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Research Communications |
a Division of Molecular Parasitology and Centre of Biological-Medical Research, Heinrich Heine University, 40225 Duesseldorf, Germany
b CNRS UPR 1524, INRA, 78352 Jouy-en-Josas, France
c Max Planck Institute for Cell Biology, 68526 Ladenburg, Germany
d Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece
e Max Planck Institute for Immunobiology, 78112 Freiburg, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: membrane receptor • androgen receptor • Ca2+ influx • sex steroids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The currently prevailing view is that testosterone cannot directly act on T cells. This view is based on reports that T cells do not contain the classical androgen receptor (AR)2 (for reviews, see refs 4, 5). The AR is a protein of approximately 110 kDa with several domains for androgen binding, nuclear localization, dimerization, DNA binding, and transactivation (12, 13). The predominant site of localization of AR in the absence of androgen is the cytoplasm, while ligand presence induces the import of AR into nuclei (12, 14).
Recent findings have revealed unconventional nongenomic surface receptors for testosterone in rat osteoblasts (15). These belong to the class of membrane receptors coupled to phospholipase C via a pertussis toxin-sensitive G-protein. Binding of testosterone to these cell surface receptors causes a rapid increase in intracellular free Ca2+ concentration ([Ca2+]i) and an increased formation of inositol 1,4,5-triphosphate and diacylglycerol. Preliminary evidence indicates that in murine T cells, testosterone also induces a rapid rise in [Ca2+]i, presumably due to Ca2+ influx triggered by binding of testosterone to receptors on the outer surface of T cells (16). However, it is also possible that testosterone diffuses through the plasma membrane, binds to intracellular AR, and secondarily induces the rise in [Ca2+]i. To exclude this possibility, we have followed the effect of testosterone conjugated to bovine serum albumin (BSA), a conjugate that cannot cross the plasma membrane, on [Ca2+]i of T cells. We have also reinvestigated the occurrence of classical AR in T cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of T cells
Spleens were aseptically removed from mice. Total nucleated spleen cells were isolated as detailed previously (9). T cells were then prepared using the nylon-wool procedure (17). This fraction contained 90–95% Thy 1.2+ T cells, as routinely examined in a FACScan (fluorescence-activated cell scanner; Becton Dickinson, Sunnyvale, Calif.). In some experiments, T cells were further purified; T cells were stained with anti-CD3-fluorescein isothiocyanate (FITC) (Pharmingen, Hamburg, Germany) and sorted in a FACScan according to standard procedures (18). For cytosol preparation, T cells were isolated as Ig- cells by magnetic cell sorting (MACS) (19). In brief, total spleen cells were labeled with biotin-conjugated anti-Ig monoclonal antibody (Boehringer, Mannheim, Germany), streptavidin-FITC, and biotinylated magnetic microparticles. The labeled cells were then separated by passage through a magnetized column obtaining the Ig- cells in the effluent. For localization of AR, the portion of cytoplasm was increased by stimulating T cells at 37°C, 5% CO2, and 98% humidity for 48 h with the monoclonal antibody anti-CD3
(5 µg/ml; Pharmingen, Hamburg, Germany) in RPMI 1640 medium supplemented with 5% fetal calf serum, 2 mM L-glutamine, 20 µM 2-mercaptoethanol, and 0.4% penicillin/streptomycin (5000 U/ml; 5000 µg/ml).
Determination of [Ca2+]i
T cells were washed twice with 20 mM HEPES buffer (pH 7.2) supplemented with 130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 1 mM Na2HPO4 and 1 mg/ml glucose. Subsequently, 107 T cells/ml were loaded with 3 µM Fura-2/acetoxymethylester (Amersham, Les Ulis, France) in the same buffer at 37°C for 20 min, diluted with 7 ml of HEPES buffer, and reincubated at 37°C for 20 min. The Ca2+ measurements were performed in a Hitachi F-2000 spectrofluorometer at a constant temperature of 37°C. Reagents were added directly to the cuvette under continuous stirring. Testosterone, testosterone 3-(O-carboxymethyl)oxime/bovine serum albumin (testosterone-BSA), nifedipine, verapamil, and neomycin were from Sigma (St. Quentin, Fallavier, France); the other testosterone and BSA derivatives were from Sigma (Deisenhofen, Germany). Cyproterone was kindly provided by Schering (Berlin, Germany). Hormones were dissolved in ethanol, the final concentration of which did not exceed 0.01% in the cuvette, and did not affect [Ca2+]i (cf. also ref 15). The Fura-2 fluorescence was measured at 340 nm (calcium-bound Fura-2) and 380 nm (free Fura-2) for excitation and 510 nm for emission. [Ca2+]i was computed from the ratio of 340/380 nm fluorescence values as described previously (20).
Labeling with testosterone-BSA-FITC
T cells (107 cells/ml) were suspended in phosphate-buffered salt solution (PBS+: 140 mM NaCl, 2.7 mM KCl, 6.4 mM Na2HPO4, 1.4 mM KH2PO4, 0.5 mM MgCl2, 0.9 mM CaCl2, pH 7.2). Aliquots of 150 µl were centrifuged, and the cell pellets were incubated at room temperature for 1 h with 200 µl of 1.5 x 10-5 M testosterone 3-(O-carboxymethyl)oxime: BSA-FITC conjugate (testosterone-BSA-FITC; Sigma, Deisenhofen, Germany). Only BSA-FITC and BSA were used in the corresponding control experiments. Colocalization was performed at a 1:50 working dilution for 20 min with concanavalin A (ConA) -rhodamine (Vector, Burlingame, Calif.). After two washings, the cells were resuspended and fixed in 0.5% paraformaldehyde (PFA) in PBS+ and allowed to adhere onto polylysine-coated glass coverslips. After 30 min, the coverslips were briefly rinsed with PBS+, mounted on slides in a 1:1 (v/v) mixture of glycerol and vectashield (Vector) containing 2% (w/v) 1,4-diazabicyclo-[2.2.2]octane (DABCO, Merck, Darmstadt, Germany).
Visualization of androgen receptors
These experiments were performed with both intact and permeabilized T cells. The latter were fixed with 0.5% PFA in PBS+ at 37°C for 30 min, then treated with 0.05% Tween-20, before diluting to 107 cells/ml in PBS+ supplemented with 0.5% BSA and 0.05% Tween-20. Aliquots of 150 µl were centrifuged, and the cell pellets were incubated at room temperature for 1 h with 200 µl of the anti-AR antibody AR (N-20) (1 µg/ml; Santa Cruz Biotechnology, Heidelberg, Germany) or the anti-AR antibody AR (C-19) (0.5 µg/ml; Santa Cruz Biotechnology). Anti-rabbit immunoglobulin G (IgG) (whole molecule) FITC conjugate (working dilution 1:320; Sigma, Deisenhofen, Germany) was used as secondary antibody for 45 min. After two washings, the cells were resuspended in 0.5% PFA in PBS+ and allowed to adhere onto polylysine-coated glass coverslips for 30 min. The coverslips were briefly rinsed with PBS+ and mounted on slides as described above.
Confocal laser scanning microscopy
Cells labeled with testosterone-BSA-FITC and ConA-rhodamine were analyzed with a Leica confocal laser scanning microscope (CLSM) unit (Leica Lasertechnik, Heidelberg, Germany) mounted on a Zeiss IM 35 microscope (Zeiss, Oberkochen, Germany). FITC and rhodamine fluorescence were excited by the 488 nm argon laser line and the 568 nm krypton laser line, respectively. Z-series optical sections were taken at 0.5 µm intervals and evaluated using AVS software (Advanced Visual Systems Inc., Waltham, Mass.) on an Indigo 2 UNIX workstation (Silicon Graphics Inc., Mountain View, Calif.) as described elsewhere (21). Cells labeled with the anti-AR antibodies AR (N-20) or AR (C-19) and FITC-labeled secondary antibody anti-rabbit IgG were analyzed with the CLSM LEICA TCS NT version 1.5.451 (Leica Lasertechnik, Heidelberg, Germany). FITC fluorescence was excited by the 488 nm argon laser line. Optical sections of 0.5 µm intervals were evaluated using Adobe Photoshop 5.0 for windows and Corel-Draw 8 for windows.
Nuclear import of AR
Human prostate carcinoma LNCaP cells (DSM ACC 256; gift of Dr. Schultz, Urological Clinic, Heinrich-Heine-University, Duesseldorf, Germany) were cultured on polylysine-coated glass coverslips in RPMI 1640 medium supplemented with 10% fetal calf serum at 37°C and 5% CO2. LNCaP cells and intact T cells were incubated with 100 nM testosterone for 105 min at 37°C and 5% CO2 before adding 100 nM testosterone again for 15 min. After two washings with PBS+, the cells were fixed with 0.5% PFA in PBS+ at 37°C for 30 min and permeabilized with 0.05% Tween-20. These cells and corresponding control cells not preincubated with testosterone were monitored for AR as described above.
Flow cytometry
Intact and permeabilized T cells were labeled with testosterone-BSA-FITC, BSA-FITC, and the anti-AR antibodies AR (N-20) and AR (C-19), as described above. In some experiments, the reactions with the anti-AR antibodies were performed in the presence of 10-fold blocking peptides AR (N-20)P and AR (C-19)P, respectively (Santa Cruz Biotechnology, Heidelberg, Germany). Labeling with monoclonal antibody to mouse CD8 conjugated with phycoerythrin (CD8-PE, 1:500; Boehringer, Mannheim, Germany) and anti-mouse CD4-PE (1:200; Becton Dickinson, Heidelberg, Germany) was performed as described previously (9). Cells were analyzed in a FACScan (Becton Dickinson) with a sample size of 10,000 cells gated on the basis of forward and side scatter. The data were stored and processed using the FACScan software as described previously (9).
Binding assay with 3H-R1881
Cytosol was prepared from T cells isolated as Ig- cells according to standard procedures (22). AR were measured using the dextran-coated charcoal method (23), with slight modifications, and 3H-R1881 (Amersham, Braunschweig, Germany) as a radioligand. As a control, parallel fractions were also assayed for glucocorticoid receptor using the dextran-coated charcoal technique method and 3H-dexamethasone (Amersham) as a radioligand (22).
Western blotting
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out according to Laemmli (24) using 8% polyacrylamide gels with approximately 20 µg total protein per lane. The slab gels were positioned on nitrocellulose membranes (0.45 µm pore size, Schleicher & Schuell, Dassel, Germany), placed in a Biometra semi-dry blot cell (Biometra, Göttingen, Germany) between three sheets of Whatman paper (Whatman Ltd., Mainstone, U.K.) and soaked with transfer buffer (48 mM Tris, 39 mM glycerin, 0.0375% sodium dodecyl sulfate, 20% methanol). After blotting, the membranes were incubated with the anti-AR antibody AR (N-20) at a concentration of 0.1 µg/ml diluted in 10 mM Tris/HCl (pH 7.5), 0.15 mM NaCl, and 0.05% Tween (TST) at 23°C for 1 h, washed three times with TST for 10 min, and incubated at 23°C for 1 h with horseradish peroxidase-conjugated goat-anti-rabbit IgG (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:50,000 in TST. After two washing steps, antibody detection was performed by the enhanced chemoluminescence plus Western blotting detection system (Amersham, Braunschweig, Germany) according to the manufacturer's instructions.
RNA isolation
Testes and spleens were taken from male C57BL/10 mice. The mouse macrophage cell line RAW 264.7 (ATCC number TIB-71) was cultivated in RPMI supplemented with 10% fetal calf serum. RNA was isolated from testes, spleens, RAW 264.7 cells, and FACSorted CD3+ T cells using the GTC/CsCl method (25).
Reverse transcription-PCR
Polymerase chain reactions (PCR) were performed using the RNA-PCR Kit from Perkin-Elmer (Weiterstadt, Germany). For the initial random primed reverse transcription (RT), 1 µg of total RNA was used for each reaction in an MJ Minicycler (MJ Research, Biozym, Hess; Oldendorf, Germany). The PCR was carried out using AmpliTaq DNA Polymerase (Perkin-Elmer) and the oligonucleotide primer pair AR1 5'-GACCTTGGATGGAGAACTACTCCG-3' and AR2 5'-GGTTGGTTGTTGTCATGTCCGGC-3' for 32 cycles, with the following parameters: 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. PCR fragments were cloned into the pMOSBlue T vector (Amersham, Braunschweig, Germany).
DNA sequencing
Clones were analyzed by automated laser fluorescent sequencing using the LICOR sequencer (MWG, Ebersberg, Germany) with Thermo Sequenase fluorescent-labeled sequencing kit (Amersham, Braunschweig, Germany).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1).
The fluorescence of testosterone-BSA-FITC colocalized with that of ConA-rhodamine, a marker of the plasma membrane ( Fig. 1). Binding of testosterone-BSA-FITC occurred on almost all intact T cells of both the CD4+ and CD8+ subset, as examined by flow cytometry ( Fig. 2).
This binding was specific for testosterone, since BSA-FITC without conjugated testosterone did not adhere to T cells ( Fig. 2).
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To test the possibility that the testosterone binding sites on the T cell surface were classical AR, intact T cells were incubated with the anti-AR antibody AR (N-20) and the anti-AR antibody AR (C-19). The AR (N-20) antibody is an affinity-purified rabbit polyclonal antibody raised against the amino acids 2–21 at the amino terminus of the AR, whereas the AR (C-19) antibody is raised against a 20 amino acid peptide of the carboxy terminus of the AR. Neither anti-AR antibody revealed any significant fluorescence on T cells either by flow cytometry ( Fig. 3, Fig. 4)
or CLSM (data not shown).
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Ca2+ influx induced by testosterone-BSA
Incubation of intact T cells with 100 nM testosterone-BSA, which does not enter T cells, induced an increase in [Ca2+]i of isolated splenic T cells by approximately 120–150 nM, whereas BSA alone had no effect on [Ca2+]i ( Fig. 5).
This increase appeared as a prolonged elevation that was not due to saturation, since subsequent addition of 100 nM testosterone-BSA induced an additional increase in [Ca2+]i ( Fig. 5A). The rise in [Ca2+]i induced by testosterone-BSA was not prevented by preincubation of T cells at 37°C for 30 min with 1 µM cyproterone, a blocker of the classical AR ( Fig. 5B). The induced increase in [Ca2+]i was obviously due solely to influx of extracellular Ca2+. Indeed, when extracellular Ca2+ was completely removed by ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), testosterone-BSA did not cause any change in [Ca2+]i of T cells ( Fig. 5C). Moreover, the Ca2+ influx progressed through Ca2+ channels, since the specific Ca2+ channel blocker Ni2+ inhibited the rise in [Ca2+]i induced by testosterone-BSA ( Fig. 5B).
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For characterization of the Ca2+ channels, testosterone, instead of testosterone-BSA, was used to avoid any possible negative effects of the BSA component. Figure 6A
shows that testosterone at the physiological concentrations of 1 and 10 nM was able to induce a rise in [Ca2+]i in T cells by about 20–25 nM and 30–60 nM, respectively. Repeated additions of 10 nM testosterone induced additional rises in [Ca2+]i. Depolarization of T cells with high K+ concentrations did not affect the subsequent testosterone-induced Ca2+ influx ( Fig. 6B). However, increasing doses of the Ca2+ channel blocker Ni2+ caused a gradual decrease and, at 5 mM Ni2+, an almost complete inhibition of the testosterone-induced Ca2+ influx ( Fig. 6C). In the presence of Mn2+, which is known to bind avidly to Fura-2 and quench Fura-2 fluorescence, testosterone led to a decrease in fluorescence to well below the resting level ( Fig. 6D). In contrast, the voltage-gated channel blockers verapamil and nifedipine did not affect the testosterone-induced Ca2+ channel-mediated Ca2+ influx ( Fig. 6E, F).
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Detection of classical AR
The presence of classical AR was examined in T cells, isolated as Ig- cells by MACS. These cells were fractionated and the cytosol was assayed for AR using 3H-R1881 as a radioligand (0.39–200 nM final concentration) in the presence or absence of a 100-fold excess of nonlabeled hormone. In parallel fractions, the glucocorticoid receptor was measured using 3H-dexamethasone as a radioligand. We were unable to detect any significant binding of 3H-R1881 to the cytosol fraction. In parallel cytosolic fractions, however, Scatchard plot analysis revealed 1020 glucocorticoid receptor sites per T cell (Kd=2.22 nM), in accordance with previous findings (22, 26).
In contrast, AR mRNAs were found in T cells. RNA was first extracted from FACSorted T cells containing more than 99% CD3+ cells. RT-PCR of this RNA using primers of the DNA binding domain of the AR revealed a band of the expected 486 nt size ( Fig. 7A).
An identical band was detected in testes and spleens, but not in cells of the mouse macrophage cell line RAW 264.7 ( Fig. 7A). DNA sequencing confirmed that the PCR fragment from T cells contained the predicted region of the DNA binding domain.
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Furthermore, Western blotting in combination with the very sensitive enhanced chemiluminescence detection system was used to identify AR in T cells and, comparatively, in the AR-expressing LNCaP cells (27). The anti-AR antibody AR (N-20) reacted with the AR band at about 110 kDa in LNCaP cells (
Fig. 7B). The AR band was not visible when the antibody reaction was done in the presence of 10-fold blocking peptide AR (N-20)P ( Fig. 7B). The latter consists of the amino acids 2–21 of the amino terminus of the AR, the epitope recognized by the anti-AR antibody. In contrast to LNCaP cells, T cells contained only very low levels of AR protein. This became evident when the same amounts of T cell and LNCaP cell proteins were comparatively subjected to Western blotting at different exposure times. Figure 7C shows that a short expression time of 1 min detected the AR-band only in LNCaP cells. However, when the expression time of the same blot was increased to 10 min, a very faint band at 110 kDa could also be revealed in T cells ( Fig. 7D).
Moreover, AR could be structurally detected only in permeabilized T cells by using flow cytometry. When permeabilized T cells were incubated with the anti-AR antibodies AR (N-20) or AR (C-19) and the corresponding fluorescent secondary antibody, fluorescence was observed in T cells (Figs. 3, 4). This fluorescence was competitively displaced by the blocking peptides AR (N-20)P or AR (C-19)P, which exhibited the same amino acid sequence as the epitope recognized by the two anti-AR antibodies, respectively.
CLSM revealed that the anti-AR antibodies AR (N-20) and AR (C-19) labeled the cytoplasm of permeabilized T cells, whereas nuclei remained unlabeled ( Fig. 8).
In these experiments, T cells were previously activated by anti-CD3 in order to increase the cytoplasmic portion. When intact T cells were preincubated with testosterone before permeabilizing with Tween-20 and labeling with the anti-AR antibody AR (N-20), the fluorescence was still located in the cytoplasm as in T cells without any testosterone preincubation (data not shown). Obviously, in the presence of testosterone, AR were not translocated into nuclei of T cells. By contrast, under the same incubation conditions, nuclear import of cytoplasmic AR was induced in more than 60% of human prostate carcinoma LNCaP cells ( Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The membrane testosterone receptors apparently are also different from the membrane-associated estradiol (E2) receptors recently identified in mouse T cells (28). Indeed, binding of E2 to E2 receptors induces both an influx of Ca2+ and a fast release of Ca2+ from intracellular stores, whereas testosterone induces solely Ca2+ influx. Moreover, the E2-induced Ca2+ influx can be blocked by approximately 50% by nifedipine, whereas nifedipine has no effect on testosterone-induced Ca2+ influx. Despite this difference, the presence of membrane receptors for both testosterone and E2 in T cells support growing evidence indicating that plasma membranes of diverse other cells are associated with receptors for steroid hormones (29–31), including androgens (32–35) and estradiol (36, 37). The nature and functioning of such membrane receptors are not yet understood. In prostate cell membranes, one possibility has been elaborated: membrane complexes consisting of sex hormone binding globulin (SHBG) bound to membrane SHBG receptor mediate rapid nongenomic effects of both androgens and E2, i.e., an increase in intracellular cAMP (38, 39).
Moreover, we were surprised to also find classical intracellular AR in splenic T cells, even though the mainstream view considers T cells to be devoid of AR (5). Absence of AR was evaluated mostly from the negative results obtained from binding assays with 3H-testosterone or 3H-R1881 in cytosolic fractions of T cells (40–43). In accordance, we have also been unable to detect appreciable amounts of AR in cytosolic fractions of T cells using the charcoal binding assay with 3H-R1881 as ligand. Nevertheless, the present data unequivocally indicate the occurrence of AR in T cells at both RNA and protein levels. Applying RT-PCR with primers spanning the DNA binding domain and Western blotting with antibodies to the NH2 terminus of the AR, we have revealed the presence of AR in FACSorted >99% pure CD3+ T cells. After permeabilization of T cells, AR are also detectable in T cells by the anti-AR antibodies AR (N-20) and AR (C-19), when using flow cytometry. CLSM localizes the AR predominantly in the cytoplasm and not in the nuclei of permeabilized T cells. This demonstration of classical AR in the cytoplasm of T cells is in accordance with other results showing AR in the cytoplasm of diverse cell types in the absence of ligand (12, 14). In contrast, however, to what happens to cytoplasmic AR in other cells, the presence of testosterone did not induce AR translocation into nuclei of intact T cells under experimental conditions sufficient to induce significant nuclear import of AR in the LNCaP human prostate cancer cell line. Incidentally, a population of estrogen receptors has been reported that is cytoplasmic, but nontranslocatable to nuclei, in human breast cancer-derived MCF-7 cells (44). The `nontranslocatability' of AR in T cells strongly suggests that these AR are not active in the genomic pathway. The testosterone binding site at the carboxy terminus of the AR might possibly be inactivated, since specific binding of 3H-R1881 in the cytosolic fraction of T cells cannot be detected. In this context, it is noteworthy that AR in T cells have been proposed to be developmentally regulated. Thus, immature T cells in the thymus possess functional AR (45, 46). However, binding of testosterone is no longer observable in mature peripheral T cells, which have therefore been thought to contain no AR (45), though this finding may also be explained by the presence of inactive AR, as reported here.
Finally, our data support earlier findings that cells contain testosterone receptors in their plasma membranes (15). These membrane receptors do not mediate the classical genomic AR response, but rather suggest a novel nongenomic testosterone signaling pathway involving Ca2+ as an intracellular mediator. However, the intracellular signaling varies between different cell types. In rat osteoblasts, for example, testosterone also induces Ca2+ release from intracellular stores (15), which is in contrast to what happens in T cells. However, the testosterone-induced nongenomic responses of intracellular Ca2+ signaling could secondarily have `genomic consequences'. Indeed, there is information available that Ca2+ is able to modulate expression of specific genes in diverse cell types including T cells (47), for example, through Ca2+-responsive promotor elements (CARE) and/or negative CARE and/or through Ca2+ sensitive transcription factors such as NF-AT, NF-
B, and c-Jun amino-terminal kinase (48).
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FOOTNOTES |
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2 Abbreviations: AR, androgen receptor; [Ca2+]i, intracellular free Ca2+ concentration; BSA, bovine serum albumin; ConA, concanavalin A; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid; MACS, magnetic cell sorting; FITC, fluorescein isothiocyanate; CLSM, confocal laser scanning microscopy; FACScan, fluorescence-activated cell scanner; E2, estradiol; PFA, paraformaldehyde; RT-PCR, reverse transcription-polymerase chain reactions; testosterone-BSA, testosterone 3-(O-carboxymethyl)oxime/bovine serum albumin; TST, 10 mM Tris/HCl (pH 7.5), 0.15 mM NaCl, and 0.05% Tween.
Received for publication March 16, 1998.
Revision received September 16, 1998.
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 J. Krucken, M. A. Dkhil, J. V. Braun, R. M. U. Schroetel, M. El-Khadragy, P. Carmeliet, H. Mossmann, and F. Wunderlich Testosterone Suppresses Protective Responses of the Liver to Blood-Stage Malaria Infect. Immun., January 1, 2005; 73(1): 436 - 443. [Abstract] [Full Text] [PDF]
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A. C. Roden, M. T. Moser, S. D. Tri, M. Mercader, S. M. Kuntz, H. Dong, A. A. Hurwitz, D. J. McKean, E. Celis, B. C. Leibovich, et al. Augmentation of T Cell Levels and Responses Induced by Androgen Deprivation J. Immunol., November 15, 2004; 173(10): 6098 - 6108. [Abstract] [Full Text] [PDF]
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 A. M. Braun and P. Thomas Biochemical Characterization of a Membrane Androgen Receptor in the Ovary of the Atlantic Croaker (Micropogonias undulatus) Biol Reprod, July 1, 2004; 71(1): 146 - 155. [Abstract] [Full Text] [PDF]
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 Y. Zagar, G. Chaumaz, and M. Lieberherr Signaling Cross-talk from G{beta}4 Subunit to Elk-1 in the Rapid Action of Androgens J. Biol. Chem., January 23, 2004; 279(4): 2403 - 2413. [Abstract] [Full Text] [PDF]
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A. M. Braun and P. Thomas Androgens Inhibit Estradiol-17{beta} Synthesis in Atlantic Croaker (Micropogonias undulatus) Ovaries by a Nongenomic Mechanism Initiated at the Cell Surface Biol Reprod, November 1, 2003; 69(5): 1642 - 1650. [Abstract] [Full Text] [PDF]
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T. Chiyo, T. Yamazaki, K. Aoshika, S. Kominami, and Y. Ohta Corticosterone Enhances Adrenocorticotropin-Induced Calcium Signals in Bovine Adrenocortical Cells Endocrinology, August 1, 2003; 144(8): 3376 - 3381. [Abstract] [Full Text] [PDF]
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M. Estrada, A. Espinosa, M. Muller, and E. Jaimovich Testosterone Stimulates Intracellular Calcium Release and Mitogen-Activated Protein Kinases Via a G Protein-Coupled Receptor in Skeletal Muscle Cells Endocrinology, August 1, 2003; 144(8): 3586 - 3597. [Abstract] [Full Text] [PDF]
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 R. M. LOSEL, E. FALKENSTEIN, M. FEURING, A. SCHULTZ, H.-C. TILLMANN, K. ROSSOL-HASEROTH, and M. WEHLING Nongenomic Steroid Action: Controversies, Questions, and Answers Physiol Rev, July 1, 2003; 83(3): 965 - 1016. [Abstract] [Full Text] [PDF]
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 G. Castoria, M. Lombardi, M. V. Barone, A. Bilancio, M. Di Domenico, D. Bottero, F. Vitale, A. Migliaccio, and F. Auricchio Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action J. Cell Biol., May 12, 2003; 161(3): 547 - 556. [Abstract] [Full Text] [PDF]
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 E. A. Papakonstanti, M. Kampa, E. Castanas, and C. Stournaras A Rapid, Nongenomic, Signaling Pathway Regulates the Actin Reorganization Induced by Activation of Membrane Testosterone Receptors Mol. Endocrinol., May 1, 2003; 17(5): 870 - 881. [Abstract] [Full Text] [PDF]
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F. C. W. Wu and A. von Eckardstein Androgens and Coronary Artery Disease Endocr. Rev., April 1, 2003; 24(2): 183 - 217. [Abstract] [Full Text] [PDF]
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T. Shakil, A. N. E. Hoque, M. Husain, and D. D. Belsham Differential Regulation of Gonadotropin-Releasing Hormone Secretion and Gene Expression by Androgen: Membrane Versus Nuclear Receptor Activation Mol. Endocrinol., November 1, 2002; 16(11): 2592 - 2602. [Abstract] [Full Text] [PDF]
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C. A. Heinlein and C. Chang The Roles of Androgen Receptors and Androgen-Binding Proteins in Nongenomic Androgen Actions Mol. Endocrinol., October 1, 2002; 16(10): 2181 - 2187. [Abstract] [Full Text] [PDF]
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Z. Guo, W. P. M. Benten, J. Krucken, and F. Wunderlich Nongenomic Testosterone Calcium Signaling. GENOTROPIC ACTIONS IN ANDROGEN RECEPTOR-FREE MACROPHAGES J. Biol. Chem., August 9, 2002; 277(33): 29600 - 29607. [Abstract] [Full Text] [PDF]
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 A. C. B. Cato, A. Nestl, and S. Mink Rapid Actions of Steroid Receptors in Cellular Signaling Pathways Sci. Signal., June 25, 2002; 2002(138): re9 - re9. [Abstract] [Full Text] [PDF]
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D. Liu and J. S. Dillon Dehydroepiandrosterone Activates Endothelial Cell Nitric-oxide Synthase by a Specific Plasma Membrane Receptor Coupled to Galpha i2,3 J. Biol. Chem., June 7, 2002; 277(24): 21379 - 21388. [Abstract] [Full Text] [PDF]
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Z. Guo, J. Krucken, W. P. M. Benten, and F. Wunderlich Estradiol-induced Nongenomic Calcium Signaling Regulates Genotropic Signaling in Macrophages J. Biol. Chem., February 22, 2002; 277(9): 7044 - 7050. [Abstract] [Full Text] [PDF]
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S. Tschuschke, H.-P. Schmitt-Wrede, H. Greven, and F. Wunderlich Cadmium Resistance Conferred to Yeast by a Non-metallothionein-encoding Gene of the Earthworm Enchytraeus J. Biol. Chem., February 8, 2002; 277(7): 5120 - 5125. [Abstract] [Full Text] [PDF]
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 A. Q. Ding and J. N. Stallone Testosterone-induced relaxation of rat aorta is androgen structure specific and involves K+ channel activation J Appl Physiol, December 1, 2001; 91(6): 2742 - 2750. [Abstract] [Full Text] [PDF]
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 C. W. Roberts, W. Walker, and J. Alexander Sex-Associated Hormones and Immunity to Protozoan Parasites Clin. Microbiol. Rev., July 1, 2001; 14(3): 476 - 488. [Abstract] [Full Text] [PDF]
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S. F. Palter, A. B. Tavares, A. Hourvitz, J. D. Veldhuis, and E. Y. Adashi Are Estrogens of Import to Primate/Human Ovarian Folliculogenesis? Endocr. Rev., June 1, 2001; 22(3): 389 - 424. [Abstract] [Full Text] [PDF]
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 G. P. H. Leung, S. B. Cheng-Chew, and P. Y. D. Wong Nongenomic effect of testosterone on chloride secretion in cultured rat efferent duct epithelia Am J Physiol Cell Physiol, May 1, 2001; 280(5): C1160 - C1167. [Abstract] [Full Text] [PDF]
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S. Morimoto, C. Fernandez-Mejia, G. Romero-Navarro, N. Morales-Peza, and V. DÍaz-Sánchez Testosterone Effect on Insulin Content, Messenger Ribonucleic Acid Levels, Promoter Activity, and Secretion in the Rat Endocrinology, April 1, 2001; 142(4): 1442 - 1447. [Abstract] [Full Text]
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W. P. M. Benten, C. Stephan, M. Lieberherr, and F. Wunderlich Estradiol Signaling via Sequestrable Surface Receptors Endocrinology, April 1, 2001; 142(4): 1669 - 1677. [Abstract] [Full Text]
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 E. Falkenstein, H.-C. Tillmann, M. Christ, M. Feuring, and M. Wehling Multiple Actions of Steroid Hormones---A Focus on Rapid, Nongenomic Effects Pharmacol. Rev., December 1, 2000; 52(4): 513 - 556. [Abstract] [Full Text] [PDF]
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F.M. Lyng, G.R. Jones, and F.F.G. Rommerts Rapid Androgen Actions on Calcium Signaling in Rat Sertoli Cells and Two Human Prostatic Cell Lines: Similar Biphasic Responses Between 1 Picomolar and 100 Nanomolar Concentrations Biol Reprod, September 1, 2000; 63(3): 736 - 747. [Abstract] [Full Text]
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 M. Estrada, J. L. Liberona, M. Miranda, and E. Jaimovich Aldosterone- and testosterone-mediated intracellular calcium response in skeletal muscle cell cultures Am J Physiol Endocrinol Metab, July 1, 2000; 279(1): E132 - E139. [Abstract] [Full Text] [PDF]
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 W. P. M. Benten, M. Lieberherr, O. Stamm, C. Wrehlke, Z. Guo, and F. Wunderlich Testosterone Signaling through Internalizable Surface Receptors in Androgen Receptor-free Macrophages Mol. Biol. Cell, October 1, 1999; 10(10): 3113 - 3123. [Abstract] [Full Text]
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J. Krucken, O. Stamm, H.-P. Schmitt-Wrede, A. Mincheva, P. Lichter, and F. Wunderlich Spleen-specific Expression of the Malaria-inducible Intronless Mouse Gene imap38 J. Biol. Chem., August 20, 1999; 274(34): 24383 - 24391. [Abstract] [Full Text] [PDF]
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X174/Hae III) on the left. Arrow indicates the predicted band of 486 nt-size from the DNA binding domain of AR. B) LNCaP cells were subjected to Western blotting using the anti-AR antibody AR (N-20) (-) or the antibody together with the corresponding blocking peptide AR (N-20)P (+). C) Western blot of LNCaP cells and T cells performed with the anti-AR antibody AR (N-20) and the ECL detection system. Short exposure of 1 min detected the AR band only in LNCaP cells. D) After increase of the exposure time of the same blot to 10 min, a very faint band could be also revealed in T cells. Arrows indicate the AR band at about 110 kDa.
