| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Institute of Clinical Pharmacology, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Heidelberg, Germany
ABSTRACT 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 1. Knock-out mice 2. Pharmacology 3. Systems where the classic receptor has not been demonstrated D. Additional Evidence For and Against Distinct Receptors 1. Glucocorticoids 2. Progesterone 3. Estrogens 4. Androgens 5. Neurosteroids 6. Mineralocorticoids 7. Vitamin D3 8. Triiodothyronine and thyroxine 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?
 |
ABSTRACT |
|---|
 Top NextReferences |
|---|
|
I. INTRODUCTION |
|---|
|
TopPreviousNextReferences |
|---|
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 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 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.
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.
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 |
|---|
|
TopPreviousNextReferences |
|---|
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 hormones, 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.
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 effects, 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 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.
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 wild-type 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 µM 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 µM 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.
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 cis-locked conformers activate the rapid, nongenomic pathways but bind poorly to the nuclear receptor and are only weak agonists for genomic responses (145, 340). In addition, 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 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.
|
The interconversion between the 6-s-trans- and 6-s-cis-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-cis-conformers 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).
|
|
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 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 closed-ring RU 26752), but at the same time prevents the high-affinity 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 detectable by several experimental approaches: immuno-staining, 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).
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 molecules, 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.
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 of binding sites for cortisol have been detected, a high-affinity 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 high-affinity 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, 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 post-translational 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 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.
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. 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 mPR-specific 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 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 
-sub-unit 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 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 28-kDa 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.
Estrogens classically exert their biological effects by activation of ERs modifying gene expression. Again, ERs, which regulate the transcriptional activity of target genes 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 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 Gi. The observed ER Gi interaction was inhibited by ICI 182780 and pertussis toxin. Cotransfection of Gi 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 µ-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, or on adenylyl cyclase and thus indirectly on PKA, uncoupling the µ-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)-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 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) (GDPS) in both cell lines, whereas the G protein activator guanosine 5'-O-(3-thiotriophosphate) (GTPS) 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.
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 Western 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 AR-cDNA 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.
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 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 t-butylbicyclophosphorothionate (TBPS) to the GABA receptor complex at concentrations between 100 nM and 10 µM. 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 preoptic 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 region-specific 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, enh
P < 0.05 versus no PT or no GDP