1 Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033; and 2 Department of Otolaryngology, School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cellular distribution of parchorin, a new chloride intracellular channel family member, was investigated in rabbit tissues by immunohistochemistry using an antibody recognizing the sequence containing a parchorin-specific repeat. Parchorin preferentially resides in the epithelium of the ducts of the lacrymal, parotid, submandibular, and mammary glands and the pancreas, prostate, and testis. In the trachea and lung, parchorin was found in the airway epithelium and type II alveolar cells. In the kidney, parchorin was distributed mainly from the thick ascending limb to the distal convoluted tubule. In the eye, both pigment and nonpigment epithelia of the ciliary body were positive, whereas only the pigment epithelium was positive in the retina. Parchorin was also present in the cochlea and semicircular canal. The amount of parchorin in the gastric mucosa, but not in the submandibular glands, increased after weaning. In the mammary gland, parchorin expression was greater in a lactating rabbit (1 wk after delivery) compared with a pregnant (3 wk) rabbit. The cellular distribution and changes in expression indicate that parchorin plays an important role, possibly in chloride transport, in the cells that create an ion gradient for water movement.
chloride channel; duct; rabbit; immunohistochemistry
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN THE VARIOUS ORGANS OF
THE body, a huge amount of water moves as cerebrospinal fluid,
urine, or digestive fluid. It has been considered that the water is
transepithelially or laterally driven by the osmotic pressure created
by ions (including Na+, K+, and
Cl) and that their movement is the consequence of
activation of various ion pumps, transporters, and channels (1,
26, 30). The regulation of fluid movement in the body is very
important for homeostasis. This has been proven by natural or
artificial diseases in which aquaporins (35, 36), cystic
fibrosis transmembrane conductance regulator (CFTR; 9),
Na+-K+-2Cl
cotransporter 1 (NKCC1; 5, 7), or ClC-K1 (19) is mutated or deleted. Subsequent abnormalities occur in urine reabsorption and
secretions from airway epithelium, sweat glands, the pancreas, and the
inner ear. Among these, Cl
conductance afforded
by CFTR is considered essential for transepithelial water transport,
since the defect in CFTR function causes CF due to an impairment in
water movement in various tissues. In addition to CFTR, ClC-2 and other
volume-sensitive Ca2+-dependent voltage-gated
Cl
channels have been suggested (1) to play
important roles in water movement in the apical side of epithelial cells.
Recently, we (21) cloned a new protein,
parchorin, which has homology to the Cl intracellular
channel (CLIC) family. In contrast to other CLIC family members, most
of which are considered to be Cl
channels in
intracellular vesicles (32), parchorin is a soluble cytosolic phosphoprotein and translocates to the plasma membrane under
stimulation. When acid-secreting parietal cells are stimulated, tubulovesicles containing H+-K+-ATPase fuse
with the apical membrane, and both K+ and Cl
permeability in the apical membrane are increased. Consequently, HCl
secretion is elicited (35). During this activation
process, it was observed (21, 33) that parchorin
translocated from the cytosol to the apical membrane. It was also
observed (21) that parchorin, transfected to LLC-PK1
cells, translocated to the plasma membrane and accelerated the
Cl
efflux rate when Cl
efflux was caused
by elimination of extracellular Cl
concentration.
Parchorin is preferentially expressed in tissues related to water
movement, i.e., the gastric mucosa, choroid plexus, salivary gland, and
kidney (21, 33). Parchorin was named according to its
characteristic distribution, i.e., the highest expression being in the
parietal cell and choroid plexus. These observations suggest strongly
that parchorin plays an important role in the regulated movement of
body fluid via Cl
transport.
In the present study, we examined the cellular localization of parchorin in various tissues using immunohistochemistry under light microscopy. We also observed development-related changes of parchorin expression in some exocrine glands.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Production of parchorin fragments as green fluorescent protein fusion protein. Full-length rabbit parchorin (22) or its fragments cut out with NaeI-EcoRI (73-637), AccI-EcoRI (159-637), BstXI-EcoRI (262-637), XhoI-EcoRI (404-637), BamHI-SalI (1-404) were subcloned into pEGFP vector (Clontech). Of these, XhoI and SalI sites were artificially introduced by PCR. COS-7 cells were transfected with these cDNA by the DEAE-dextran method. The cells were then homogenized with 120 mM NaCl and 20 mM HEPES-Tris, pH 7.4, and the 100,000 g supernatant was concentrated with Centriprep 30 (Amicon). The fusion protein was lastly purified with HPLC using a gel-filtration column (Diol-150).
Western blotting. To estimate the contents of parchorin by immunoblotting, various rabbit tissues were homogenized with 120 mM NaCl and 20 mM HEPES-Tris, pH 7.4, and centrifuged at 800 g for 15 min, and the supernatant was harvested. After the protein assay, the sample was separated by SDS-PAGE according to the method of Laemmli (15) and blotted to a polyvinylidene difluoride membrane (Bio-Rad) using a semidry apparatus at 1 mA/cm2 for 50 min. The membrane was blocked with 5% skim milk in 0.5% Tween-PBS at room temperature for 1 h. We incubated the membrane with anti-parchorin mouse monoclonal antibody (33) or anti-green fluorescent protein (GFP) rabbit polyclonal antibody (Clontech) (1:5,000 dilution each) at 4°C overnight. The membrane was then further incubated with a second antibody, horseradish peroxidase-conjugated anti-mouse IgG (Sigma, 1:2,000 dilution) or anti-rabbit IgG (Santa Cruz, 1:2,000 dilution), at 37°C for 1 h and visualized using the Renaissance Western blot chemiluminescence reagent kit (NEN).
Immunohistochemistry. Each organ dissected from a Japanese White rabbit (Shiraishi Tokyo) was immediately fixed with 10% formalin in PBS at 4°C for 2 h to overnight, depending on the thickness of the tissue. The tissue was then immersed for 24 h in 15% (wt/vol) sucrose in 0.1 M phosphate buffer for cryoprotection and frozen in Tissue-Tek OCT compound (Sakura Finetechnical). Cryostat sections (10 µm) were obtained and mounted on slides coated by 3-aminopropyltriethoxysilane. For the inner ear sample, the tissue was decalcified in 10% EDTA-Tris for 7 days before freezing. Endogenous peroxidase activity was inhibited by 30 min preincubation in 0.3% H2O2-methanol followed by a 10-min wash in PBS, and blocking was performed with 8% skim milk in PBS for 1 h. The section was probed with anti-parchorin mouse monoclonal antibody (33) (1:1,000 dilution) in PBS-Tween at 4°C overnight. Except for mammary glands in which endogenous biotin appeared to be high, the section was incubated with biotin-conjugated anti-mouse IgG (Sigma; 1:3,000 dilution) and subsequently with horseradish peroxidase-conjugated biotin-avidin complex (Vectastain ABC kit, Vector Laboratories) at 37°C, both for 50 min. For mammary glands, horseradish peroxidase-conjugated anti-mouse IgG (Sigma; 1:50 dilution) was used as the second antibody. The section was developed for an appropriate time in 0.025% diaminobenzidine, 1 µM H2O2, and 10 mM Tris · HCl, pH 7.5, and counterstained with 1% methyl green and 0.1 M barbital-acetate, pH 4.0, at room temperature for 2 h. The images were taken by microscopy (Olympus BX-50) connected to a digital charge-coupled device camera (Fujix HC-2500 3CCD, Fujifilm).
For double staining, the section was permeabilized with 1% Triton X-100 in PBS at room temperature for 30 min and then blocked with 5% skim milk in PBS-Tween for 1 h. To detect parchorin and Tamm-Horsfall glycoprotein, the section was incubated with anti-parchorin mouse monoclonal antibody (1:200 dilution) and anti-Tamm-Horsfall glycoprotein (COSMO-BIO; 1:100 dilution) at 4°C overnight, followed by Cy3-anti-mouse IgG (Amersham Pharmacia Biotech; 1:50 dilution) and FITC-anti-sheep IgG (Sigma; 1:20 dilution) at 4°C overnight. For parchorin and cytokeratin, only mouse antibodies were available. Therefore, the section was sequentially incubated with anti-cytokeratin (AE1/AE3, Dako, ready to use), FITC-anti-mouse IgG (Sigma; 1:50 dilution), anti-parchorin, and Cy3-anti-mouse IgG. We took special care to ensure that the section with anti-cytokeratin, FITC-anti-mouse IgG, and Cy3-anti-mouse IgG (but without anti-parchorin) did not show any signal for Cy3. This confirmed that the second antibody for parchorin did not bind to uncovered mouse IgG probed for cytokeratin. The sections were examined with a microscope (Nikon Eclipse) with a confocal laser scanning system (µRadiance, Bio-Rad). FITC was excited at 488 nm (argon-ion laser) and detected with HQ 515/30 filter, and Cy3 was excited at 543 nm (green helium-neon laser) and detected with E570LP. A sequential acquisition mode was employed to avoid "bleed through" of the staining. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of epitope of anti-parchorin antibody.
To ensure the specificity of the antibody for recognizing parchorin
among the CLIC family members, we first identified the epitope of the
presently used monoclonal anti-parchorin antibody (33). As
shown in Fig. 1A, we created
full-length and deletion mutants of parchorin as GFP fusion protein,
transiently expressed them in COS-7 cells, and partially purified them.
All the fragments were successfully expressed, and it was confirmed
that they were recognized by anti-GFP antibody (Fig. 1B,
right). As shown in Fig. 1B, left, the monoclonal
anti-parchorin antibody recognized GFP-parchorin1-637
(full length), GFP-parchorin73-637, GFP-parchorin159-637, and
GFP-parchorin1-404, as well as native parchorin
partially purified from rabbit gastric mucosa, but it did not recognize
GFP-parchorin262-637 or
GFP-parchorin404-637 (CLIC homology domain). It is
obvious that the antibody recognized any fragments having the amino
acid sequence 159-262, but it did not recognize fragments without
that sequence. This indicates that the epitope for the antibody exists
in the sequence that contains the characteristic GGSVDA repeat and is
specific for parchorin (21). We conclude that the antibody
could specifically distinguish parchorin from other CLIC family members
reported to be widely distributed in the organs.
|
Cardiovascular system. Parchorin immunoreactivity was completely absent in the aorta and heart (data not shown). Cardiac tissue is reported to be enriched in other CLIC family members (2, 6, 17, 24, 32), and thus parchorin is exceptional in that regard. The arteriole, capillary, and vascular endothelium were all negative for parchorin, which is obvious from staining of the various tissues mentioned below.
Digestive system.
As previously reported (21), within the fundic mucosa,
only parietal cells showed highly positive staining for parchorin antibody (Fig. 2A). In the
pyloric mucosa where parietal cells are absent, no reactivity was
noticed (data not shown). In the intestine, from duodenum to rectum, no
positive staining was obtained, as shown in the case of the
rectum (Fig. 2B).
|
Respiratory system.
In the trachea, parchorin was exclusively expressed in the mucous
epithelial cells that secrete airway fluid (Fig.
3, A and B). In
previous reports (21, 33), parchorin was not detectable in
the lung by Western blotting, but in the present study it was found by
immunohistochemistry, in the surface epithelium of bronchiole and type
II alveolar surface cells (Fig. 3, C and D). It
was reported (10) that these cells drive the absorption of
body fluid within the alveolae using osmotic pressure created by ion
transport in which Na+-K+-ATPase in the
basolateral membrane and the amiloride-sensitive epithelial
Na+ channel in the apical membrane are involved.
|
Urinary system.
In the kidney, staining for parchorin was strongly positive in the
outer part of the medulla, and dense positive dots were also visible in
the cortex. Some weak staining was present near the calyx in the inner
medulla (Fig. 4A). Under
higher magnification, it was found that some of the tubular cells,
including the macula densa, was parchorin positive (Fig. 4,
B and C). Within the medulla, some of the
papillary ductal cells near the calyx and the epithelium of the calyx
were parchorin positive (Fig. 4D).
|
|
Genital organs.
In the prostate, glandular epithelial cells of the urethra were
positively stained with anti-parchorin antibody (Fig.
6, A and B). Also,
in the testis, the epithelial cells of rete testis were strongly
positive (Fig. 6, C and D).
|
Sensory organs.
In the eyeball, both pigment and nonpigment cell layers of the ciliary
body were positive for parchorin (Fig. 7,
A and B). In the retina, pigment epithelium,
which is known to deliver water to the side of the choreal membrane,
was exclusively positive (Fig. 7, A and C),
whereas no positive staining was noted in the visual nerves or
photoreceptor cell layer.
|
|
Correlation between levels of parchorin and secretory capacity.
In general, the acid-secreting capacity of the mammalian stomach is
scant after birth until weaning, and thereafter it drastically increases. It has been shown (4) that
H+-K+-ATPase activity parallels this pattern of
increased acid secretion in the rabbit stomach. To examine whether
developmental changes in acid secretory capacity are parallel to
parchorin expression, we quantified parchorin in the 800 g
supernatant of mucosal homogenate obtained from rabbit fetus (3 wk of
gestation), suckling rabbits (1 wk after birth), and adult rabbits by
Western blotting. As shown in Fig.
9A, the content of parchorin
increased with development. In contrast, no difference was observed
between suckling and adult in the submandibular glands, reflecting the
fact that salivary secretion does not increase after weaning
(17).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The CLIC family proteins have a highly conserved region in the
COOH terminus (ca. 230 amino acids), which is considered to be involved
in Cl conductance (32). At present, more
than seven members of the CLIC family have been identified. Among them,
CLIC-1 and CLIC-3 have short NH2-terminal regions with this
consensus sequence. CLIC-1 (NCC27) was first identified
(31) as a nuclear Cl
channel but then was
found in extranuclear vesicular structures in the cells, with
relatively widespread distribution. CLIC-3 was identified
(23) as a mitogen-activated protein kinase-associated intracellular Cl
channel, and it was found in the heart
and lung with highest levels in the placenta. Of the CLIC family
proteins with longer NH2-terminal regions, CLIC-4 (p64H1)
was found to be relatively widespread in distribution and to exist in
the intracellular vesicular structure (6). CLIC-5 was
originally purified from placental microvilli as a protein that bound
to actin and ezrin, and its expression was reported (2) to
be high in striated muscle. However, recent work (Shanks RA, Berryman
M, Edwards JC, Navarre J, Urushidani T, and Goldenning JR, unpublished
observations) revealed that the highest level of this molecule (now
termed as CLIC-5A) was in the lung. The first discovered
member, p64, is widely distributed to various tissues and localized
mainly in intracellular vesicles (16). There has been no
success in discerning any common physiological role for the CLIC family
of proteins based on their tissue distribution. Recently, a
putative human homolog of bovine p64 was identified (Shanks et al.,
unpublished observations) as a splicing variant of CLIC-5 and
designated as CLIC-5B. This protein is exclusively present in
the Golgi apparatus of intestinal mucosal cells, suggesting it has a
specialized role in intestinal function. The intestinal mucosa, which
is known to transport a huge amount of water, lacks parchorin,
indicating that CLIC-5B may play the role instead of parchorin.
Among the members of the CLIC family, parchorin has a characteristic
feature. This protein has an exceptionally long, hydrophilic, and
acidic NH2-terminal sequence and a peculiar quality in that it translocates from the cytosol to the plasma membrane in association with stimulation. Recent work (32) demonstrated that
CLIC-1, which only has a little of this NH2-terminal
sequence, worked as a Cl channel alone when it was
expressed in Escherichia coli and reconstituted in an
artificial membrane. This means that the consensus sequence itself has
the ability to form a Cl
channel. This argues against the
hypothesis that CLIC family proteins work together to activate a
Cl
channel. However, parchorin is definitely a soluble
protein because of its highly hydrophilic nature. Further investigation
is necessary to elucidate the molecular mechanism by which parchorin
itself forms an ion channel within the membrane, if indeed this is
really possible. It might be more likely that the protein binds either directly or via other proteins to the channel proteins. In any event,
the cellular distribution of parchorin revealed in the present study
strongly suggests its specialized physiological role in ion transport
as related to water movement.
In the present study, we elucidated that the epitope of anti-parchorin monoclonal antibody exists in the sequence containing the parchorin-specific GGSVDA repeat in the middle of the NH2-terminal region. This ensures specific recognition of parchorin to distinguish it from the other CLIC members present in the tissues. In previous reports (21, 33), parchorin was not detectable in the lung and pancreas by Western blotting. In the present study, however, the expression of parchorin was high but quite restricted to the epithelial cells of the bronchiole and type II alveolar cells in the lung and to the epithelial cells of the pancreatic duct. Consequently, the content of parchorin in their total homogenate is lower than the detection limit by Western blotting. Care should be taken in discussing the physiological role of proteins based on their tissue distribution according to Western or Northern blot analysis. To our knowledge, the present work is the first report for the cell-specific expression of the CLIC members by histochemistry of normal tissues. It would be worthwhile to perform the histochemical analysis of other CLIC family members to clarify their physiological roles.
Parchorin was found in the apical membrane of epithelial cells in various organs, including the gallbladder, pancreas, airway, and prostate, and it also exists mainly in the cytosol of cells such as type II alveolar cells and the retinal pigment epithelium in the eye. The observation (21, 33) that parchorin translocates from the cytosol to the membrane in association with stimulation in parietal or transfected LLC-PK1 cells might reflect the degree of activation of water movement in each tissue. In the present study, the rabbits were simply killed under anesthesia without any pretreatments, so the physiological state of the tissues might have varied. We postulate that parchorin appears in the apical membrane in tissue where water transport is constitutively or accidentally activated. To obtain conclusive results, detailed examination by electron microscopy in which the stimulatory level of the tissues in vivo would be completely controlled is necessary.
Currently, CFTR is considered to be one of the most important
Cl channels in the apical membrane of water-transporting
epithelium (1). Although the tissue distribution of CFTR
has something in common with that of parchorin, that is, in the airway
epithelium, salivary gland, pancreas, and gallbladder (9),
there were obvious differences. The heart, one of the main organs for
CFTR, completely lacked parchorin. Comparing cellular distribution,
CFTR was found in both acinar and ductal cells in the submandibular
glands and pancreas (9), whereas the expression of
parchorin was restricted to ductal cells in both tissues. CFTR is known
to be present at high levels in the base of the submandibular gland but
only slightly present in the surface epithelium of the airway
(22), whereas parchorin was exclusively present in the
mucous epithelial cells. Therefore, it is suggested that
parchorin plays a different role than CFTR in Cl
transport, whether its function is actually as channel or activator. Parchorin was found to be present in ductal cells in all the exocrine glands tested (submandibular glands, parotid glands, lacrymal glands,
pancreas, prostate, testis, and mammary glands), suggesting that it
plays a role in the transport system specific for ducts. It is evident
that parchorin is a good marker for ducts.
In the kidney, parchorin was found mainly in the thick ascending limb
of Henle's loop and the distal convoluted tubule. Various ion
transporters and channels working in the kidney comprise the complex
system for urine concentration. NKCC was reported (11) as
a Cl transporter in the apical side of the thick
ascending limb of Henle's loop, whereas the
Na+-Cl
cotransporter acts in the distal
convoluted tubule. It was also reported (24) that the
distal convoluted tubule contained CFTR. Further study is required to
find out how parchorin cooperates with these proteins. It could be
strongly suggested at this stage that parchorin involves physiological
Cl
absorption, since the corresponding regions have a
very high level of Cl
absorption in the kidney.
The inner ear has a highly controlled water-movement system to ensure
hearing and body balance. NKCC1 is well known as a Cl
transporter expressed in the stria vascularis (5, 25). It has also been reported (12, 25) that ClC-1, ClC-2, ClC-3, and ClC-K1 were present in outer hair cells. Parchorin was highly expressed in epithelial cells near the organ of Corti in the cochlea. This strongly suggests that parchorin is involved in the transport of
endolymph fluid. On the basis of the study (5) that
NKCC1-deficient mice exhibited diseases due to impediments in the inner
ear, it can be inferred that ion transport in the inner ear must be
quite important in maintaining ear function. The inner ear should be an
interesting area of study in future investigations of parchorin.
Recently (3), the ClC2-deficient mouse was reported.
Although a diverse phenotype had been expected from the wide
distribution of this channel, tissue-specific diseases emerged: male
infertility and blindness, because of the loss of cells maintaining the
blood-testis barrier and the blood-retina barrier, respectively. These
results suggest the pathophysiological significance of Cl
transport in Sertoli's cells and retinal pigment epithelium. Considering the finding that parchorin exists in both the epithelium of
the testis and the retinal pigment epithelium, the male genital organs
and the eye should also be interesting areas for future study.
To elucidate the physiological significance of parchorin in secretion, we studied its level of expression in organs in which secretory capacity drastically changes. We chose the gastric mucosa and mammary gland for this purpose. In the gastric mucosa, the content of parchorin increased after weaning. This was in contrast to an unchanged amount of parchorin in the submandibular gland before and after weaning, reflecting the fact that salivary secretion does not change with weaning. In the mammary gland, parchorin content was increased in the lactating period compared with the pregnant period. These results were consistent with our idea that the amount of parchorin correlated with the secretory capacity of the exocrine glands. It would be important for future study to know whether expression of parchorin is essential for an increase of secretory capacity.
In summary, we showed that parchorin is specifically expressed in cells
that are thought to be involved in ion transport for various organs
participating in water movement. Along with the observation that its
expression was proportional to physiological function, we suggest that
parchorin plays an indispensable role in ion transport, possibly in
Cl transport, essential for water movement. The molecular
mechanism by which parchorin participates in ion transport, possibly in Cl
channel activity, requires further study to be
understood. Future study should also probe the physiological status of
the organs expressing parchorin and include the development of a
gene-targeting animal.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. H. Jennings for manuscript editing.
![]() |
FOOTNOTES |
---|
This study was supported in part by the Japanese Ministry of Education, Science, Sports, and Culture Grants 13470511 and 13557220.
Address for reprint requests and other correspondence: T. Urushidani, Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, The Univ. of Tokyo, Tokyo 113-0033, Japan (E-mail: urushi{at}mol.f.u-tokyo.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00239.2001
Received 29 May 2001; accepted in final form 19 November 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Begenisich, T,
and
Melvin JE.
Regulation of chloride channels in secretory epithelia.
J Membr Biol
163:
77-85,
1998[ISI][Medline].
2.
Berryman, M,
and
Bretscher A.
Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli.
Mol Biol Cell
11:
1509-1521,
2000
3.
Bösl, MR,
Stein V,
Hübner C,
Zdebik AA,
Jordt SE,
Mukhopadhyay AK,
Davidoff MS,
Holstein AF,
and
Jentsch TJ.
Male germ cells and photoreceptors, both dependent on close cell-cell interactions, degenerate upon ClC-2 Cl channel disruption.
EMBO J
20:
1289-1299,
2001
4.
Crothers, JM,
Reenstra WW,
and
Forte JG.
Ontogeny of gastric H+-K+-ATPase in suckling rabbits.
Am J Physiol Gastrointest Liver Physiol
259:
G913-G921,
1990
5.
Delpire, E,
Lu J,
England R,
Dull C,
and
Thorne T.
Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter.
Nat Genet
22:
192-195,
1999[ISI][Medline].
6.
Edwards, JC.
A novel p64-related Cl channel: subcellular distribution and nephron segment-specific expression.
Am J Physiol Renal Physiol
276:
F398-F408,
1999
7.
Evans, RL,
Park K,
Turner RJ,
Watson GE,
Nguyen HV,
Dennett MR,
Hand AR,
Flagella M,
Shull GE,
and
Melvin JE.
Severe impairment of salivation in Na+/K+/2Cl cotransporter (NKCC1)-deficient mice.
J Biol Chem
275:
26720-26726,
2000
8.
Goto, K,
and
Ishikawa H.
Differential distribution of actin and cytokeratin in isolated full-length rabbit renal tubules.
Cell Struct Funct
23:
73-84,
1998[ISI][Medline].
9.
Grubb, BR,
and
Boucher RC.
Pathophysiology of gene-targeted mouse models for cystic fibrosis.
Physiol Rev
79 Suppl:
S193-S214,
1999[Medline].
10.
Hamann, S,
Zeuthen T,
Cour ML,
Nagelhus EA,
Ottersen OP,
Agre P,
and
Nielsen S.
Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye.
Am J Physiol Cell Physiol
274:
C1332-C1345,
1998
11.
Hebert, SC.
Roles of Na-K-2Cl and Na-Cl cotransporters and ROMK potassium channels in urinary concentrating mechanism.
Am J Physiol Renal Physiol
275:
F325-F327,
1998
12.
Kawasaki, E,
Hattori N,
Miyamoto E,
Yamashita T,
and
Inagaki C.
Single-cell RT-PCR demonstrates expression of voltage-dependent chloride channels (ClC-1, ClC-2 and ClC-3) in outer hair cells of rat cochlea.
Brain Res
838:
166-170,
1999[ISI][Medline].
13.
Kawasaki, E,
Hattori N,
Miyamoto E,
Yamashita T,
and
Inagaki C.
mRNA expression of kidney-specific ClC-K1 chloride channel in single-cell transcription-polymerase chain reaction analysis of outer hair cells.
Neurosci Lett
290:
76-78,
2000[ISI][Medline].
14.
Kimura, M,
Suzuki T,
and
Hishida A.
A rat model of progressive chronic renal failure produced by microembolism.
Am J Pathol
155:
1371-1380,
1999
15.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
16.
Landry, D,
Sullivan S,
Nicolaides M,
Redhead C,
Edelman A,
Field M,
al-Awqati Q,
and
Edwards J.
Molecular cloning and characterization of p64, a chloride channel protein from kidney microsomes.
J Biol Chem
268:
14948-14955,
1993
17.
Leeson, CR,
and
Forman DE.
Postnatal development and differentiation of the secretory elements of the rabbit parotid and submandibular glands.
Anat Anz
149:
210-225,
1981[ISI][Medline].
18.
Levesque, PC,
Hart PJ,
Hume JR,
Kenyon JL,
and
Horowitz B.
Expression of cystic fibrosis transmembrane regulator Cl channels in heart.
Circ Res
71:
1002-1007,
1992[Abstract].
19.
Matsumura, Y,
Uchida S,
Kondo y Miyazaki H,
Ko SBH,
Hayama A,
Morimoto T,
Liu W,
Arisawa M,
Sasaki S,
and
Marumo F.
Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel.
Nat Genet
21:
95-98,
1999[ISI][Medline].
20.
Matthay, MA,
Flori HR,
Conner ER,
and
Ware LB.
Alveolar epithelial fluid transport: basic mechanism and clinical relevance.
Proc Assoc Am Physicians
110:
496-505,
1998[ISI][Medline].
21.
Nishizawa, T,
Nagao T,
Iwatsubo T,
Forte JG,
and
Urushidani T.
Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells.
J Biol Chem
275:
11164-11173,
2000
22.
Pilewski, JM,
and
Frizzell RA.
Role of CFTR in airway disease.
Physiol Rev
79 Suppl:
S215-S255,
1999[Medline].
23.
Qian, Z,
Okuhara D,
Abe MK,
and
Rosner MR.
Molecular cloning and characterization of a mitogen-activated protein kinase-associated intracellular chloride channel.
J Biol Chem
274:
1621-1627,
1999
24.
Rubera, I,
Tauc M,
Verheecke-Mauze C,
Bidet M,
Poujeol C,
Touret N,
Cuiller B,
and
Poujeol P.
Regulation of cAMP-dependent chloride channels in DC1 immortalized rabbit distal tubule cells in culture.
Am J Physiol Renal Physiol
276:
F104-F121,
1999
25.
Sakaguchi, N,
Crouch JJ,
Lytle C,
and
Schulte BA.
Na-K-Cl cotransporter expression in the developing and senescent gerbil cochlea.
Hear Res
118:
114-122,
1998[ISI][Medline].
26.
Speake, T,
Whitwell C,
Kajita H,
Majid A,
and
Brown PD.
Mechanisms of CSF secretion by the choroid plexus.
Microsc Res Tech
52:
49-59,
2001[ISI][Medline].
27.
Spicer, SS,
and
Schulte BA.
The fine structure of spiral ligament cells relates to ion return to the stria and varies with place-frequency.
Hear Res
100:
80-100,
1996[ISI][Medline].
28.
Spicer, SS,
and
Schulte BA.
Evidence for a medial K+ recycling pathway from inner hair cells.
Hear Res
118:
1-12,
1998[ISI][Medline].
29.
Spicer, SS,
Thomopoulos GN,
and
Schulte BA.
Structural evidence for ion transport and tectorial membrane maintenance in the gerbil limbus.
Hear Res
143:
147-161,
2000[ISI][Medline].
30.
Spring, KR.
Routes and mechanism of fluid transport by epithelia.
Annu Rev Physiol
60:
105-119,
1998[ISI][Medline].
31.
Tulk, BM,
and
Edwards JC.
NCC27, a homolog of intracellular Cl channel p64, is expressed in brush border of renal proximal tubule.
Am J Physiol Renal Physiol
274:
F1140-F1149,
1998
32.
Tulk, BM,
Schlesingers PH,
Kapadia SA,
and
Edwards JC.
CLIC-1 functions as a chloride channel when expressed and purified from bacteria.
J Biol Chem
275:
26986-26993,
2000
33.
Urushidani, T,
Chow D,
and
Forte JG.
Redistribution of a 120 kDa phosphoprotein in the parietal cell associated with stimulation.
J Membr Biol
168:
209-220,
1999[ISI][Medline].
34.
Urushidani, T,
and
Forte JG.
Signal transduction and activation of acid secretion in the parietal cell.
J Membr Biol
159:
99-111,
1997[ISI][Medline].
35.
van Os, CH,
and
Deen PM.
Aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus.
Proc Assoc Am Physicians
110:
395-400,
1998[ISI][Medline].
36.
Verkman, AS,
Yang B,
Song Y,
Manley GT,
and
Ma T.
Role of water channels in fluid transport studied by phenotype analysis of aquaporin knockout mice.
Exp Physiol
85 Suppl:
233S-241S,
2000[Abstract].