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1 Department of Physiology, The mammalian
urinary bladder exhibits transepithelial
Na+ absorption that contributes to
Na+ gradients established by the
kidney. Electrophysiological studies have demonstrated that
electrogenic Na+ absorption across
the urinary bladder is mediated in part by amiloride-sensitive
Na+ channels situated within the
apical membrane of the bladder epithelium. We have used a combination
of in situ hybridization, Northern blot analysis, and
immunocytochemistry to examine whether the recently cloned epithelial
Na+ channel (ENaC) is expressed in
the rat urinary bladder. In situ hybridization and Northern blot
analyses indicate that
amiloride-sensitive sodium channel; sodium transport
THE MAMMALIAN urinary bladder functions as a transient
reservoir for water and various solutes filtered and excreted by the kidneys. The epithelium of the urinary bladder serves as a barrier to
retain these solutes and water. In addition, the bladder epithelium can
modify the urinary contents by reabsorbing or secreting specific solutes. The urinary bladder epithelium has been shown to undergo dramatic changes in architecture in response to changes in bladder volume. In the contracted bladder, cells lining the bladder lumen are
typically goblet shaped and are characterized by a folded apical
membrane and a prominent population of subapical vesicles (22, 25, 34).
During filling, the bladder epithelium accommodates the volume increase
by stretching the cells so that the luminal cells become more cuboidal
in shape with a smooth apical membrane and by the insertion of
subapical vesicles into the apical membrane (25, 34). Ultrastructural
analysis has demonstrated that, during stretching, the number of
subapical vesicles decreases with an increase in the luminal membrane
(34). Bladder stretching results in an ~70% increase in apical
surface area because of the addition of new membrane from this
subapical pool, as measured by an increase in membrane capacitance
(25).
Electrophysiological studies have demonstrated that epithelial
Na+ channels are present in the
apical plasma membrane of the mammalian urinary bladder, where they
function in transepithelial Na+
reabsorption. These Na+-selective
channels mediate the movement of
Na+ out of the urinary space (2,
17, 39, 42) and are selectively inhibited by submicromolar
concentrations of the diuretic amiloride (21). Both the hormone
aldosterone and stretch of the bladder in response to changes in
urinary volume have been shown to increase the number of functional
Na+ channels in the apical
membrane (13, 25, 26). Maximal Na+
transport rates across rabbit urinary bladder have been estimated to be
as high as ~80 µmol/h (27), suggesting that
Na+ transport across the urinary
bladder may contribute to reducing Na+ concentration in the final
urine in Na+-retaining states.
The epithelial Na+ channel (ENaC)
recently has been cloned from colon, kidney, and lung and consists of
three homologous subunits ( Tissue preparation.
Normal Sprague-Dawley rats were kept on a standard laboratory diet and
had free access to fresh water. For in situ hybridization and Northern
blot analysis, animals were euthanized by deep anesthesia with 100% CO2. Urinary bladders
were punctured with a hypodermic needle, and the urine was removed to
collapse the bladder. Urinary bladders were subsequently removed, cut
into small pieces, and placed in ice-cold phosphate buffered saline
(PBS). For immunocytochemistry, animals were killed, and bladders were
rapidly excised. Bladders were subsequently rinsed in ice-cold PBS, cut
into small pieces, and placed in ice-cold PBS before embedding for
frozen sectioning.
Antibodies.
An anti- In situ hybridization.
Small pieces of urinary bladder were fixed in 3% paraformaldehyde
prepared in diethyl pyrocarbonate-treated PBS containing 5% sucrose.
Samples were subsequently cryoprotected by infiltration in PBS
containing increasing concentrations of sucrose, with the final sucrose
concentration being 20%, placed in gelatin capsules (Polysciences,
Warrington, PA), filled with Tissue-Tek OCT compound (Miles, Elkhart,
IN), and snap frozen in liquid nitrogen. Samples were stored
at
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-, 
-, and 
-rat ENaC
(rENaC) are expressed in rat urinary bladder epithelial cells. Quantitation of the levels of -, -, and -rENaC mRNA expression in rat urinary bladder, relative to -actin mRNA
expression, indicates that, although comparable levels of - and
-rENaC subunits are expressed in the urinary bladder of rats
maintained on standard chow, the level of -rENaC mRNA expression is
5- to 10-fold lower than - or -rENaC mRNA. Immunocytochemistry,
using an antibody directed against -rENaC, revealed that ENaCs are
predominantly localized to the luminal membrane of the bladder
epithelium. Together, these data demonstrate that ENaC is expressed in
the mammalian urinary bladder and suggest that amiloride-sensitive
Na+ transport across the apical
membrane of the mammalian urinary bladder epithelium is mediated
primarily by ENaC.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-ENaC, -ENaC, and -ENaC) (2, 6).
Significant amino acid sequence similarities across species suggest
that ENaCs belong to a common gene family and are structurally related
to putative mechanosensitive ion channels found in the nematode
Caenorhabditis elegans (9), a
Phe-Met-Arg-Phe-NH2-activated
Na+ channel cloned from the snail
Helix
aspersa (29), and amiloride-sensitive Na+ channels expressed in brain
(16, 37, 45). To date, limited information is available regarding the
biochemical and molecular characteristics of ENaCs expressed in the
mammalian urinary bladder. In this report, we demonstrate by a
combination of Northern blot analysis, in situ hybridization, and
immunocytochemistry that ENaCs are expressed in the epithelium of the
mammalian urinary bladder.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-rENaC antibody was generated by Lofstrand Laboratories
(Gaithersburg, MD), using a synthetic peptide corresponding to residues
44-57 (GLGKGDKREEQGLG) within the
NH2-terminal intracellular domain
of the -subunit of rENaC (5). The peptide was conjugated to bovine
serum albumin through a COOH-terminal cysteine residue before being
used as an immunogen. Antibodies were purified, using a protein
A-agarose column (Pierce, Rockford, IL), following the manufacturer's
protocol. An
anti-Na+-K+-adenosinetriphosphatase
(anti-Na+-K+-ATPase)
antiserum was a generous gift of Dr. Stephen Ernst (Univ. of Michigan)
and has previously been described (10, 41).
80°C until use. Five-micrometer-thick sections were
sectioned and collected on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA).
-, -, and -ENaC
subunits (390 bp, 207 bp, and 244 bp, respectively) were utilized (3).
Each cDNA was linearized, and 2 µg were used as a template for the
synthesis of antisense or sense complementary RNAs (cRNAs) [4
µl transcription buffer (Promega, Madison, WI), 0.5 µl 100 mM
dithiothreitol, 8 µl of a nucleotide triphosphate mix
(including digoxigenin-UTP), 1 µl ribosomal RNasin (40 units/µl), and the appropriate RNA polymerase (20 units) in a total volume of 20 µl at 37°C for 2-3 h]. The length and amount of each
cRNA were verified, after phenol-chloroform extraction and ethanol precipitation, by ethidium bromide staining in a formaldehyde-agarose gel. Digoxigenin-labeled cRNAs were detected in situ with the use of an
anti-digoxigenin antibody conjugated to alkaline phosphatase (8).
Briefly, each cRNA probe [~2 ng/µl, in a hybridization solution containing 1× SSC (0.15 M NaCl and 0.015 M sodium
citrate, pH 7.0), 5× Denhardt's solution (0.1%
polyvinylpyrrolidone, 0.1% Ficoll, and 0.1% bovine serum albumin),
salmon DNA (500 µg/ml), yeast tRNA (250 µg/ml), and 50%
formamide] was allowed to hybridize overnight at
50°C to tissue sections, followed by washing twice with 2×
SSC at room temperature for 5 min and then once with STE at room
temperature for 5 min. Sections were subsequently treated with
ribonuclease A at 37°C for 30 min to remove unhybridized probe and
washed at high stringency with 2× SSC, 50% formamide at 50°C
for 5 min, 1× SSC at room temperature for 5 min, and 0.5× SSC at room temperature for 5 min. Sections were then blocked with 2%
goat serum for 30 min, and bound digoxigenin was detected by incubation
with alkaline phosphatase-conjugated anti-digoxigenin antibodies
(Boehringer Mannheim, Indianapolis, IN) at room temperature for 1 h,
followed by color development using nitro blue
tetrazolium-5-bromo-4-chloro-3-indolyl phosphate
p-toluidine salt as the substrate.
Sense cRNAs were used to evaluate the specificity of probe
hybridization.
Northern blot analysis. Total RNA was isolated from rat urinary bladder, using the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (7), and size fractionated in a 1.2% agarose-formaldehyde gel. The RNA was transferred onto a nylon membrane and probed with 32P-random prime labeled ~1.5-kb cDNA inserts of each ENaC subunit (15). The blots were washed at a high stringency (final wash, 0.1× SSC at 65°C) and apposed to a phosphor screen for imaging (Molecular Dynamics, Sunnyvale, CA).
Immunofluorescence microscopy.
Urinary bladder samples were placed unfixed in gelatin capsules
containing tissue-freezing compound (Triangle Biomedical Services, Durham, NC), frozen by submersion in liquid nitrogen, and stored at
80°C until use. Five-micrometer-thick sections were cut,
using an IEC Minotome cytotome, and collected on either Superfrost Plus or gelatin-subbed glass slides. Before processing for
immunofluorescence microscopy, sections were fixed for 1 min in
ice-cold methanol and then allowed to air dry. Sections were rehydrated
in PBS and then incubated for 1 h in PBS containing 10% normal goat
serum (NGS) (Sigma, St. Louis, MO). Sections were incubated overnight at 4°C in either rabbit polyclonal anti--rENaC antibody (1:200 dilution; 6.5 µg/ml final concentration) or rabbit polyclonal anti-Na+-K+-ATPase
antiserum (1:500 dilution) in PBS containing 10% NGS. After incubation
in primary antibodies, sections were sequentially washed in PBS (twice
for 5 min), phosphate-buffered NaCl containing 1% bovine serum albumin
(three times for 5 min; twice for 30 min), and PBS (twice for 5 min).
Sections were then incubated in a 1:200 dilution of Texas
red-conjugated goat anti-rabbit immunoglobulin G (Cappel/Organon
Teknika, Durham, NC) for 1 h at room temperature. Control consisted of
preincubation of primary antibody with excess free peptide (15 µg/ml
final concentration). After six washes in PBS, sections were mounted in
glycerol-PBS (9:1 vol/vol) containing 0.1% phenylenediamine (Sigma),
covered with glass coverslips, and sealed with nail polish. Sections
were photographed on a Nikon Optiphot UD microscope equipped for
epifluorescence and differential interference microscopy.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Expression of ENaC subunit mRNAs in rat urinary
bladder epithelium.
The expression of ENaC subunit mRNA in the rat urinary bladder (Fig.
1) derived from animals maintained on a
standard feed was examined by Northern blot analysis. Radiolabeled
-, -, and -ENaC probes hybridized to mRNAs of ~3.7, 2.6, and
3.2 kb in size, respectively, in agreement with the reported size in
rENaC mRNAs in colon and kidney (Fig. 1) (6). In addition, slot blot Northern analyses were performed to examine the abundance of -, -, and -rENaC mRNAs in rat urinary bladder, relative
to -actin expression (Table
1). Similar levels of expression of -
and -rENaC mRNAs were observed. Interestingly, levels
of expression of -rENaC mRNA were ~5- to 10-fold lower than -
and -rENaC mRNAs.
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|
- and -rENaC mRNAs were detected in the urinary
bladder epithelia of animals maintained on standard rat chow (Fig.
2, A and
C). No significant signal was
obtained in parallel sections hybridized with control sense cRNA
probes, demonstrating the specificity of the hybridization with the
ENaC probes (Fig. 2, B and
D). A weak specific signal for
-rENaC mRNA was also detected in rat urinary bladder epithelia (Fig.
2, E and
F).
|
Immunolocalization of -ENaC in the
urinary bladder.
To further demonstrate expression of ENaC in the urinary bladder, the
distribution of -rENaC was examined by indirect immunofluorescence microscopy (Fig. 3), using an
antibody generated against a peptide corresponding to amino acids
44-57 within the NH2 terminus
of -rENaC. Immunostaining for -rENaC was predominantly localized to the apical membranes of cells lining the bladder lumen (Fig. 3). In addition, a diffuse subapical
intracellular staining was observed (Fig. 3). Immunostaining was
specific, as preincubation of the antibody with excess free immunogenic
peptide blocked antibody binding to the epithelial cells (Fig. 3).
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Immunolocalization of
Na+-K+-ATPase
in the rat urinary bladder.
Na+-K+-ATPase
is expressed in the basolateral plasma membrane of most epithelial
cells (18, 20). An
anti-Na+-K+-ATPase
-subunit antibody was used to localize this transporter in the rat
urinary bladder to provide a better definition of the architecture of
this epithelium and to help delineate the basolateral plasma membrane
of the urinary bladder epithelium. As shown in Fig. 4,
Na+-K+-ATPase
localized to the basolateral membrane of the bladder epithelial cells.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Several ENaCs have recently been described. These include a family of
structurally related two-membrane-spanning-domain polypeptides (-,
-, and -ENaC), which assemble into an oligomeric
Na+ channel. A guanosine
3',5'-cyclic monophosphate-regulated amiloride-sensitive cation channel is expressed in renal and airway epithelia and is
structurally related to a cation channel expressed in rod outer segment
(19). An apical membrane protein cloned from a
Xenopus renal epithelial cell line,
termed Apx, is an amiloride-sensitive cation channel (36) or a cation
channel regulator (43). Although previous electrophysiological studies
have indicated that amiloride-sensitive Na+-selective ion channels (13,
24, 26-28, 35) are functionally expressed in the apical plasma
membrane of the urinary bladder epithelium, their molecular identity
was unknown.
We provide data in this paper that the recently cloned ENaCs are
expressed in the rat urinary bladder. In situ hybridization and
Northern blot analyses indicate that -, -, and -rENaC are expressed in the rat urinary bladder. Quantitation of the levels of
-, -, and -rENaC mRNA expression in the rat urinary bladder, relative to -actin mRNA expression, suggests that, although
comparable levels of - and -rENaC subunits are expressed in the
urinary bladder of rats maintained on standard chow, the level of
-rENaC mRNA expression is 5- to 10-fold lower than either - or
-rENaC. This resembles the pattern of ENaC mRNA expression in human
airway epithelia (4) and the M-1 cortical collecting duct cell line (23), where -ENaC subunit mRNA expression is significantly lower
than the expression of - and -ENaC mRNAs. Differential expression
of the three ENaC subunits has been observed in several tissues,
including lung, liver, skin, and colon (1, 4, 14, 30, 32, 33, 38, 40).
In light of ENaC being viewed as a heteromeric channel being composed
of three subunits (6), the functional consequences of different levels
of ENaC subunit mRNA expression within the same tissue are presently
not understood.
Amiloride-sensitive Na+ transport
across the mammalian urinary bladder is stimulated by the
mineralocorticoid hormone aldosterone or by feeding animals an
Na+-restricted diet (13, 27).
Several candidate mechanisms for the regulation of
Na+ channel by aldosterone have
been described, and regulatory mechanisms may differ among different
tissues or cell types. These mechanisms include posttranslation
modification of a channel subunit or an associated regulatory protein
(i.e., G proteins or membrane cytoskeleton) and increases in ENaC mRNA
and protein expression (reviewed in Refs. 2 and 31). With respect to
the latter, aldosterone (or low dietary
Na+) increases the level of
expression of - and -ENaC mRNA in the rat colon (1, 30). However,
in kidney and primary cultures of cortical collecting duct and of inner
medullary collecting duct cells, responses to aldosterone (or low
dietary Na+) have included no
change in ENaC subunit mRNA expression (38) or a small increase in
-ENaC mRNA (1, 44) or in -ENaC mRNA expression (11). In light of
the foregoing discussion, our observations of low levels of -ENaC
mRNA expression in the urinary bladders from animals fed a standard
diet raise the possibility that the aldosterone-induced increase in
Na+ transport across the urinary
bladder may be, at least in part, mediated by increases in -ENaC
mRNA and protein levels.
To further corroborate expression of ENaC in the urinary bladder, we
examined the distribution of the -subunit of ENaC by immunofluorescence microscopy. -ENaC was localized primarily to the
apical membranes of the cells lining the lumen of the rat urinary
bladder. This localization of ENaC to the apical membrane domain is in
agreement with recent immunocytochemical studies demonstrating that
ENaCs are localized to the apical membrane in the kidney, distal colon,
lung, sweat ducts, and parotid salivary glands (12, 38). In addition, a
diffuse subapical cytoplasmic staining of the luminal cells was
observed, suggesting that ENaC may also be localized to a population of
intracellular vesicles. Lewis and de Moura (25, 26) have demonstrated
that during urinary bladder distension, there is a change in membrane
capacitance, presumably resulting from an increase in apical membrane
area induced by vesicle fusion. In addition, they have shown that there is a greater than 10-fold increase in the density of
amiloride-blockable Na+ channels
within the apical membrane of the bladder in response to stretch. Based
on these data, Lewis and de Moura (25, 26) postulated that during
urinary bladder distension, there is a fusion of
Na+ channel-containing cytoplasmic
vesicles to apical membrane.
In summary, we have demonstrated that -, -, and -ENaC subunits
are expressed in the rat urinary bladder. Expression of ENaC in the
urinary bladder suggests that the ENaC gene family represents the
amiloride-sensitive Na+
conductance previously characterized in the mammalian urinary bladder
by electrophysiological methods (13, 24, 26-28, 35).
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51391, DK-07006, and DK-46705 and the Department of Veterans Affairs. B. J. Harte was a recipient of a Medical Student Research Award from the American Heart Association. Y. J. Ahn was the recipient of a Postdoctoral Fellowship Award from the American Heart Association, Southeastern Pennsylvania Affiliate. This work was performed during the tenure of an Established Investigatorship Award from the American Heart Association (T. R. Kleyman).
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FOOTNOTES |
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Address for reprint requests: T. R. Kleyman, Medical Research (151), Veterans Affairs Medical Center, University and Woodland Ave., Philadelphia, PA 19104.
Received 20 May 1997; accepted in final form 11 September 1997.
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Abstract
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