Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan
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ABSTRACT |
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Although aquaporin 3 (AQP3) is expressed in many tissues in the kidney, gastrointestinal tract, lung, and other organs, its physiological significance in the body still remains to be clarified. To determine whether AQP3 expression is regulated by dexamethasone in human airway epithelium, we studied mRNA expression, protein expression, and water permeability of the cell membrane in a human airway epithelial cell line (A549 cells). Expression of AQP3 mRNA and protein was studied by Northern blot analysis and immunoblot analysis, and osmotic water permeability (Pf) was measured by a stopped-flow light-scattering method. Expression of AQP3 mRNA and protein was detectable in A549 cells and was stimulated by dexamethasone. Pf in A549 cells after incubation with dexamethasone was ~2.5-fold greater than that without dexamethasone. Moreover, this dexamethasone-induced increase in Pf was inhibited after treatment with HgCl2. In conclusion, the present study shows that A549 cells express AQP3 and that dexamethasone upregulates the expression of AQP3 and increases the water permeability of the cell membrane. Dexamethasone-regulated AQP3 expression might be important in certain forms of pulmonary diseases accompanied by airway hypersecretion that are treated by corticosteroid administration.
water channel; osmotic water permeability; steroid
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INTRODUCTION |
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AQUAPORINS (AQPs) have been postulated to play an important role in the pathway for water transport in highly water-permeable membranes. Five types of AQP have been cloned so far in mammals, namely AQP1 (18), AQP2 (7), AQP3 (5, 11, 15), AQP4 (8, 13), and AQP5 (19).
Our understanding of the structure, tissue distribution, and membrane localization of AQP is becoming clearer. AQP1 is expressed in erythrocytes, kidney proximal tubule, and other water-permeable epithelia. AQP2 is the vasopressin-regulated water channel and is selectively expressed in renal collecting ducts. AQP3 is expressed in kidney, colon, small intestine, liver, pancreas, spleen, prostate, and lung. AQP4 is most abundantly expressed in brain. AQP5 is expressed in submandibular, parotid, and sublingual salivary glands, lacrimal gland, eye, and lung. In respiratory organs, AQP1 is expressed in pulmonary capillary endothelium rather than in the alveolar epithelium (16). AQP3 is localized to the basolateral membrane of tracheal epithelial cells (6). AQP4 is localized to the basolateral membrane of medium-sized bronchi (6).
The function of AQP3 has been reported to differ from the functions of other AQPs. In addition to water, glycerol and urea can also move through the cell membrane in AQP3 (5, 11), although water does not seem to share the same pathway with glycerol or urea (4). However, the physiological significance of AQP3 in the body is not clear at present.
In AQP, maternal corticosteroid was demonstrated to increase expression of AQP1 in fetal rat lung (14). Although airway epithelium tends to be a target for steroid therapy in many pathological states and the expression of AQP1 is upregulated by corticosteroid, the regulatory effect of corticosteroid on AQP3 expression has not been examined.
To determine whether AQP3 expression in human airway epithelium is regulated by corticosteroid, we studied mRNA and protein expression and water permeability of the cell membrane in a human airway epithelial cell line (A549 cells). We found that AQP3 expression and water permeability of the cell membrane are stimulated by corticosteroid in A549 cells.
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MATERIALS AND METHODS |
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Cell culture. A549 cells derived from lung adenocarcinoma were obtained from the Japanese Cancer Research Resources Bank. The cells were plated at a density of 105 to 106 cells/ml in 90 (Sumitomo Bakelite)- or 150 (Nunc)-mm culture dishes and then were incubated at 37°C in 5% CO2 in Eagle's minimal essential medium (MEM; BRL, Gaithersburg, MD) with 10% fetal bovine serum (FBS; BRL), 100 U/ml penicillin (BRL), and 100 µg/ml streptomycin (BRL). Medium was changed every 2 days. The cells were used when they reached a near-confluent state.
Northern blot analysis. For studies of
mRNA expression, A549 cells were cultured in MEM without FBS in 90-mm
culture dishes. After a 24-h culture, A549 cells were incubated with
dexamethasone (109 to
10
6 M; Sigma, St. Louis,
MO) for 0-24 h. In a separate set of studies, A549 cells were
preincubated with actinomycin D
(10
6 M; Sigma) or
cycloheximide (10
6 M;
Sigma) for 2 h and then were incubated with dexamethasone (10
7 M) for 6 h at
37°C.
At the end of incubation, total RNA was isolated by acid guanidinium
thiocyanate-phenol-chloroform extraction using Isogen (Nippon gene).
After the cells were lysed in 1.5 ml of Isogen, 0.3 ml of
chloroform-isoamyl alcohol (49:1) were added and mixed well. RNA was
precipitated from the aqueous phase with 1 vol isopropanol at
70°C. RNA was washed with 75% ethanol and was dissolved in ribonuclease-free water.
Ten micrograms of total RNA were separated by electrophoresis on a 1%
agarose gel containing formaldehyde (Wako) and
3-(N-morpholino)propanesulfonic acid (Sigma). The RNA was
transferred onto nylon membranes (Magna Graph; MSI, Westborough, MA)
with 10× saline-sodium citrate (SSC) and was cross-linked by
ultraviolet light. The cDNA probe (1.4 kb) for human AQP3, which
includes its entire open reading frame, and that for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; American Type Culture
Collection) were labeled by random priming using
[32P]dCTP (Amersham).
After prehybridization for 3-4 h, the membrane was hybridized for
24 h at 42°C in 50% formamide (Wako), 5× sodium chloride-sodium phosphate-EDTA, 5× Denhardt's solution, 0.16% sodium dodecyl sulfate (SDS; BRL), and 20 µg/ml salmon sperm DNA (Sigma). The membrane was washed two times for 20 min at 65°C in
0.2× SSC and 0.1% SDS and then was exposed to a film for
24-72 h at 70°C.
Autoradiograms were analyzed by densitometric scanning using software (NIH Image). AQP3 mRNA expression was shown relative to the GAPDH band measured in the same lane by quantitative densitometry. Densitometric analysis was expressed as mean ± SD from four sets of studies for each group. The value of the control group was designated as one. The effects of dexamethasone were assessed with unpaired t-tests. Significance was established as P < 0.05.
Immunoblot analysis. For studies of
immunoblot analysis, A549 cells were incubated with
107 M dexamethasone
for 24-48 h after a 24-h culture in MEM without FBS in 90-mm
culture dishes. Cells were washed two times with Ca2+/Mg2+-free
phosphate-buffered saline (PBS), treated with lysis buffer (Promega,
Madison, WI), and centrifuged. Total protein concentration was
determined by the Bradford protein assay (Bio-Rad, Hercules, CA) using
bovine serum albumin (Sigma) as a standard.
Twenty micrograms of cellular total protein mixed with sample buffer
(Daiichi pure chemicals) were heated to 70°C and were electrophoresed on a 10-20% gradient SDS-polyacrylamide gel
(Daiichi pure chemicals). The gel was transferred to
Immobilon-polyvinylidene difluoride membranes (Millipore, Bedford, MA)
in a semidry blotter and were stained with Coomassie brilliant blue
(Katayama Chemical) to confirm the equivalence of the samples. The
membranes were blocked with 2% skim milk in 0.2 M
tris(hydroxymethyl)aminomethane · HCl, 1.5 M
NaCl, pH 7.5, and 0.05% Triton X-100 (TBS-T) overnight at 4°C and
then were blotted with specific antibodies (anti-human AQP3 antibody at
1:100 dilution). After three washes with 0.2% skim milk in TBS-T, the
membranes were incubated with
125I-labeled protein A (Amersham),
washed three times with TBS-T, and exposed to a film for ~72 h at
70°C.
Autoradiograms were analyzed by densitometric scanning in the same manner as Northern blots. Densitometric analysis is expressed as mean ± SD from three sets of studies for each group. The value of the control group was designated as one. The effects of dexamethasone were assessed with unpaired t-tests. Significance was established as P < 0.05.
Assay for plasma membrane water
transport. For studies of water permeability across the
cell membrane, we measured osmotic water permeability
(Pf) in freshly
suspended A549 cells by a stopped-flow light-scattering method using a
stopped-flow apparatus (SX.18MV; Applied Photophysics, Leatherland,
UK). After a 24-h incubation with
107 M dexamethasone in
150-mm culture dishes, A549 cells were detached from the dishes by
incubation in PBS containing 50 mM EDTA for 10 min. The cells were
washed two times with PBS and were suspended at a density of 2.5 × 105 to 5 × 105 cells/ml. Cell suspension was
mixed for <1 ms with hyperosmotic solution (PBS containing 600 mM
mannitol) at 10°C to give a 300 mosM inwardly directed osmotic
gradient. The osmotic gradient is known to cause water efflux, cell
shrinkage, and an increase in scattered light intensity. The time
course of 90° scattered light intensity at 530-nm wavelength was
measured. Measurements were performed five to six times in each sample
for signal averaging. Data were fitted to a biexponential function
using software provided by Applied Photophysics; water transport rates
were calculated from the initial curve slope normalized to the total
signal amplitude. Pf was calculated
from the water transport rates and the cell surface-to-volume ratio.
The diameter of A549 cells was estimated to be 6 µm by a particle
size distribution analyzer (Sysmex CDA-500; Toa). In some samples,
cells were incubated with 50 µM
HgCl2 for 5 min, followed by 5 mM
-mercaptoethanol for 5 min before measurements.
Expression of AQP in A549 cells. To examine whether AQPs other than AQP3 are expressed in A549 cells, 1 µg of total RNA was reverse transcribed and amplified by polymerase chain reaction (PCR) with five sets of primers. The sequences of each primer were as follows: 1) AQP1, sense primer 5'-ATCATCGCCCAGTGCGTG-3' and antisense primer 5'-GTAGATGAGTACAGCCAG-3'; 2) AQP2, sense primer 5'-ATGAGATCACGCCAGCAG-3' and antisense primer 5'-AGTGACGACA GCTGGAGC-3'; 3) AQP3, sense primer 5'-TGCCTGGGGACCCTCATC-3' and antisense primer 5'-GATCATATCCAAGTGTCC-3'; 4) AQP4, sense primer 5'-GCAGGAATCCTCTATCTG-3' and antisense primer 5'-TTCAACATCTGGACA GAA-3'; and 5) AQP5, sense primer 5'-GGTGTGGCACCGCTCAAT-3' and antisense primer 5'-ACTCAGGCTCAGGGAGTT-3'. The PCR was carried out in the following profile: 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min (25 cycles). The PCR products were electrophoresed on a 1% agarose gel.
AQP3 gene in A549 cells. To confirm that cDNA and the promoter region of AQP3 were identical in both A549 cells and the normal human genome, we determined the sequence of AQP3 in A549 cells. One microgram of total RNA from A549 cells was reverse transcribed and amplified by PCR with a sense primer (5'-CCCGCCATGGGTCGACAGAAGGAG-3') and an antisense primer (5'-CACTCAGATCTGCTCCTTGTGCTT-3') (12) to make PCR products (0.9 kb) that contain the entire open reading frame of human AQP3. The PCR was carried out with a long and accurate PCR kit (Takara, Japan) in the following profile: 98°C for 20 s and 68°C for 2 min (30 cycles).
One microgram of genomic DNA extracted from A549 cells was amplified by PCR with a sense primer (5'-TCTGCCTAAGCCTCTCAGCCCCCT-3') and an antisense primer (5'-GTCGACCCATGGCGGGGCAGGCGG-3') to make the 5'-flanking region (318 bp) of human AQP3 (10). The PCR was carried out in the following profile: 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min (35 cycles).
Each PCR product was subcloned into pGEM-T Vector (Promega) and then was sequenced using a fluorescence DNA sequencer (373A; Applied Biosystems).
Effect of dexamethasone on the promoter activity of
AQP3. To examine the effect of dexamethasone on the
promoter activity of the 5'-flanking region in the AQP3 gene, we
utilized the luciferase assay. The 5'-flanking region of AQP3
(318 bp; the first nucleotide of the ATG codon was designed as
+1) was subcloned into pGL2-basic vector (Promega). A549 cells were
grown in 15-cm dishes, and ~5 × 107 cells growing in the
exponential phase were harvested by 0.25% trypsin and were resuspended
in (in mM) 30.8 NaCl, 120.7 KCl, 1.46 KH2PO4,
8.1 Na2HPO4,
and 10 MgCl2. Transfection was
performed by electroporation under 400 V at 960 µF in a 0.4-mm-wide
cuvette. To evaluate the efficiency of transfection,
pSV-
-galactosidase vector was transfected together. Transfected
cells were seeded on 3-cm dishes, incubated with full medium for 24 h,
and then incubated with dexamethasone
(10
7 M) for 8 or 24 h.
At the end of incubation, the cells were washed with PBS two times and were incubated in cell lysis buffer (Promega) at room temperature for 15 min. Ten microliters of the cell lysate were mixed with 50 µl of luciferase assay reagent (PicaGene, Toyoink, Japan) at room temperature, and the light intensity of the sample was counted in a luminometer (Lumat LB9501).
To measure -galactosidase activity, 50 µl of the cell lysate were
mixed with 50 µl of
-galactosidase assay buffer (Promega), incubated at 37°C for 30 min, and mixed with 150 µl of 1 M sodium carbonate. The absorbance of the sample was measured at 405 nm.
The data of luciferase activity are expressed as mean ± SD from five sets of studies for each group. The luciferase activity of the control group was designated as 100.
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RESULTS |
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Induction of AQP3 mRNA by
dexamethasone. To examine the concentration-dependent
induction of AQP3 mRNA by dexamethasone, A549 cells were incubated with
dexamethasone (109 to
10
6 M) for 8 h. AQP3 mRNA
was upregulated in a concentration-dependent manner, with a maximal
induction when A549 cells were incubated with
10
6 M dexamethasone (Fig.
1). Densitometric analysis showed that AQP3
mRNA expression in the dexamethasone-treated group
(>10
8 M
dexamethasone) increased to more than three times the level of
that in the control group. To examine the time course of induction of
AQP3 mRNA, A549 cells were incubated with
10
7 M dexamethasone for
0-24 h. The peak induction of AQP3 mRNA was observed 8 h after
incubation (Fig. 2). Densitometric analysis showed that AQP3 mRNA expression in the dexamethasone-treated group
(10
7 M for 8 h) increased
to approximately four times the level of that in the control group.
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To examine the effects of a transcription inhibitor and a
translation inhibitor on the expression of AQP3 mRNA, A549 cells were
preincubated with 106 M actinomycin D or
10
6 M cycloheximide for 2 h and then were
incubated with 10
7 M dexamethasone for 6 h at
37°C. Although dexamethasone-induced expression of AQP3 mRNA was
inhibited by actinomycin D, it was not inhibited by cycloheximide (Fig.
3). These findings suggest that AQP3 is a
primary response gene that is induced without any intervening protein
synthesis.
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In addition, we have confirmed that the expression of AQP3 mRNA in A549 cells has not been affected with serum by Northern blot analysis (data not shown).
Immunoblot analysis. The effect of
dexamethasone on the expression of AQP3 protein was assessed by
immunoblotting. The expression of AQP3 protein was increased by
dexamethasone, and its peak expression was observed 24 h after
incubation (Fig. 4). Densitometric analysis showed that AQP3 protein expression in the dexamethasone-treated group
(107 M for 24 h) increased
to approximately six times the level of that in the control group.
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Water permeability. To examine the
effect of dexamethasone on
Pf, A549 cells
were incubated with 107 M
dexamethasone for 24 h. A typical recording of scattered light intensity in suspended A549 cells is shown in Fig.
5A. The
diameter of A549 cells did not change even after the cells were
incubated with dexamethasone (control 6.5 ± 0.2 µm, dexamethasone
6.2 ± 1.02 µm; not significant).
Pf in A549 cells
after incubation with dexamethasone [159.5 ± 38.1 (SD)
µm/s, n = 7] was about
2.5-fold greater than that without dexamethasone (62.5 ± 5.7 µm/s, n = 13; Fig.
5B). Although
Pf in
dexamethasone-treated cells was decreased after incubation with 50 µM
HgCl2 (33.8 ± 8.1 µm/s,
n = 8), the inhibition by
HgCl2 was reversed by
2-mercaptoethanol (129.7 ± 19.6 µm/s,
n = 3). These findings suggest that
AQP is expressed even in control A549 cells and that the
dexamethasone-induced increase in water permeability is due to the
increased expression of AQP, which in this case seems to be AQP3.
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Expression of AQP in A549 cells. Total RNA of A549 cells was reverse transcribed and amplified by PCR to confirm whether the AQPs other than AQP3 are expressed in the A549 cells. The size of the PCR products amplified with each set of primers was expected to be 360 ~ 390 bp. Although PCR products of AQP3 could be observed at the expected size, none could be detected for the other AQPs (AQP1, AQP2, AQP4, and AQP5; Fig. 6.).
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AQP3 gene in A549 cells. cDNA and the promoter region of AQP3 in A549 cells were sequenced for comparison with those in the normal human genome. The sizes of the PCR products representing cDNA and the promoter region of AQP3 were identical in both A549 cells and the normal human genome. Moreover, the entire sequence of cDNA and the promoter region in A549 cells were the same as those in the normal human genome.
Effect of dexamethasone on the promoter activity of
AQP3. To examine the effect of dexamethasone on the
promoter activity of the 5'-flanking region in the AQP3 gene, we
measured the luciferase activity in A549 cells that had been
transfected with the reporting vector containing the 5'-flanking
region. Compared with the level in the control group, luciferase
activity was not changed in transfected A549 cells, which were
incubated with dexamethasone
(107 M) for 8 h; however,
luciferase activity was upregulated ~1.3-fold in cells incubated for
24 h (Fig. 7).
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DISCUSSION |
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This study shows that the human airway epithelial cell line (A549 cells) expresses AQP3 and that dexamethasone stimulates mRNA and protein expression of AQP3 and increases cell membrane water permeability as measured by a stopped-flow light-scattering method.
There are many kinds of channels in humans. They are regulated by various substances and are considered to link with various types of pathogenesis of several disease states. For example, AQP2 (AQP-CD) is a vasopressin-regulated AQP that is responsible for the massive reabsorption of water in renal collecting ducts. In nephrogenic diabetes insipidus (NDI), a male patient with an autosomal-recessive form of NDI was found to be a compound heterozygote for two mutations in the AQP2 gene, each of which resulted in nonfunctioning AQP molecules (3). In the rat experimental models, AQP2 expression was highly upregulated by dehydration for several days (9).
Glucocorticoid receptor is present in both fetal and adult lungs (1), and glucocorticoid is postulated to be one of the factors at work in maturing the fetal lung. Around birth, a large volume of pulmonary fluid must be cleared to make an air-conducting system. This process is reported to be largely dependent on active sodium transport by the pulmonary epithelium (17), which is consistent with the finding that the amiloride-sensitive sodium channel is upregulated by steroid hormones (2). AQP1 expression in fetal rat lung was reported to increase from the last gestational day to the first postnatal day and to persist at high levels into adulthood. In the report by King et al. (14), AQP1 expression was increased in corticosteroid-treated rat lung, which suggests that maternal internal corticosteroids may increase the expression of AQP1 in the fetal lung. AQP4 and AQP5 were also reported to be expressed in the perinatal rat lung (22), although the effect of corticosteroid on their expression remains unclear. Although the effect of corticosteroid on AQP3 expression in the human native lung has not been examined, the present study raises the possibility that dexamethasone-regulated AQP3 may play several roles in the clearance of pulmonary fluid. In addition, corticosteroid is administered in many lung diseases. Corticosteroid is an especially critical drug for improvement of clinical symptoms in bronchial asthma. Because hypersecretion in the airway is one of the major pathophysiological findings in bronchial asthma, increased expression of AQP3 by dexamethasone might help in reabsorption of the secreted fluid. Clearly, further studies are needed to examine the role of AQP3 in lung diseases.
Although dexamethasone was demonstrated to upregulate the promoter
activity of AQP3, glucocorticoid response elements (GREs) could not be
identified in the proximal promoter region (318 bp) of the AQP3
gene. These findings suggested that another element that
dexamethasone acts upon might be present in this promoter. However, since promoter activity in dexamethasone-treated cells increased less than the expression of mRNA and protein, it is possible
that a GRE is present at a site other than this proximal promoter
region of the AQP3 gene. More extensive examination of the regulation
of AQP3 expression by corticosteroid is needed.
AQP3 is localized to the basolateral membrane of tracheal epithelial cells (6). In this study, we could not clarify the AQP3 localization because A549 cells do not have tight junctions or separate apical and basolateral membranes (20).
The present study shows that AQP3 expression is upregulated by dexamethasone in the human airway epithelial cell line. To our knowledge, no cell line other than A549 cells is known to express AQP3 endogenously, although one cell line (immortalized rat proximal tubule cells) is known to express AQP1 (21). A549 cells might be a useful tool to study the regulatory mechanism of AQP3 expression.
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ACKNOWLEDGEMENTS |
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This work was supported in part by research grants from the Japanese Ministry of Education, Science, and Culture.
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FOOTNOTES |
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Address for reprint requests: N. Inase, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan.
Received 9 May 1997; accepted in final form 14 August 1997.
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