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Department of Human Anatomy and Genetics (L.C., A.N., J.F.M., H.C.C.), University of Oxford, Oxford OX1 3QX, United Kingdom; and Department of Neuroendocrinology (J.C.B.), Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College of Science Technology and Medicine, Hammersmith Hospital Campus, London W12 0NN, United Kingdom
Address all correspondence and requests for reprints to: Dr. H. C. Christian, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, United Kingdom. E-mail: helen.christian{at}anat.ox.ac.uk.
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Introduction |
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The initial effect of glucocorticoids in several tissues, including the anterior pituitary, is to cause externalization of annexin I from the cytoplasm to the outer cell surface where it is retained by a Ca2+-dependent mechanism (5, 7, 9). Subsequently the steroids induce de novo annexin I synthesis to replenish the intracellular stores of the protein (5). Functional and binding studies suggest that the glucocorticoid-induced translocation of annexin I is an important mechanism that enables the protein to access binding sites on the surface of the endocrine cells and thereby exert paracrine regulation on the release of ACTH and other pituitary hormones. In support of this hypothesis, we have identified annexin I-binding sites on the surface of all the main types of endocrine cells in the rat anterior pituitary gland (14) and shown that recombinant annexin I protein mimics the acute inhibitory actions of glucocorticoids on pituitary hormone release.
FS cells are the nonendocrine, agranular cells in the anterior pituitary. These star-shaped cells cluster together to form follicles and extend cytoplasmic processes that connect with processes of endocrine cells and other FS cells creating an extensive three-dimensional network within the anterior pituitary. The precise functions of the enigmatic FS cells in the anterior pituitary gland have not been well characterized. Several functions have been ascribed to FS cells including supportive and trophic effects, roles in ion transport, phagocytic and catabolic activities (15), and many paracrine functions. With their long cytoplasmic processes rich in gap junctions extending between other endocrine cell types (16), the FS cells are in an ideal position to play a role in intercellular communication mechanisms. Indeed there is substantial evidence that FS cells modulate the release of pituitary hormone secretion from surrounding endocrine cells through the release of several bioactive molecules (e.g. follistatin, IL-6, nitric oxide, basic fibroblast growth factor, leptin) (17). Moreover, Fauquier et al. (18) have recently demonstrated that the FS cell network forms an extensive functional intrapituitary circuitry in which information, Ca2+ and small diffusible molecules, can be transferred via gap junctions over long distances. Our finding that the FS cells are the principal source of annexin I in the anterior pituitary gland (6) and the observations that FS cells are rich in glucocorticoid receptor (19) have led us to propose that annexin I is a paracrine mediator of glucocorticoid action and that the FS cells are an important target for glucocorticoid action. On a temporal basis, the glucocorticoid-induced translocation of annexin I in the rat pituitary parallels the onset of the steroid inhibition of ACTH release (7).
To prove the role of FS cells and annexin I in glucocorticoid regulation of pituitary hormone release, pure FS cell populations in vitro are needed. However, studies of homogeneous FS cell populations have been impeded by the inability to obtain pure populations of these cells, although enriched populations of FS cells for in vitro studies can be obtained by biophysical methods (20). The TtT/GF cell line is a stable mouse pituitary-derived FS cell line that has been well characterized. TtT/GF cells display the morphological features of FS cells (processes, follicle formation) and express the FS cell marker proteins S100 and glial fibrillary acidic protein and the regulatory peptides transforming growth factor ß, leptin, and IL-6 (21, 22, 23) as well as annexin I (6). In the present study, we have therefore investigated whether TtT/GF cells externalize annexin I and have quantified in vivo the intercell contacts made by native FS cells in the anterior pituitary.
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TtT/GF cell culture
TtT/GF cells were cultured in DMEM-Ham’s F12 medium containing 10% fetal calf serum, 2.5 mM L-glutamine, 100 IU/ml-penicillin, and 100 µg/ml streptomycin (all from Sigma, Dorset, UK). Flasks containing approximately 2 million to 5 million TtT/GF cells were allowed to reach confluence and were treated with 12 ml control Earle’s balanced salts solution (EBSS) (Sigma), 56 mM K+ EBSS, or 0.1 µM dexamethasone sodium phosphate (Sigma). The rationale for testing K+ was as a negative control because a depolarizing stimulus was not expected to stimulate release. Cells were exposed to drugs for 3 h at 37 C after which cell proteins were extracted for Western blot analysis. Cell viability was checked at the end of each experiment by trypan blue exclusion (cell viability was always >95%). Dexamethasone was initially dissolved in a small amount of ethanol and subsequently diluted with EBSS immediately before use; the final concentration of ethanol did not exceed 0.1%.
To visualize by confocal microscopy the intensity and localization of annexin I immunoreactivity after the various treatments, TtT/GF cells were plated at 50 cells/mm2 on glass culture slides (Nunc, Life Technologies, Inc., Paisley, UK) and grown until the cells reached confluence. Cells were treated as above with control EBSS, 56 mM K+ EBSS, or 0.1 µM dexamethasone in 500 µl for 3 h. The cells were then fixed for 10 min in a mixture of freshly prepared 3% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M PBS, pH 7.4 at 37 C (6) for immunostaining of annexin I.
Detection of annexin I by Western blotting
Annexin I in TtT/GF cells was detected by SDS-PAGE and Western blot analysis.
Initial protein extraction.
For total cell annexin I extraction, TtT/GF cells cultured in T-150 flasks were washed with PBS and scraped off with a rubber policeman into 500 µl PBS (Sigma) containing EDTA (10 mM, Sigma), Triton (1% vol/vol, BDH Chemicals Ltd., Poole, UK), phenylmethylsulfonyl fluoride (1 mM, Sigma), and aprotinin (1 µg/ml) followed by sonication on ice. In separate flasks proteins bound to the outer surface of the TtT/GF cell membranes by Ca2+-dependent mechanisms (surface annexin I) were removed before the extraction by washing the cells for 1 min in PBS (500 µl) containing EDTA (1 mM) and protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 µg/ml aprotinin) (7). Annexin I in the remaining tissue (i.e. intracellular annexin) was then extracted as described above. The EDTA washes and extracts were analyzed immediately after the total protein concentration of the extracts had been determined by use of a BCA protein assay reagent (Pierce Chemical Co., Chester, UK).
SDS-PAGE and Western blot analysis.
Briefly, proteins extracted from TtT/GF cells were separated by SDS-PAGE [4 µg/channel (EDTA washes) and 20 µg/channel (cell extracts) in a volume of 20 µl by use of a midget gel Hoeffer electrophoresis system and power pack, LKB, Milton Keynes, UK] and transferred electrophoretically (64 mA, 1 h, Hoeffer system) to nitrocellulose paper (Bio-Rad Laboratories, Inc. Ltd., Hemel Hempstead, UK). To concentrate proteins harvested in the EDTA cell washes, each wash (500 µl) was precipitated with 10% trichloroacetic acid for 30 min on ice. The protein pellet was washed with a 1:1 mixture of ethanol and ether and resuspended in gel-loading gel buffer (200 nM Tris-HCl, pH 8.8; 1 M sucrose; 5 mM EDTA; 0.01% bromophenol blue; 5 mM dithiothreitol; and 2% SDS). Annexin I was detected by overnight incubation (4 C) with antiannexin I (diluted 1:5000) and visualized by sequential exposure to a peroxidase-conjugated donkey antisheep antiserum (diluted 1:5000) and diaminobenzidine (50 ml, 0.05% wt/vol) containing H2O2 (20 µl, all from Sigma). A well-characterized sheep polyclonal antiannexin I antiserum (gift from Dr. Jamie Croxtall, William Harvey Research Institute, London, UK) against full-length human recombinant annexin I (annexin 11–346) was used for both Western blot analysis and immunostaining (24). The housekeeping protein ß-actin was also detected with a monoclonal anti-ß-actin clone AC-15 (1:5000) and peroxidase-conjugated antimouse secondary antibody (both from Sigma, Poole, UK). The molecular weights of immunoreactive bands were determined by comparison with the migration of molecular mass standards (high-molecular-weight range rainbow labeled, Amersham International, Buckinghamshire, UK). The ODs of bands were measured semiquantitatively, using a Fujix Bas 1500 imaging system with a low-level, light-sensitive camera and TINA software (Raytek, Sheffield, UK). Intensity values were normalized relative to control values.
Immunostaining
For visualization of intracellular annexin I immunoreactivity, cells were permeabilized with 0.2% Triton X-100 (in PBS at room temperature) for 5 min; this step was omitted for detection of surface annexin I immunoreactivity. Nonspecific antibody binding sites were blocked with 3% BSA in PBS at room temperature for 60 min and incubated with the antiannexin I antibody diluted 1:6000 (4 C, overnight). Immunoreacted sections were washed with PBS and then incubated with fluorescein-conjugated donkey antisheep secondary antibody (Vector Laboratories, Inc., Burlingame, CA) for 1 h at room temperature. All sera were diluted in PBS containing 3% BSA. The culture chambers were removed and the slides mounted in Vectashield mounting medium (Vector Laboratories, Inc.) containing propidium iodide to counterstain cell nucleic acids. Cells were then examined using a TCS confocal microscope (Leica Corp. Microsystems, Wetzlar GmbH, Germany). Nonspecific immunostaining and background labeling were assessed by substitution of normal sheep serum for primary antisera. Preincubation of the antiannexin I serum with an excess of human recombinant annexin I (a gift from Dr. J. Browning, Biogen Research Corp., Cambridge, MA) abolished the immunostaining.
Quantification of annexin I immunofluorescence
For quantitative immunofluorescence, several confocal images of random fields containing approximately 40 cells were collected. At least 30 random cells in each image were then selected by a systematic random procedure and the fluorescence intensity measured per cell. All values were adjusted by subtraction of the fluorescence intensity of control cells incubated with normal sheep serum. Intracellular annexin I was quantified in permeabilized cells, whereas the amount of surface annexin I was measured in nonpermeabilized cells. The number of immunofluorescent foci on the surface of nonpermeabilized cells were counted from the same images converted to gray scale format in which the fine TtT/GF cytoplasmic processes could be viewed more clearly.
Electron microscopy
For immunoelectron microscopy, cryoprotected mouse pituitary tissue was cut into 300-µm-thick slices with a Vibratome (Camden Instruments, Sileby, UK), slam frozen, freeze substituted at -80 C in methanol for 48 h and embedded at -20 C in LRGold acrylic resin (London Resin Company Ltd., Reading, UK) in a Reichert freeze substitution system (Reichert MM80E, Leica Corp., Milton Keynes, UK). Ultrathin sections (50–80 nm) were prepared by use of a Reichert Ultracut S microtome, mounted on 200-mesh nickel grids, incubated for 2 h with antiannexin I for annexin I localization (dilution 1:200) and for 1 h with donkey antisheep IgG-15 nm gold complex (British Biocell, Cardiff, UK), and then lightly counterstained with uranyl acetate and lead citrate. All antisera were diluted in 0.1 M phosphate buffer containing 0.1% egg albumin. Controls included preincubation of the antiannexin I antiserum with a 100-fold excess of recombinant human annexin I. Sections were viewed with a JEM-1010 transmission microscope (JEOL USA, Inc., Peabody, MA).
Quantitation of FS cell-endocrine cell contacts by electron microscopy
Endocrine cells were identified on the basis of their secretory granule populations (shape, electron density, size, and distribution), and organelle structure, nucleus size, and chromatin characteristics (25). Immunogold labeling for PRL, LH, and GH was also performed to assist with the identification of lactotrophs, gonadotrophs, and somatotrophs, respectively (Ref. 26 and data not shown). Ultrathin sections were incubated for 2 h with rabbit antirat PRL (1:5000), guinea pig antirat LH (1:3000), or monkey antirabbit GH (1:5000; all from the National Hormone and Pituitary Program, Gaithersburg, MD) and subsequently were incubated for 1 h with protein A-15 nm gold complex (British Biocell). FS cells were identified and the number and identity of cells with which the FS cell made contact was counted. Contacts made by 30 FS cells from 3 animals were quantified.
Data analysis
Semiquantitative measures of annexin I expression were made by comparisons of Western blot band ODs (arbitrary units) to give a relative numerical guide to the ratios of the band intensities and their variance. Responses to dexamethasone and K+ were calculated as a percentage of the corresponding drug-free control and expressed as the mean ± SD (n = 3 gels); statistical comparisons between the normally distributed data from groups were made by the standard t test. Differences were considered significant if P was less than 0.05. Each of the studies was repeated several times (for specific details, see legends), and in all instances the data profile was similar.
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Results |
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Figure 1
illustrates the effects of dexamethasone (0.1 µM, 3 h) and K+ (56 mM, 3 h) on the cellular location of annexin I in TtT/GF cells. Typical Western blots are shown in Fig. 1, A–C, and densitometry data in Fig. 1F. Dexamethasone and K+ markedly increased (P < 0.01) the amount of annexin I detected in the EDTA washes (control lanes 1–2 vs. K+ lanes 3–4 vs. dexamethasone-treated lanes 5–6, Fig. 1A). Whereas K+ marginally but significantly (P < 0.05) reduced the amount of annexin I in both the washed tissue lysate and total tissue lysate (K+-treated cells, lanes 3–4, Fig. 1, B, C, and F), dexamethasone treatment significantly increased (P < 0.01) annexin I in these compartments (dexamethasone-treated lanes 5–6, Fig. 1, B, C, and F). No change was observed in the amount of ß-actin detected in intracellular extracts (Fig. 1D), and no ß-actin immunoreactive bands were detected in cell surface EDTA washes (Fig. 1E).
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), which extended throughout the cytoplasm of all TtT/GF cells and appeared somewhat concentrated at the ends of processes but had no obvious association with specific organelles (as described by us previously) (6). TtT/GF cells were also immunopositive for S100 (Ref. 21 and data not shown). In TtT/GF cells that had been fixed, but not permeabilized with detergent, annexin I immunofluorescence was concentrated in several peripheral patches of immunoreactivity at the tips of the processes that the TtT/GF cells develop in culture (Fig. 2, B and C). Labeling did not coincide with propidium iodide nucleic acid staining suggesting that the annexin I antibody was detecting cell surface annexin I (Fig. 2B). Figure 2C shows clearly that the annexin I immunofluorescence is in continuity with the FS cell processes. Dexamethasone (0.1 µM) and K+ (56 mM) treatment of TtT/GF cells significantly (P < 0.01) increased the intensity of fluorescent foci on the surface of cells that were not detergent treated before immunostaining (Fig. 3). Furthermore, the number of immunoreactive foci was significantly (P < 0.01) greater in dexamethasone-treated cells (5.5 ± 0.4 foci/cell) vs. control (2.2 ± 0.5 foci/cell). Dexamethasone also significantly (P < 0.01) increased total annexin I immunoreactivity quantified in fixed and permeabilized cells, whereas K+ had no significant effect (Fig. 3).
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demonstrates how in ultrathin sections of freeze-substituted anterior pituitary after perfusion fixation, the ultrastructural identification of FS cells with elongated cytoplasmic processes but with no secretory granules can be seen clearly and the specific cells that the processes contact can be visualized. In nonperfused pituitary tissue, the intimate cell-to-cell contacts make visualization of FS cell processes difficult (Fig. 4A). However, in perfusion-fixed material, the cells become separated (perhaps by perfusion pressure) allowing the processes to be seen clearly (Fig. 4B). Immunogold localization of annexin I confirmed (as described previously) (6) diffuse annexin I immunoreactivity over the cytoplasm of FS cells and also revealed accumulation of immunogold at the ends of processes contacting endocrine cells in the extracellular space (Fig. 5). However, surface annexin 1 immunoreactivity was not observed at junctional contacts between FS and endocrine cells at points along the body of the FS cell (Fig. 5). FS cell processes were found to make contact with all of the endocrine cell types and also with other FS cells (Fig. 6).
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Discussion |
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Visualization by confocal immunofluorescence microscopy of annexin I associated with the surface membrane of TtT/GF cells in culture showed that annexin I appears as punctate foci of immunoreactivity at the tips of the processes that the TtT/GF cells produce in culture. This finding was not entirely unexpected because, in other well-studied annexin I-externalizing cells, surface annexin I immunoreactivity also appears to be localized to one or more patches on the surface of the cells, for example in A549 lung adenocarcinoma cells (28, 29) and human neutrophils (30). Interestingly, in A549 cells, the points on the surface at which annexin I is localized are also strongly immunoreactive for tubulin and cytokeratin 8 but not actin (29). The similarity in terms of restricted distribution suggests that annexin I externalization is limited to certain locations in the cell cytoskeleton in all cells. Rat anterior pituitary cells express membrane-associated tubulin (31) and human FS cells express cytokeratin 8 (32) such that similar colocalization may occur in TtT/GF cells and FS cells in vivo. The annexin I protein may be targeted to specific areas of the cytoskeleton rich in tubulin and cytokeratin 8 once synthesized. Interestingly, in response to dexamethasone annexin I was not externalized generally from the surface of the TtT/GF cells, but rather annexin I immunoreactivity was increased specifically at the foci present on cell processes. Furthermore, exposure to dexamethasone increased the number of immunoreactive annexin I foci as well as increasing the intracellular content of annexin I. Within TtT/GF cells, we confirmed our previous findings that intracellular annexin I is not packed or concentrated into any detectable form of subcellular organelle but appears to be free within the cytosol (6).
Contrary to our expectation, 56 mM K+ also stimulated externalization and release of annexin I as measured by Western blotting and immunofluorescence. These data are consistent with studies demonstrating externalization of another member of the annexin family, annexin V, in response to 56 mM K+ in insect cells (33). The mechanism by which 56 mM K+ stimulates externalization of annexin I is unknown. One clear effect of this treatment is membrane depolarization, which will alter a number of membrane channel and transporter properties that may result in externalization of annexin I. If annexin I is transported by a membrane potential-sensitive transporter or channel, then depolarization would alter the rate of transport. Another effect of sustained depolarization is to cause reorganization of the membrane such that phospholipids move from one side of the lipid bilayer to another in a flip-flop manner (34). Theoretically, because annexin I binds to phospholipids, this process could drag the protein across the membrane simulating externalization. However this process of phospholipid movement is probably too slow, even under sustained depolarization (1–6 translocations per hour) (35) to account for the large increase in external protein observed (
2- to 3-fold in 30 min). Furthermore, annexin I binds preferentially to anionic phospholipids such as phosphatidylinositol and phosphatidylserine (36) whose translocation to the external surface would be slowed by depolarization, which causes the outer surface of the cell to become less positively charged. Another possibility is that sustained exposure of the membrane to high external K+ is causing release by activation of a voltage-dependent second messenger system, but what this might be is unknown. The high molar K+ nonspecific depolarizing stimulus, however, would not be expected to affect synthesis of annexin I by TtT/GF cells because membrane depolarization would not be expected to influence protein synthesis in a genomic manner. Indeed, the small but significant decrease in the amount of annexin I protein inside the cells as detected by Western blotting would be consistent with some externalization in the absence of synthesis. This decrease was not detected by immunofluorescence, but this is not surprising because the immunocytochemistry method of analysis employed fewer cells than Western blotting.
These data clearly suggest that some form of membrane transport mechanism is localized in specific areas of the plasma membrane in the processes of the FS cells. Consistent with the lack of apparent association of annexin I with intracellular organelles, annexin I lacks the hydrophobic signal sequence that targets proteins to the classical secretory pathway (37). Furthermore, drugs that block various steps in the exocytotic pathway do not alter the cellular export of annexin I that occurs in response to glucocorticoid treatment (38, 39). Proteins that are externalized from cells but are not packaged into intracellular vesicles and released by exocytosis must either diffuse through the membrane or be transported across the membrane in some way. Given the size and charge of annexin I, diffusion would be minimal. Other cytoplasmic proteins released by cells include growth factors and cytokines (40, 41), which are externalized quite slowly in comparison with the very fast exocytotic release of vesicle-packaged proteins. There is some evidence to suggest that such proteins are translocated by ATP-binding cassette transporters (41) of which a large family is known to exist in eukaryote cells (42).
Despite considerable investigation of the functions of FS cells, a full understanding of their role is still lacking (15). Overall it appears that the network of FS cells fulfills a number of important housekeeping roles in the anterior pituitary and influences hormone secretion by various mechanisms. Recently, Fauquier et al. (18) have shown that FS cells are excitable and capable of synchronizing their electrical activity and intercellular calcium signals in a three-dimensional manner. The communicating FS cell network is therefore well positioned to exert paracrine and juxtacrine influences on hormone secretion (17). Annexin I is one of the regulatory proteins produced by FS cells and plays a primary role in the acute inhibitory actions of glucocorticoids on ACTH, PRL, GH, and TSH release (2). In freeze-substituted sections of mouse anterior pituitary, immunogold electron microscopy revealed annexin I immunoreactivity throughout the cytoplasm of FS cells but also accumulated at the extracellular surface of FS cell processes contacting adjacent endocrine cells. It is probable that this surface annexin I immunogold reactivity reflects the annexin I foci visualized on the surface of TtT/GF cells by immunofluorescence microscopy and detected in EDTA cell washes by Western blotting. As we have demonstrated, the FS stellate projections lie in close apposition with all the main classes of endocrine secretory cell and the concentration of annexin I at extracellular foci on these projections suggests a juxtacrine mode of action directly onto neighboring cells. Contacts were made mostly with PRL, GH, and FS cells and only a few LH, TSH, and ACTH cells, but this probably reflects the relative proportions of these cells in the anterior pituitary. Often putative gap junctions were observed between the FS processes and other FS cells and endocrine cells, but surface annexin I immunogold reactivity was not detected at these contacts. FS cells express connexin 43 (43) and functional gap junctions have been previously characterized between FS cells and endocrine cells with a similar profile of contact communication primarily among PRL, GH, and FS cells (17).
Overall our data suggest that some regulatory actions of steroids on endocrine cells are exerted indirectly via the FS cells. Glucocorticoids promote the translocation of annexin I protein from the cytoplasm to the cell membrane in TtT/GF FS cells in which it adheres to the cell membrane by a Ca2+-dependent mechanism. The cell-surface adherence is suggested to be critical to annexin I actions because it provides a means whereby the protein may gain access to receptors on the outer surface of cells and thereby initiate a physiological response via juxtacrine actions. This concept is supported by several lines of evidence. Firstly, antisense probes that specifically reverse the inhibitory actions of glucocorticoids on GH, PRL, and ACTH release inhibit de novo annexin I synthesis and thus also prevents the exportation of the newly synthesized annexin I protein induced by glucocorticoid (9, 11, 13). Secondly, the antiannexin I antiserum that, like the antisense oligonucleotides, readily neutralize the antisecretory actions of the steroids, would not be expected to penetrate cell membranes readily but could effectively sequester annexin I at an extracellular site. Finally, we have demonstrated the presence of high-affinity (Kd 
13 nM), saturable annexin I-binding sites on the surface of several endocrine pituitary cell types (14); these sites resemble those on human peripheral leukocytes, which are essential for annexin activity (44). The signaling mechanisms activated by these putative receptors are unknown.
In conclusion, these results indicate that dexamethasone rapidly regulates the expression (within 3 h) and subcellular localization of annexin I in FS TtT/GF cells such that the protein can mediate some of the rapid actions of the glucocorticoids in the anterior pituitary in vivo. Annexin I is externalized at patches on the processes of TtT/GF cells that appear to correspond to the tips of the processes of FS cells contacting adjacent endocrine cells in vivo. The mechanism of annexin I externalization remains to be determined.
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Abbreviations: EBSS, Earle’s balanced salts solution; FS, folliculo-stellate cells; PRL, prolactin.
Received May 20, 2002.
Accepted for publication July 24, 2002.
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receptors. Biochem Biophys Res Commun 14192:351–358This article has been cited by other articles:
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  C. D. John, V. Sahni, D. Mehet, J. F. Morris, H. C. Christian, M. Perretti, R. J. Flower, E. Solito, and J. C. Buckingham Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides FASEB J, April 1, 2007; 21(4): 1037 - 1046. [Abstract] [Full Text] [PDF]
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E. Theogaraj, C. D. John, H. C. Christian, J. F. Morris, S. F. Smith, and J. C. Buckingham Perinatal Glucocorticoid Treatment Produces Molecular, Functional, and Morphological Changes in the Anterior Pituitary Gland of the Adult Male Rat Endocrinology, November 1, 2005; 146(11): 4804 - 4813. [Abstract] [Full Text] [PDF]
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, surface Ax1 (EDTA wash); 
, intracellular lysate (cells extracted after EDTA wash); 
, total cell lysate. Values are expressed as mean ± SEM, n = 3 experiments. **, P < 0.01; *, P < 0.05, standard t test.
