1 2. Physiologisches Institut, Osmotic stress in ducklings (Anas
platyrhynchos) results in salt secretion and adaptive
cell proliferation and differentiation in the nasal glands. We
investigated whether osmotic stress in vivo or muscarinic ACh receptor
activation in vitro changed the expression levels of the cellular
protooncogene products Fos and Jun, which may play a role in the
initiation of the adaptive processes. Using Fos- and Jun-specific
polyclonal antisera in Western blot experiments, we demonstrated that
Jun is constitutively expressed in nasal gland tissue, whereas Fos is
not detectable in tissue from unstressed (naive) animals. Under
conditions of osmotic stress imposed by replacing the drinking water of
the animals with a 1% NaCl solution, Jun protein remains constant in
nasal gland tissue, whereas Fos protein is transiently upregulated.
Treatment of cultured nasal gland tissue with muscarinic agonists
results in a transcriptionally regulated expression of Fos in an
atropine-sensitive manner. Immunohistochemical experiments show that
Fos accumulation occurs in the nuclei of the secretory cells. These
results indicate that the activation of the
c-fos gene induced by muscarinic ACh receptor-mediated signaling pathways may play an important role in the
initiation of adaptive growth and differentiation processes in nasal
glands of osmotically stressed ducklings.
avian nasal salt gland; adaptive cell growth; cell differentiation; c-jun; regulation of transcription; muscarinic acetylcholine receptor; Anas
platyrhynchos
TRANSCRIPTION OF THE protooncogene
c-fos is rapidly activated in animal
cells by a variety of different external stimuli. Its protein product
Fos dimerizes with (in many cases constitutively expressed) Jun
proteins to form the transcription factor AP-1, which binds with high
affinity to AP-1 sites in the regulatory regions of "late" genes,
thereby altering their transcription rates. Many of these genes under
control of AP-1 are important in developmental processes, e.g., cell
proliferation, cell growth, and cell differentiation (1).
When marine or potentially marine birds ingest excess salt, secretion
of an NaCl-rich fluid from their nasal glands is activated, and this
serves to maintain the osmotic balance of the body fluids (7, 22). In
addition to salt secretion, long-term osmotic stress induces cellular
differentiation in the secretory cells of the gland, which is
characterized, among other factors, by the amplification of the plasma
membrane area at the basolateral cell surface (5) and upregulation of
membrane-associated transport proteins involved in salt secretion (2,
4, 10, 16). In addition to these processes, adaptive hyperplasia is
induced when animals become osmotically stressed for the first time (9, 14, 15). Cell proliferation, especially of the peripheral cells that
line the blind ends of the secretory tubules, leads to tubule
elongation and optimizes the salt-excretory capacity of the organs
(15).
We are interested in the cellular signaling mechanisms that mediate the
initiation of these adaptive processes in the avian salt gland.
Denervation of the nasal glands or administration of atropine, a potent
antagonist at muscarinic receptors, has been shown to abolish salt
secretion as well as the adaptive responses to osmotic stress (reviewed
in Ref. 21), indicating that induction of cell proliferation and
differentiation in the gland may depend, at least in part, on the same
signaling mechanisms as the onset of salt secretion. Salt secretion is
under the control of parasympathetic nerves, which have terminals in
the salt gland parenchyma (7). In isolated salt gland cells, muscarinic
ACh receptor activation results in inositol lipid breakdown (6, 26),
inositol phosphate production and metabolism (12, 25), and generation
of a sequence of intracellular calcium signals (reviewed in Ref. 24).
The latter control the opening of plasma membrane ion channels, the activation of which is required for salt secretion (17-19).
In this study we attempted to identify protooncogenes in the nasal salt
gland of ducklings that are activated by exposing the animals to
osmotic stress and may be involved in mediating the initiation of the
adaptive responses, cell proliferation and cell differentiation,
observed in the glands of osmotically stressed animals.
Animals.
One-day-old ducklings (Anas
platyrhynchos) were purchased from a commercial
hatchery, placed in cages in a room with a 12:12-h light-dark cycle,
and fed chick starter crumbs with drinking water ad libitum. These
ducklings are referred to as "naive" to discriminate the adaptive
condition of these animals from that of a second group of experimental
animals whose drinking water had been replaced with saline (1% NaCl in
tap water) for different periods of time. This group is termed
"stressed." All experimental animals were 6-12 days old.
Preparation of cellular extracts for Western blot experiments.
Ducklings were decapitated, and the nasal glands were exposed and
injected with ice-cold HEPES-buffered saline (25), rapidly dissected
out, and frozen in liquid nitrogen. The glands were pulverized in a
porcelain mortar chilled with liquid nitrogen and subsequently
homogenized in intracellular saline containing (in mmol/l) 100 KCl, 20 NaCl, 2 MgCl2, 0.96 NaH2PO4,
0.84 CaCl2, 1 EGTA, 25 HEPES, 0.2 Pefablock (Roth), and 0.1 Na3VO4
and 0.5% (vol/vol) igepal CA-630 (Sigma Chemical), pH 7.2, using glass homogenizers. The homogenate was kept on ice for 15 min. After centrifugation (13,000 g, 5 min,
4°C), supernatants were transferred to fresh polypropylene tubes,
and aliquots were frozen at Western blots.
A total amount of 15 µg of protein was loaded onto each lane of 10%
SDS gels in a Bio-Rad minigel apparatus. A premixed protein cocktail
(Sigma Chemical) was used as a molecular weight standard. Electrophoresis was performed at 120 V for 90 min. Proteins were blotted onto a nitrocellulose membrane (Schleicher and Schüll) for 4 h at 0°C and 26 V using a Bio-Rad wet-blotting apparatus for
minigels. Nonspecific binding sites on the blots were blocked using 3%
(wt/vol) dry milk powder (Bio-Rad) in Tris-buffered saline containing
0.05% (vol/vol) Tween 20 (TTBS) for 1 h at 4°C. Blots were
incubated with a 1:1,000 dilution of primary antibody overnight at
4°C with gentle movement on a rocking table. Antibodies against a
peptide sequence (amino acids 128-152) of the human Fos protein (SC-253) were obtained from Santa Cruz Biotechnology. Antibodies against a peptide sequence (amino acids 91-105) of the murine Jun
protein (SC-45) were also purchased from Santa Cruz Biotechnology. Specificity of antibody binding was shown by preincubation of the
antisera with their respective antigenic peptides, which abolished Fos-
and Jun-related signals on Western blots. Blots were washed three times
(20 min each) in TTBS before the secondary antibody (horseradish
peroxidase-conjugated donkey anti-rabbit antiserum, Amersham) was added
at a dilution of 1:6,000 for 1 h at 4°C. Finally, blots were washed
three times (20 min each) in TTBS, incubated with enhanced
chemiluminescence reagents (Amersham) according to the manufacturer's
instructions, and exposed to X-ray film. Multiple exposures were
performed to ensure that signals were in the linear range of the X-ray
film.
Organotypic culture of nasal gland tissue.
Tissue slices were prepared and organotypically cultured for 2 h in
serum-free medium as described previously (10). Drugs were added
directly to the culture medium as 100-1,000× stock solutions
in distilled water or DMSO. The DMSO concentration never exceeded 0.1%
(vol/vol) in the culture medium and was inefficient by itself in
altering protein levels of Fos or Jun. Activation of muscarinic ACh
receptors in nasal gland tissue was achieved by adding carbachol (500 µmol/l) to the medium. Atropine was used at a concentration of 100 µmol/l to block the muscarinic receptor. Actinomycin D (5 µmol/l)
was used as an inhibitor of transcription.
Northern blot analysis of c-fos transcription.
Total RNA was isolated from freshly prepared nasal gland tissue of
naive animals or animals that had been drinking 1% NaCl solution for 2 or 24 h. Aliquots of 200 µg of total RNA were precipitated with
ethanol and stored at Fos immunocytochemistry.
Naive ducklings (n = 6) and ducklings
that had been drinking 1% NaCl solution for 4 h
(n = 8) were injected
through the leg vein with low doses of pentobarbital sodium in
physiological saline (Ceva) until they were unresponsive to pinching
the web of their feet. The ducklings were transcardially perfused
(hydrostatic pressure of 150 cmH2O) with 250 ml of cold PBS to
remove the blood from the body and, subsequently, with 250 ml of
freshly prepared 4% (wt/vol) paraformaldehyde solution in PBS for
rapid fixation of the organs. Salt glands were dissected out, cut into
~1-mm3 pieces, and transferred
to fixation solution for 1 h. Tissue was transferred to beakers
containing 20% sucrose solution in PBS and stored at 4°C until all
tissue fragments had settled at the bottom of the beakers. The tissue
was gently blotted dry and rapidly frozen on pulverized dry ice. All
gland fragments of one animal were frozen in different orientations
into a single block using Tissue-Tek (Cambridge Institute) as the
freezing medium. Blocks were cut in 20-µm sections in a cryotome
(Reichert and Jung) with the chamber temperature set to
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
80°C after protein concentration
had been determined and SDS sample buffer had been added.
80°C. Agarose gel electrophoresis of
total RNA (40 µg/lane) was performed on 1% agarose/formaldehyde gels. Electrophoresis, blotting of the RNA, hybridization, and washing
processes were performed as described previously (13). Two
oligonucleotide probes [FOS1 (nt 671-693) and FOS2 (nt
1,765-1,801)] were synthesized (Pharmacia) according to the
chicken c-fos mRNA sequence (20) and
labeled by addition of a poly(A) tail using [
-32P]ATP (NEN;
6,000 Ci/mmol). Hybridization was performed simultaneously with both
probes at 53°C overnight. Signals were visualized by autoradiography on X-ray film. Blots were rehybridized using an oligonucleotide probe for mammalian
-actin (NEN) to control for even
loading of the gel lanes.
18°C
and the sample block temperature set to
22°C. Sections were
transferred onto poly-L-lysine-coated microscopic
slides. The slides were washed in PBS containing 0.1% (vol/vol) Triton
X-100 for 2 h at 4°C and subsequently placed face down on Plexiglas
plates, the rims of which had been taped with 0.15-mm-thick plastic
tape. This created a space between the surface of the Plexiglas plates
and the slides, which was filled with 200 µl of blocking buffer (PBS
containing 0.3% Triton X-100 and 5% FCS) for 1 h at room temperature.
During the incubation periods the Plexiglas plates with the slides were put in a moist chamber to prevent evaporation of solutions. For the
incubation with primary antibodies (same antisera and dilutions as used
in the Western blot experiments, 24 h, 4°C), slides were placed on
fresh Plexiglas plates. To remove unbound primary antibodies, the
slides were washed three times for 10 min at room temperature in a
cuvette containing PBS plus 0.1% Triton X-100. Slides were then
incubated on Plexiglas plates with a biotinylated secondary antibody
(Vector) for 2 h at room temperature. After the slides were washed
three times for 10 min each at room temperature in PBS containing 0.3%
Triton X-100, they were placed on fresh Plexiglas plates and incubated
for 45 min at room temperature with an avidin/biotin-horseradish peroxidase mix (Vector) that had been prepared according to the manufacturer's recommendations. In an attempt to reduce background staining, the tissue sections in some experiments were treated with
avidin/biotin-blocking reagents (Vector) before the primary antibody
was added. Slides were washed in a cuvette at room temperature for 10 min once in PBS plus 0.3% Triton X-100, once in PBS, and once in 50 mmol/l Tris · HCl, pH 7.6. The following enzymatic reaction with 0.03% diaminobenzidine and 0.005% hydrogen peroxide in
Tris · HCl buffer as substrates was performed in the
dark for 8 min at room temperature. Reactions were terminated by
placing the slides in distilled water. Tissue sections were dehydrated by washing with increasing concentrations of ethanol and finally with
xylene. Tissue sections were covered with Entellan (Merck) and glass
coverslips.
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RESULTS |
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Western blot experiments using cellular extracts from salt gland tissue of ducklings (A. platyrhynchos) indicated that Fos and Jun proteins were specifically recognized by antibodies directed against peptide sequences of mammalian origin, indicating a significant degree of similarity in avian and mammalian protooncogene products in some portions of the proteins. Especially the polyclonal antisera directed against amino acids 128-152 of the human Fos protein and against amino acids 91-105 of murine Jun were suitable to detect the avian proteins, whereas other antibodies (Santa Cruz Biotechnology monoclonal antibodies against amino acids 1-111 of human Fos or against amino acids 56-69 of human Jun) did not recognize the avian proteins.
Nasal gland tissue isolated from naive animals or from animals osmotically stressed for different periods of time was tested for Jun and Fos reactivity in Western blot experiments. Fos protein was not detectable in tissue preparations from naive animals, whereas Jun protein was constitutively expressed and did not change under conditions of osmotic stress in the animals (Fig. 1). Fos protein, however, initially appeared in nasal gland tissue after 1 h of osmotic stress in the animals, increased to maximum levels between 4 and 6 h, and faded up to 15 h (Fig. 1).
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Because nasal gland tissue is composed of different types of cells (secretory cells lining the secretory tubules, connective tissue, blood vessels with endothelial cells), we used an immunocytochemical approach to verify that the transient Fos expression was actually taking place in the secretory cells. As shown in Fig. 2, the diaminobenzidine reaction product due to the presence of Fos protein was detectable only in nasal glands from animals that had been drinking 1% NaCl solution for 4 h (Fig. 2A), but not in tissue from naive control animals that had been drinking fresh water (Fig. 2B). The immunoreaction occurred preferentially in the nuclei of the secretory cells, indicating that accumulation of Fos protein in these cells may be responsible for transcriptional changes related to adaptive processes. To a lesser extent, however, other cell types (cells in the connective tissue and endothelial cells) were also labeled, indicating that these cells are involved in the functional and structural changes taking place in the glands under osmotic stress as well. Nevertheless, the major portion of gland tissue is made up of secretory cells, and it is therefore clear that the signals detected on Western blots are mainly due to the strong accumulation of Fos protein in the secretory cells. Immunohistochemical tests using antibodies directed against Jun protein showed a more generalized cytosolic staining pattern, which did not change with the osmotic condition of the animals (not shown).
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To characterize the mechanism by which Fos protein is elevated in nasal gland tissue of stressed ducklings, we performed Northern blot analyses of total RNA isolated from glands of naive and salt-stressed birds. Because the c-fos sequence of ducks is not known, we used oligonucleotide probes, the nucleotide sequences of which were derived from the chicken c-fos gene, assuming that the sequences of chicken and duck c-fos are very similar if not identical. As shown in Fig. 3, hybridization signals were detected only with mRNA from nasal glands of ducklings that had been drinking 1% NaCl solution for 2 h. Nasal gland RNA from naive animals or from animals osmotically stressed for 24 h did not contain any detectable c-fos mRNA. These data confirm the results from the Western blot experiments that Fos expression in nasal gland cells is a transient phenomenon after the onset of osmotic stress. Furthermore, these data show that Fos expression is transcriptionally regulated.
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To more specifically identify the stimulus for the c-fos induction in nasal gland cells, we utilized an organotypic tissue culture system using serum-free medium (10), which enabled us to activate the cultured cells with agonists for selected receptor systems. Nasal gland tissue slices were cultured for 2 h in the presence of 500 µmol/l carbachol to activate muscarinic ACh receptors. These receptors have been reported to occur at high densities on salt gland cells (11, 13) and to be required for the initiation of the adaptive growth and differentiation processes in the gland (21). As shown in Western blot experiments (Fig. 4), carbachol treatment of cultured nasal gland tissue resulted in a substantial increase in Fos expression, a response that could be blocked by simultaneous application of 100 µmol/l atropine, a high-affinity antagonist at muscarinic receptors. These results indicate that it is specifically the activation of muscarinic receptors that is responsible for the induction of Fos accumulation in the secretory cells. In another set of Western blot experiments, the transcriptional inhibitor actinomycin D was added to the tissue culture medium at a concentration of 5 µmol/l in parallel with carbachol. Actinomycin D blocked the carbachol-mediated Fos expression completely (Fig. 4), confirming the result from the Northern blot experiments that Fos expression in nasal gland cells is transcriptionally regulated.
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Immunocytochemical detection of Fos protein in nasal gland tissue slices organotypically cultured and stimulated for 2 h with carbachol revealed patterns of staining almost identical to those observed in freshly prepared nasal gland tissue from osmotically stressed ducklings with nuclear Fos accumulation in the secretory cells only in the carbachol-treated slices (Fig. 2C). No specific staining was observed in the unstimulated control cells (not shown), and very much reduced levels of Fos expression were seen in slices that had been treated simultaneously with atropine and carbachol (Fig. 2D). No changes in signal density or distribution were detected with Jun-specific antisera (not shown). These results indicate that muscarinic receptor activation is the central stimulus in nasal gland secretory cells for the induction of the c-fos gene and for the transient Fos protein accumulation in nasal gland cell nuclei in osmotically stressed ducklings.
Interestingly, all samples of cultured tissue showed Fos immunoreactivity in the connective tissue, most likely related to nuclei of fibroblasts. These signals were independent of muscarinic receptor activation and were insensitive to atropine treatment (Fig. 2, C and D). This indicates that the slicing procedure or the culture conditions of the slices in serum-free media induced c-fos in the connective tissue. The secretory cells, however, were not affected by the culture conditions per se. However, there may be a low level of nonspecific binding of the primary antibody or very low levels of diffuse Fos protein in the tissue sections, since there was always some background staining (Fig. 2, A-F) that was not seen in tissue sections not treated with the primary antiserum (Fig. 2G). Specific signals due to the presence of antigen in the nuclei, however, were only visible in activated cells as dark brown or black precipitates of reaction products.
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DISCUSSION |
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In animals subjected to environmental stress, the regulation of gene expression in effector organs is crucial for the initiation of adaptive growth and differentiation processes that serve to optimize organ function and to enable the animal to maintain its homeostasis. Cells in the nasal gland of domestic ducklings can be induced in vivo to proliferate (15) and to differentiate (2, 5, 10) simply by replacement of the animal's drinking water with a 1% NaCl solution. We used the nasal gland as a model system to study early changes in gene expression after the onset of osmotic stress. In this study we focused on the changes in the expression levels of the immediate early gene products Fos and Jun. These nuclear proteins have a special function in growth and differentiation of many cell types, since they form a heterodimeric structure called AP-1, which functions as a transcription factor by binding to special DNA stretches in the regulatory regions of growth- and differentiation-related genes, thereby changing their transcription rates (1).
Western blot experiments using cellular extracts of freshly prepared nasal gland tissue from naive or osmotically stressed ducklings revealed that Jun is a constitutively expressed protein in the gland, whereas Fos is virtually absent in naive cells but is transiently expressed after the onset of osmotic stress in the animals, reaching maximum levels between 4 and 6 h (Fig. 1). This time course indicates that Fos expression most likely plays a role in the initiation of the adaptive growth and/or differentiation processes, which become apparent 12-15 h after the onset of osmotic stress (10, 15), whereas salt secretion from the gland starts at 2 h of osmotic stress, even before Fos reaches maximum levels.
Fos expression is especially pronounced in the secretory cells of the nasal gland (Fig. 2), but other cell types, particularly vascular endothelial cells, show Fos expression as well. This demonstrates that virtually all cell types in the gland are involved in the adaptive changes and the improvement of organ function. However, inasmuch as the secretory cells constitute the major portion of cells in the gland, it is assumed that the changes in overall nasal gland Fos expression detected in Western blot experiments (Fig. 1) are primarily due to changes in Fos expression in the secretory cells. Most of the nuclei labeled by Fos-specific antibodies in the tissue sections belong to principal cells in the distal portions of the secretory tubules (Fig. 2, A and C). In some experiments, however, nuclei of peripheral cells seemed to be stained as well. Because it has been suggested that adaptive cell proliferation may be restricted to peripheral cells, whereas cell differentiation may occur in all parts of the gland except the peripheral cells (5, 15), it remains unclear at this stage which of these adaptive processes may be the physiological target of Fos accumulation.
It is well known that salt secretion in the avian salt gland is under control of the parasympathetic nervous system. Release of ACh from nerve terminals onto the surface of the secretory cells stimulates salt secretion via activation of muscarinic receptors, inositol phosphate accumulation, and calcium signaling (reviewed in Ref. 23) but may also be a component of the signaling pathways inducing the adaptive growth and differentiation processes. This was concluded from experiments in which atropine had been systemically administered to ducklings before they were osmotically stressed. Under these conditions, salt secretion as well as the adaptive growth and differentiation processes in the nasal glands were completely suppressed (reviewed in Ref. 21).
We utilized a previously developed organotypic tissue culture system (10) to test whether the muscarinic ACh receptor plays a role in the induction of c-fos in nasal gland cells. Carbachol enhanced Fos expression in cultured tissue in an atropine-sensitive manner (Fig. 4), indicating that the Fos accumulation was a specific response to muscarinic receptor activation. As shown in Fig. 2C, the atropine-sensitive carbachol effect on Fos expression was observed exclusively in the secretory cells of the gland. Fos-specific staining of cell nuclei in the connective tissue, however, was independent of muscarinic ACh receptor activation and was not blocked by atropine. We conclude that Fos in the connective tissue was induced by the slicing procedure or by the serum-free culture conditions. These observations match those from the Western blot experiments, where Fos protein could never be detected in freshly isolated naive tissue. Cultured naive tissue slices, however, always showed a low level of Fos expression (cf. control lanes in Fig. 4), presumably representing Fos protein in cells from the connective tissue. The Fos-specific staining pattern (Fig. 2C) in the secretory cells of carbachol-treated cultured tissue slices, however, resembled that observed in nasal gland tissue of animals osmotically stressed for 4 h (Fig. 2A), indicating that osmotic stress induces c-fos expression in nasal gland cells via activation of muscarinic receptors.
As indicated by the results from Northern blot experiments with total RNA isolated from nasal glands of naive and osmotically stressed ducklings (Fig. 3) and, moreover, by results obtained from experiments with cultured tissue treated with actinomycin D (Fig. 4), Fos expression seems to be controlled by modulation of the transcriptional rate of the c-fos gene. This is concluded from the facts that mRNA for Fos was detected only for a short period of time in nasal glands of osmotically stressed ducklings and that the carbachol-mediated Fos expression in cultured tissue was entirely suppressed by the transcriptional blocker actinomycin D.
The promoter region of the mammalian c-fos gene contains at least two elements, the serum response element (SRE) (28) and the cAMP response element (8), which are potential target sites for intracellular signaling cascades activated by muscarinic receptor subtypes coupled to the phospholipase C pathway. The SRE can be activated by protein kinase C-mediated signaling (3), whereas the SRE and cAMP response element are subject to regulation by the free cytosolic calcium concentration (27, 29). Which of these pathways activates c-fos transcription in nasal gland cells of osmotically stressed ducklings remains to be clarified in future studies.
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ACKNOWLEDGEMENTS |
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We thank Dr. I. Schulz, who provided lab space and some of the equipment used in this study. The help provided by Daniela Brenner and Panagiotis Patronas in the immunohistochemical experiments is gratefully acknowledged.
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FOOTNOTES |
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J.-P. Hildebrandt is the recipient of a Heisenberg stipend from the Deutsche Forschungsgemeinschaft. Financial support was provided by Deutsche Forschungsgemeinschaft Grants Hi 448/3-1 and 4-1.
Experiments with animals were conducted according to German animal welfare regulations and approved by the Landratsamt Homburg (LIII/180-07) or the Landratsamt Darmstadt (II17a-19c20/15-B2138).
Address for reprint requests: J.-P. Hildebrandt, 2. Physiologisches Institut, Medizinische Fakultät, Universität des Saarlandes, Haus 58, D-66421 Homburg/Saar, Germany.
Received 12 September 1997; accepted in final form 8 June 1998.
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