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INTRODUCTION |
NKCC1,1 the basolateral
or secretory isoform of the Na-K-2Cl cotransporter (1, 2), is a member
of the cation-coupled chloride transporter family (3, 4), which
includes the apical Na-K-2Cl cotransporter of the renal thick ascending
limb (5, 6), the NaCl cotransporter of the renal distal convoluted
tubule (6), and four KCl cotransporters (7-9). NKCC1 mediates the coupled electroneutral transport of 1 Na+, 1 K+, and 2 Cl
ions across the plasma membrane
(4), driven by the inwardly directed Na+ and
Cl gradients that occur under physiological conditions.
It has a broad tissue distribution (1, 2) and is expressed in both nonepithelial and epithelial cell-types. Regulation of cell volume is a
major function of NKCC1 (3), and in vascular endothelial cells, it may
play an important role in controlling cytosolic concentrations of
Cl and K+ during agonist stimulation (10). In
epithelial cells, it is restricted to basolateral membranes (11, 12),
and there is strong pharmacological evidence that it provides an entry
pathway for ions that are secreted across the apical membrane.
Studies of tissue samples of wild-type and CFTR-deficient mice have
shown that cAMP-induced Cl secretion in intestinal and
nasal epithelial cells is sharply inhibited by treatment with
bumetanide, an inhibitor of NKCC1 (13). This observation suggests that
basolateral Cl uptake via NKCC1 is important in
maintaining CFTR-mediated Cl secretion across the apical
membrane. There is recent evidence that NKCC1 might also provide a
basolateral Cl entry mechanism that is required for the
maintenance of high levels of HCl secretion by gastric parietal cells
(14), which is generally thought to be dependent on
Cl/HCO3 exchange. In the
inner ear, NKCC1 is expressed at high levels on the basolateral
membrane of marginal cells of the stria vascularis (15, 16).
K+ secretion by marginal cells is responsible for the high
concentrations of K+ in the endolymph and for the
endocochlear potential, which is eliminated by perilymphatic or
systemic application of bumetanide or furosemide (17-19). These
observations and the ototoxicity of loop diuretics (20) suggest that
NKCC1 contributes to the high rates of K+ uptake needed to
maintain K+ currents across the apical membrane of the
marginal cell and that NKCC1 plays a critical role in hearing.
Many of the conclusions regarding the functions of NKCC1 in secretory
epithelial tissues are based on studies in which loop diuretics have
been used as inhibitors. However, these compounds inhibit other members
of the cation-coupled cotransporter family, such as the apical Na-K-2Cl
cotransporter of the thick ascending limb and isoform 1 of the KCl
cotransporter (7), and the existence of additional members of this
family is a distinct possibility (21). To obtain a better understanding
of the physiological functions of NKCC1, we have prepared a mouse line
carrying a null mutation in the nkcc1 gene (locus
Slc12a2) and have performed an initial analysis of the phenotype.
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EXPERIMENTAL PROCEDURES |
Generation of Mutant Mice by ES Cell/Gene Targeting
Technology--
Nkcc1 genomic clones were isolated from a
strain 129/SvJ mouse phage library and partially characterized by
restriction endonuclease mapping, DNA sequencing, and polymerase chain
reaction analysis using primers based on mouse NKCC1 cDNA sequences
(1). The targeting vector, MJK+KO, which was described in
previous studies (22, 23), contained both the neoR
gene for positive selection of targeted ES cells and the herpes simplex
virus thymidine kinase gene for negative selection. A 2.7-kb
NotI-EcoRV fragment, beginning in intron 3 (with
the NotI site originating in the vector cloning site) and
terminating at codon 401 in exon 6, was inserted 3' to the
neoR gene in the targeting vector; a 4.2-kb
EcoRV/BamHI fragment, beginning with codon 402 and extending to near the end of intron 8, was inserted 5' to the
neoR gene. Generation and identification of targeted
ES cells, blastocyst-mediated transgenesis, and breeding of chimeric
mice and their ES cell-derived agouti offspring were carried out as
described previously (22, 23).
The probes used for Southern blot analysis, generated by polymerase
chain reaction amplification of the Nkcc1 gene sequences, were a 1.7-kb outside probe extending from the beginning of exon 9 to
the end of exon 10 and a 1.0-kb inside probe spanning the region from
exon 4 to exon 5. ES cells were first analyzed using the 1.7-kb probe,
which contained sequences from outside the region used in preparing the
targeting construct, and then cells that appeared to be correctly
targeted were confirmed by analysis with the 1.0-kb inside probe.
Genotype Analysis--
DNA from tail biopsies was analyzed by
Southern blot hybridization using the probes described above or by
polymerase chain reaction. For polymerase chain reaction genotyping,
three primers were included in the reaction mixture. Forward
(5'-GGAACATTCCATACTTATGATAGATG-3') and reverse
(5'-CTCACCTTTGCTTCCCACTCCATTCC-3') primers corresponding to sequences
from the 5'- and 3'-ends of exon 6, respectively, amplified a 105-base
pair product from the wild-type gene. For the mutant gene, a reverse
primer (5'-GACAATAGCAGGCATGCTGG-3'), complementary to sequences from
the untranslated region of the neoR gene, and the
forward primer from the 5'-end of exon 6 amplified a 156-base pair product.
Body Weight Analysis--
For growth curves of young animals,
mice of all three genotypes from six heterozygous matings were weighed
at 3, 5, 6, 7, 14, 21, and 28 days following birth. For adult animals,
7.5-week-old (5 mice of each genotype) and age-matched 10-15-week-old
(21 Nkcc1/, 14 Nkcc1+/, and 22 Nkcc1+/+) mice were weighed. Data presented in
the growth curve included Nkcc1/ mice that
died around the time of weaning; exclusion of these mice, which tended
to be slightly smaller in body weight than surviving mutants, had
little noticeable effect on the growth curve.
Histology and Morphometry--
A histological survey of tissues
from 15-17-day-old Nkcc1+/+,
Nkcc1+/ and Nkcc1/
mice (two of each genotype) was performed using tissues that were fixed
in 10% neutral buffered formalin, embedded in paraffin, and stained
with hematoxylin and eosin. Stomach, duodenum, jejunum, ileum, cecum,
and colon from 3-week-old (two of each genotype) and 8-week-old (five
of each genotype) Nkcc1+/+ and
Nkcc1/ mice were prepared for detailed light
and transmission electron microscopy as described previously (23).
Intestinal segments from 15-21-day-old Nkcc1+/+
and Nkcc1/ mice (7 of each genotype) were
either fixed in 4% paraformaldehyde, embedded in plastic, sectioned,
and stained with toluidine blue or fixed in 10% neutral buffered
formalin, embedded in paraffin, sectioned, and stained with hematoxylin
and eosin. Morphometry of the intestinal epithelium was determined by
measuring villus tip enterocytes for the height of the brush border,
height of both apical and basolateral cytoplasm (relative to the
position of the nuclei), total epithelial cell height, width of the
lamina propria, and total villus width. Intact inner ears were isolated from 4-5-week-old Nkcc1+/+ and
Nkcc1/ mice (two of each genotype) that were
perfused for 5 min with 2% glutaraldehyde/2% paraformaldehyde in 0.1 M sodium-cacodylate buffer, pH 7.3. The inner ears were
decalcified in EDTA, postfixed in buffered 1% osmium tetroxide,
dehydrated in ethanol and propylene oxide, and embedded in plastic.
Serial 1- and 2-µm sections were cut with a diamond knife and stained
with toluidine blue. Sections of ears from one
Nkcc1+/+ and 3 Nkcc1/
mice were prepared in a similar manner but were not decalcified in EDTA.
Blood Pressure Measurements--
Adult mice of all three
genotypes were anesthetized with intraperitoneal injections of inactin
(100 µg/g of body weight) and ketamine (50 µg/g of body weight),
and mean arterial pressure was determined using a femoral artery
catheter as described previously (24).
Suckling Mouse Intestinal Secretion Assay--
Escherichia
coli heat-stable enterotoxin, STa, was reconstituted to 10 µg/ml
in sterile saline, with 1% Evan's Blue dye added as a tracer. 1 µg
of STa was administered to 4-day-old suckling mice by intragastric
injection (25). Mice were allowed to suckle until toxin administration,
but not afterward. After 3 h incubation at room temperature, mice
were euthanized, and the entire intestinal tract from proximal duodenum
to distal colon was excised. The weights of the fluid-filled intestine
and remaining carcass were measured, and the gut weight to carcass
weight ratio was determined. Animals into which STa was injected
successfully were identified by their blue-stained stomachs.
Bioelectric Measurements--
Electrical potential difference
measurements were performed under short-circuit current
(ISC) recording conditions. Freshly isolated jejunum and
cecum from 12-16-week-old mice were mounted in voltage-clamped Ussing
chambers (13, 26) and bathed in standard Ringer solution for 20 min to
record basal ISC. Next, the ISC response
resulting from addition of 10 µM forskolin (bilateral) was recorded for 20 min, followed by a recording of the ISC
response after addition of 100 µM bumetanide to the
serosal side. ISC responses were also measured using
cultured tracheal epithelial cells from both genotypes. Epithelial
cells from tracheal ring explants were cultured to confluency on
permeable collagen matrix supports as described previously (27).
Tracheal epithelial monolayers were mounted in Ussing chambers and
bathed in standard Ringer solution for 20 min while basal
ISC was recorded. ISC responses following the
consecutive addition of 10 µM amiloride (luminal), 10 µM forskolin (bilateral), and 100 µM
bumetanide (serosal) were recorded for 5, 10, and 10 min, respectively,
following each addition. Forskolin-stimulated ISC responses
for intestinal segments and cell monolayers were normalized to tissue area.
pH Measurements of Stomach Contents--
Wild-type and
homozygous mutant mice were fasted for 4 h, injected
subcutaneously with histamine HCl (2 µg/g of body weight), and
euthanized 15 min later. Dissection of stomachs, transfer of stomach
contents to 2 ml of nitrogen-saturated normal saline, and measurement
of pH was carried out as described previously (23).
Northern Blot Analysis--
Total RNA was isolated from stomach,
small intestine, cecum, colon, brain, lung, heart, kidney, and skeletal
muscle of 4-week-old mice using Tri-ReagentTM (Molecular
Research Center, Inc., Cincinnati, OH). Each tissue sample was pooled
from three mice. Northern blots were prepared and hybridized as
described previously (22), using rat or mouse cDNA probes for
NKCC1, NHE3, CFTR, AE2, and glyceraldehyde-3-phosphate dehydrogenase.
Auditory Brainstem Response--
ABR measurements of
avertin-anesthetized 5-8-week-old Nkcc1+/+,
Nkcc1+/, and Nkcc1/
mice were recorded as described previously (28) using three subcutaneous electrodes, one inserted under each ear and one at the top
of the skull. The mice were positioned between high frequency transducers in a sound-attenuated chamber, and sound stimuli were directed into the ear by tubing. The ABR was computer-averaged (time-locked with onset of 128-1024 stimuli, at 20/s) out of the continuous electroencephalographic and electrocardiographic activity. The ABR peak amplitudes were averaged in the 0.5-8.0 µV range; the
electroencephalographic and electrocardiographic activity ranged to
>30 µV but averaged <0.5 µV due to their randomness with regard
to onset of the acoustic stimuli. All electroencephalographic and
electrocardiographic activity that exceeded 30 µV was rejected from
the averaging technique. The sound stimuli used were a 0.1-ms broad
band click and 3-ms pure tones of 8, 16, and 32 kHz at 20-100 db. The
threshold of hearing was defined as the minimum sound intensity, in db,
required to elicit a characteristic waveform.
Statistics--
Statistical significance was determined by
single-factor analysis of variance, analysis of variance-protected
Bonferroni's test, or unpaired t test where appropriate.
Data are presented as the mean ± S.E.
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RESULTS |
Generation of NKCC1-deficient Mice and Gross Phenotype--
To
disrupt the gene encoding NKCC1, we used a construct in which the
neoR gene was inserted into exon 6 (Fig.
1A). Targeted ES cells were identified by Southern blot analysis similar to that shown in Fig.
1B and used for blastocyst-mediated transgenesis. Chimeric mice derived from two ES cell clones transmitted the mutant gene to
their offspring, and these mice were bred to establish NKCC1-deficient mouse lines. Matings of heterozygous mice yielded live offspring of all
three genotypes (Fig. 1B). Northern blot analysis using probes corresponding to coding sequences from regions 3' (Fig. 1C) and 5' to the neoR insertion site
(data not shown) demonstrated that expression of the 6.5-kb
Nkcc1 mRNA was reduced in
Nkcc1+/ tissues and eliminated in
Nkcc1/ tissues.
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Fig. 1.
Generation of NKCC1 null mutant mice.
A, targeting strategy for disruption of gene encoding NKCC1.
Top, wild-type allele showing exons 2-10 and unique 12-kb
StuI restriction fragment; middle, targeting
construct with the neoR gene disrupting exon 6;
bottom, targeted allele and 4.5- and 9.5-kb StuI
restriction fragments identified with the inside and outside probes,
respectively. Restriction enzymes are labeled as follows: B, BamHI; E, EcoRV; P, PstI; S, StuI; N, NotI (used for linearization of construct). TK,
herpes simplex virus-thymidine kinase gene used for negative selection
of targeted ES cells. B, Southern blot analysis of tail DNA
from wild-type (+/+), heterozygous (+/ ), and mutant (/) mice. DNA
was digested with StuI and hybridized separately with the
outside and inside probes (diagrammed in A). C,
Northern blot analysis of total RNA (10 µg per lane) from tissues of
wild-type (+/+), heterozygous (+/), and homozygous mutant (/)
mice. Top, hybridization with an NKCC1 probe from the region
3' to the neoR insertion. Bottom,
hybridization with glyceraldehyde-3-phosphate dehydrogenase as a
loading control.
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Genotype analysis of 560 offspring from heterozygous matings showed
that wild-type, heterozygous, and homozygous mutant offspring were born
in approximately a 1:2:1 Mendelian ratio (30% +/+; 46% +/; 24%
/). Relative to both wild-type and heterozygous mice, Nkcc1/ mice experienced growth retardation
(Fig. 2) that was readily apparent within
1 week of birth and particularly severe during the third week of life.
At weaning on day 21, Nkcc1/ mice (5.5 ± 0.3 g) were significantly smaller than both wild-type (12.8 ± 0.3 g) and heterozygous (12.8 ± 0.2 g)
mice. Homozygous mutants gained weight well after weaning; however,
they remained smaller as adults (~80% of wild-type mice) and, in
general, had less body fat. Extrapolation of the growth patterns to
their day of birth suggested that there was little, if any, growth
retardation of Nkcc1/ mice in
utero. To further test for the possibility of in utero growth retardation, 12 offspring from a heterozygous mating were removed by Caesarian section on postcoital day 17 and weighed. No
significant difference in body weight was observed in this small sample
of mice (Nkcc1+/+ and
Nkcc1+/ mice, 0.64 ± 0.02 g,
n = 8; Nkcc1/ mice,
0.64 ± 0.01 g, n = 4).
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Fig. 2.
Nkcc1/ mice
exhibit growth retardation. A, 19-day-old female
Nkcc1+/+ (top) and
Nkcc1/ (bottom) littermates.
B, growth curves for Nkcc1+/+,
Nkcc1+/, and Nkcc1/
mice.
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Nkcc1/ mice frequently exhibited unusual
head postures, in which the head was tilted to one side or tilted
upward and backward with the nose held high. The mutants also exhibited
circling behavior accompanied by a tendency to engage in rapid
spinning, which persisted throughout the life of the animal. In some
instances, Nkcc1/ mice engaged in repetitive
circling in a vertical direction, in which they climbed the underside
of the downward sloping end of the cage top, where food was placed,
moved upside down along the underside of the cage top, dropped to the
floor and ran back to the sloping end of the cage top to continue the
process. While spinning, mutant mice sometimes lost their balance, but
quickly righted themselves. Loss of balance occurred occasionally when the mutants were walking in a straight line.
A histological survey of sections from brain, heart, lung, liver,
spleen, salivary gland, skeletal muscle, eye, and pancreas of
15-17-day-old Nkcc1+/+,
Nkcc1+/, and Nkcc1/
mice revealed no significant lesions, with the exception of a single
homozygous mutant in which cells of the choroid plexus were vacuolated
and swollen. However, the choroid plexus of another young mutant
appeared normal, and no abnormalities were observed in the choroid
plexus of two adult mutant mice that were examined. Although gross
histological abnormalities were not observed in the intestinal tract of
young mutant mice that appeared relatively healthy, abnormal intestinal
histopathology was observed among homozygous mutants that sickened or
died around the time of weaning (discussed below). Also, in enterocytes
of one of the sick homozygous mutant mice, there appeared to be very
little cytoplasm between the nucleus and the basal surface of the cell
when compared with enterocytes of an age-matched wild-type mouse. To
assess the significance of this observation, morphometry of the
intestinal epithelium of viable young (21 days of age or less) and
adult (8 weeks old) Nkcc1+/+ and
Nkcc1/ mice was performed. For the adult
mice, there were no significant differences between the two genotypes
in any of the measurements (described under "Experimental
Procedures"). For the young mice, there were significant
differences in total epithelial cell height (Nkcc1/, 21.2 ± 1.4 µm,
n = 8; Nkcc1+/+, 25.3 ± 1.2 µm, n = 11; p < 0.03) and in the
height of the basal cytoplasm (Nkcc1/,
3.59 ± 0.39 µm, n = 8;
Nkcc1+/+, 5.01 ± 0.33 µm,
n = 11; p < 0.01).
Increased Incidence of Death among Null Mutants--
Almost 30%
of Nkcc1/ mice died just before or just
after weaning at 21 days of age (Fig. 3).
The animals were active and seemed fully viable one day and then were
found dead in their cages the next day, suggesting a rapid onset of
morbidity and then death. We were able to identify and examine seven
Nkcc1/ mice between the ages of 18 and 26 days that had hunched backs, were relatively immobile, and seemed to be
near death. All of the mice showed evidence of bleeding in the
intestine, and one mouse had a fecal blockage of the large intestine.
Fig. 4 shows a 23-day-old mutant that
exhibited bleeding in the cecum and a severe blockage of the colon
(Fig. 4, A and B) and a 26-day-old mouse that
exhibited bleeding in the small intestine and cecum (Fig.
4C). The cecum of Nkcc1/ mice
often had a worm-like appearance (Fig. 4C) similar to that described for CFTR-deficient mice (29).
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Fig. 3.
Survival rate for
Nkcc1/ and
Nkcc1+/+ mice. The percentage of
survival among Nkcc1/ and
Nkcc1+/+ mice is plotted against age. After 28 days of age, Nkcc1/ mice did not exhibit an
increased incidence of death relative to
Nkcc1+/+ mice.
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Fig. 4.
Gross pathology of intestinal tract.
A and B, intestinal tract of a 23-day-old
Nkcc1/ mouse with blood in the cecum
(A) and a severe blockage of the colon (A and
B). In these and subsequent panels, the single
arrow indicates the cecum, double arrows indicate the
colon, and arrowheads indicate the small intestine.
C, small intestine, cecum, and colon of
Nkcc1/ mouse that died at 26 days of age.
Note blood in the small intestine and in the wormlike cecum.
D and E, intestinal tract from 13-month-old
Nkcc1/ mouse found dead in its cage. Note
the wormlike extension (asterisk in D) of the
cecum and severe obstructions of the cecum (D) and colon
(E) by feces. F, intestinal tract of 21-day-old
heterozygous mutant found dead in its cage; it had blood in the lumen
of its cecum, obstruction of both cecum and colon by feces,
intussusception of the small intestine (at the site marked by the
arrowhead), and a wormlike extension (asterisk)
of its cecum. It should be noted that obstructions were observed in
only 5 of 31 (16%) of the dead or dying mice.
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We also identified a 13-month-old null mutant mouse that had seemed
healthy throughout its life but was found dead in its cage one morning.
It had a worm-like extension of its cecum (Fig. 4D) and
exhibited a severe blockage of both its cecum (Fig. 4D) and
colon (Fig. 4E) by feces. A 21-day-old heterozygous mouse was found dead with blood in the lumen of its cecum, a blocked colon,
and enteric intussusception, a condition in which part of the small
intestine prolapses into the lumen of an adjacent region (Fig.
4F). Like homozygous mutants, its cecum had an extension with a coiled, worm-like appearance (see Fig. 4F). Fig.
5 shows the intestinal tract and sections
through the cecum of an 18-day-old null mutant that was identified
while still alive but in a morbid state; it died naturally just before
it was examined. Bleeding throughout the small intestine was observed,
and a large focal hemorrhage was visible in the intact cecum (Fig.
5A). Blood was within both the lumen of the cecum (Fig.
5B) and the interstitium (Fig. 5C and
D), and in some regions, there were pools of blood (Fig.
5D). There was no evidence of a significant inflammatory response. These observations raise the possibility that a defect in the
circulatory system might have contributed to the increased death rate
of null mutants.
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Fig. 5.
Histological analysis of hemorrhage in the
wall of the cecum of Nkcc1/ mouse. A, intestinal tract of 18-day-old mutant
mouse that was found in a morbid state and examined just after it had
died. Note the large focal hemorrhage in the cecum (arrow).
B, section through cecum of the same mouse showing blood
within the lumen (arrow). C and D,
higher magnification views of the regions boxed in
B with broken (C) or solid
(D) lines. Note red blood cells within the lumen
(arrowhead in C) and evidence of extensive
bleeding and pooling of blood in the interstitium (C and
D).
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Mean Arterial Blood Pressure Is Reduced in NKCC1-deficient
Mice--
NKCC1 has been found in both endothelial cells (10) and
vascular smooth muscle (30), consistent with the possibility that NKCC1
deficiency might cause a vascular defect. Blood pressures of
anesthetized mice were measured using a femoral artery catheter (Fig.
6). Relative to that of wild-type mice
(91.8 ± 3.9 mm Hg, n = 5), mean arterial pressure
was significantly reduced in both Nkcc1+/
(77.7 ± 4.1 mm Hg, n = 6) and
Nkcc1/ (68.6 ± 2.8 mm Hg,
n = 6) mice.
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Fig. 6.
Mean arterial blood pressure. Blood
pressure of anesthetized mice was measured using a femoral artery
catheter. Blood pressures for Nkcc1+/
(n = 6) and Nkcc1/
(n = 6) mice were significantly different from those of
Nkcc1+/+ (n = 5) mice at
p < 0.03 (*) and p < 0.001 (**),
respectively.
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Analysis of Secretory Function of NKCC1-deficient Epithelial
Tissues--
As an initial test of secretory function in the
intestinal tract, the suckling mouse secretion assay was performed to
determine whether cGMP-mediated secretion was impaired in the intestine of Nkcc1/ mice. Injection of E. coli heat-stable enterotoxin STa into the stomachs of 4-5-day-old
mice normally induces fluid accumulation in the intestinal lumen,
yielding an increased gut weight/carcass weight (G/C) ratio. As shown
in Fig. 7, comparisons of the G/C ratios
between maximally stimulated and unstimulated mice of all three
genotypes indicated that Nkcc1/ mice (G/C
ratio = 0.117 ± 0.007) were able to secrete fluid as well as
Nkcc1+/+ (G/C ratio = 0.122 ± 0.004)
and Nkcc1+/ (G/C ratio = 0.127 ± 0.006) mice.
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Fig. 7.
Suckling mouse intestinal secretion
assay. Gut-carcass ratios of Nkcc1+/+,
Nkcc1+/, and Nkcc1/
suckling mice were measured following intragastric administration of
E. coli STa. Genotypes were determined after the assay had
been completed. STa-stimulated secretion was statistically significant
(p < 0.001) in mice of all three genotypes
(n = 21 Nkcc1+/+, 15 Nkcc1+/, and 8 Nkcc1/ mice) when compared with unstimulated
controls (n = 7 Nkcc1+/+, 15 Nkcc1+/, and 7 Nkcc1/ mice). There were no significant
differences in STa-stimulated secretion among the three
genotypes.
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To test whether cAMP-mediated Cl secretion was defective
in the adult Nkcc1/ intestine, samples of
jejunum and cecum from 8-week-old Nkcc1+/+ or
Nkcc1/ mice were mounted in Ussing chambers,
and both basal and forskolin-stimulated secretion were measured under
short-circuit current (ISC) conditions (Fig.
8). As illustrated by the tracing for
cecum (Fig. 8A), after stimulation with forskolin,
application of bumetanide had no effect on ISC in tissues
from Nkcc1/ mice but did cause a reduction
in Nkcc1+/+ tissues. Although basal currents
were nearly identical for the two genotypes in both tissues,
cAMP-stimulated ISC in both jejunum and cecum of
Nkcc1/ mice was ~50% of that observed in
wild-type tissues (Fig. 8, B and C). Following
the addition of bumetanide, the ISC of
Nkcc1+/+ jejunum was sharply reduced, whereas
the ISC of Nkcc1/ jejunum was
slightly reduced but remained near cAMP-stimulated levels. (As can be
seen in Fig. 8A, some reduction of current occurred in
tissues of both genotypes during the 20-min period of stimulation with
forskolin, but addition of bumetanide to
Nkcc1/ tissues did not cause a further
decrease in current.) Addition of bumetanide reduced ISC
significantly in wild-type cecum but not in
Nkcc1/ cecum. The ISC in
Nkcc1+/+ and Nkcc1/
cecums following bumetanide treatment were essentially identical and
were significantly elevated relative to basal levels.
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Fig. 8.
Short-circuit currents in jejunum and cecum
of wild-type and mutant mice. ISC was measured using
voltage-clamped Ussing chambers under basal conditions and during
sequential addition of forskolin and bumetanide. A, typical
ISC recordings from the cecum of
Nkcc1+/+ (left) and
Nkcc1/ (right) mice. Current
deflections result from repetitive voltage spikes used to measure
tissue resistance. B and C, summary of
experiments with jejunum (B) and cecum (C) of
Nkcc1+/+ (n = 6) and
Nkcc1/ (n = 6) mice showing
basal ISC (Basal), forskolin-stimulated
ISC (cAMP) (forskolin is a cAMP-mediated
secretagogue), and bumetanide-resistant ISC
(Bumet). *, p < 0.05 versus
basal ISC for same genotype;  , p < 0.05 versus cAMP-stimulated ISC of
Nkcc1+/+ mice; +, p < 0.05 versus Nkcc1+/+ of same treatment
group.
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Short circuit currents were also measured in cultured tracheal
epithelial cells (Fig. 9). Basal
ISC was significantly lower in tracheal cell monolayers
from Nkcc1/ mice. Treatment with amiloride
to inhibit the epithelial Na+ channel caused a significant
reduction of ISC in Nkcc1+/+ cells
but not in Nkcc1/ cells. Stimulation with
forskolin caused ISC to increase in both sets of samples,
but cAMP-stimulated ISC was significantly lower in
Nkcc1/ cells than in
Nkcc1+/+ cells. Treatment with bumetanide
reduced ISC significantly in wild-type cells but did not
reduce ISC in Nkcc1/ cells.
Short circuit currents in Nkcc1+/+ and
Nkcc1/ tracheal cells that had been treated
with amiloride, forskolin, and bumetanide were both significantly
elevated relative to those recorded after treatment with amiloride
alone.
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Fig. 9.
Short-circuit currents in cultured tracheal
epithelial monolayers from wild-type and mutant mice.
ISC in cultured tracheal epithelial cells from
Nkcc1+/+ (n = 6) and
Nkcc1/ (n = 6) mice was
measured using Ussing chambers under basal conditions
(Basal) and after sequential additions of amiloride
(+Amil), forskolin (+Forsk), and bumetanide
(+Bumet). *, p < 0.05 versus
basal ISC of same genotype; , p < 0.05 versus amiloride-treated group of same genotype; +,
p < 0.05 versus Nkcc1+/+ of same treatment group.
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There is evidence that NKCC1 plays a major role in the secretion of HCl
in amphibian gastric mucosa (14). To determine whether the lack of
NKCC1 in the mouse impairs gastric acid secretion, wild-type and
homozygous mutant mice were treated with histamine, and the pH of the
stomach contents was measured. As shown in Fig. 10A,
Nkcc1/ mice were able to acidify their
stomach contents as well as Nkcc1+/+ mice
(pH = 2.85 ± 0.15 in Nkcc1/;
3.44 ± 0.30 in Nkcc1+/+). These data are
consistent with the results of histological analyses of stomach
sections and ultrastructural analyses of gastric parietal cells. When
examined at high magnification by light microscopy (Fig.
10B, left panels), parietal cells of homozygous
mutants were abundant and appeared normal. Electron microscopy revealed
a well developed secretory canaliculus (Fig. 10B,
right panels) and no apparent ultrastructural abnormalities
that might be indicative of reduced secretion of acid or impaired
viability of the parietal cell.
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Fig. 10.
Gastric acid secretion and histology of
Nkcc1+/+ and
Nkcc1/ parietal cells.
A, 15 min after stimulation with histamine, stomach contents
were diluted in 2 ml of nitrogen-saturated normal saline, and the pH
was determined (n = 5 Nkcc1+/+
and 6 Nkcc1/ mice). The differences were not
statistically significant. B, light microscopy of toluidine
blue-stained sections of oxyntic mucosa of wild-type (+/+) and
homozygous mutant (/) stomachs (left panels) revealed no
obvious histopathology in Nkcc1/ stomachs or
alterations in the abundance of mature parietal cells
(arrows); electron micrographs of parietal cells
(right panels) revealed no differences in ultrastructure.
Note the active secretory caniliculi (SC) in both genotypes.
M, mitochondria; BM, basement membrane.
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Northern Blot Analysis of Ion Transporters Involved in Secretion
and Absorption in the Gastrointestinal Tract--
It was of interest
to determine whether the lack of NKCC1 might lead to alterations in
mRNAs encoding other transporters involved in secretion or
absorption in the gastrointestinal tract. As shown in Fig.
11, mRNA encoding NHE3 was reduced
in both small intestine and colon of Nkcc1/
mice (63 and 50% of wild-type, respectively, when measured by phosphorimager analysis). mRNAs encoding CFTR and AE2 appeared to
be slightly reduced in colon of mutant mice, but not in small intestine
or stomach.
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Fig. 11.
Northern hybridization analysis of ion
transporters involved in secretion and absorption in the
gastrointestinal tract. Northern blots of total RNA (10 µg/lane)
from stomach, small intestine, and colon of wild-type (+/+),
heterozygous (+/), and homozygous mutant (/) mice were
hybridized with probes for NHE3, CFTR, AE2, and
glyceraldehyde-3-phosphate dehydrogenase (as a loading control).
mRNA sizes are shown on the right. The smaller AE2
mRNA in stomach encodes the AE2c variant (31), which is expressed
only in stomach.
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Auditory Brainstem
Responses--
Nkcc1/ mice did not give an
ear twitch in response to a hand clap (Preyer's reflex), indicating
that their hearing might be impaired. To test the possibility and the
extent of any hearing loss, ABR measurements were performed. As shown
in Fig. 12A,
Nkcc1+/+ mice exhibited the characteristic ABR
waveform at sound pressure levels as low as 20-30 db. The ABR wave
form was not observed in Nkcc1/ mice for any
of the sound stimuli used, even at 99 db, demonstrating that these mice
were deaf. The mean values for ABR thresholds in response to each sound
stimulus was elevated in Nkcc1+/ mice relative
to that observed in Nkcc1+/+ mice (Fig.
12B); however, the differences were not statistically significant.
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Fig. 12.
Auditory brainstem responses.
A, representative ABR recordings of
Nkcc1/ and Nkcc1+/+
mice exposed to a broad band click. Top,
Nkcc1/ mouse shows no response at 99 db;
bottom, responses of Nkcc1+/+ mouse
in the 20-50 db range. Note the difference in scales. B,
summary of ABR studies performed on Nkcc1+/+
(open bars, n = 8) and Nkcc1+/
(hatched bars, n = 9) mice exposed to a broad band
click or pure tone frequencies of 8, 16, and 32 kHz. Threshold sound
pressure levels at which the characteristic ABR waveform was detected
are shown. The differences between wild-type and heterozygous mutants
were not statistically significant.
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Histology of the Inner Ear--
In wild-type ears (Fig.
13A), the lumen of the
cochlear duct (scala media), containing the K+-rich
endolymph generated by the stria vascularis, was separated by
Reissner's membrane from the scala vestibuli, which contains perilymph
with the typically low K+-concentrations of most
extracellular fluid. In contrast, in most sections of
Nkcc1/ ears, the lumen of the cochlear duct
was collapsed, and Reissner's membrane was lying against the
interdental cells of the spiral limbus, the tectorial membrane, and the
stria vascularis (Fig. 13B), rather than being in its usual
position separating a patent lumen of the scala media from the scala
vestibuli. Although complete collapse of the cochlear duct was a common
feature, some exceptions were observed. One mutant had a relatively
wide scala media in both ears. In another mutant, the scala media of
the right ear was collapsed, but the scala media of the left ear was
wide. The left ear of this mutant was found to have a ruptured
Reissner's membrane in deeper serial sections (Fig. 13C).
Such a torn membrane could allow influx of perilymph from the scala
vestibuli and might account for the wider lumen in other regions of the
cochlear duct. In sections of mutant ears with a wide scala media
lumen, Reissner's membrane was collapsed partially, lying against the
spiral limbus region, the tectorial membrane, and only the uppermost
portion of the stria vascularis.
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Fig. 13.
Histology of the auditory system.
Sections of the cochlea of Nkcc1+/+
(panels A and D) and
Nkcc1/ (panels B, C, E, and
F) mice. Reissner's membrane (RM) separates the
scala media (SM) and the scala vestibuli (SV) in
wild-type cochlea (panel A). In a mutant cochlea duct with
collapsed lumen, Reissner's membrane is pressed against the spiral
limbus (L), tectorial membrane (TM), and stria
vascularis (S) (panels B, E, and F),
or may be ruptured (panel C) as seen in a mutant ear with a
wide scala media lumen. Intercellular spaces in the stria vascularis of
mutant mice (panels B and C) are greater than
those of wild-type mice (panel A). The typical architecture
of the organ of Corti is seen in wild-type mice (panel D),
with the tunnel of Corti (T) formed by pillar cells
(P) and flanked by outer (OHC) and inner
(IHC) hair cells. A nerve (N) can be seen
traversing the tunnel of Corti. The organ of Corti is abnormal in
mutant mice (panels E and F), although pillar
cells and occasional hair cells can be seen. Calcification
(Ca++) at the tectorial membrane was observed in
sections of mutant ears that had not been decalcified during processing
of tissues (panel E), and a dense fibrous material was
observed on the tectorial membrane of mutant ears that had been
decalcified (panel F). Myelinated nerve fibers and Schwann
cells fill Rosenthal's canals (R). (The light staining of
nerves in Rosenthal's canal in E is due to incomplete
osmication during sample preparation.) Spiral ganglion (G)
neuronal cell bodies were reduced in number in some mutants
(panel B). The scala tympani (ST) is a perilymph
channel below the flexible basilar membrane, on which the organ of
Corti rests. Scale bar, 100 µm in A-C, 50 µm
in D-F.
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The organ of Corti from a wild-type mouse is shown in Fig.
13D. Well developed pillar cells define the tunnel of Corti,
and outer and inner hair cells and their support cells are visible on
either side of the tunnel. Efferent nerve fibers can be seen passing
through the tunnel en route toward the outer hair cells, and
Rosenthal's canal was filled with Schwann cells and myelinated nerve
fibers. The tectorial membrane overlies the organ of Corti and contacts
the sterocilia of the hair cells. The structure of the organ of Corti
in Nkcc1/ mice was perturbed in several
ways. Our analysis included the evaluation of sections from calcified
(e.g. inner ears that had not been decalcified in EDTA) and
decalcified tissues. Sections of calcified
Nkcc1/ ears (Fig. 13E) revealed
an accumulation of calcified crystalline material at the tectorial
membrane. In some sections from decalcified mutant ears (Fig.
13F), the tectorial membrane had densely stained thick
fibers at the periphery. Neither the calcified tectorial membrane-associated material nor the densely stained thick fibers were
observed in sections of nondecalcified or decalcified
Nkcc1+/+ ears, respectively. Although pillar
cells could be identified in most sections of
Nkcc1/ ears, the tunnel of Corti was absent
often, and inner hair cells and particularly outer hair cells could be
identified only infrequently. Neuronal cell bodies in the spiral
ganglion were reduced in numbers in some sections of
Nkcc1/ ears (compare panels A and
B of Fig. 13). Rosenthal's canals were filled with
myelinated nerve processes and Schwann cells in ears of both wild-type
and mutant mice.
The stria vascularis (Fig. 13, A-C and Fig.
14) is a specialized region of the
epithelial lining of the cochlear duct that is essential for the
production of endolymph, and it consists of marginal cells,
intermediate cells, basal cells, and capillaries. In the stria
vascularis of wild-type mice (Fig. 14A), marginal cells,
which normally secrete K+ and form a continuous sheet in
contact with the endolymph, are columnar and have elaborate extensions
of the basal region of the cell that results in an extensive
plasmalemma surface area. In the stria vascularis of homozygous mutants
(Fig. 14B), the marginal cells lacked the numerous basal
processes seen in Nkcc1+/+ marginal cells and
were separated from basal and intermediate cells by intercellular
spaces that were considerably wider than those observed in wild-type
mice (compare panels A-C of Fig. 13 and panels A
and B of Fig. 14).
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Fig. 14.
Histology of the stria vascularis.
Sections of the stria vascularis of Nkcc1+/+
(panel A) and Nkcc1/ (panel
B) mice. In the wild-type, marginal cells (M) are in
contact with the endolymph of the scala media (SM). In the
mutant, Reissner's membrane (RM), bordered on the
left by the scala vestibuli (SV), is pressed
against the marginal cell layer. Nkcc1/
marginal cells have fewer extensions of their basal surfaces than those
of wild-type mice. The intercellular spaces between the marginal cell
layer and deeper layers of the stria vascularis are wider in mutant
than in wild-type ears. Basal cells (B) are present in both
genotypes. Scale bar, 25 µm.
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Reissner's membrane is composed of a squamous epithelium on the scala
media side that is backed by an even thinner squamous mesothelium on
the scala vestibuli side, and it normally separates the endolymph in
the cochlear duct from the perilymph in the scala vestibuli. In regions
of Nkcc1/ ears that had virtually no scala
media lumen, the collapsed Reissner's membrane was often in direct
contact with the marginal cell layer of the stria vascularis (Fig.
14B).
The vestibular organs include the semicircular ducts, the saccule, and
the utricle, each of which has an epithelial lining with specialized
regions of innervated sensory epithelium. In the ampulla of each of the
three semicircular ducts, this innervated area is termed the crista
ampullaris, and in both the saccule and utricle, it is termed a macula.
In the semicircular ducts of wild-type mice, the membranous labyrinth
was open, consistent with its having been filled with endolymph (Fig.
15A). Associated with the
crista ampullaris was a tall gelatinous cupula that extended toward the
epithelial lining opposite the sensory epithelium. In the ampullae of
Nkcc1/ semicircular ducts, the membranous
labyrinth was collapsed upon the cupula (Fig. 15B), and the
perilymphatic space was expanded. The saccule (Fig. 15C) and
utricle (data not shown) of Nkcc1+/+ mice
appeared normal, with the otolithic membrane carrying calcium carbonate
crystals, the otoconia, on its lumenal surface and contacting the
stereocilia of the sensory hair cells in the underlying maculae. The
membranous labyrinth in both saccule and utricle of
Nkcc1+/+ mice was open. In contrast, the
membranous labyrinth of the saccule (Fig. 15D) and the
utricle (data not shown) of Nkcc1/ mice was
irregular and generally collapsed so that the epithelial lining
opposing the macula was lying against the otolithic membrane, somewhat
encapsulating the otoconia. In optimal planes of section of the
Nkcc1/ maculae and cristae (Fig. 15,
B and D), hair cells with their tufts of
stereocilia could be found.
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Fig. 15.
Histology of the vestibular system. In
the ampulla of the semicircular ducts of
Nkcc1+/+ mice (panel A), the
gelatinous cupula (C) overlying the crista ampullaris
(CA) sensory epithelium is bathed in endolymphatic fluid. In
the Nkcc1/ ampulla (panel B), the
epithelial lining that normally encloses the endolymph is collapsed
upon the cupula. Remnants of trabeculae, strands of connective tissue
cells that normally attach the epithelial lining of the semicircular
duct to the bone, can be seen on both the basal surface of the
epithelial lining and on the bone (panel B, upper left). In
panel C, the Nkcc1+/+ saccule
otoconia (O), embedded in the gelatinous otolithic membrane
(OM) that overlies the neuroepithelium of the macula
(M), are bathed in endolymph. In the
Nkcc1/ saccule (panel D), the
epithelial lining of the saccule has collapsed onto the otoconia.
Scale bars: A, 80 µm; B, 100 µm;
C and D, 50 µm.
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DISCUSSION |
Our major objective was to develop an NKCC1-deficient mouse that
could be used to assess the role of this transporter in the secretion
of ions and fluid by epithelial tissues. Northern blot analysis
demonstrated that the wild-type mRNA was expressed at reduced
levels in heterozygotes and was absent in homozygous mutants, confirming that disruption of exon 6 had produced a null mutation. Offspring of heterozygous matings were born in a normal Mendelian ratio
and exhibited no evidence of growth retardation in utero or
structural malformations in major organs. These data suggest that NKCC1
does not play an essential role during embryonic or fetal development,
although they do not rule out more subtle functions in development.
The causes of the increased death rate and growth retardation of
Nkcc1/ mice have not been established, but
it seems likely that the two are related. A competitive disadvantage in
suckling relative to their normal siblings might have contributed
to the growth retardation among null mutants, which were deaf and had
impaired balance. In CFTR-deficient mice, a high incidence of death
resulting from meconium ileus and intestinal obstructions was observed
during the first 5 days after birth and the week after weaning (29). We
anticipated that the lack of NKCC1 might cause a similar defect, but
this was not observed. The absence of meconium ileus and intestinal obstructions among NKCC1-deficient mice during the first 5 days after
birth correlated well with the lack of an apparent defect in the
STa-stimulated secretion in 4-5-day-old mice. Some of the mutants had
fecal obstructions of the intestine at around weaning, but this was not
a consistent finding and could have contributed to death in only a
small subset of the animals that died.
Mean arterial pressure was significantly reduced in the mutants,
and hemorrhage in the intestinal tract was a consistent finding, suggesting that a vascular defect might have played a role in both
growth retardation and the increased frequency of death. Growth
retardation, for example, could have been caused by impaired nutrient
absorption secondary to reduced blood flow in the intestine. During
agonist stimulation of endothelial cells, NKCC1 activity is coupled to
that of Ca2+-dependent K+ channels
(10), and in smooth muscle, its activity is up-regulated in response to
angiotensin II (30). Those findings and the reduced mean arterial
pressure in both heterozygous and homozygous mutants suggest that NKCC1
plays an important role in the control of blood pressure. Given the low
blood pressure in Nkcc1/ mice, it is
conceivable that a further deficit in blood pressure, for example, from
hypovolemia occurring as a result of insufficient consumption of milk,
might lead to circulatory shock. Additional studies will be needed to
test this hypothesis.
Earlier studies demonstrated that cAMP-stimulated, CFTR-mediated short
circuit currents in jejunum, cecum, and trachea are bumetanide-sensitive (13) and that treatment of T84 cells with forskolin increases bumetanide-sensitive 86Rb-uptake
activity (32). This suggests that Cl uptake via NKCC1
contributes to cAMP-stimulated Cl secretion. Our studies
showing that cAMP-stimulated ISC in jejunum, cecum, and
tracheal epithelium of adult Nkcc1/ mice was
limited to ~50% of the levels observed in
Nkcc1+/+ tissues provide direct evidence that
NKCC1 serves as an entry pathway for Cl secreted by the
CFTR. Nevertheless, cAMP-stimulated short circuit currents in jejunum,
cecum, and tracheal epithelium of NKCC1-deficient mice were
significantly elevated relative to basal levels, indicating that other
basolateral transport mechanisms contribute to ion uptake needed for
maximally stimulated secretion. It is possible that much of the
remaining cAMP-stimulated ISC in
Nkcc1/ tissues is due to
HCO3 secretion, as recent studies have
shown that a portion of the agonist-stimulated ISC is due
to CFTR-dependent HCO3
secretion (34-36).
In contrast to the results obtained in Ussing chamber studies of
isolated adult tissues, a deficit in STa-stimulated secretion was not
observed in the intestines of 4-5-day-old NKCC1-deficient mice. In
adult tissues, anion secretion in response to STa or to guanylin or
uroguanylin, the endogenous ligands for the receptor that is activated
by STa, has been shown to be dependent on CFTR activity (33-37) and to
be inhibited by bumetanide (35). It is possible that the differences
observed between the Ussing chamber and suckling mouse assays are
related to the age of the animals, with NKCC1 contributing little to
electrolyte uptake needed for stimulated secretion in the 4-5-day-old
mouse but contributing significantly to this process in the adult. If
this is the case, then other basolateral transport mechanisms must
mediate uptake of anions and Na+ (which is needed for the
Na+,K+-ATPase activity required to maintain the
electrical driving force for anion secretion) during STa-stimulated
secretion. Coupled Cl/HCO3 and
Na+/H+ exchange is a possible alternative
mechanism for basolateral uptake of Cl and
Na+ in intestinal epithelium of the NKCC1-deficient
suckling mouse. Another possibility is that
Na+-HCO3 cotransport
contributes to the basolateral transport processes needed to maintain
secretion in 4-5-day-old Nkcc1/ mice, as
maximum uroquanylin-stimulated HCO3
secretion in duodenum is dependent on the presence of serosal HCO3 (37). The activity of one or both
of these basolateral transport systems may be responsible for the
absence of a severe intestinal secretory defect in
Nkcc1/ mice, such as that observed in
CFTR-deficient mice, and may also account for the remaining
cAMP-stimulated secretion that is observed in isolated tissues of adult
Nkcc1/ mice.
When compared with the significant reduction in ISC that
occurred in Nkcc1+/+ tracheal epithelial cells,
amiloride had only a minor effect at most on ISC in
Nkcc1/ cells, indicating that the activity
of the epithelial Na+ channel is reduced in cells lacking
NKCC1. We observed a reduction in the mRNA encoding NHE3, the major
absorptive Na+/H+ exchanger in both small
intestine and colon. Changes occurring in tracheal cells in
vitro, of course, may not be reflective of events that occur
in vivo, and alterations in Nhe3 mRNA levels are not necessarily indicative of alterations in NHE3 activity; nevertheless, these results provide suggestive evidence that a reduction in Cl secretion may be accompanied by
compensatory reductions in Na+ absorption.
The secretion of ~160 mM HCl and ~17 mM KCl
by gastric parietal cells requires the coordinated activities of ion
transporters on both apical and basolateral membranes. There is strong
evidence that the major mechanism for Cl uptake and
HCO3
,
,
TOP
ABSTRACT
INTRODUCTION
