21-carbon steroids unsubstituted at carbon 11. In a mammalian cell line transfected with the novel receptor, progestins activate a MAPK pathway. This finding together
with changes in protein abundance in response to hormones in the course of oocyte development suggests that
this is a novel, membrane progesterone receptor with a
defined action. Other homologous genes have been demonstrated, and members of this gene family have been
detected in human tissues (548).
For progesterone-induced effects, much evidence
points to the existence of nonclassic receptors, particularly for the well studied induction of the acrosome reaction. Evidence for classic receptors is mainly drawn from
immunochemical studies, with the exception of Xenopus,
where two recently published studies (14, 34; see sect. IV)
convincingly demonstrate a role of the classic PR in nongenomic events.
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FIG. 5. Role of G proteins in prostaglandin E2 (E2)-stimulated
endothelial nitric oxide synthase (eNOS) activity. A: effect of exogenous
guanosine 5⬘-O-(2-thiodiphosphate) (GDP␤S) on eNOS stimulation in
endothelial cell plasma membranes. The conversion of L-[3H]arginine to
3
L-[ H]citrulline was measured in purified plasma membranes incubated
for 30 min under basal conditions or in the presence of 10⫺8 M E2 in
buffer alone or buffer plus 2 mM GDP␤S. B: effect of pertussis toxin
(PT) on eNOS stimulation in COS-7 cells. Values are means ⫾ SE for
NOS activity in fmol L-[3H]citrulline/well for intact cells and in pmol
citrulline䡠mg protein⫺1䡠min⫺1 for plasma membranes (n ⫽ 3). *P ⬍ 0.05
versus basal; †P ⬍ 0.05 versus no PT or no GDP␤S. [From Wyckoff et al.
(534), copyright 2001 The American Society for Biochemistry and Molecular Biology.]
or on adenylyl cyclase and thus indirectly on PKA, uncoupling the ␮-opioid receptors from the ion channels (229).
Many of the findings on MAPK activation by estrogens were obtained in tissues containing both ER␣ and
ER␤. To individually explore the role of each ER in the
rapid activation of the MAPK signaling pathway, ER-negative RAT-2 fibroblasts were transfected with DNA clones
encoding either ER␣ or ER␤. For both receptors, 17␤estradiol induced a rapid phosphorylation of MAPK. Differences were found for the time course of activation:
after activation, MAPK dropped within 15 min to control
levels in ER␣ cells, whereas in ER␤-transfected cells,
MAPK remained activated for at least 1 h. This may be
important, since the timing and duration of MAPK activity
seems to be essential for nerve growth factor (NGF)-
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kinases, or MAPK inhibited the stimulation of eNOS by
17␤-estradiol (442). Similar results were obtained in human umbilical vein endothelial cells: 17␤-estradiol and the
17␤-estradiol-BSA-conjugate activated MAPK as well as
cGMP synthesis and NO release, whereas ICI 182780
blocked these effects (412).
In bovine aortic endothelial cells (BAEC), 17␤-estradiol rapidly increases cGMP release; this effect is sensitive to the ER antagonist ICI 164384 (182). In the same cell
system, NO synthesis is augmented rapidly by both 17␤estradiol and BSA-17␤-estradiol-conjugate, and the ER␣
antagonist ICI 182780 completely blocked estrogen-stimulated NO release, which supports the assumption that a
plasma membrane receptor similar to ER is involved in
these effects (231).
Results of a recently published investigation indicate
that G proteins could be involved in the signaling of the
nongenomic actions of 17␤-estradiol (Fig. 5). 17␤-Estradiol induced increases of NOS activity in intact endothelial cells that were fully blocked by the G protein inhibitor
pertussis toxin. Coimmunoprecipitation investigations of
the plasma membrane of COS-7 cells transfected with
ER␣ and specific G␣ proteins demonstrated that 17␤estradiol induced interactions between ER␣ and G␣i. The
observed ER␣ G␣i interaction was inhibited by ICI 182780
and pertussis toxin. Cotransfection of G␣i into COS-7
cells expressing ER␣ and eNOS led to a pronounced
increase in 17␤-estradiol-mediated eNOS stimulation
(534).
Further insights in the molecular mechanisms involved in the activation of eNOS could be obtained in a
human endothelial cell hybridoma line. 17␤-Estradiol rapidly induced phosphorylation and activation of eNOS
through the PI 3-kinase-Akt pathway. One of the downstream targets of the PI 3-kinase pathway is the serine/
threonine kinase Akt, also referred to as protein kinase B
(PKB). Moreover, the 17␤-estradiol-induced NO release
could be blocked by the PI 3-kinase inhibitor LY 294002,
and 17␤-estradiol and the BSA-17␤-estradiol-conjugate
both increased the phosphorylation of Akt and eNOS. The
functional involvement of Akt could be confirmed using
adenoviral approaches, since a dominant-negative Akt
abolished the 17␤-estradiol-stimulated NO release (208).
Other findings that point to the modulation of a G
protein-coupled receptor by an ER have been reported
earlier (243). Here, estradiol attenuated the hyperpolarizing action of ␮-opioids on hypothalamic neurons. Interestingly, both the ER agonist diethylstilbestrol and the
antagonist ICI 164384 blocked the estradiol effect. Because the response could be mimicked by protein kinase
A (PKA) activators and blocked by the corresponding
inhibitors, PKA is apparently involved. More detailed
studies revealed the involvement of PKC, that may act
directly to uncouple GABAB receptors from K⫹ channels,
NONGENOMIC STEROID ACTION
Physiol Rev • VOL
that the membrane receptor of 17␤-estradiol belongs to
membrane receptors that are coupled to phospholipase C
via a pertussis toxin-sensitive G protein. Moreover, the ER
blockers tamoxifen and raloxifene did not inhibit the
rapid increase of [Ca2⫹]i of IC-21 cells induced by both
17␤-estradiol and its BSA-conjugate. The membrane receptor properties of 17␤-estradiol found in IC-21 cells
seem to be similar to other G protein-coupled receptors
including sequestration after ligand binding. Also, the particular receptor becomes sequestrated within minutes after binding of 17␤-estradiol. The reason for the 17␤-estradiol receptor internalization remains unknown (47).
Moreover, the assumption that many of the effects of
17␤-estradiol involve membrane-associated mechanisms
independent of classical ERs has recently been revisited.
It was demonstrated that 17␤-estradiol-BSA-conjugate induced time-dependent increases in PKC in resting zone
(RC) and growth zone chondrocytes (GC) from female rat
costochondral cartilage within minutes. These effects
could be prevented in the presence of the G protein
inhibitor guanosine 5⬘-O-(2-thiodiphosphate) (GDP␤S) in
both cell lines, whereas the G protein activator guanosine
5⬘-O-(3-thiotriophosphate) (GTP␥S) increased PKC in
17␤-estradiol-BSA-conjugate incubated GC cells but had
no effect in RC cells. In both RC and GC cells, the phospholipase C (PLC) inhibitor U 73122 blocked 17␤-estradiol-BSA-conjugate stimulated PKC activity, whereas the
phospholipase D (PLD) inhibitor wortmannin had no effect. Neither the classical ER antagonist ICI 182780 nor
the agonist diethylstilbestrol had any inhibitory or stimulatory effects. The nongenomic mechanism of the 17␤estradiol-BSA conjugate seems to be dependently mediated by G protein-coupled PLC (474).
For many of the nongenomic estradiol effects, evidence suggests the participation of either classic ER itself
or a closely related form located at the membrane. However, receptors seem to exist that extensively differ in
their properties and are unlikely to be related to ER.
4. Androgens
In the past 25 years, membrane androgen binding
sites that are discussed to be responsible for rapid androgen signaling have been described in several tissues or
cells (for review, see Ref. 144). But of late, the nature of
these binding sites has been controversial.
Recently, binding sites for testosterone on the surface of T cells and IC-21 macrophages have been detected
with testosterone-BSA-FITC that are likely to be involved
in the context of nongenomic testosterone action on
[Ca2⫹]i (44, 46). However, in IC-21 macrophages, the absence of the classic AR has been demonstrated even by
independent experimental approaches. Incubation of the
cells with an anti-AR antibody (directed against the NH2
terminus) did not significantly label the cells, and in West-
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induced cellular differentiation. Moreover, the activation
of MAPK in ER␣ transfected cells could be partially or
completely blocked by tamoxifen and ICI 182780. In contrast, in ER␤-transfected cells both compounds were less
sensitive to inhibit that effect (502).
In addition to its function in the female reproductive
system, 17␤-estradiol plays an important role also in the
development and regulation of the male reproduction
system. Results of ligand blot analysis indicate a specific
estradiol binding protein in human sperm. The same protein band could be found by an antibody directed against
the steroid binding domain of the classic ER (␣H222). The
binding of 17␤-estradiol was correlated with a rapid and
sustained increase of [Ca2⫹]i. These effects on [Ca2⫹]i
could also be obtained by the use of the BSA-17␤-estradiol-conjugate, which is incapable to penetrate the plasma
membrane (18). Furthermore, the receptor seems to be
involved in the activation of two different signal transduction pathways. In addition to the increase of [Ca2⫹]i,
17␤-estradiol stimulated tyrosine phosphorylation of several sperm proteins, resulting in inhibition of progesterone-induced calcium influx and acrosome reaction (268).
The data indicate that 17␤-estradiol might be able to
modulate the nongenomic effects of progesterone in human sperm during the fertilization. The effects of progesterone as a well-known stimulus for human spermatozoa
are described in detail in section IIIB.
As mentioned above, nongenomic estrogen effects
seem to be mediated by a membrane-bound ER similar to
the classical intracellular ER. Interestingly, some studies
indicate binding sites which could be different from ER.
Some years ago, it could be demonstrated that 17␤-estradiol leads within few minutes to a rapid and sustained (2
h) tyrosine phosphorylation and activates MAPKs as well
as ERK1 and ERK2 in PC12 cells. These effects can be
blocked by the MAPK/ERK1 inhibitor PD 98059, but not
by the classic ER antagonist ICI 182780. Moreover, the
ability of estradiol to phosphorylate ERK persisted in ER␣
KO mice (483). Up to now, it remains unclear if these
effects of estrogen on ERK activation are mediated by the
activation of ER␤ or by an yet unidentified ER. 17␤Estradiol can potentiate kainate-induced currents in isolated hippocampal neurons of the ER␣ KO mouse or
wild-type mice. The preservation of these rapid 17␤-estradiol actions in presence of ICI 182780, which blocks both
ER␣ and ER␤, suggests the existence of a functional membrane ER that is distinct from the intracellular nuclear form
(502). Further evidence could be obtained recently in the
mouse macrophage cell line IC-21. 17␤-Estradiol signaling
as measured by a rapid increase in [Ca2⫹]i was initiated at
the plasma membrane: the plasma membrane-impermeable 17␤-estradiol-BSA-conjugate also induced this rise in
[Ca2⫹]i. Again, this rise in [Ca2⫹]i could be inhibited by
pertussis toxin, and the phospholipase C inhibitor U
73122 blocked the release of intracellular Ca2⫹, indicating
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5. Neurosteroids
Many adrenal and gonadal steroids as well as some of
their synthetic derivatives can modulate neuron excitability and synaptic communication in the central nervous
system. For these compounds the term neuroactive steroids has been coined (274) or neurosteroids for those
which mainly glial cells can synthesize de novo (31).
These are traditional definitions, under which most steroids discussed in other sections would be covered as
well. However, this section focuses on phenomena that
are tightly linked to receptors occurring mainly in the
central nervous system. Neuroactive steroids engage the
“classical” receptors of progesterone and androgens regulating transcription through interaction with these intracellular receptors. However, nongenomic ways of action
are known for a variety of neurosteroid effects that involve other than the classical steroid receptors. Neuroactive steroids may modulate an array of neurotransmitter
receptors and regulate gene expression with an intracellular cross-talk between nongenomic and genomic effects. The most prominent among these neurotransmitter
Physiol Rev • VOL
receptors, G protein-coupled receptors, and ligand-gated
ion channels are the GABAA, the NMDA, the Sigma1, the
5-HT3, and the glycine receptors.
Just as diverse as the array of receptors and compounds involved in neurosteroid action, are the physiological effects they exert. Neuroactive steroids influence
sleep patterns, the reaction to stress, sex recognition,
memory function, brain plasticity, vigilance, and mood
(33, 410). Hence, they may have a therapeutic potential far
beyond today’s applications.
A) GABAA RECEPTOR. Due to their specific potential to
allosterically enhance or block the action of GABA or
other GABAergic substances like barbiturates, and benzodiazepines by interaction with the GABAA receptor,
neuroactive steroids may exert extremely diverse neuropsychological effects.
Depending on the specific structure of the GABAA
receptor with its subunits forming ligand-gated ion channels, neuroactive steroids can alter the excitability of
GABAergic neurons (523). GABA receptors are heteroligomeric proteins containing a number of allosterically
interacting binding sites. The neurotransmitter GABA as
well as benzodiazepines, or barbiturates, can bind to
these structures (62, 454). 3␣,5␣-Tetrahydroprogesterone
(3␣,5␣-THP) and 3␣,5␣-tetrahydrodeoxycorticosterone
(3␣,5␣-THDOC) were the first steroids that have been
shown to modulate neuronal excitability by interaction
with GABAA receptors (274). As potent barbiturate-like
ligands of the GABA receptor-chloride ion channel complex, both steroids inhibit binding of the convulsant tbutylbicyclophosphorothionate (TBPS) to the GABA receptor complex at concentrations between 100 nM and 10
␮M. As coagonists at the GABAA receptor, they also increase the binding of flunitrazepam (169). In addition, the
GABAA receptor is involved in 3␣,5␣-THP- and 3␣,5␣THDOC-stimulated chloride uptake into isolated brain
vesicles, as well as neurosteroid-potentiated inhibitory
actions in cultured rat hippocampal and spinal cord neurons (386). The pharmacological activity of benzodiazepines differs with the ␣-subunit composition and necessitates the presence of a ␥-subunit of the GABA receptor.
In contrast, the effects of neuroactive steroids do not
depend on such strictly defined basic requirements for
their structure-activity relationship. The potentiating and
direct actions of the steroids used by Puia et al. (385)
were expressed with every combination of subunits
tested. Recent studies suggest that anabolic steroids may
induce subunit-specific rapid modulation of GABAA receptor-dependent mediated currents in brain regions essential for neuroendocrine function (223). Stanozolol,
17␣-methyltestosterone, and nandrolone, steroids which
are significant drugs of abuse in adolescent girls, induced
rapid and reversible alterations in GABAergic currents.
This was demonstrated in neurons of the ventromedial
nucleus of the hypothalamus (VMN) and the medial pre-
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ern blot analyses using this antibody no immunoreactive
band was found. In addition, RT-PCR approaches using
primers spanning the DNA-binding domain and three different regions from the COOH terminus of the AR-cDNA
did not reveal the expected PCR products, whereas the
correct amplificates were found in the control RNA from
mouse testes (46). Moreover, in T cells where rapid androgen signaling has been shown (see also sect. IIC3),
anti-AR antibodies directed against the NH2 or COOH
terminus of AR did not reveal any significant fluorescence
on the cell surface. However, in contrast to the common
view that the classic AR is absent in these cells (348), an
inactive form (with regard to genomic events) of AR was
demonstrated in the cytoplasm of T cells (44).
On the other hand, a participation of the classic AR is
discussed in the dihydrotestosterone-induced rapid activation of MAPK. Only PC3 cells transfected with ARcDNA showed this effect; cells transfected with the sham
plasmid were inactive (370).
Moreover, the sex hormone binding globulin (SHBG)
is considered to play a role in permitting certain steroids
to act without entering the cell (405). Upon ligand binding, the SHBG interacts with a high-affinity membrane
receptor. This SHBG-receptor complex has been described to cause a rapid generation of cAMP after exposure to the respective steroid. For example, in prostate
cell membranes, an increase in intracellular cAMP in
response to both androgens and estradiol, via binding to a
membrane SHBG receptor, has been shown (120, 405).
For androgens, evidence is mixed, pointing to either
classic AR or a nonclassic receptor, or even participation
of additional proteins.
NONGENOMIC STEROID ACTION
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have indeed been characterized as essential for a steroidinduced positive allosteric interaction at the GABAA receptor. Only those steroids containing a 3␣-OH-group
within the A-ring of its molecule are apparently able to
enhance GABA or benzodiazepine action at the GABAA
receptor (171, 362). All steroids with a 3␤-OH conformation that have been investigated so far have been ineffective in increasing GABAA receptor-mediated Cl⫺ conductance or flux (362, 386). These results harbor speculations
that 3␣-hydroxysteroids have a distinct stereoselectivity
at the GABAA receptors. Analogously, 3␣-reduced pregnane steroids (e.g., DHEA-S, PREG-S) have been shown to
exert GABA-antagonistic effects at the GABAA receptor
(244, 409, 445). In the search for synthetic steroids that
effectively enhance GABA action at the GABAA receptor,
Anderson and co-workers (6) refined this aspect. Steroid
in vitro and in vivo activities similar to 3␣-hydroxypregnan-20-ones all had an ether oxygen on the ␤-face of the
steroid D-ring. This suggests that for effective coagonism
at the GABAA receptor, the hydrogen bond-accepting substituent should be near perpendicular to the plane of the
D-ring on the ␤-face of the steroid (6). Recent work has
identified the structural basis for steroids augmenting
GABAA receptor function while inhibiting NMDA neurotransmission (296). The combination of these properties
would make a clinically useful anesthetic steroid.
Much effort has been spent on characterizing a specific steroid binding site on the GABAA receptor. Today,
there is considerable evidence that steroid recognition
sites reside on the GABAA receptor, and not in the bilayer
surrounding it. In steroid-insensitive Xenopus oocytes a
temporary expression of GABAA receptor subunits caused
steroid responsiveness (449) even though desensitization
did not show rigorous stereoselectivity (528). In addition,
3␣,5␣-dihydroprogesterone has been shown to modulate
ligand binding to solubilized GABAA receptors in a fashion compatible with membrane-bound receptors (179).
The coagonism as well as the allosteric antagonism
of neurosteroids to GABA at the GABAA receptor mediate
multiple functional effects. These are briefly discussed
below but have been extensively reviewed elsewhere
(244, 362, 411, 509).
B) NMDA RECEPTOR. The N-methyl-D-aspartate (NMDA)
class of glutamate receptors (NMDAR) are essential for
learning, memory, and development in the central nervous system (317, 428). NMDARs are multimeric complexes formed from both NMDA receptor subunit (NR1)
and modulatory NR2 subunits (316, 328).
The subunit composition is thought to be essential
for receptor functionality (154) and its pharmacological
characteristics, e.g., single-channel conductance and deactivation kinetics (147, 466) as well as sensitivity to Mg2⫹
(308), Zn2⫹, (213) and ifenprodil (525).
Subunit composition of the NMDAR has also been
proposed as critical for the effects of neuroactive ste-
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optic area (mPOA). These two brain regions are critical
for the expression of reproductive behavior. In doses
compatible with steroid abuse, all three steroids significantly enhanced peak synaptic current amplitudes and
prolonged synaptic current decays in neurons of the VMN.
In contrast, peak current amplitudes of synaptic currents
from neurons of the mPOA were significantly diminished,
suggesting a GABAA receptor subunit- and brain regionspecific alteration of neuroendocrine function mediated
by neuroactive anabolic steroids (223). This finding substantiates other in vitro investigations examining GABAA
receptor binding by steroids. The blockade of GABAA
receptors by dehydroepiandrosterone sulfate (DHEAS)
was found to be different in the posterior and the intermediate pituitary, and in the latter even varied from cell to
cell (201). The steroid may thus have a role in neuropeptide secretion, and the differing properties of GABA receptors may participate in the regulation of peptidergic
systems. However, these results were obtained in a rat
model with concentrations of circulating DHEAS several
orders of magnitude lower than in humans. With the use
of recombinant GABAA receptors, expressed in an insect
cell line, enhancement by steroids of [35S]TBPS binding
was sensitive to the presence of the ␥2-subunit and the
characteristic composition of the ␣-subunit (␣1 ␤2 ␥2S ⬎
␣1 ␤2, ␣6 ␤2, ␣6 ␤2 ␥2S, and ␣6 ␤2 ␦). Addition of RU 5135,
a GABA antagonist, reduced the inhibitory phase and
exposed a small enhancement of TBPS binding. The subunit-dependent interactions of steroid and GABA site ligands are in line with a three-state model in which the
receptor having bound one molecule of GABA or the
respective steroid has a different affinity for TBPS than
the resting state, while the receptor occupied by two
molecules of GABA, steroid, or both has little affinity for
TBPS (463). Corticosterone, which is elevated under
stress conditions, has been found to decrease the negative
modulation of TBPS binding in the hippocampus by both
GABA and 3␣,5␣-THDOC, while augmenting the enhancement of flunitrazepam binding by these steroids in the
dentate gyrus (349). In addition, striking regional differences in the modulation of TBPS binding were found after
prolonged corticosterone treatment.
Neuroactive steroids may also alter GABAA receptor
subunit composition per se. Withdrawal from 3␣,5␣-THP
was associated with an upregulation of the ␣4-subunit of
the GABAA receptor in rat hippocampal slices. This was
functionally linked to increased anxiety and benzodiazepine insensitivity (196, 458). It is possible that GABAA
receptor subunit expression is regulated by the hormonal
environment and that neuroactive steroids themselves are
involved in this complex issue (106). If these findings can
be substantiated, a concept embracing nongenomic as
well as genomic mechanisms may be developed by which
steroids alter brain function.
On the other side, structure prerequisites for steroids
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ter receptor. It embodies a unique binding site in the
central nervous system and peripheral organs. Nevertheless, distribution within the central nervous system and
molecular structure have not been characterized satisfactorily. After the ␴1-receptor protein was purified and its
cDNA cloned in several species (200, 228, 440), specific
regions involved in ␴1-receptor function, analyzed by expression patterns of receptor mRNA in mice brain, were
detected. Substantial mRNA expression was found in cerebral cortex, hippocampus, and Purkinje cells of the
cerebellum (441). Ligands of the ␴1-receptor may exert a
potent neuromodulation on excitatory neurotransmitter
systems, including the noradrenergic, glutamatergic, and
cholinergic systems (49, 184, 185, 287, 309). It has been
proposed that neurosteroids modulate ␴-receptor activity
by a G protein coupling at the neurosteroid binding site
(491). DHEAS stimulated the [35S]GTP␥S binding in synaptic membranes of mouse neurons. This effect was
blocked by NE-100, an experimental ␴-receptor antagonist. The DHEAS-induced stimulation was blocked by
pertussis toxin (PTX) treatment and completely recovered by reconstitution of PTX-treated membranes with
recombinant Gi1 (491). Another possible mode of action
of steroids at the ␴-receptor is a stimulation of Ca2⫹
influx. However, this has not been shown for central
nervous system-located ␴-receptors but in sperm. Prostaglandin E1 and progesterone induced Ca2⫹ entry and acrosome reaction, an effect that was blocked by the steroidal ␴-receptor ligand RU 1968 (421). It remains to be
determined if these findings can be reproduced in neuronal ␴-receptors.
Neuroactive steroids have the potential to inhibit the
binding of central nervous system-bound ␴1-receptor radioligands in vitro and in vivo. This seems to imply a
direct communication between neuroactive steroids and
␴1-receptors (289).
Interestingly, the progesterone-binding membrane
protein isolated from liver (cf. sect. IID2) shares some
properties with ␴-receptors (302).
D) 5-HT3 RECEPTOR. Neuronal receptors of the 5-HT3
type are members of the Cys loop family of ligand-gated
ion channels. This family of receptors also includes the
GABAA, the glycine, and nicotinic acetylcholine receptors. All of these receptors are pentamers, usually formed
by the coassembly of one to four different subunits (281).
Evidence suggests that the Cys loop family of receptors is
modular in design, with the extracellular NH2-terminal
domain containing the ligand binding site and the transmembrane regions containing the pore (128). Recent data
substantiate the hypothesis that there is a high degree of
structural and functional homology between receptors in
the Cys loop receptor family (397). Consequently, the
gonadal steroids, 17␤-estradiol and progesterone, have
been described as functional antagonists not only at the
above-mentioned receptors but also at the 5-HT3 receptor.
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roids. The modulatory action of steroids on NMDA receptors and excitatory postsynaptic transmission was studied in outside-out patches of rat motoneurons. NMDAinduced responses were significantly enhanced by 20oxopregn-5-en-3␤-yl sulfate (PS), while in the presence of
20-oxo-5␣-pregnan-3␣-yl sulfate (3␣,5␣ S) and 20-oxo-5␤pregnan-3␣-yl sulfate (3␣,5␤S) they were significantly diminished. PS and 3␣,5␤S both altered the relative distribution of the openings to individual conductance levels.
In controls, the most frequent openings of the NMDAR
channels were at the 70-pS conductance level, whereas in
the presence of PS or 3␣,5␤S, the most frequent openings
were at the 55-pS conductance level (1). These actions of
neuroactive steroids are most likely mediated nongenomically due to their rapid onset of action which does not
allow for the classic way of action of steroids via intracellular steroid receptors.
Pregnenolone, a rather well investigated neuroactive
steroid, has been shown to enhance memory performance
in various test paradigms in rodents (285). This effect has
been linked to a nongenomic allosteric modulation of the
NMDAR (304). The hypothesis of nongenomically mediated effects of pregnenolone at the NMDA is corroborated
by findings that pregnenolone sulfate blocks learning and
memory impairment induced by D-2-amino-5-phosphonovalerate, a competitive NMDA receptor antagonist (286).
Furthermore, pregnenolone sulfate has also been shown
to act as a coagonist at the NMDA receptor, allosterically
modulating receptor currents within seconds (65).
Apart from these well-characterized nongenomic effects at postsynaptic receptors, pregnenolone sulfate has
also been shown to act at glutamatergic synapses in the
CA1 region of the hippocampus of rats. It enhanced
paired-pulse facilitation of glutamate excitatory postsynaptic potentials at ionotropic NMDA receptors with an
EC50 ⬍1 ␮M (358). Similarly, Monnet et al. (310) demonstrated that pregnenolone sulfate enhances NMDAR-dependent norepinephrine release in hippocampal slices via
the sigma1-receptor (310). These results point to a further
site of action in addition to postsynaptic receptors at
which neurosteroids may have an effect on nervous system plasticity (see sect. IID5C).
Contrasting the fast, almost instantaneous nongenomic
effects of neuroactive steroids, these compounds have also
been shown to act via the classical intracellular steroid
receptor cascade, e.g., influencing subunit composition of
NMDAR. High doses (5 and 15 mg/kg) of nandrolone
decanoate administered over 14 days intramuscularly produced a significant decrease in the mRNA expression of
the NR2A receptor subunit both in the hypothalamus and
hippocampus of male rat brain (249). A decrease in the
level of NR2B receptor mRNA was observed in hypothalamus at the lower dose of nandrolone.
C) SIGMA1 RECEPTOR. The sigma1 (␴1)-receptor seems
to be distinctly different from any other known transmit-
NONGENOMIC STEROID ACTION
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Neuroactive steroids have been shown to modulate
glycine receptors, but data on effective concentrations
are sparse. Potencies have been published for pregnenolone sulfate and progesterone in chick embryonic
spinal cord (531, 532) and Xenopus laevis oocytes (275).
Maksay et al. (275) also report on effects of DHEAS. The
mechanism by which pregnenolone sulfate and progesterone bind to the receptor and mediate their respective
effect do not seem to be identical. Progesterone exerted
incomplete and noncompetitive inhibition of glycine receptor currents, whereas data suggest that pregnenolone
sulfate inhibited glycine-induced receptor currents fully
and competitively (531, 532). This effect has been shown
to be dose dependent. Antagonism of the glycine response
by pregnenolone sulfate was neither voltage nor agonist
dependent, suggesting that pregnenolone does not act as
an open-channel blocker. In addition, inhibition of glycine-induced currents by pregnenolone sulfate appears to
be mediated competitively. The steroid brings about a
parallel, rightward shift of the glycine dose-response
curve (531). In contrast, the neuroactive steroid 5␣-pregnan-3␣-ol-20-one had no effect on glycine-invoked currents in Xenopus oocytes expressing human recombinant
glycine receptors (377). This heterogeneity of effects was
also seen in experiments with other neurosteroids. In
addition, structure-activity requirements of glycine receptors have been suggested to be different from GABAA
receptors with inhibition dominating over allosteric enhancement (176, 382). This proposal has been corroborated by findings investigating structure-activity requirements of androstane steroids, used to address the role of
chirality at the C-3 position, and pregnene steroids and
their respective 3␣ and 3␤ substituents. Neuroactive steroids do seem to possess stereoselectivity at carbon 3. On
the other hand, strict structural requirements of the 3-substituents for potentiation versus inhibition of glycine receptor chloride channel function were demonstrated
(275). It has been hypothesized that these data advocate a
role for neuroactive steroids in neuronal development,
since the subunit-specific effects are particularly pronounced in receptors that most closely resemble perinatal
glycine receptor composition (275).
F) OTHER MECHANISMS. Both the ␮-opioid and the
GABAB receptors in hypothalamic neurons have been
shown to be uncoupled from K⫹ channels by estradiol.
This action, however, does not proceed as allosteric interaction at the receptors directly, but requires a signaling
cascade comprising PKA and PKC (229). In a similar
fashion, estradiol or its membrane-impermeant derivative
coupled to BSA potentiated kainate-induced currents in
hippocampal neurons (195) by modulation of non-NMDA
glutamate receptors. The steroid action is attributed to a
Gs protein-coupled receptor and cAMP-dependent phosphorylation in the cytosol (194, 195). Remarkably, the
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This has been demonstrated in cells stably expressing the
5-HT3 receptor using whole cell voltage-clamp recordings.
In contrast to pregnenolone sulfate and cholesterol,
functional antagonistic characteristics at the 5-HT3 receptor have also been shown for 17␣-estradiol, 17␣-ethinyl17␤-estradiol, testosterone, and allopregnanolone (523).
From comparative studies using various alcohols, it has
been proposed that the modulation of 5-HT3 receptor
function by steroids is dependent on their respective molecular specificity. Steroid-induced antagonistic effects at
the 5-HT3 receptor are not mediated via the serotonin
binding site. This has been suggested because the investigated steroids did not alter the binding affinity of [3H]GR
65630 to the 5-HT3 receptor. In addition, kinetic experiments were able to demonstrate a different response
pattern to 17␤-estradiol when compared with the competitive antagonist metoclopramide (523). Gonadal steroids
have been suggested to interact allosterically with the
5-HT3 receptor at the receptor-membrane interface. This
has been shown for progesterone (533). A noncompetitive, voltage- and agonist-independent mechanism that
was distinct from that of open-channel blockers has also
been proposed for progesterone-induced inhibition of the
5-HT3 response (533).
Furthermore, it has been demonstrated that neuroactive steroids may have effects at both the 5-HT3 receptor
channel and adjacent voltage-gated sodium channels. The
relationship between most of the compounds’ lipophilicity and their cation channel inhibiting properties is compatible with the mechanistic principle of steroid-induced
inhibition of the two channels, i.e., a nonspecific hydrophobic interaction with certain membrane lipids in the
neighborhood of the two channels (26). However, despite
a considerable body of evidence, a satisfactory picture of
neuroactive steroids action at the 5-HT3 receptor remains
elusive. A concept integrating noncompetitive and competitive nongenomic effects of neuroactive steroids and
effects that may be mediated via classical intracellular
steroid receptors in various types of neuronal cells has
still got to be developed. Such a concept should also
incorporate the interaction with other neurotransmitter
receptors and feedback mechanisms regulating receptor
density and subtype composition as well as concentration
levels of circulating and tissue-bound steroids.
E) GLYCINE RECEPTOR. The glycine receptors belong to
the same superfamily of transmitter-gated ion channels as
the GABAA receptor (240, 459). Analogously to nicotinic
acetylcholine, GABAA and serotonin 5-HT3 receptors, the
glycine receptor consists of five subunits each with four
transmembrane segments (189). Two types of glycine receptor subunits have been cloned so far (␣1-␣4 and ␤;
also see detailed review by Harvey et al., Ref. 206). The
functional properties of the inhibitory receptors are likely
to be heterogeneous and may be brain region or even
neuron specific (377).
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6. Mineralocorticoids
In addition to the evidence presented from KO experiments and pharmacology, some limited information
about receptors for nongenomic aldosterone action is
available. Specific aldosterone binding has been demonstrated using 3H-labeled aldosterone derivatives in human
mononuclear lymphocytes (HML) (9) and later in plasma
membrane fractions thereof, in pig kidney and liver, as
well as in microsomes from pig liver (99, 299, 518). Radioligand binding to plasma membranes was saturable
with a KD of ⬃0.1 nM as confirmed by displacement
experiments with unlabeled aldosterone. While canrenone and cortisol could not displace the ligand up to
micromolar concentrations, fludrocortisone and desoxycorticosterone exhibited intermediate affinity. Thus aldosterone binding to HML membranes is in good agreement
with respect to kinetics and steroid selectivity with functional data on rapid effects such as ion transport and
second messengers, which therefore may be mediated by
these sites.
Membrane binding sites from microsomal preparations of pig liver have been studied and revealed a maximum binding capacity of ⬃700 fmol/mg protein (299).
Binding of tritiated aldosterone was saturable with two
apparent dissociation constants of ⬍11 and 118 nM, respectively. Further purification has not been achieved due
to the low stability of the protein in its solubilized form.
Purification from other sources such as HML has not been
possible yet due to low abundance.
A different mechanism that may lead to nongenomic
Physiol Rev • VOL
actions of aldosterone in epithelia has been reported from
the laboratory of Brian Harvey. The ␣-isoform, but not the
␦-, ⑀-, and ␨-isoforms of PKC, has been found to be directly
activated by aldosterone in vitro (204). The activation was
significant at 10 pM, a concentration which is within the
physiological range.
Evidence for an aldosterone receptor system with
properties markedly different from the classic MR may
also be drawn from studies in the isolated working rat
heart (29). In this system, perfusion with aldosterone at
nanomolar concentration has an immediate positive inotropic effect, which is also seen with spironolactone applied instead of aldosterone. The most striking finding,
however, was the inability of spironolactone to blunt
aldosterone action when perfused simultaneously; instead, an additive inotropic action of both agents was
found. This strongly indicates that the nongenomic action
of aldosterone, and the action of spironolactone, are mediated by independent receptors.
Mineralocorticoid action probably is the topic with
the most active controversy about receptors. Although
some aldosterone-induced nongenomic responses share
properties with genomic action of classic MR, other properties in the same systems render MR participation rather
unlikely (see sect. IIC2B). Most of nongenomic mineralocorticoid action thus is probably mediated by nonclassic
receptors.
7. Vitamin D3
After rapid effects of vitamin D3 have been demonstrated for many years, membrane binding sites potentially transmitting these effects have been subsequently
described in chick intestine (329, 330) and in ROS 24/1
cells (20, 22). In chick intestine, studies with analogs of
vitamin D3 [particularly 1,25(OH)2-7-dehydrocholesterol
and 1,25(OH)2-lumisterol3] have provided convincing correlations between binding to the solubilized membrane
receptor and the ability to initiate transcaltachia.
Larsson et al. (247) tested the ability of the vitamin D
metabolite 24R,25(OH)2D3 to specifically bind to basal
lateral membranes isolated from intestinal epithelium of
Atlantic cod (a seawater fish), carp (a freshwater fish),
and chicken. Specific saturable binding was shown in
membranes from all three species. In addition, in isolated
Atlantic cod and carp enterocytes, 24R,25(OH)2D3 but not
24S,25(OH)2D3, suppressed Ca2⫹ uptake into cells in a
dose-dependent manner, suggesting that their binding
molecule(s) is receptor-like mediating rapid, nongenomic
responses in intestinal cells (247).
In competition experiments, both 1␣,25(OH)2D3 and
1␤,25(OH)2D3 displaced [3H]-1␣,25(OH)2D3 from membranes while 25(OH)D3 did not. The KD value was 0.8 ␮M
for 1␣,25(OH)2D3 and 0.5 ␮M for the 1␤ epimer. These
binding sites perfectly match with rapid effects of vitamin
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impermeant derivative needed simultaneous application
to both sides of the plasma membrane to be active.
Estradiol also acts directly on the striatum of ovariectomized rats to enhance dopamine release, e.g., induced by amphetamine (40, 41). Furthermore, the steroid
inhibits L-type Ca2⫹ channels within seconds in striatal
neurons when present at picomolar concentrations, an
effect probably mediated by a G protein-coupled receptor
(298).
The neuronal nicotinic acetylcholine receptor(s) has
been shown to be targets of steroids as well (355). Blockade of this receptor occurred with the synthetic steroid
3␣-hydroxy-5␣-androstane-17␤-carbonitrile at 1.5 ␮M.
The inhibition exhibited little selectivity for stereochemistry at the 3 and 5 positions, which is in contrast to its
importance for GABAA receptor modulation. The potency
to block nicotinic receptors correlated with their ability to
produce anesthesia in Xenopus.
For neurosteroids, little controversy exists about the
nature of receptors. Many of the neurotransmitter receptors described above are well known, unrelated to classic
steroid receptors, and susceptible to modulation by steroids.
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8. Triiodothyronine and thyroxine
Several mechanisms are described in the literature
through which thyroid hormones may nongenomically
induce cellular responses. However, receptors mediating
such phenomena have not been definitely characterized.
Some enzymes have been reported to be modulated in
their activity by thyroid hormones, such as pyruvate kinase (12), where triiodothyronine (T3) is more effective
than thyroxine (T4) in preventing tetramer formation and
concomitant inhibition of enzyme activity. In another example, the less iodinated 3,5-diiodothyronine (3,5-T2)
binds to cytochrome-c oxidase, counteracting ATP inhibition of oxidase activity (10). T3 is less effective, while T4
and the noniodinated thyronine have no effect at all. This
mechanism may explain the short-term effect of thyroid
hormones on mitochondrial respiration.
The activation of a crucial signaling enzyme, PKC,
has been shown in vitro in erythrocytes (248). However,
these experiments have not been performed with isolated
PKC, so indirect effects are still possible.
High-affinity (KD in the low nanomolar range or below) membrane binding sites for T3 or T4 have been
described in mammalian liver (379), placenta (3), thymocytes (429), and others as well as in avian embryonic
synaptosomes (178). The latter system exhibits several
properties characteristic for G protein-coupled receptors
(GPCR), such as the reduction in T3 binding capacity by
GTP␥S (177). In turn, T4 and T3 lower the GTPase activity,
and upon ADP-ribosylation the thyroid hormones increased the formation of inactive G␣0-GDP. The studies
were done, however, on whole cell preparations, and the
postulated GPCR was not identified.
Additional evidence for the possible involvement of a
GPCR has come from the investigation of signal transduction pathways. T4 activates the MAPK signaling line, but
the effect is abolished by GTP␥S or PTX (259). Here, only
T4, but not T3, is active at physiological concentrations.
Partial identification of nonclassic thyroid receptor
proteins has been reported only a few times. The affinity
labeling reagent N-bromoacetyl-125I-T3 was found to react
specifically with a 65-kDa protein, as judged from SDSPAGE, in placenta (3). The protein, solubilized in its
native state, is possibly a 140- to 150-kDa dimer as observed by gel filtration. On the other hand, the same
reagent detected a 27-kDa protein in neuroblastoma
plasma membranes (183).
A 43-kDa mass protein has been described in mitochondria that is related to the ligand binding domain of
thyroid receptor (TR) ␣1 (529). This protein could bind to
a thyroid responsive element and mitochondrial DNA sequences and was shown to bind T3 with high affinity (86).
No link has been made, however, to direct nongenomic
action, which may be mediated through a 28-kDa protein
(530).
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D3 regarding their selectivity for vitamin D3 analogs, mirroring the activity of these compounds in rapid effects on
electrolyte transport.
In this context, a 1␣,25(OH)2D3 binding site located
in the basolateral membrane of vitamin D-replete chick
intestinal epithelium has been described that was functionally correlated with transcaltachia. This protein exhibited saturable binding for 3H-labeled 1␣,25(OH)2D3
(KD ⫽ 0.72 nM, Bmax ⫽ 0.24 pmol/mg protein) (330). A
functional correlation between the 1␣,25(OH)2D3 membrane binding site and transcaltachia was observed in
three experimental situations: 1) vitamin D deficiency,
which suppresses transcaltachia, resulted in reduced specific binding of 3H-labeled 1␣,25(OH)2D3 to the basolateral membrane relative to corresponding fractions from
vitamin D-replete chicks; 2) the 1␣,25(OH)2D3 membrane
binding site exhibited downregulation of specific 3H-labeled 1␣,25(OH)2D3 binding following exposure to the
nonradioactive ligand; and 3) the relative potencies of
two 6-s-cis-analogs of 1␣,25(OH)2D3 [1␣,25-(OH)2-7-dehydrocholesterol and 1␣,25-(OH)2-lumisterol3] to bind to the
1␣,25(OH)2D3 membrane protein and their ability to initiate transcaltachia were congruent (329). Further experiments done with basolateral membranes of vitamin Dreplete chick intestinal epithelium showed that a polyclonal antiserum (Ab99) directed against this membrane
receptor was able to block the binding of the radioligand
and to recognize a single protein band of 64.5 kDa in
Western blot analyses (332). A protein of similar size
was also labeled by the affinity ligand [14C]-1␣,25(OH)2D3bromoacetate. The labeling was reduced in the presence
of an excess of unlabeled secosteroid. In contrast, the
nuclear VDR could not been detected in these basoateral
membrane fractions by a monoclonal antibody against the
nuclear VDR (9A7) in Western blot analyses (332). The
same affinity reagent was found to label a membrane
protein in ROS24/1 cells that was identified to be annexin
II (21). In addition, antibodies to annexin II blocked the
vitamin D-induced increases in [Ca2⫹]i and diminished the
binding of 1␣,25(OH)2D3 to the protein in partially purified plasma membranes. However, these findings still
await confirmation. An antibody directed against a chick
1,25(OH)2D3 binding protein identified a 66-kDa protein in
rat chondrocytes (331) and also blocked the 1␣,25(OH)2D3dependent increase in PKC activity, supporting the hypothesis that a membrane receptor is involved in the
initiation of rapid nongenomic effects.
Interestingly, a rapid increase of [Ca2⫹]i has also been
described in the osteoblastic cell line ROS 24/1 which
lacks VDR (24), indicating that a nonclassic receptor is
involved.
In summary, a substantial body of evidence now exists
to indicate that at least some of the rapid 1␣,25(OH)2D3induced effects are transmitted by a membrane receptor,
distinct from the classic VDR.
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LÖSEL ET AL.
In summary, although some hints on the identity of
receptors for nongenomic thyroid effects have been acquired, complete identification or characterization is not
yet achieved by far.
The possibility of classic receptors, or proteins
closely related to, participating in the mediation of rapid
effects has rarely been studied. However, many characteristics of responses or ligand binding observed are incompatible with classic receptors.
III. EFFECTS, SIGNALING PATHWAYS,
AND CLINICAL IMPLICATIONS
For about half a century, glucocorticoids play an
important role in the treatment of autoimmune diseases,
allergic diseases, and more recently in conditions that
require the suppression of immune response, as for instance organ transplantation. Genomic effects of glucocorticoids have been extensively investigated during
the past decades. They normally take place at low steroid
concentrations in nanomolar ranges, as is the case under
physiological conditions, and are mediated by cytosolic
receptors that alter the expression of specific genes.
Rapid glucocorticoid effects can be further divided into
specific and nonspecific nongenomic effects (80). Both
occur within seconds, but the latter ones only at high
dosages of the glucocorticoid and are assumed to function via direct effects on cellular membranes and subsequent changes in ion transport. Specific nongenomic effects can be observed within seconds to minutes and
seem to be mediated by steroid-selective membrane receptors (516). Rapid effects of glucocorticoids are investigated to a much lower extent than rapid effects of the
other classes of steroid molecules, but they have been
described at cellular levels, at the level of whole organ
function, as well as at the level of whole physiological
response.
Direct membrane effects of glucocorticoids have
been shown already in 1974 in isolated hypothalamic
synaptosomes (125). They were considered as the cellular
counterpart for the negative feedback mechanism between plasma cortisol and ACTH. In hippocampal slice
preparations of CA1 pyramidal cells, corticosterone influences the excitability as shown by increased spike amplitudes (398). In vitro experiments using guinea pig ganglion neurons demonstrated that glucocorticoid can hyperpolarize the membrane potential within 2 min (215).
Incubation of rat cerebral cortex synaptosomes and of the
human neuroblastoma line SK-N-SH with different glucocorticoids showed a rapid and dose-dependent increase
of glutamate uptake (544). Because the effect could be
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A. Glucocorticoids
blocked by an inhibitor of G protein activation, the authors conclude that G proteins might be involved in glucocorticoid activation in these cells. In Ishikawa human
endometrial cells, Koukouritaki et al. (237) demonstrated
rapid alterations in actin polymerization dynamics after
treating cells for 15 min with 0.1 ␮M dexamethasone. In
parallel, they observed a decreased cAMP content in
treated cells and concluded a regulation of the processes
by cAMP-dependent pathways. Because the glucocorticoid antagonist RU 486 completely abolished the effect, it
seems to be mediated via specific glucocorticoid binding
sites. However, transcriptional mechanisms were unlikely
to be involved, since actinomycin D did not block the
dexamethasone-induced actin polymerization.
Acute actions of dexamethasone on nicotine-induced
intracellular Ca2⫹ transients, as well as nicotine-induced
catecholamine secretion, were examined in porcine adrenal chromaffin cells (503). In the presence of 1 ␮M dexamethasone, both the nicotine-induced Ca2⫹ transient and
catecholamine secretion were reduced by about 18%
within 5 min. Interestingly, in these cells a long-term
exposure (48 h) to glucocorticoids leads to the opposite
effect, namely, a potentiation of Ca2⫹ signaling (162).
Similarly, corticosterone was found to inhibit the nicotine-induced Ca2⫹ influx with a half-maximal inhibitor
concentration (IC50) of 0.61 ␮M in the pheochromocytoma cell line PC12. Because this effect was mimicked by
the PKC activator phorbol 12-myristate 13-acetate, antagonized by PKC inhibitors, and blocked by pertussis toxin,
it was concluded that corticosterone acts through the
pertussis toxin-sensitive G protein-PKC pathway (388). In
a similar manner, corticosterone inhibited the change in
[Ca2⫹]i induced by high extracellular K⫹ in PC12 cells, but
with the maximal effect seen at a relatively high concentration of 10 ␮M of the steroid (265). The effects of
corticosterone in PC12 cells were also detected in the
presence of a corticosterone-BSA conjugate, suggesting
that corticosterone might act via membrane receptors.
Recently, again in PC12 cells, rapid nongenomic effects of
corticosterone on neuronal nicotinic acetylcholine receptor have been shown (447). With the use of whole cell
clamp technique the influence of corticosterone on the
acetylcholine-induced current (IACh) was studied. Preincubation of these cells with corticosterone for 4 min had
an inhibitory effect on the peak of the acetylcholineinduced inward current. The effect was reversible, concentration dependent, and voltage independent. Intracellular application of corticosterone did not affect the IACh.
Extracellular application of corticosterone to the preincubated cells inhibited the curve amplitudes by ⬃49%.
Corticosterone conjugated to BSA showed an inhibition
similar to that caused by unconjugated corticosterone.
Neither actinomycin D nor cycloheximide influenced the
inhibition of acetylcholine-induced current by corticoste-
NONGENOMIC STEROID ACTION
Physiol Rev • VOL
uptake of glutamate (544). Corticosterone was also found
to rapidly inhibit arginine vasopressin release in hypothalamic slices of rats at the smallest dose of 100 nM (262).
It has been suggested that rapid glucocorticoid actions
may be involved in these phenomena because arginine
vasopressin plays an important role in the regulation of
hypothalamo-pituitary-adrenal axis activity. Rapid effects
of corticosterone in the rostral ventrolateral medulla, especially on barosensitive cardiovascular and bulbospinal
presympathetic neurons, have been described that result
in a very rapid (within 45 s) change in the firing rate.
These effects are likely to be nongenomic ones because of
the rapid onset of the responses, but they are attenuated
by the classic corticosteroid receptor antagonist RU 486
(402). Corticosterone was found to rapidly affect 45Ca2⫹
uptake upon depolarization by high K⫹ in synaptic plasma
membranes as well as to modulate calmodulin binding,
with the maximal effect occurring at a concentration of 1
␮M (475).
Corticosteroids are assumed to influence behavioral
changes associated with stressful events. Environmental
perturbations can cause behavioral responses within minutes, and there is strong evidence that corticosteroids
may induce changes by acting through nongenomic mechanisms. Taricha granulosa is known for rapid behavioral
responses to stress, in a way that stress can suppress the
courtship behaviors of Taricha (a prolonged amplectic
clasp to facilitate receptivity in the female). This rapid
effect is dependent on corticosterone and can be mimicked by corticosterone injection (311). Intraperitoneal
injection of male Taricha with 32 nmol corticosterone
suppressed sexual behavior within 8 min (352). In whitecrowned sparrows, noninvasive corticosterone administration caused an increase in perch hopping within 15
min, indicating elevated locomotor activity that is consistent with behavioral responses to natural perturbations
(70). In further experiments, it could also be demonstrated that the rapid behavioral responses to corticosterone varied with photoperiod and dose (72). Sparrows
exposed to a long-day photoperiod showed increased activity in perch hopping at intermediate levels of corticosterone (⬃24 ng/ml), whereas in short-day exposed sparrows, the same dose level had no behavioral effect. The
authors propose a less sensitive neural mechanism that
regulates the behavioral response to corticosterone during a winter (short-day) photoperiod. Although intermediate levels of 24 and 40 ng/ml corticosterone increased
the activity to threefold background values in the sparrows exposed to the long-day photoperiod, high physiological levels of corticosterone (65 and 97 ng/ml) did not
induce any behavioral change. A model for nongenomic
actions of glucocorticoids on behavior is shown in Figure 6.
The locomotor responses of rats in a novel environment could be increased by systemic corticosterone in-
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rone, thus suggesting a nongenomic mechanism mediating the above effects. The authors propose the binding of
corticosterone to a specific site on the outer cell membrane, probably on the neuronal nicotinic receptor-coupled channel. The resulting inhibition of IACh is supposed
to control the immediate catecholamine release from
sympathetic cells.
Rapid nongenomic effects that occur with high doses
of dexamethasone, stabilizing membranes, could be
shown in rat liver lysosomal membranes (212). Marker for
lysosomal membrane integrity was the release of ␤-glucuronidase. Ten minutes after intravenous administration
of 3 or 10 mg/kg dexamethasone, ␤-glucuronidase release
was inhibited by 38 and 33%, respectively. A comparable
inhibition was observed after 24 h. Pretreatment of rats
with the glucocorticoid receptor antagonist RU 486 did
not antagonize the short-term effect but almost completely prevented the 24-h membrane protection. The authors suggest a dual mechanism of high-dose dexamethasone on membrane integrity by rapid nongenomic effects and long-term receptor-dependent genomic events.
The nervous system is a field intensively investigated
in regard to rapid glucocorticoid action. Rapid effects
have been shown with regard to excitability, neuroendocrine responses, and behavioral tasks. Systemic glucocorticoid administration yielded rapid effects on neural activity in the hypothalamus of rat, rabbit, and cat (13, 149).
In the limbic system of lizard serotonergic responses to
systemically administered corticosterone were detected
within 20 min after treatment, suggesting a possible mechanism for mediation of changing social behavioral events
(468). Rapid corticosterone effects on neurophysiological
processes and behavioral changes have also been extensively investigated in the amphibian Taricha granulosa.
Multiple neurophysiological effects have been observed
within 3 min of corticosterone injection, including a decline or cessation of firing of sensory responsive neurons
and reduced excitability of reticulospinal neurons (403,
404).
In freely moving rats, either adrenalectomized or
sham-operated, an intraperitoneal injection of 2.5 mg/kg
corticosterone induced a transient increase in extracellular aspartate and glutamate levels in the CA1 area of the
hippocampus within 15 min. The same fast and reversible
effects were achieved by intrahippocampal corticosterone administration. Classic antagonists of intracellular
glucocorticoid receptors or inhibitors of protein synthesis
did not abolish the effect. Similar results were obtained
with dexamethasone, whereas nonglucocorticoid steroids
did not affect amino acid transmission in this hippocampal area (500). In rat cerebral cortex synaptosomes and
human neuroblastoma cells, the addition of several glucocorticoids including corticosterone and dexamethasone 21-phosphate at a concentration of 1 ␮M rapidly (4
and 15 min, respectively) stimulated the Na⫹-dependent
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B. Progesterone
jections within 7.5 min (420). The doses administered (2.5
or 5.0 mg/kg) are described to mimic plasma concentrations produced by substantial stress (464). NO seems to
be involved in this neurochemical mechanisms, since NO
synthesis inhibitors prevent these rapid effects. Inhibition
of protein synthesis and blocking of intracellular receptors by specific receptor antagonists did not inhibit the
rapid effects (419).
Apart from genomic glucocorticoid effects, membrane receptor-mediated mechanisms of glucocorticoids
are assumed to participate in cellular processes leading to
apoptosis. These mechanisms seem to involve damage of
mitochondrial function by disruption of the mitochondrial
membrane potential and decreased ATP availability (79).
Functionally, there is evidence that at least in mouse S49
lymphoma cells, the human CCRF-CEM cell line and in
lymphocytes of leukemic patients, membrane glucocorticoid receptors are necessary to initiate glucocorticoidinduced therapeutic apoptosis (117, 164, 168, 511). Membrane glucocorticoid receptors are present in normal and
in cancerous lymphoid cells, and their actions may have
important consequences for function and diseases of the
immune system.
Further reports show a role of nongenomic glucocorticoid action in the inhibition of phosphate uptake in
primary rabbit proximal tubule cells in a PLC/PKC-dependent way (357).
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FIG. 6. Model for nongenomic actions of glucocorticoids through an
opioid-like receptor. Glucocorticoids are shown to signal through an
opioid-like receptor to inhibit the release of corticotropin releasing
hormone (CRH) and vasotocin (AVT) and to inhibit Ca2⫹ currents and to
effectively inhibit sex behavior and locomotor behavior. All of these
have been shown to be activities of opioid receptor agonists. [From
Evans et al. (136), copyright 2000 The Endocrine Society.]
Rapid effects of progesterone have already been described in 1942 when Hans Selye found that an intraperitoneal application of progesterone induces a prompt onset of anesthesia in rats (437). Since then, many reports
on nongenomic progesterone action followed, mostly in
germ cells like sperm or oocytes.
Xenopus oocytes naturally arrest at the G2 phase of
meiosis I. In response to progesterone, these cells are
induced to enter the M phase. This process, known as
meiotic maturation, is not affected by actinomycin D and
occurred even in enucleated oocytes (284), suggesting a
nongenomic effect. Only the early maturation events are
discussed here, subsequent effects including a MAPK cascade and a Cdc2 activation are reviewed elsewhere (151).
The initial biochemical responses of progesterone involve
an inhibition of an oocyte adenyl cyclase and reduction of
intracellular cAMP (152, 416). This decrease is thought to
translate into a decrease in the activity of PKA (277). In
addition, the protein kinase Eg2 becomes phosphorylated
very early in the maturation process (7). These events of
progesterone-induced maturation seem not to involve the
␣-subunits of classical G proteins, but rather the G␤␥
subunits as the principal mediators (269, 446). In addition
to Xenopus, progesterone also triggers the oocyte maturation in Rana pipiens oocytes involving a transient release of Ca2⫹ followed by a decrease in intracellular
cAMP (236) and a transient rise in cGMP (235; for review,
see Ref. 319).
Moreover, nongenomic progesterone effects have
been intensively studied in human sperm but also in spermatozoa from other mammals, e.g., mice (210), hamster
(263), dog (453), and stallion (96).
In addition to zona pellucida glycoprotein (ZP3), it
has been demonstrated that progesterone is a natural
stimulus of the acrosome reaction in sperm (292, 353).
This event is mediated by a rapid increase of intracellular
[Ca2⫹]i occurring within seconds after addition of the
steroid (15, 293). The maximal increase was detected at
relatively high progesterone concentrations of 1–10 ␮M
(58) which, however, naturally occur in the cumulus matrix surrounding the oocyte (17). The signal transduction
mechanisms that are involved in progesterone-induced
effects on [Ca2⫹]i and the acrosome reaction have been
reviewed in detail elsewhere (360, 384). Interestingly, the
potent antagonist of the intracellular PR, RU 486 (10 ␮M),
has been found to decrease [Ca2⫹]i when given alone thus
partially antagonizes the effect of progesterone on [Ca2⫹]i
and sperm acrosome reaction (535). However, due to the
similar inhibition of the thapsigargin-induced Ca2⫹ elevation by RU 486, it has been speculated that the antagonist
does not necessarily compete with progesterone for the
same binding sites to exert its inhibitory action (439). In a
former study, the progesterone-induced [Ca2⫹]i increase
NONGENOMIC STEROID ACTION
Physiol Rev • VOL
and inward currents in thymic epithelial cells (209). In
addition, similar concentrations of the steroid cause an
immediate rise in [Ca2⫹]i in a murine Leydig tumor cell
line (mLTC-1) which seems to be devoid of PR (129).
Several other reports deal with rapid progesterone
effects on ion fluxes. However, their physiological significance is uncertain since unphysiologically high steroid
concentrations were used in the studies. For example, it
has been described that progesterone (50 ␮M) decreases
Ca2⫹ currents in a human intestinal smooth muscle cell
line (54). Moreover, micromolar concentrations of progesterone rapidly block voltage-gated and Ca2⫹-activated
K⫹ channels, resulting in depolarization of the membrane
and inhibition of Ca2⫹ signaling in T lymphocytes (127).
This provides a nongenomic mechanism for progesterone-mediated immunosuppression in the placenta. For
the sake of completeness, it should be mentioned that
rapid electrophysiological effects of progesterone have
been described in Leydig cells (406), rat hepatocytes
(504), natural killer cells (278), and CA1 hippocampal
neurons (222).
Progesterone has been demonstrated to modulate
vasorelaxation after acute application. For example, progesterone at micromolar concentrations induces a dosedependent relaxation of rat uteri (precontracted with
KCl) (81, 197) and saphenous artery segments (precontracted with norepinephrine) within 10 min (225) and
rapidly decreases the contractile activity of murine jejunum (347). However, acute treatment with progesterone
at physiological concentrations reduces bradykinin- and
calcium ionophore A23187-mediated relaxations in porcine coronary artery rings (477).
Progesterone is considered to be involved in maintaining pregnancy by depressing the uterotonic action of
the peptide hormone oxytocin. In this context, it was
postulated that progesterone binds to the rat oxytocin
receptor (OTR), a member of the GPCR family, with high
affinity (KD ⬃20 nM). Moreover, application of progesterone (10 nM-1 ␮M) to cells expressing this receptor caused
an inhibition of the oxytocin-induced Ca2⫹ response
(187). Interestingly, in CHO cells expressing recombinant
human OTR, no inhibition of oxytocin binding by progesterone (up to 10 ␮M) was observed. Instead, a progesterone metabolite, 5␤-pregnane-3,20-dione, inhibited oxytocin binding with a inhibition constant of 32 nM (187). In
contrast, Burger et al. (77) found that within minutes high
concentrations of progesterone (⬎10 ␮M) attenuated or
blocked the signaling of several GPCRs, including the
OTR.
Nongenomic progesterone effects have also been described in conjunction with sexual behavior (115). For
example, activation of sexual receptivity in rats occurs
within 10 min of intravenous infusion of progesterone
(20 – 400 ␮g) (303). In ovariectomized, estradiol-primed
mice, an intravenous progesterone (200 ␮g) infusion sig-
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was not affected by RU 486, but the concentration of 1 ␮M
(15) perhaps was too low.
Moreover, progesterone also promotes Na⫹ (155,
361) and Cl⫺ fluxes (490) in spermatozoa which may be
linked to the depolarizing effect of progesterone (360).
The finding that progesterone induces the human
sperm acrosome reaction has pushed speculations on the
clinical significance of this effect. In this regard it has
been shown that sperm responsiveness to progesterone is
reduced in oligozoospermic and infertile patients (16).
Moreover, progesterone binding to the acrosomal membrane has been suggested to be a valuable predictor for
outcome of in vitro fertilization. High progesterone binding to sperm was linked to a higher success rate of
fertilization (161).
In addition to germ cells, a number of nongenomic
progesterone effects have also been described in several
other tissues or cells. For example, in isolated neocortical
slices, progesterone (0.1 ␮M) augmented forskolin-induced cAMP production within 15 min without prior treatment with estrogen. In these studies progesterone alone
was ineffective, the steroid also failed to affect norepinephrine- or isoproterenol-induced cAMP increase (2). In
contrast, preincubation of rat hypothalamic slices with
progesterone (20 nM) for 5 min significantly suppressed
norepinephrine-stimulated cAMP formation by ⬎60%.
This effect was estrogen dependent in that progesterone
in vitro did not inhibit NE-stimulated cAMP accumulation
in slices from ovariectomized rats not pretreated with
estradiol (371). In addition, progesterone was found to
decrease cAMP levels in quiescent adipocytes or in adipocytes stimulated by isoproterenol (470).
Rapid Ca2⫹ fluxes in response to progesterone (10
pM to 10 nM) which are modulated by PLC-␤1 and PLC-␤3
and involve the G␣q/11 subunit have been demonstrated
in female and male osteoblasts. 17␣-Hydroxyprogesterone was as active as progesterone and medroxyprogesterone had no effect. In addition, a rapid progesteroneinduced increase in the formation of inositol 1,4,5trisphosphate (IP3) and 1,2-diacylglycerol (DAG) was
observed in these cells (191, 251). Similarly, progesterone
triggers an immediate and transient peak in [Ca2⫹]i and an
induction of IP3 in porcine granulosa cells (257, 272). The
mentioned effects in osteoblasts and granulosa cells have
been shown to be insensitive to RU 486 and were also
triggered by progesterone bound to BSA, although at
100-fold higher concentrations. This always opens the
possibility that a 1% impurity of free steroid is responsible
for the rapid responses, rather than the impermeable
compound (see also sect. V).
Recently, progesterone (0.5 ␮M) was found to blunt
KCl-induced elevation of [Ca2⫹]i and to decrease L-type
calcium channel current in vascular smooth muscle cells
(VSMC) (28). The steroid (1 ␮M) has also been described
to rapidly increase and induce membrane depolarizations
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LÖSEL ET AL.
C. Estrogens
One of the first observations on nongenomic actions
of estrogens has been produced in the female reproductive system. The endometrium is a major target for ovarian steroids, and steroids play an essential role in regulating growth and differentiation in the endometrium
leading to dynamic morphological and functional
changes. In cultured endometrial cells, 17␤-estradiol rapidly (⬍10 min) stimulates Ca2⫹ influx (375), which was
confirmed recently (366). Accordingly, putative membrane estrogen receptors on the cell surface could be
detected in endometrial cells (376). 17␤-Estradiol also
elicits functional changes in the endometrial cells by increasing the number and density of microvilli. These results could be supplemented by findings that 17␤-estradiol-induced structural changes are biphasic: they appear to
be secondarily enhanced after 7 min (391). Rapid increases in [Ca2⫹]i could also be found in maturing human
oocytes and granulosa cells after addition of only 0.1 nM
17␤-estradiol (318, 479). These effects could be induced
by 17␤-estradiol, estrone, 17␣-estradiol, and estriol,
whereas progesterone, pregnenolone, testosterone, androstendione, and 5␣-dihydrotestosterone were ineffecPhysiol Rev • VOL
tive. Moreover, the estrogen-induced [Ca2⫹]i increase was
not affected by the ER agonist/antagonist tamoxifen or by
the RNA and protein synthesis blockers actinomycin D or
cycloheximide (400), which is in contrast to findings in
other tissues. The estrogen-induced [Ca2⫹]i increase in
granulosa cells is inhibited by pretreatment with inhibitors of the inositol phospholipid hydrolysis like neomycin
and U 73122 (444). Further investigations revealed that
17␤-estradiol may directly influence the quality of maturing oocytes. The addition of 17␤-estradiol did not influence the progression of oocytes through meiotic maturation, but it improved the fertilization potential of the in
vitro matured oocytes (479).
As already mentioned in section IID3, estrogen binding sites are also present on human sperm. Because spermatozoa are almost devoid of protein de novo synthesis,
and sperm protein synthesis is confined to the mitochondria, effects mediated by estrogens can be linked to nongenomic actions. Previous studies have shown that 17␤estradiol induces functional changes in sperm by the enhancement of sperm motility (39, 95). Moreover, in
spermatozoa of fertile and infertile men, the addition of
17␤-estradiol is claimed to improve the results of the
zona-free hamster ova penetration test (91). A recently
published study revealed that 17␤-estradiol alters the testicular androgen production by causing a decrease in
gonadotropin-stimulated 11-ketotestosterone synthesis in
testes of Micropogonias undulatus (264).
The detection of estrogen receptors in the brain and
estrogen-concentrating cells in the pituitary gland, hypothalamus, and other brain regions (372) has shifted the
interest to estrogen effects, which are not directly connected to reproduction, and there is increasing evidence
that estrogen actions in the brain are mediated in part by
nongenomic mechanisms. Within seconds after application, 17␤-estradiol led to a brief hyperpolarization and
increased potassium conductance in rat medial amygdala
neurons, an effect which persisted after suppression of
protein synthesis. In addition, a greater proportion of the
neurons from females than males were affected by 17␤estradiol (325, 537). The rapid time course and the insensitivity to protein synthesis inhibitors led to the assumption that genomic effects may not be involved. Moreover,
it is now well established that estrogens act via interaction with specific receptors at the plasma membranes of
neurons (69, 484 and others), followed by the activation of
intracellular signal transduction pathways. Further investigations have shown that estrogens stimulate the formation of cAMP (194), the phosphorylation of CREB (543),
and the formation of IP3 (148) and activate the MAPK
signaling pathway in a neuroblastoma cell line (513).
Previous studies have revealed that estrogens may
have an important role in the differentiation of mouse
midbrain dopaminergic neurons via nongenomic mechanisms, which includes the interaction with a membrane
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nificantly increased lordosis within 10 min. Interestingly,
this effect was seen in both PR KO and wild-type mice
(160). Recently, it has been shown that the actions of
progesterone in the ventromedial hypothalamus require
intracellular PR but, interestingly, in the ventral tegmental
they do not. In the ventral tegmental, progesterone may
facilitate lordosis after metabolism to and/or biosynthesis
of 3␣,5␣-THP, which may have subsequent actions at
other receptors like GABAA/benzodiazepine receptor
complexes (159).
Recently, it has been described that 17␤-estradioland progesterone-BSA bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In this context, progesterone (100 nM) was found to rapidly decrease Vmax of the
enzyme, whereas Michaelis constant (Km) was not
changed (221).
Like other steroids, progestins also activate MAPK.
In T47D cells, which are rich in PRA and PRB and also
contain ER, treatment with R 5020 (10 nM) resulted in a
rapid stimulation of ERK-2 activity, which was already
detectable after 2 min. The effect was inhibited by the use
of the antiprogestin RU 486 (1 mM) and, surprisingly, by
various anti-estrogens. In T47D-Y cells, which lack PR but
are endowed with high ER levels, no stimulation of ERK-2
activity by R 5020 was found, suggesting that the PR is
required for this effect (305). The rapid activation of the
c-Src/p21ras/MAPK signaling pathway has been described
to depend on the ability of the intracellular PR to interact
with SH3 domains (61).
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Physiol Rev • VOL
critical functions outside the nucleus. The potential biological effects are considerably broadened because of the
PI 3-kinase/Akt pathway and its effects in various cell
functions (156).
The rapid nongenomic effects of estrogens raise the
important question about the regulation of estrogen synthesis via the regulation of the enzyme aromatase in the
brain. It has been assumed that changes in brain aromatase activity are mediated by changes in enzyme concentrations, which are relatively slow. A recently published study demonstrated that brain aromatase activity
can be modulated within minutes by Ca2⫹-dependent processes that presumably involve the phosphorylation of
aromatase or another regulatory protein, which indicates
rapidly changing levels of locally produced estrogens
available for nongenomic actions (19).
In addition to the effects of estrogens on brain differentiation and neuroprotection, estrogens seem to have
also an important impact on the endocrinological function of the brain. As reported years ago, GH3/B6 rat pituitary tumor cells exhibit a rapid prolactin release with
nanomolar amounts of estrogen within minutes (354).
These effects could be confirmed recently, and a membrane form of ER␣ could be identified as mediator using
different antibodies directed against ER␣, which are too
large to penetrate into cells. An antibody (Ab R4) against
the hinge region of ER␣ increased prolactin release in a
time- and concentration-dependent manner, whereas Ab
H151 targeted against a different hinge region epitope
decreased prolactin release and blocked the effects of
17␤-estradiol (337).
Rapid and specific effects of 17␤-estradiol have been
also demonstrated in isolated duodenal cells, initiated on
the cell surface and resulting in the activation of the
cAMP pathway and Ca2⫹ influx through dihydropyridinesensitive Ca2⫹ channels (373). Furthermore, the PLC inhibitors neomycin and U 73122 suppressed the stimulatory effect of 17␤-estradiol on the Ca2⫹ influx into duodenal cells, indicating an involvement of the PLC (374).
Recently, evidence could be provided that 17␤-estradiol indirectly stimulates the cytosolic PKA activity in
female rat distal colonic epithelium. Basal PKA activity
was stimulated by cAMP, which was abolished by the
addition of the specific inhibitor PKI and the adenylyl
cyclase inhibitor SQ-22536. In contrast, the PKC was directly stimulated by 17␤-estradiol; the specific PKC inhibitor GF 109203X prevented the 17␤-estradiol-induced PKA
activation. The PKC and PKA inhibitors abolished the
increase in [Ca2⫹]i. These effects were only demonstrated
in female rats; 17␤-estradiol failed to induce a similar
effect in membrane or cytosolic fractions of male rats
(121). Gender-related differences could be also observed
in the regulation of Cl⫺ secretion in the rat distal colonic
epithelium. 17␤-Estradiol inhibited colonic Cl⫺ secretion,
which involved intracellular Ca2⫹ and PKC. The ability of
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receptor coupled to intracellular signaling pathways. In
previous studies, the addition of 17␤-estradiol evoked a
selective increase in newly synthesized dopamine (359)
and dopamine release (40). In cultured embryonic dopaminergic neurons, 17␤-estradiol evoked a rapid Ca2⫹ release from intracellular stores in cultured embryonic dopaminergic neurons reaching a peak rise within 5 s. The
BSA-estrogen conjugate was equally effective in triggering the typical rise in [Ca2⫹]i (53). Moreover, estrogens
modified within seconds after application basal firing
rates (97) and led to rotational and stereotypic behavior in
adult rodents (40). It could be further demonstrated that
17␤-estradiol and the BSA-estrogen conjugate enhanced
neurite growth and arborization of dopaminergic neurons.
This effect was inhibited by antagonists of cAMP/PKA and
Ca2⫹ signaling pathways, but not by the ER antagonist ICI
182780 (52). With the use of quantitative double-labeling
immunocytochemistry and gel shift assay, it could be
demonstrated that 17␤-estradiol exposure induced the
phosphorylation of CREB in midbrain dopaminergic cells.
Again, this effect was antagonized by the simultaneous
application of inhibitors of the cAMP/PKA or Ca2⫹ pathways, but not the ER antagonist ICI 182780 (52).
Several lines of evidence indicate that estrogens possess neuroprotective properties, which may be induced
by genomic as well as nongenomic mechanisms. Physiological concentrations of 17␤-estradiol rapidly intensified
the release of secreted amyloid ␤-precursor protein (APP)
in mouse hippocampal HT22 and human neuroblastoma
SK-N-MC cells. The synthesis of amyloid ␤ and its precursor APP is a central step in the pathogenesis of Alzheimer’s disease. Again, the rapid secretion of APP is mediated through 17␤-estradiol via the phosphorylation of
ERK1/2, which are both members of the MAPK pathway
(279), indicating a cross-talk between genomic and nongenomic actions. Interestingly, the activation MAPK signaling pathway and the increased release of APP induced
by 17␤-estradiol was independent of the ERs, as it occurred also in neuronal cells lacking a functional ER
(279). In a recent study it could be shown that estrogen
has an antiapoptotic effect on primary cortical neurons
that was mediated through the PI 3-kinase/Akt signal
transduction pathways. The PI 3-kinase/Akt signals
evoked the phosphorylation of CREB at a serine residue
and the upregulation of the antiapoptotic protein Bcl-2.
The neuroprotection of estradiol could be inhibited by the
suppression of these signals. The rapid time course, an
effect was seen within 5 min, suggests a nongenomic
response. In contrast to the study mentioned above, the
ER antagonist ICI 182780 attenuated the phosphorylation
of PI 3-kinase/Akt, indicating an involvement of ER in this
effect (214). A direct interaction of the ER with the regulatory subunit of PI 3-kinase in endothelial cells was
already shown earlier (451), and linking the ER to the PI
3-kinase pathway suggests that the ER may be involved in
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onset, the attenuation of the 17␤-estradiol response, and
the observation that the effect was not accompanied by
an increase of eNOS protein expression suggest that these
effects were caused by a nongenomic action of 17␤-estradiol. The BSA-17␤-estradiol conjugate also increased the
NO concentration, and the ER␣ antagonist ICI 182780
completely blocked estrogen-stimulated NO release,
which supports the assumption that a plasma membrane
receptor similar to ER␣ is involved in these effects (231).
A model for the mode of nongenomic modulation of NO
synthesis in caveolae is given in Figure 7.
Further effects of estrogens on vascular tissues have
been already described in section IID3.
In addition to the estrogen effects described in his
section, estrogen binding sites in plasma membranes
could be found in various tissues like myometrium, liver,
a breast cancer cell line (MCF-7), and on the cell surface
of osteoblast and osteoclast-like cells (51, 153, 376).
As described in detail in section IIIB, progesterone
faciliates the human sperm acrosome reaction, an effect
which could broaden the therapeutic options for male
infertility. The discovery of functional ERs in the surface
of human sperm and the inhibition of progesterone-induced acrosome reaction by estrogens might be pathophysiologically relevant for the appropriate timing of capacitation and the acrosome reaction. A future therapeutic concept for male infertility could be the inhibition of
the sperm ER while enhancing the progesterone effect.
For many years, hormone replacement therapy appeared to be a potential cardioprotective agent in coronary heart disease, the leading cause of death in developed countries. Beneficial therapeutic effects could be
derived from the 17␤-estradiol and progesterone actions
to immediately relax small and large arteries. However,
recent clinical trials have not elicited any advantageous
effects of hormone replacement therapy on risk for major
cardiovascular events among women with established
coronary heart disease.
The investigations of steroids in the nervous system
have emphasized the fact that estrogens possess neuroprotective, antiapoptotic, and antioxidant effects that
may be in part induced by nongenomic mechanisms. The
study of estrogen actions in the brain might be a challenging topic and a potential target for pharmaceutical development.
D. Androgens
Rapid effects of androgens on [Ca2⫹]i have been
intensively studied in several systems. In male rat osteoblasts, testosterone (10 pM to 10 nM) increased [Ca2⫹]i
within 5 s via Ca2⫹ influx as well as via Ca2⫹ mobilization
from the endoplasmic reticulum. In addition, IP3 and DAG
formation was induced within 10 s after addition of sim-
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17␤-estradiol to reduce the Cl⫺ secretion in the female
colon may probably contribute to the clinically important
17␤-estradiol-induced salt and water retention in females
(105).
Clinical and experimental data suggest that estrogens
may play a role in the genesis and perpetuation of colorectal cancer (130, 367). The mechanism by which estrogen exerts proliferative properties has been assumed to
be exclusively mediated by genomic actions. Interestingly, 17␤-estradiol induced a rapid cellular alkalinization
of crypts and cancer cells that was sensitive to Na⫹/H⫹
exchanger (NHE) blockade or PKC inhibition. Thymidine
incorporation drastically increased both in crypts and
cancer cells, which, again, could be inhibited by NHE
blockade, indicating a nongenomically mediated mitogenic effect of 17␤-estradiol (527).
In a previous investigation, 17␤-estradiol has been
shown to increase the pancreatic insulin secretion and to
decrease the insulin resistance antagonizing the effects of
menopause on glucose and insulin metabolism (74). In
male and ovariectomized mice, 17␤-estradiol seems to be
able to prevent the development of diabetes mellitus
(126), an effect which could be probably mediated by a
nongenomic mechanism. At physiological concentrations,
17␤-estradiol rapidly and reversibly closes KATP channels
from pancreatic ␤-cells and depolarizes the plasma membrane resulting in increased [Ca2⫹]i, which in turn enhances the insulin secretion (327).
In human and pig coronary arteries and rat aortas,
17␤-estradiol and 17␣-estradiol induce an acute and clinically relevant relaxation (399, 418). Furthermore, 17␤estradiol leads to an acute relaxation of rat arteries from
females, but not males (291). Within minutes, 17␤-estradiol increases basal intracellular cAMP in pulmonary vascular smooth muscle cells, which is probably caused by
an estrogen-induced activation of membrane adenylate
cyclase (146). In PC12 cell membranes, the increased
intracellular cAMP concentration is paralleled by enhanced activation of membrane-bound guanylate cyclase
and also of the atrial natriuretic factor-stimulated guanylate cyclase activity (94). Furthermore, in PC12 cells,
17␤-estradiol rapidly reduces the catecholamine secretion
by inhibiting L- and N-type Ca2⫹ channels and P2X2 receptors (232).
As demonstrated in BAEC, 17␤-estradiol induces a
translocation of eNOS from the plasma membrane to
intracellular sites close to the nucleus. This effect of
17␤-estradiol was detected at concentrations as low as 1
pM. Moreover, cGMP release could be rapidly increased
by 17␤-estradiol, and this effect could be blocked by the
ER antagonist ICI 164384 (182). Also in BAEC, 17␤-estradiol (5 nM) increased NO production through ER␣ localized in specific plasma membrane domain caveolae. The
NO production reached its maximum at 5 min before
falling to near basal levels over the next 30 min. The rapid
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991
ilar concentrations of the steroid (255). Interestingly,
comparable amounts of testosterone did not elicit an
increase in [Ca2⫹]i in female rat osteoblasts (256). In
primary cultures of neonatal rat myotubes, testosterone
(50 –100 nM) induced an increase of [Ca2⫹]i within seconds. This response was frequently accompanied by irregular oscillations. In this system, testosterone also produced a rapid increase in IP3 levels that reached a peak
threefold higher than basal level (133). Moreover, physiological concentrations (1–10 nM) of testosterone were
described to rapidly raise [Ca2⫹]i in activated T cells.
Contrary to the effects in osteoblasts, the testosteroneinduced increase of [Ca2⫹]i in these cells is solely resulting from Ca2⫹ influx (44, 45). In this context it is worthwhile to note that classic ARs in splenic T cells appear to
be genomically unresponsive to testosterone. On the
other hand, the postulated cell surface receptors for testosterone are described to be functionally active.
The finding that T cells respond to testosterone in a
nongenomic manner suggests an as yet unexploited option for a modulation of T-cell activity with possible apPhysiol Rev • VOL
plications in proliferative and infectious diseases. For
example, the binding of the androgen to surface receptors
of T cells followed by an immediate Ca2⫹ influx may be
the initial event in the testosterone-induced susceptibility
to Plasmodium chabaudi malaria in mice (45). If this
finding could also be demonstrated in humans for other
plasmodium species, it may be speculated that selective
inhibition of testosterone T-cell surface receptors may
open a new path of preventing malaria infections.
The effects of testosterone on [Ca2⫹]i described
above are not inhibited by nuclear AR antagonists like
hydroxyflutamide or cyproterone, suggesting an action
independent of classic AR. This argumentation is further
supported by findings in mouse IC-21 macrophages where
the classic AR is absent. In these cells, testosterone likewise induced a [Ca2⫹]i increase that was found to be
predominately due to release of Ca2⫹ from intracellular
stores (46).
Moreover, studies with regard to androgen-induced
Ca2⫹ fluxes have been done in immature rat Sertoli cells,
which contain the normal AR, in the human prostatic
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FIG. 7. Composite model of nongenomic
activation of eNOS. Recent studies have shown
localization of estrogen receptor (ER)␣ to
caveolae, direct interaction of ER␣ with the p85
subunit of phosphatidylinositol (PI) 3-kinase,
and activation of the PI 3-kinase-Akt-eNOS pathway. [From Mendelsohn (295), copyright Lippincott Williams & Wilkins.]
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(0.1–100 nM) blocked the adenosine vasodilator effect
and increases vascular resistance (89). Although the studies were done in a time range of 1 h, actinomycin D and
cycloheximide were ineffective in blocking these effects.
On the contrary, similar concentrations of testosterone were found to induce a vasodilation in canine coronary conductance and resistance arteries in vivo (98).
With the use of supraphysiological concentrations of testosterone (25–300 ␮M), direct vasorelaxing effects on rat
thoracic aorta have been described (107). Similarly, the
androgen 5␤-dihydrotestosterone has been found to produce relaxation of rat aorta rings precontracted by norepinephrine or K⫹ (368). However, because of the high
concentrations used, the specificity is doubtful.
Several other rapid androgen responses have been
described. For example, the effects on the electrical activity of neurons have already been reviewed elsewhere
(144, 516). More recently, the testosterone-induced modulation of Cl⫺ secretion in cultured rat efferent duct
epithelia was analyzed by the use of the short-circuit
current (Isc) technique (252). Testosterone rapidly reduced forskolin-stimulated Isc with an IC50 value of 1 ␮M.
Cyproterone acetate or flutamide did not block the effect.
Furthermore, micromolar concentrations of testosterone
reduced the forskolin-induced rise of intracellular cAMP
in these cells (252). In addition, it has been demonstrated
that physiological concentrations of testosterone or testosterone-BSA (0.1 nM to 1 ␮M) can elicit significant
prolactin release from lactotrophs (type 2) in the male rat
pituitary within 5 min (101). Testosterone (100 nM), like
progesterone and 17␤-estradiol, was also found to depress gonadotropin releasing hormone receptor mediated
stimulation of low-Km GTPase activity in anterior pituitary membranes from male rats (393) and to increase
glycogen phosphorylase activity in the liver (119).
E. Neurosteroids
Neuroactive steroids have been suggested to influence physiology and pathophysiology of the central nervous system in various fields. They control regulation of
gene expression after binding to intracellular receptors,
which may act as transcription factors. Neuroactive steroids, however, have also been shown to be potent modulators of an array of ligand-gated ion channels and specific G protein-coupled receptors nongenomically. As the
foundation for their effects, an intracellular cross-talk
between nongenomic and genomic actions of neuroactive
steroids has been suggested. The possible physiological
and pathophysiological role of neuroactive steroids has
been proposed and explored in circumstances such as
sleep, memory function, neuroprotection, depression and
anxiety disorders, reaction to stress, premenstrual syndrome, and addiction diseases. These aspects will be
briefly reviewed in the following paragraphs.
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tumor cell line (LNCaP), which contains a mutated AR,
and the human prostatic cell line PC3, which does not
contain AR (270). Testosterone as well as the synthetic
androgen R 1881 (both 1–1,000 pM) rapidly induced transient Ca2⫹ increases in all cell types. These responses
could be inhibited by hydroxyflutamide but, in contrast to
effects on the nuclear receptor, were not inhibited by
cyproterone acetate.
In addition, testosterone, although at nonphysiological high concentrations (0.3–3 ␮M), increased [Ca2⫹]i in
freshly isolated Sertoli cells in a hydroxyflutamide-sensitive manner (186). A similar effect has been shown by
using other androgens like mibolerone (dimethylnortestosterone) and 5␣-dihydrotestosterone in LNCaP cells
(465).
Furthermore, androstenedione, the main intrafollicular androgen, rapidly increased [Ca2⫹]i in human granulosa luteinizing cells (273) and porcine ovarian granulosa
cells (257). This increase was dose dependent, with maximum activity at 1 nM androstenedione in pig and 10 nM
in human cells. In both cases, comparable concentrations
of testosterone had no effect, and the nuclear androgen
receptor antagonist flutamide was ineffective. In human
granulosa cells, the increase in [Ca2⫹]i resulted from both
Ca2⫹ influx and mobilization from endoplasmic reticulum,
whereas in porcine cells, this effect is solely dependent on
intracellular stores. In both cases, G protein-mediated
PLC activation appears to be involved.
Similar to estrogens and progestins, androgens were
found to stimulate MAPK (also called ERK) in several
cells. The androgen R 1881 stimulates activity of ERK
promptly after addition of the steroid (1 nM) in human
PMC42 breast cancer cells (545). Unlike anti-estrogens
and anti-progestins, which inhibit agonist-induced ERK
activation (305), the anti-androgen flutamide stimulates
ERK activity in the same manner as R 1881. In this context, dihydroxytestosterone (100 nM) was also shown to
rapidly induce phosphorylation of ERK1 and ERK2 in
primary prostatic stromal cells and LLNCaP cells but not
in human genital skin fibroblasts, another androgen-responsive cell (370). This dihydrotestosterone-induced
stimulation of ERK was also found in human prostate PC3
cells, which were stable transfected with wild-type AR,
but not in mock-transfected cells, suggesting an involvement of the classic AR. Like in PMC42 cells, the antiandrogens hydroxyflutamide and casodex (100 nM and 10
␮M) alone were able to induce ERK phosphorylation
(370). Therefore, the antiandrogens currently available as
therapeutics may not totally abrogate all androgenic activity in target cells. These observations may help to
explain the clinical phenomenon of withdrawal responses
observed in patients suffering from prostate cancer who
become refractory to these antagonists (294, 422).
Androgen effects have also been described in cardiac
tissue. In the isolated and perfused rat heart, testosterone
NONGENOMIC STEROID ACTION
Physiol Rev • VOL
investigating transdermal estrogen replacement compared with placebo in 62 postmenopausal women (380).
Women receiving estrogen, however, reported a subjectively better sleep quality than women on placebo, suggesting effects beyond a modulation of GABA or glutaminergic receptors shown for other neuroactive steroids. This
finding slightly contrasts the results of another small
study investigating sleep patterns and corresponding EEG
changes in 11 postmenopausal women. Transdermal estrogen enhanced REM sleep duration, reduced time
awake during the first two sleep cycles, and restored
normal sleep EEG patterns compared with women not
receiving estrogen replacement therapy (8). The differences of these two studies may have originated in different baseline stress levels of women in the two studies.
The procedure of taking the EEG may have elevated the
baseline stress level with an associated increase in cortisol levels. This is suggested by a study of Prinz et al. (383),
who found that elevated urinary free cortisol is associated
with impaired sleep and earlier awakening in postmenopausal women without estrogen replacement therapy, but
not in women on estrogen replacement therapy. These
studies illlustrate the current dilemma of evaluating the
effects of neuroactive steroids in sleep studies in humans.
Diverse study populations, and the lack of in vitro investigations for the steroids tested in vivo, have so far hardly
been able to accurately test the proposed hypotheses.
Instead, the complexity of the matter has only just become obvious with studies generating more questions
than answers.
Memory involves a complex interaction of many different types of receptors and thus is modulated by several
pathways. DHEA and its sulfate DHEAS have been described to enhance memory, an effect that can in part be
explained by the modulation of sleep patterns via the
GABAA receptor. Other pathways include interaction at
the NMDA receptor, as suggested for pregnenolone. This
compound as well as its sulfate and DHEA caused improvements in avoidance training and other test paradigms in rodents (285). As pregnenolone sulfate also reduces scopolamine-induced learning deficits and memory
impairment caused by a selective NMDA antagonist, an
allosteric action at this receptor is likely (286, 304). Sigma1 receptors also participate in memory, as the memoryenhancing effect of DHEAS and pregnenolone sulfate can
be blocked by a sigma1 receptor antagonist (395). However, memory enhancing and neuroprotection (see below)
cannot always be separated from each other, particularly
in elder individuals, and many neuroactive steroids exhibit both effects. A different mechanism underlying steroid action in memory processes may be the modulation
of excitatory synaptic transmission by estrogens, particularly of glutamate receptors in the hippocampus (195).
In the context of neuroprotection, the administration
of antagonists at the NMDA receptor, such as the syn-
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In sleep disorders, clinical experience has been gathered in studies investigating GABAA receptor modulating
neuroactive steroids. Progesterone has been proposed to
shorten sleep latency and to increase non-rapid-eyemovement sleep (non-REM) as well as sleep duration
(157). The hypothesis that allosteric modulation of the
GABAA receptor may play a significant role also in sleep
disturbances in humans is backed by the finding that
DHEA-S, an allosteric inhibitor of GABAA receptors, is
markedly decreased in patients with chronic fatigue syndrome (241).
Investigators have also made progress in examining
the sleep-modulating properties of DHEA. After oral administration of DHEA, an increase in REM sleep can be
observed while simultaneously EEG activity in the sigma
frequency range during REM sleep is enhanced. This finding which may seem puzzling at first sight is compatible
with a mixed GABA-agonistic/antagonistic profile of
DHEA as a modulator of the GABAA receptor (158). However, high doses of systemic DHEAS, the sulfate ester of
DHEA, did not affect sleep structure in rats in another
study, whereas a dose-dependent modulation of electroencephalogram (EEG) patterns was seen during non-REM
sleep (423). DHEAS (50 mg/kg) significantly increased
EEG power in the frequency range of sleep spindles, and
100 mg/kg depressed EEG power in the slow-wave frequency bands. It has been hypothesized that additionally
to GABAA receptor modulation, glutamate-induced currents may be responsible for conferring this effect of
DHEAS. The GABA agonistic properties of neurosteroids
have led researchers to investigate a possible role of these
compounds as therapeutic modulators of unfavorable
sleep-wake rhythms. Compared with placebo, the group
receiving 15 mg/kg allopregnanolone over a treatment
period of 5 days exhibited shorter non-REM sleep latencies, prolonged REM sleep latencies, and longer non-REM
sleep episodes in rats (109). This finding is comparable to
the action of other agonistic modulators of the GABAA
receptor such as short-acting benzodiazepines. However,
in this study, no development of tolerance with respect to
sleep-modulating characteristics was observed, which
would have been expected for other known drugs targeting the benzodiazepine binding site of the GABAA receptor.
One of the major problems in this field of research is
the lack of information how neuroactive steroids may
modulate sleep-wake behavior and EEG patterns in humans. This is particularly troublesome since the steroids
that have been shown to modulate these aspects in the
animal model have hardly been investigated at all in humans. The limited evidence gathered so far derives primarily from rather small studies in postmenopausal
women with and without hormone replacement therapy.
Sleep latency, distribution of sleep stages, sleep efficiency, and total sleep time were very similar in a study
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are supposed to downregulate GABAergic neurotransmission and induce anxiety-like states (55). Changes in the
levels of neuroactive steroids are supposed to occur in
response to such events, indicating a close relation of
stress, neurosteroid levels, and GABA receptor function
(27). For example, elevated levels of corticosterone,
which may be present during stress conditions, have been
shown to affect the modulation of ligand binding to
GABAA receptors by THDOC and GABA itself (349) and
altering the hippocampal inhibitory tone.
There are numerous and sometimes contradictory
reports on the etiology of premenstrual syndrome (PMS).
While some studies established a correlation between a
decline of the levels of the anxiolytic, GABA-modulating
allopregnanolone and adverse symptoms such as anxiety,
other studies failed to find such a relationship (457).
Replacement therapy with allopregnanolone or its precursor progesterone were not reliably successful. Estradiol
levels have been correlated with PMS symptoms as well,
because this steroid has been associated with excitatory
effects, possibly experienced as anxiety. Estradiol effects
may occur, however, at increased steroid levels as well as
on withdrawal and appear to be complex. Interestingly,
the action of selective serotonin uptake inhibitors may be
mediated at least in part by a direct increase in allopregnanolone synthesis from progesterone (190).
For addiction diseases, the data obtained on improvement of mood disorders and other pysychological
syndromes by steroid administration suggest their possible use for alleviating the problems associated with drug
withdrawal. In a clinical study, however, progesterone
was ineffective in improving symptoms of benzodiazepine
withdrawal in patients having taken diazepam for more
than 1 yr. In contrast, animal models of drug dependence
identified allopregnanolone as effective in reducing anxiety and hyperlocomotion (396). Interesting results on the
mechanism of clinical addiction development were obtained in rodent models. Gender differences were observed in the rapid augmentation of behavioral responses
to stimulants such as amphetamine and cocaine (41).
While estrogen enhanced dopamine activity in the striatum of female rats, male rats were not affected. These
findings may at least partly explain the higher risk of
developing addiction to cocaine in females as well as the
effect of the menstrual cycle on addiction behavior. The
elevation of allopregnanolone levels in the cerebral cortex by ethanol (494) may explain in part not only the
hypnotic effect, but also mild anxiolysis that may contribute to the development of ethanol addiction.
F. Mineralocorticoids
Early work on the rapid action of aldosterone, the
principal mineralocorticoid, has been reported occasion-
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thetic 5␤-pregnan-3-ol-20-one 3-hemisuccinate, has been
shown not only to inhibit NMDA-induced currents, but
also to prevent cell death in primary cultures of rat neurons. The same steroid when administered intravenously
reduces infarct size after focal cerebral ischemia (514).
Protection against NMDA-induced neuronal death is also
inferred by 17␤-estradiol by direct inhibition of the NMDA
receptor (515). The clinical importance of such phenomena is supported by epidemiological studies that revealed
a decreased risk and severity of dementia in women on
estrogen replacement therapy (321). Other studies have
correlated decreased levels of neurosteroids to various
forms of dementia (32). The NMDA receptor complex also
is targeted by many nonselective sigma receptor ligands,
although selective sigma ligands show neuroprotective
action as well, presumably by preventing the release of
excitotoxic amino acids (288). Other mechanisms of neuroprotection may involve the activation of MAPK by estrogens and several progestins (334). The relevance of
this pathway is underlined by the abolishment of estrogen
neuroprotection by MAPK inhibitors (452). The clinical
use of steroids for memory enhancement and neuroprotection is currently under investigation (246).
Depression as a disorder of mood often is asssociated with anxiety. In this context, anxiolytic properties
that have been demonstrated for several steroids are of
great physiological importance. In rodents, the administration of positive allosteric GABAA modulators comprising allopregnanolone (56, 549), tetrahydrodeoxycorticosterone (THDOC; Ref. 108), alphaxolone (73), and ganaxolone (42) all showed such action under various
conditions. Remarkably, anxiolysis seems to be independent from sedative properties associated with some of the
steroids. Despite the numerous studies done in rodents,
hardly any clinical trials of possible anxiolytic properties
of this class of compounds have been reported.
During one of the few controlled, double-blind studies, progesterone was shown to improve many symptoms
of women suffering from premenstrual syndrome (116),
including alleviation of depression. Because progesterone
is metabolized to many different neuroactive steroids, it is
difficult to determine which hormone(s) ultimately confer
the effect. Some evidence points to the GABAA modulator
5␣-pregnanolone (495), but there is no clear correlation
between steroid levels and clinical symptoms (426). However, the alleviation of symptoms of depression by tricyclic antidepressants has been attributed to the correction
of a disequilibrium of neuroactive steroids (401).
Beneficial effects on depressive syndromes have also
been shown for steroid agonists at the sigma1-receptor
(395).
Apart from depressive disorders, anxiolysis is also
beneficial in stress conditions. Consequently, allopregnanolone and THDOC elicit marked antistress effects in
addition to their anxiolytic properties. Stress conditions
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NONGENOMIC STEROID ACTION
same group reported a rapid modulation of plasma membrane H⫹ conductance (173) and activation of the ERK
pathway (172) that subsequently affects NHE activation in
Madin-Darby kidney cells (344). In the same system, nuclear signaling initiated by exposure to aldosterone has
been studied. Atomic force microscopy was used in this
study to detect the contraction of nuclear pore complexes
in response to a Ca2⫹ signal, that in turn is known to be
elicited by aldosterone. Furthermore, the shrinkage of
nuclei containing fluorescently stained DNA within 2 min
upon addition of aldosterone has been demonstrated;
however, there were two different patterns of response.
The author concluded that Ca2⫹ accumulated in the nucleoplasm at high concentration during contraction,
thereby preparing the pores to specifically import the
activated (ligand-bearing) MR into the nucleus, which
eventually leads to genomic action. This model therefore
integrates nongenomic and genomic action into a dualaction model. Recently, the rapid responses of skeletal
muscle cells to aldosterone at 10 –100 nM (besides testosterone) were studied (133). In this system, aldosterone
induced a rapid transient rise in intracellular Ca2⫹ which
peaks at 3 min, followed by a second slow increase being
maximal at 30 min after hormone addition. The fast response occurs as a series of rapid oscillations in Ca2⫹
concentration on the single-cell level. Interestingly, Ca2⫹
oscillations distinct from the events in the cytosol occur
in the nuclei, which may have physiological significance
for muscle function. A transient increase of IP3 precedes
the Ca2⫹ peak. The Ca2⫹ response remained unchanged
upon preincubation with 1 mM spironolactone, and estradiol, progesterone, and dexamethasone were were unable
to elicit a response even at 1 mM.
In another recent study, nongenomic aldosterone effects on cortical collecting duct cells have been investigated (205). In these cells, aldosterone at 1 nM produced
a transient increase of intracellular Ca2⫹ within 5 min.
FIG. 8. Calcium images of porcine endothelial cells
before and 5 min after addition of 100 nM aldosterone,
as quantified by fura 2 ratiometric imaging. Bright areas correspond to high intracellular calcium. [From
Wehling et al. (521), copyright Academic Press.]
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ally. Studies include clinical work on hemodynamics
(233) and on Na⫹ efflux from erythrocytes (462). Two
decades later, a dual effect on Na⫹ efflux in rat tail
arteries was found that consisted of an early, ouabaininsensitive effect occurring within 15 min and a delayed
Na⫹-K⫹-ATPase-dependent part (323). The rapid action
was not affected by actinomycin D and thus is likely to be
nongenomic. As suggested by the two latter studies, other
ion fluxes are modulated rapidly as well in different cell
types. Our laboratory demonstrated increases of [Na⫹]i,
[K⫹]i, and [Ca2⫹]i in human mononuclear lymphocytes
upon aldosterone stimulation that were half-maximal at
0.1 nM (for review, see Refs. 144, 519). The increase in cell
volume that accompanies ion fluxes has been reported in
both HML and endothelial cells (427), thus suggesting a
more general role for cell volume regulation. Rises in
[Ca2⫹]i have been demonstrated in many different cell
types by our laboratory; an instructive example is shown
in Figure 8.
The Na⫹ influx is probably induced by a rapid activation of the membrane-bound NHE, which also causes a
pH rise (alkalinization) in the cell. This phenomenon has
also been demonstrated by BCECF spectrofluorometry
(517) in VSMC. Similar results have been reported recently in strips of human chorionic and uterine arteries,
where alkalinization occurred within a few seconds (5).
This work also reports instructive studies on agonists and
antagonists (discussed in sect. IIC2B). Cortisol was active
as an agonist only after addition of the hydroxysteroid
dehydrogenase inhibitor carbenoxolone. The rise in pH
probably reflects activation of the NHE, as judged by the
ability of amiloride derivatives to abolish the response.
Rapid aldosterone-induced changes of pH have been
reported in the distal tubule of toad kidney (345). The
intracellular alkalinization was amiloride sensitive and
thus probably mediated by the NHE; however, the time
frame of 15–20 min does not rule out genomic action. The
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LÖSEL ET AL.
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A modified follow-up study revealed changes in SVR during the first 45 min after steroid administration (425).
After exhaustive exercise, aldosterone was found to
be increased with the level positively correlated to work
load, possibly reflecting increased volume demand. However, physical training, which leads to a substantial gain in
working capacity, does not affect aldosterone levels at
rest or at exercise (175). A possible explanation is the
suppression of the renin-angiotensin-aldosterone system
(RAAS) secondary to training to counteract stress-induced activation (211). The action on baroreflex mentioned above and the reducing effect on heart rate variability (271) may point to an adrenergic-like action of
aldosterone. A direct interaction has been shown as well
between aldosterone and modulators of the ␣1- and ␤-adrenergic system (424).
Increased mortality in patients with chronic heart
failure or coronary heart disease has been correlated with
activation of the RAAS. Downregulation of the latter or of
the adrenergic system has both been successful lowering
mortality in such patients. While angiotension-converting
enzyme inhibitors are an established part of therapy, administration of the MR antagonist spironolactone has
proven beneficial only recently during the Randomized
Aldactone Evaluation Study (RALES) (378). While being
an antagonist at the classic MR, spironolactone itself as
well as its closed-ring metabolites form an equilibrium
with their open-ring derivatives (see sect. IIC2B) in vivo.
Based on findings reported by Alzamora et al. (5), some
open-ring derivatives appear to have antagonist activity
for nongenomic aldosterone action in vitro, although
more comprehensive data are not available. As the dose
(25 mg daily) was much lower than usually employed to
effect diuresis, the therapeutic benefit may have arisen, at
least in part, by the inhibition of nongenomic mechanisms.
A different mechanism for the action of spironolactone has been proposed by recent findings in the isolated
working rat heart (29). Here, spironolactone exerts an
autonomous positive inotropic action and does not antagonize but adds to the inotropic action of aldosterone, with
spironolactone being roughly 10 times more potent than
aldosterone. This might explain much of the beneficial
action of the antagonist seen in the RALES study.
G. Vitamin D3
Starting from early observations in intestinal epithelia, rapid effects of 1␣,25(OH)2D3 on transepithelial movements of calcium have been termed “transcaltachia.” For
rapid effects of vitamin D3 on intracellular calcium, both
release from intracellular stores and influx have been
identified as sources, with their relative contributions
depending on the cell type studied (333). In chicken myo-
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Again, spironolactone was ineffective at 10,000-fold excess as was actinomycin D. The same laboratory suggested PKC␣ as a direct target for nongenomic aldosterone action (203, 204). In this scenario, PKC␣ activation
leads to Ca2⫹ influx. The increased intracellular Ca2⫹
concentration together with a direct PKC␣ effect activates the NHE, which leads to alkalinization of the cell.
This in turn stimulates KATP channels. The release of
arachidonic acid by phospholipase A2 action and subsequent PGE2 formation is also thought to participate in
mediating Ca2⫹ entry (204). The physiological meaning of
these phenomena in Na⫹ absorbing epithelia may be the
maintenance of a sufficient ion potential to promote Na⫹
entry by activation of basolateral K⫹ conductance (202,
203). Although PKA was not found to be activated in distal
colon cells (202), in bronchial epithelium PKA seems to
participate in aldosterone signaling, as well as adenylate
cyclase, Ca2⫹-ATPase, and a G protein-coupled receptor
(492). In contrast to many other systems, however, aldosterone leads to a decrease of intracellular Ca2⫹ in these
cells.
In vitro studies on rapid aldosterone action frequently utilize steroid concentrations that are significantly above the levels found in humans at rest. However,
under certain conditions such as physical exercise, aldosterone may reach nanomolar levels (478). Therefore, the
physiological relevance of many phenomena reported in
vitro can be assumed. Furthermore, local aldosterone
synthesis has been demonstrated in tissue, such as the
heart (450), thus posing the possibility of much higher
local concentration. This is particularly important for cardiovascular pathophysiology as discussed below.
Although the quick response of plasma aldosterone
levels to posture changes is well known, in vivo studies in
search for rapid effects corresponding to these changes
have been conducted only with lingering. Aldosterone has
been shown to significantly facilitate phosphocreatinine
recovery in human calf muscle after physical exercise
within minutes after intravenous administration (539).
The effect depends on participation of the anaerobic pathway, being absent when aerobic metabolism is used
alone. In the dog, convincing in vivo evidence for rapid
aldosterone action in vivo has been found for effects on
baroreceptor neuron discharge frequency (510), where
effects are detectable as early as 15 min after steroid
administration. These findings were confirmed in humans
in a clinical study, where aldosterone was found to blunt
the baroreflex response (536). High aldosterone levels
may therefore impair baroreflex function in hypertension
or heart failure. The above-mentioned early studies done
by Klein and Henk (233) have been confirmed in our
laboratory using invasive techniques. Within 3 min after
intravenous administration of 0.5 mg aldosterone, the
systemic vascular resistance (SVR) increased (520) but
returned to or even declined below baseline after 10 min.
NONGENOMIC STEROID ACTION
Physiol Rev • VOL
the lowest effective concentration of 100 pM (505, 506).
Similar 1␣,25(OH)2D3-induced effects on the generation
of DAG and IP3 have also been found in a variety of other
cells including typical vitamin D effector cells like enterocytes (64), dispersed porcine parathyroid cells (254), osteoblasts (461), ROS 17/2.8 cells (102), and keratinocytes
(476), but also in cells like primary chick-embryo muscle
cells (315) and hepatocytes (23). In rat skeletal muscle,
the involvement of PLC and a PTX-sensitive G protein in
the rapid 1␣,25(OH)2D3-induced release of IP3/inositol
phosphates and DAG has been shown (137, 314). In addition, 1␣,25(OH)2D3 was found to rapidly activate phospholipase D in Caco-2 cells (a human colon cancer cell
line) (230), chick myoblasts (315), and rat skeletal muscle
(138).
Because DAG can cause the activation of PKC, the
influence of 1␣,25(OH)2D3 on this protein has also been
examined in several tissues and cells, respectively. In
growth zone chondrocytes, 1␣,25(OH)2D3 (10 nM) causes
a rapid increase in PKC activity that was insensitive to
actinomycin D and cycloheximide (67, 473). A recent
study showed that the activation of PKC by 1␣,25(OH)2D3
and 24R,25(OH)2D3 involves rapid increases in diacylglycerol via a phospholipase D (PLD)-dependent mechanism.
24R,25(OH)2D3, but not 24S,25(OH)2D3 or 1␣,25(OH)2D3,
stimulated PLD activity in resting zone cells within 3 min
via nongenomic mechanisms. Neither 1␣,25(OH)2D3 nor
24R,25(OH)2D3 affected PLD in growth zone cells. Inhibition of PI 3-kinase, PKC, PI-specific PLC (PI-PLC), and
phosphatidylcholine (PC)-specific PLC (PC-PLC) had no
effect on PLD activity (472).
Analogs of 1␣,25(OH)2D3, which exhibit ⬍0.1% of the
binding to the classic receptor found for the parent compound, elicit comparable increases in PKC specific activity (188). In addition, a rapid activation of PKC by
1␣,25(OH)2D3 has been reported in rat epithelium cells
(506). Also, 1␣,25(OH)2D3 has been shown to directly
activate PKC, indicating the possibility of a 1␣,25(OH)2D3
binding domain on the enzyme (455). A recent study on
Ca2⫹ mobilization from inner stores and extracellular
Ca2⫹ entry in chick skeletal muscle cells after stimulation
with 1␣,25(OH)2D3 could show that these effects were
abolished by the PKC inhibitors bisindolylmaleimide and
calphostin. The authors presume that 1␣,25(OH)2D3 activates and translocates PKC␣ to the membrane, suggesting
that this isozyme accounts for PKC-dependent 1␣,25(OH)2D3
modulation of Ca2⫹ entry. With the help of antisense technology, the expression of PKC␣ was selectively knocked out
by intranuclear microinjection of an antisense oligonucleotide against PKC␣ mRNA and the authors could demonstrate a reduction of the Ca2⫹ influx component of the
response to 1␣,25(OH)2D3. These results pointed out that
1␣,25(OH)2D3 induced activation of PKC␣ enhances extracellular Ca2⫹ entry, partially contributing to mainte-
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blasts, IP3 and DAG have been found to be involved in
triggering the calcium response (315), while rapid cGMP
responses have been seen in human fibroblasts (30).
Moreover, 1␣,25(OH)2D3 (10⫺8 M) significantly increased
MAPK phosphorylation, with the earliest response being
detectable at 30 s (460). None of these immediate effects
requires gene transcription or protein synthesis (145).
In this section we emphasize the signaling pathway
and point out the possibly related clinical implications.
Rapid actions on Ca2⫹ fluxes in bone-forming osteoblasts which function as an integral part of the vitamin D
endocrine system and are main target cells for
1␣,25(OH)2D3 have been intensively studied in recent
years. An increase in [Ca2⫹]i occurring within minutes in
response to treatment with 1␣,25(OH)2D3 (10 –100 pM)
has been found in primary cultures of mouse osteoblasts
(254). Similar results have been described in rat osteosarcoma cells (ROS 17/2.8; Ref. 82 and others). Baran et al.
(25) found an increase in [Ca2⫹]i accompanied the increased expression of osteocalcin mRNA steady-state levels in rat osteosarcoma cells. In a subsequent experiment
they inhibited the rapid increase in [Ca2⫹] by the inactive
epimer 1␤,25(OH)2D3 and found a reduction of osteocalcin mRNA. The results demonstrate the functional importance of the rapid, nongenomic actions of 1␣,25(OH)2D3
in the genomic activation of the osteocalcin gene by the
hormone in rat osteoblast-like cells (25).
Further studies on the rapid 1␣,25(OH)2D3-induced
increase of [Ca2⫹]i in osteoblasts or related cell lines
showed that this effect involved both release of Ca2⫹ from
intracellular stores and extracellular Ca2⫹ influx probably
via an L-type voltage-sensitive Ca2⫹ channel (82, 254).
A rapid increase in [Ca2⫹]i following 1␣,25(OH)2D3
stimulation has been described in several other cells or
tissues not commonly addressed as vitamin D target tissues. For example, in skeletal muscle cells (499) and in both
cardiac muscle tissue and cultured chick cardiac muscle
cells, physiologically relevant doses of 1␣,25(OH)2D3 have
been found to induce an increase of [Ca2⫹]i within a few
minutes, an effect which was independent of mRNA or
protein synthesis. Similar to osteoblasts, this effect seems
to involve a voltage-dependent Ca2⫹ channel (112, 435).
The clinical implication of this calcium increase, e.g.,
possible changes in electrophysiological parameters, remains to be clarified.
In addition to the activation of Ca2⫹ influx, 1␣,25(OH)2D3
rapidly leads to hydrolysis of membrane lipids to produce
second messengers that cause release of Ca2⫹ from intracellular stores, especially the endoplasmic reticulum. In
female rat osteoblasts, 1␣,25(OH)2D3 (100 pM) transiently
increases [Ca2⫹]i within 5 s via IP3 formation, an effect
involving PLC␣ (250). Moreover, rapid, 1␣,25(OH)2D3-induced hydrolysis of membrane phosphoinositides generating DAG and IP3 has been described in rat colonocytes
and Caco-2 cells. The response was dose dependent, with
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LÖSEL ET AL.
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thermore, an inhibition of renal 25(OH)D3-1-hydroxylase
induced by 1␣,25(OH)2D3 was found (118).
In a pancreatic ␤-cell line, 1␣,25(OH)2D3 induces oscillations of [Ca2⫹]i mediated through nonselective calcium channels that are blocked by lanthanum ions (438).
Fluorescent digital ratiometric video imaging at the singlecell level was used to study the effects of 1␣,25(OH)2D3 on
[Ca2⫹]i in a pancreatic ␤-cell line, RINr1046 –38. In these
cells equilibrated at a steady-state glucose concentration
of 5.5 mM, 1␣,25(OH)2D3 (2–20 nM) rapidly, within 5–10 s,
increased [Ca2⫹]i and evoked sinusoidal [Ca2⫹]i oscillations. The [Ca2⫹]i oscillations were acutely dependent on
extracellular Ca2⫹, but not on extracellular glucose. The
1␣,25(OH)2D3-evoked [Ca2⫹]i oscillations were mediated
by nonselective Ca2⫹ channels, which are permeable to
Mn2⫹ and suppressed by extracellular La3⫹. Blockage of
voltage-dependent Ca2⫹ channels by nifedipine significantly decreased the amplitude of the oscillations. Unlike
its effect on the aldosterone-induced response of [Ca2⫹]i
in VSMC, depletion of intracellular Ca2⫹ stores with thapsigargin did not affect the 1␣,25(OH)2D3-stimulated Ca2⫹
entry estimated by the fura 2 fluorescence method. This
demonstrates that the hormone directly activates nonselective Ca2⫹ channels. The 1␣,25(OH)2D3-evoked increase
in the Ca2⫹ influx appears to generate [Ca2⫹]i oscillations
by triggering Ca2⫹ release through the ryanodine receptor/Ca2⫹ release channel, but not through activation of
the IP3 receptor. These findings are in line with a role of
a plasmalemmal vitamin D receptor coupled to the plasma
membrane Ca2⫹ channels in mediating rapid effects of the
hormone (438). They also show that rapid steroid effects
may share major similarities with regard to effects, e.g.,
on [Ca2⫹]i; however, details of signaling pathways involved in effects of different steroids in various tissues
may be widely variable. A summary of some possible
pathways for nongenomic vitamin D action, with emphasis on MAPK activation, is shown in Figure 9.
Although there are many studies on the effect of
vitamin D on insulin secretion by pancreatic ␤-cells, the
role of vitamin D in cell function is not yet clear. Kajikawa
et al. (224) have demonstrated the insulinotropic effect of
a vitamin D analog through nongenomic signal transduction. This result has not yet been evaluated in respect to
its clinical relevance, but it is tempting to speculate about
a specific interaction of vitamin D or its analogs with a
putative membrane vitamin D receptor of pancreatic
␤-cells that may enhance insulin secretion without side
effects of currently used drugs. Kajikawa et al. (224) could
show in a recent study that the vitamin D analog,
1,25(OH)2lumisterol3, enhanced high concentration of
glucose-induced insulin release together with enhancing
the Ca2⫹ influx in pancreatic ␤-cells within 15 min. In
addition, the effect was completely abolished by the antagonist of nongenomic action [1␤,25(OH)2D3], strongly
suggesting a nongenomic vitamin D effect on insulin re-
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nance of the sustained phase of the Ca2⫹ response to the
sterol (85).
Because PKC activation is linked to the activation of
MAPK, the influence of 1␣,25(OH)2D3 on this signaling
protein was analyzed. MAPK is known to be able to
integrate multiple intracellular signals transmitted by various second messengers so as to regulate many cellular
functions by phosphorylation of numbers of cytoplasm
kinases and nuclear transcription factors including the
epidermal growth factor receptor, c-Myc, and c-Jun. Recently, it was shown that 1␣,25(OH)2D3 is able to stimulate both Raf and MAPK pathways in keratinocytes (180,
280), chick enterocytes (114), and NB4 cells (460), indicating that the rapid response signal transduction components of 1␣,25(OH)2D3 may be involved in the regulation
of cell growth. In NB4 cells, 6-s-cis locked analogs of
1␣,25(OH)2D3 (10 nM) increased MAPK phosphorylation
with the same efficacy as 1␣,25(OH)2D3 itself. The translocked analogs were inactive, and 1␤,25(OH)2D3 had antagonist-like properties (460). Because in other systems
the MAPK pathway has been shown to play a role in
transducing the ligand signal from the outer cell membrane to the nucleus, it was postulated that the rapid
1␣,25(OH)2D3-induced MAPK activation may represent a
form of cross-talk that could modulate the genomic pathways of 1␣,25(OH)2D3.
Moreover, the participation of nongenomic actions of
1␣,25(OH)2D3 in the cAMP/PKA signaling pathway was
analyzed. Several studies indicated that the rapid stimulation of Ca2⫹ influx by 1␣,25(OH)2D3 in various cells was
abolished by specific inhibitors of adenylate cyclase and
PKA (113, 282, 498). In vitamin D-deficient chick soleus
muscles, 1␣,25(OH)2D3 (10 pM to 10 nM) elevated tissue
cAMP levels within 45 s to 5 min and increased adenylate
cyclase activity (150). Similarly, intracellular cAMP levels
rose in chick cardiac muscle and rat enterocytes after
stimulation with 1␣,25(OH)2D3 (283, 434).
Aside from the effects described above, 1␣,25(OH)2D3 has
also been shown to elicit various other rapid nongenomic
responses. Electrophysiological studies were done in ROS
17/2.8 cells where 1␣,25(OH)2D3 but not 1␤,25(OH)2D3
promoted the rapid enhancement of Cl⫺ current in a
concentration-dependent manner (538). The hormone has
also been reported to be involved in the stimulation of
alkaline phosphatase activity (43) and phosphate transport (226, 471).
Moreover, 1␣,25(OH)2D3 has been found to induce a
reorganization of VDR from the cytosol to the nucleus
occurring within 15 s to 30 min. This response was accompanied by an accumulation of cGMP (30). Edelman et
al. (124) described a rapid change (within seconds) in the
membrane potential of the renal proximal tubule from the
amphibian Necturus resulting from the modification of
Ca2⫹-dependent K⫹ channels by 1␣,25(OH)2D3 (124). Fur-
NONGENOMIC STEROID ACTION
999
lease (224). It remained unclear why augmentation of the
[Ca2⫹] rise induced by 1,25(OH)2lumisterol3 was observed only in the presence of a high concentration of
glucose. It may be due to the linkage of signal transduction of 1,25(OH)2lumisterol3 to modulation of the voltagedependent Ca2⫹ channels by the intracellular glucose metabolism. These striking experiments show the direct clinical implications of rapid nongenomic effects of a vitamin
D analog.
H. T3 and T4
Rapid nongenomic responses to thyroid hormones
may be divided into several categories. In addition to
modulation of ion fluxes and intracellular kinase activities
which are commonly seen with steroids, intracellular protein trafficking can be a target, along with the cytoskeleton, both being phenomena that are observed more rarely
with steroids.
Among the earliest reports of rapid thyroid effects, T3
was reported to increase the 2-deoxyglucose uptake in
chick embryo heart cells (433) and later also in various
tissues of the rat (430, 431). The effect occurred in the
lower nanomolar range and, in heart slices, was associated with a spike of intracellular Ca2⫹ that was maximal
after 30 s and originated from intracellular stores, as
Physiol Rev • VOL
judged by its independence from extracellular Ca2⫹.
Rapid increases in intracellular Ca2⫹ caused by T3 have
been described frequently (432 and others). In red blood
cells and other cell systems, T4 and T3 have been shown
to stimulate plasma membrane (110,408) and sarcoplasmic Ca2⫹-ATPase, thus increasing Ca2⫹ efflux from the
cytosol. The activation of Ca2⫹-ATPase(s) is probably not
a direct one (111) but is likely to be mediated by PKC,
which itself is activated by thyroid hormones (see sect.
IID8), but without any particular PKC isoform identified
so far. The enhancement of PKC activity has been shown
to follow PLC action (261), which forms DAG from phosphatidylinositol. IP3, which is the second product in this
reaction, is known to liberate Ca2⫹ from intracellular
stores (50). Recently, T4 has been shown to initiate a dual
DAG liberating phospholipase pathway, involving PLC
and PLD sequentially in liver cells (227). Activation of
PLD apparently is regulated by PKC, as judged from its
sensitivity toward PKC inhibitors, thus indicating a possible feedback mechanism.
The cAMP-dependent PKA has also been shown to be
activated by T4 in HeLa cells (261), and both PKA and
PKC activation are required for T4 enhancement of interferon-␥-induced antiviral activity.
Furthermore, the MAPK signal cascade may be driven
by T4, e.g., via a putative GPCR (259), finally exerting
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FIG. 9. Schematic diagram of mitogen-activated protein kinase (MAPK) cascade activation. 1␣,25(OH)2D3 may bind
to one or more of three putative classes of membrane receptors, resulting in MAPK activation by different mechanisms:
1) through membrane receptors with intrinsic tyrosine kinase activity; 2) through membrane receptors without intrinsic
tyrosine kinase activity, utilizing src; 3) to generate either membrane-associated Ras-GTP that phosphorylates Raf
isoforms; 4) more indirectly through activation of PI 3-kinase or phospholipase C (PLC), linked to diacylglycerol (DAG)
activation of protein kinase C (PKC) isoforms. All pathways ultimately lead to Raf phosphorylation and subsequent
activation of the MAPK cascade. [From Norman et al. (340). Copyright 2001, with permission from Elsevier Science.]
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LÖSEL ET AL.
usually different for nongenomic effects. This difference,
in particular the insensitivity toward inhibitors of classic
receptors as discussed above, is a useful criterion for
distinction of pathway and receptor. To obtain a desired
physiological response, it is obviously sufficient to apply a
drug that preferentially acts on one or the other pathway.
Knowledge of the receptor involved is not necessary. This
in turn, however, points to a shortcoming in the research
done to date. In contrast to the classic, genomically acting
receptors, hardly any selective agonists or antagonists for
nongenomic steroid actions are known, and we still have
some way to go before such drugs can be used in clinical
practice. Probably the only clinical application of nongenomic steroid action that has gained some popularity
were anesthetic steroids (Althesin and others, now withdrawn for side effects unrelated to the steroid). However,
the RALES study (378) provided hints to a possible nongenomic inhibition effect that has been extensively but
unconsciously used. As discussed in section IIC2B, spironolactone is metabolized to canrenone, which forms an
equilibrium with its open-ring congener canrenoate.
Given the data about nongenomic inhibition of aldosterone effects by the open-ring compound RU 28318, one
may assume a possible inhibitory activity of canrenoate as
well. The doses given in the RALES study were very low,
hardly any effect on diuresis (a known genomic effect)
has been observed, so that inhibition of the classic MR
was probably limited as well. On the other hand, significant improvements in cardiovascular parameters have
been achieved by this low-dose treatment, which may, at
least in part, involve inhibition of nongenomic aldosterone effects. In this context, spironolactone would have
to be regarded as a “super” aldosterone antagonist, inhibiting both genomic and nongenomic effects. However,
this is a model that merits further investigation before
definitive mechanisms are identified.
I. Does Receptor Type Matter for Clinical Impact?
Despite the many differences between genomic and
nongenomic pathways that have been laid out in the
previous chapters, there are several points where interaction between each other might occur. Steroids have been
shown to rapidly modulate the levels of intracellular cAMP.
Recently, steroid-induced transcription of genes has been
reported to be influenced by changes of cAMP levels (258,
336, 407). Integrating these findings into one concept, a
two-step model of steroid action has been proposed (100,
516). In this model, originally developed for aldosterone
but expandable to other steroids, the rapid nongenomic
as well as the delayed genomic steroid action are comprised together with possible mechanisms of modulation
of classic receptor-induced gene transcription by nongenomic signal transduction pathways (Fig. 10).
As described in the previous sections, much evidence
has been accumulated for the involvement of nonclassic
receptors in many nongenomic phenomena, and many
proteins are now known to act as such. This particularly
applies to many receptors that are modulated by steroids
in a coagonist fashion, such as the GABAA and similar
receptors. Despite these data, sometimes the existence of
receptors beyond those mentioned before is still doubted.
From the clinician’s point of view, the physiological and
pathophysiological phenomena that can be influenced by
administration of drugs is of utmost importance. The
pharmacological profiles, defined as the relative potency
of substances to evoke or antagonize hormone action, are
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IV. INTERACTION OF NONGENOMIC
AND GENOMIC PATHWAYS
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multiple effects, such as modulating the phosphorylation
of STAT (signal transducer and activator of transcription)
proteins (259, 260) and p53 as well as nuclear TR␤1 (448).
The latter is an example of nongenomic modulation of a nuclear, i.e., genomically acting, receptor for the same hormone.
Other nongenomic thyroid hormone effects described include a rapid stimulation of the Na⫹/H⫹ antiporter in L6 myocytes, resulting in a dose-dependent alkalinization of the cell (220), and the shortening of the
action potential in hypothyroid ventricular myocytes by
comparatively high levels of T3 (469). Interestingly, this
effect did not occur in untreated euthyroid cells.
The clinical impact of nongenomic thyroid hormone
action has been investigated in humans in vivo. Intravenous application of T3 significantly increased cardiac output while decreasing systemic vascular resistance (SVR),
while heart rate and blood pressure remained unaffected
in heart failure patients with diminished free T3 plasma
levels (199). Here the short time frame was incompatible
with genomic mechanisms. Direct modulation of vasoregulation, independent from the endothelium, may account for decreased SVR (356). T3 may affect Na⫹ currents in isolated ventricular myocytes possibly linked to
positive inotropic changes (123). The concomitant modulation of Na⫹/Ca2⫹ exchange increases contractility
(508). T3 treatment was also found to lower the prevalence of atrial fibrillation in artery bypass graft patients,
an effect that may be explained by the rapid response of
some voltage-gated K⫹ channels to thyroid hormones
(234, 417). An early nongenomic, cycloheximide-insensitive modulation of ␤-adrenoreceptor density by T3 has
been seen in cultured embryonic cardiomyocytes (496),
while the late-phase ␤-adrenoreceptor upregulation was
cycloheximide sensitive. This adrenoreceptor modulation
may explain the T3-dependent sensitization of ␤-adrenergic inotropy (507) by increasing ␤-adrenoreceptor signal
transduction and cAMP production, whereas T3 alone
does not affect cAMP (508).
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1001
One of the most important universal signals in terms
of cross-talk seems to be cAMP, the formation of which
has been shown to be rapidly increased by steroids. This
second messenger, formed from ATP by activated adenylyl cyclase, in turn activates PKA. The latter directly
phosphorylates CREB and is thought to activate the
MAPK pathway, presumably leading to SRC1 phosphorylation. The phosphorylated forms pCREB and pSRC1 cooperate in coactivation of steroid receptors as reported
for the chicken PR isoform A (407).
An outline of a rather complex, additive, and maybe
even synergistic interaction between nongenomic and
genomic effects on estrogen-induced gene transcription
has been reported recently in a neuroblastoma cell line
(497). With the use of a two-pulse stimulation schedule,
the early membrane effect elicited with the first pulse was
found to be mandatory for full transcriptional activation
by the subsequent estrogen pulse. Specific inhibitors indicated that PKA and PKC participate in the nongenomic
part, whereas Ca2⫹ seems to be involved in both the
nongenomic and the genomic events.
The amphibian homolog of the mammalian PR,
termed XPR, was found to be localized at cell membranes
of Xenopus oocytes as its 82 kDa form in small amounts
(14). Progesterone stimulation caused XPR to associate
with PI 3-kinase activity, and, subsequently, with p42MAPK.
Physiol Rev • VOL
Considering the in vitro ability of the latter to phosphorylate the 110-kDa form of XPR, regulation by the nongenomic p42MAPK pathway may be hypothesized.
In addition, the human PR was shown to possess a
polyproline motif that interacts in a ligand-dependent way
with Src homology 3 (SH3) domains of various intracellular proteins (61). Thus PR, upon binding the agonist R
5020, can activate Src tyrosine kinase, which in turn may
drive the MAPK pathway. Ultimately, MAPKs alter transcription factors in the nucleus, thus modulating the expression of genes not connected to steroid hormone responsive elements.
The latter two studies suggest a dual role for classic
steroid receptors, modulating gene expression through signal cascades in addition to direct transcriptional activity.
However, at least in the case of Xenopus, XPR does probably not explain all steroid effects on maturation, as other
steroids such as RU 486, cortisol, and deoxycorticosterone,
though inducing maturation, do not activate the XPR pathway.
V. SYNOPSIS: WHAT DO WE KNOW ABOUT
NONCLASSIC RECEPTORS?
From the evidence discussed in the previous sections, nongenomic actions of steroid hormones appear to
be mediated by three different mechanisms.
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FIG. 10. Cross-talk between nongenomic and genomic pathways for the action of steroids and steroidlike molecules
such as 3,3⬘,5-triiodothyronine (T3)/thyroxine (T4). This scheme summarizes several pathways, although not all may be
detectable for a particular steroid or in a given cell type. [Modified from Lösel and Wehling (264a).]
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LÖSEL ET AL.
Physiol Rev • VOL
weight carriers have been used very often to demonstrate
the presence of specific steroid receptors on the solventaccessible membrane. Although this procedure is straightforward and convincing, there are two shortcomings: first,
conjugates inherently may contain free, unconjugated steroid that has to be removed (131). Some commercial
preparations are not even labeled as to whether they
contain free steroid. Often, the potency of steroid conjugates to evoke effects is 30- to 100-fold lower than free
steroid, which correlates well with the amount of free
steroid present in many preparations (1–3%). Second, recent reports have shown that the presumably cell-impermeable conjugates have been detected inside the cell
(307, 335), so physiological actions exerted by steroid
conjugates pose some difficulties for interpretation. However, studies that demonstrate exclusive membrane localization of fluorescently labeled steroid conjugates [e.g.,
confocal techniques (44)] still give strong evidence for
steroid receptors located in the membrane.
The identification of binding sites for a specific steroid in a biological membrane does not necessarily imply
any receptor property in the sense of signaling. There are
many proteins known that bind steroids in a more or less
specific way, often just due the steroids’ hydrophobicity.
Even if a steroid initiates a signaling cascade involving
second messengers, there is not a compulsory link to
membrane binding, as long as the putative receptor is not
identified and available for investigation. However, some
examples still strongly suggest a functional connection,
such as the demonstration of interference, in both binding
and function with other receptor systems, e.g., the ␥-adrenergic receptor (326), which unfortunately is poorly
understood itself yet, or the GABA receptors in the nervous system (410).
From the view of physiology, many studies in search
for nongenomic steroid effects use synthetic, nonphysiological steroids. A popular example is dexamethasone, a
fluorinated glucocorticoid exhibiting ⬃30-fold potency,
compared with the physiological ligand cortisol, as measured by classic genomically mediated responses. It has
been shown, however, that the potency ranking for nonclassic effects is markedly different (78). This is also true
for the progesterone effects on spermatozoa, which
hardly correlate with affinities toward the classic cytosolic PR as convincingly demonstrated by Blackmore et al.
(59) for a very large set of substances. If there are proteins, unrelated to classic receptors, mediating nonclassic
responses, it is fair to assume that they may have a
different selectivity pattern for their ligands. Instead of, or
at least in addition to, using analogs that have been selected for their high potency to genomic receptors and
responses, employing the physiological hormones could
contribute to avoid false-negative results.
In summary, there is a considerable body of information about physiological events on the cellular level initi-
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First, a “side effect” of the well-known classic steroid
receptors is discussed, as convincingly demonstrated for
some effects of estradiol and progesterone. In this context, the ligand selectivity profile remains largely identical
for genomic and much of the nongenomic action according to current knowledge.
The second mechanism relates to nonspecific, direct
effects at the membrane level. This mechanism may at
least participate in the rapid, nongenomic action of glucocorticoids, because these steroids occur at rather high
concentration. The selectivity pattern differs significantly
between genomic and nongenomic action (78). However,
no direct experimental proof has been made to link the
steroids’ effects on membrane fluidity to intracellular signaling. A recent study has shown significant influences of
rather high steroid concentration on the fluidity of artificial bilayers (524), but despite the divergent fluidity effects, all hormonal steroids tested activated the model
enzyme Ca2⫹-ATPase, which better correlated to the mobility (increased in the presence of steroids) of the protein
itself in the lipid bilayer than to the lipid mobility itself,
without apparent steroid specificity. On the other hand,
spin-labeled surface proteins on murine spermatozoa exhibited decreased mobility upon addition of progesterone
(387), but no other steroids were investigated. Obviously,
such data that measure bulk behavior of either lipid or
protein do not closely correlate to physiological phenomena so far.
The third mechanism involves novel or nonclassic
receptors that are not closely related to the classic receptors. There are several properties that differ considerably
between the current knowledge about classic receptors
and the experimental data obtained about many rapid,
nongenomic effects. Classic receptors are thought to be
soluble proteins located in the cytosol or in the nucleus.
Many experiments, however, point to the existence of
membrane intrinsic receptors for some steroids. Antibodies directed against the ligand binding domains of classic
receptors sometimes stain cell membranes or detect
bands in Western blots of membrane preparations, in the
latter case often with considerably lower molecular
weight. Because the use of antibodies always carries the
risk of cross-reaction, additional confirmation is needed.
Some very recent studies have demonstrated membrane
location of classic receptors (14).
Some experimental approaches used for the identification of membrane-bound steroid receptors or steroid
binding proteins may have led to confusion. Many different steroid derivatives have been used, either radioactively or fluorescently labeled or bearing photoaffinity
moieties. In the steroid skeleton, the positions 3, 6/7, 11,
17 and 21 (if present) are easily derivatized. Sometimes,
derivatives substituted at different positions are implied
to be interchangeable, which is most probably not true.
Steroids conjugated to proteins or other high-molecular-
1003
NONGENOMIC STEROID ACTION
TABLE
1. Selected evidence for involvement of classic or nonclassic receptors in nongenomic steroid action
Classic
Nonclassic
Rapid ion channel responses not present in knock-out mice
(VDR)
Overexpression of classic receptor augments nongenomic
responses (ER)
Pharmacological profile largely identical to classic receptors
(many ER, AR, PR experiments)
Antibodies against classic receptors detect proteins in
membranes (ER, PR, AR)
Nongenomic effects present in knock-out mice (MR, ER)
VDR, vitamin D receptor; ER, estrogen receptor; AR, androgen receptor; PR, progesterone receptor; MR, mineralocorticoid receptor; NMDA,
N-methyl-D-aspartate.
ated by steroids on the nongenomic pathway. The wide
variety of cells and tissues in which such effects have
been demonstrated show that it is a universal phenomenon rather than a rare object. Some receptors for nongenomic steroid action are well-known proteins, e.g., the
receptors for neurosteroids. On the other hand, little is
known about the nonclassic receptors that stand at the
very beginning of most events, and the controversy that
has been outlined in the previous sections is not yet
settled. Some evidence for and against the participation of
classic and nonclassic receptors is summarized in Table 1.
The clinical implication and relevance of nongenomic steroid action is occasionally glittering through
the experimental data; however, its thorough investigation and exploitation is hampered by the lack of selective
agonists and inhibitors for nongenomic action, with the
exception of vitamin D. Phenomena such as rapid glucocorticoid action in shock, anesthetic and other neurotropic steroids, progesterone-induced acrosome reaction
and, possibly, aldosterone-mediated aggravation of cardiovascular disease are fields that will probably be worthwile for closer investigation.
Address for reprint requests and other correspondence: M.
Wehling, Institut für klinische Pharmakologie, Klinikum Mannheim, Theodor-Kutzer-Ufer, D-68167 Mannheim, Germany (Email: [email protected]).
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