Induction of epithelial Na+
channel in rat ileum after
proctocolectomy
Kaori
Koyama1,
Iwao
Sasaki1,
Hiroo
Naito1,
Yuji
Funayama1,
Kouhei
Fukushima1,
Michiaki
Unno1,
Seiki
Matsuno1,
Hisayoshi
Hayashi2, and
Yuichi
Suzuki2
1 First Department of Surgery,
Tohoku University School of Medicine, Sendai 980-0872; and
2 Laboratory of Physiology, School
of Food and Nutritional Sciences, University of Shizuoka, Shizuoka
422-8526, Japan
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ABSTRACT |
In patients with colectomy, epithelial transport
function in the remnant small intestine can be regulated in response to
the increased fecal electrolyte and fluid loss. Using a rat colectomy model, we investigated the Na+ and
K+ transport mechanisms underlying
the intestinal response. Proctocolectomy with ileoanal anastomosis was
performed on rats. The small intestinal mucosa was mounted in Ussing
chambers; then short-circuit currents and
22Na+
fluxes were measured. mRNA expression of the epithelial
Na+ channel (ENaC) was determined
by Northern blotting. Amiloride-sensitive, electrogenic
Na+ absorption appeared in the
ileum after proctocolectomy. This functional change was accompanied by
the chronological induction of mRNAs for
-,
-, and
-subunits
of the ENaC in the ileum. Tetraethylammonium-sensitive short-circuit
current was also activated. We conclude that electrogenic
Na+ absorption and probably
K+ secretion are induced in the
ileum after proctocolectomy. This induction of electrogenic
Na+ absorption is probably
mediated by the increase in the mRNA levels for all three types of
subunits of the ENaC and may contribute to the recovery from the
increased fecal Na+ loss.
aldosterone; potassium secretion; sodium absorption; intestinal
adaptation; intestinal absorption
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INTRODUCTION |
EVIDENCE IS ACCUMULATING that intestinal transport of
fluid and electrolytes is regulated not only to cope with intestinal functions but also for the purpose of maintenance of fluid and electrolyte balance in the whole body (27, 29). This can be clearly
seen in patients undergoing colectomy whose fluid and electrolyte
balance is disturbed because of loss of the absorptive function of the
colon (37, 41). Episodes of fluid and electrolyte depletion in such
patients are associated with a decrease in the volume and the
Na+ concentration of the effluent
and an increase in K+
concentration (10, 19, 32, 37). Some evidence suggests that this
"intestinal adaptation" is a result of the regulation of
epithelial Na+ and
K+ transport in the remnant small
intestine, particularly in the ileum (10, 25, 28, 37, 50, 51), but the
precise mechanism involved in this regulation remains to be clarified.
It is speculated that increases in the aldosterone level may mediate
this regulation (10, 21, 33). Detailed characterization of this
pathophysiological regulation of intestinal transport after colectomy
may result in further insight into the role of the small intestine in
control of the overall fluid and electrolyte balance of the organism. Also, it may result in improvement of the clinical treatment of patients requiring colectomy.
Aldosterone has been known to activate amiloride-sensitive,
electrogenic Na+ absorption as
well as K+ secretion in the colon
and distal nephron (4, 7, 40). The regulation of electrogenic
Na+ absorption by aldosterone
involves the activation of the amiloride-sensitive, epithelial
Na+ channel (ENaC), which mediates
Na+ entry through the apical
membrane, due to induction of the ENaC and other regulatory proteins
(2, 3, 8, 9, 11, 13, 20, 34, 35, 39, 44). The ENaC consists of three
homologous subunits,
,
, and
, and it has been shown that the
simultaneous expression of these three subunits causes a large
Na+ current (5, 9, 36, 38a).
Reports have shown that an amiloride-sensitive, electrogenic
Na+ absorption can be evoked in
the lower small intestine of rats and chickens when the animals are
salt restricted or treated with aldosterone (22, 48). Evidence for the
presence of a mineralocorticoid receptor, as well as
11
-hydroxysteroid dehydrogenase type II, has implicated the small
intestine, particularly the lower small intestine, as a
mineralocorticoid-targeted tissue (17, 18, 38, 43). These findings
suggest that both amiloride-sensitive, electrogenic
Na+ absorption and possibly
K+ secretion can be induced by
aldosterone in the remnant ileum and contribute to the intestinal
adaptation in colectomized patients.
The purpose of this study was to verify these possibilities. Using a
rat model, in which proctocolectomy with ileoanal anastomosis was
performed, both Na+ absorption and
K+ secretion in the remnant small
intestine were examined in vitro using Ussing chambers. Changes in mRNA
levels of the three ENaC subunits in the remnant small intestine were
also examined.
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MATERIALS AND METHODS |
Animals and surgical procedures.
Male Sprague-Dawley rats were housed in a temperature-controlled room
on a 12:12-h light-dark cycle and fed with standard laboratory chow
(Funabashi FII, Funabashi Farm, Funabashi, Japan) and water ad libitum.
When rats reached 8 wk old (300-350 g), proctocolectomy was
performed under pentobarbital anesthesia (pentobarbital sodium, 50 mg/kg body wt, intraperitoneal injection). The intestinal segment was
excised from 0.1 cm proximal to the ileocecal junction to the distal
end of the rectum (at the dentate line). Then the cut end of the ileum
was anastomosed with the anus using a single-layer interrupted suture
(5-0 proline) by the pull-through method.
The operated rats were administered saline (50 ml/kg body wt ip) and
housed individually in metabolic cages. After 3 days of solid meal (but
not water) deprivation so that rats could heal from the operation, the
animals were given free access to water and laboratory chow in the same
manner as before the operation for up to 8 wk after the operation.
Age-matched, nonoperated rats were used as controls. One group of rats
was fed a low-Na+ diet for
7-10 days. Experimental protocols used in this study were strictly
followed according to the guidelines of the Committee for the Care and
Use of Laboratory Animals of Tohoku University and the University of Shizuoka.
Intestinal and urinary excretions and serum corticosteroid levels.
Freshly discharged feces were collected between 9:00 AM and 11:00 AM.
Part of the sample was used for determining water content and the rest
for determining Na+ and
K+ concentrations in the fecal
water. The water content of feces was determined from the decrease in
the fecal weight after samples were dried in an oven at 105°C for
24 h. To determine Na+ and
K+ concentrations, feces were
diluted with H2O by fourfold
(wt/wt), agitated on a Vortex mixer, and centrifuged (for 5 min at
1,000 rpm). With the resultant supernatants,
Na+ and
K+ concentrations were determined
by ion chromatography using an ion-exchange column (Shim-pack IC-C1,
Shimadzu, Tokyo, Japan) and a conductivity detector (CDD6-A, Shimadzu).
Urine was collected for 24 h, and its volume was determined.
Na+ and
K+ concentrations were determined
as mentioned above.
To determine the plasma corticosteroid level, the animals were
anesthetized with ether and blood was collected from abdominal aorta
between 12:00 AM and 2:00 PM (to avoid diurnal variation). Concentrations of plasma aldosterone and corticosterone were determined by RIA kits obtained from Dainabot (Tokyo, Japan).
Short-circuit current and
22Na+ flux measurements.
The short-circuit current
(Isc) was
measured in vitro in Ussing chambers. The rats were killed by a blow to
the head followed by exsanguination. Segments (2 cm) were isolated from
areas 1-3 cm (designated as the terminal ileum), 14-16 cm,
and 24-26 cm from the end of the ileum (i.e., from the ileocecal
junction in the nonoperated group and from the ileoanal anastomosis in
the proctocolectomy group, respectively). The last segment was the area
approximately two-thirds of the distance from the ligament of Treitz to
the end of the ileum. The isolated segment was opened and rinsed free
of intestinal contents, and the external muscle layer was removed by
blunt dissection. The tissue was then mounted vertically between
acrylic resin chambers with an internal surface area of 0.5 cm2. Bathing solution in each
chamber was 10 ml and was kept at 37°C in a water-jacketed
reservoir. The mucosal solution contained (in mM) 119 NaCl, 21 NaHCO3, 2.4 K2HPO4,
0.6 KH2PO4,
1.2 CaCl2, 1.2 MgCl2, and 8.5 mannose. The
serosal solution had the same composition as that of the mucosal
solution, except that it contained 2.5 mM glutamine, 5 mM glucose, and
1 mM
-hydroxybutyrate (Na+ salt) instead of mannose.
Each solution was gassed with 95%
O2 and 5%
CO2 (pH 7.4).
Tissues were continuously short-circuited, with a compensation for
fluid resistance between the two potential-sensing bridges, using a
voltage-clamping amplifier (CEZ9100, Nihon Kohden, Tokyo, Japan). The
transepithelial potential was measured through 1 M KCl-agar bridges
connected to a pair of calomel half-cells, with the transepithelial
current applied across the tissue through a pair of Ag-AgCl electrodes
kept in contact with the mucosal and serosal bathing solutions using a
pair of 1 M NaCl-agar bridges. The asymmetric potential from the pair
of calomel half-cells used for the potential measurement was less than
±3 mV and changed by less than ±0.2 mV during each experiment.
The Isc value was expressed as positive when the current flowed from the mucosa to
serosa. Transmural tissue resistance
(Gt) was
calculated from the change in current in response to voltage pulses
according to Ohm's law.
Unidirectional transmural
22Na+
fluxes were measured in Ussing chambers under short-circuit conditions.
The mucosal-to-serosal (Jm
s)
and serosal-to-mucosal
(Js
m)
fluxes were measured in the adjacent tissues that had
Gt values
differing by <30%. Thirty minutes were allowed for isotopic steady
state to be reached after labeling either the serosal or mucosal side
of the bathing solution with 100 kBq of
22Na+.
Six samples (0.5 ml each) were taken from the unlabeled side at 15-min
intervals and replaced with an equal volume of the unlabeled solution.
Medium samples were assayed for
22Na+
in a gamma-well spectrometer.
22Na+
was purchased from DuPont NEN (Boston, MA).
RNA extraction and Northern blotting.
Total RNA was extracted from three equally divided whole intestinal
segments (i.e., upper third, middle third, and lower third) and colon
as described previously (46). The quality and quantity of RNA were
determined by absorbance at 260 nm.
Complementary DNA probes were prepared using reverse-transcribed cDNA
of rat colon as the PCR template. Primer sets specific for the
-subunit [sense: 5'-ATGGTAGCGATGTCCCGGTCAAGAA-3'
(1327-1351), antisense:
5'-AGCAGATCGTTAGCCCCTGTCCTCT-3'(2330-2306)],
-subunit [sense: 5'-CTTTGCCTGCTGGGGAGAAATACTG-3'
(1286-1310), antisense: 5'-GGAGTCATAGTTGGGAGGTGGTGGAGTG-3' (1932-1908)],
and
-subunit [sense:
5'-TCCTCTATCATCGCCCGCCGTCAGT-3' (1773-1797),
antisense: 5'-TCTCCAAACATGATCCCCAGGCTCT-3'
(2573-2549)] of the rat epithelial Na+ channel (rENaC) were
synthesized on the basis of each cDNA sequence (8, 9, 34, 35). PCR was
performed at 94°C for 30 s, at 56°C for 2 min, and at 72°C
for 2 min for 35 cycles followed by a final extension at 72°C for 5 min. The PCR products were electrophoresed, purified, and subcloned
into pBluescript (Stratagene, La Jolla, CA). Finally, we confirmed the
sequences of the PCR products using a sequencing kit (United States
Biochemical, Cleveland, OH). Complementary DNA probe for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was prepared as
described previously (1).
Forty micrograms of total RNA were electrophoresed on 1% agarose gel
and transferred onto nylon membrane (Hybond-N, Amersham). Complementary
DNA probes for
-,
-,
-rENaC and GAPDH were labeled with
32P using the Megaprime DNA
labeling system (Amersham). After a 2-h prehybridization, the blot was
hybridized overnight with labeled cDNA probes at 42°C in
hybridization buffer containing 50% formamide, 5× sodium
chloride-sodium phosphate-EDTA (SSPE), and 200 µg/ml denatured herring DNA. The blot was subjected to the following procedures: 1) washed with 2×
sodium chloride-sodium citrate (SSC) and 0.1% SDS at room
temperature for 15 min (twice), 2)
washed with 2× SSPE and 0.1% SDS or 0.1× SSC and
0.1× SDS at 42°C for 15 min, and
3) rinsed with 0.1× SSC and
0.1% SDS at room temperature. The blot was exposed to autoradiography
at
70°C or visualized using a BAS imaging analyzer (Fuji
Film, Kanagawa, Japan). The same blot was washed in 0.02× SSC-1%
SDS solution at 100°C and rehybridized with different probes.
Statistics.
Values are presented as means ± SE, with
n representing the number of animals.
Statistical comparisons between two means were made with the Student's
t-test (paired or unpaired, as
appropriate), whereas multiple comparisons were made with one-way ANOVA
followed by Fisher's test. Significance was accepted at
P < 0.05.
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RESULTS |
Body weight, food and water intake, and
Na+,
K+, and water
excretion in urine and feces after proctocolectomy.
The body weight of proctocolectomized rats was decreased to 68 ± 5% of preoperative levels at 2 wk (P < 0.05) and was maintained until 8 wk after surgery (72 ± 5% of preoperative weight,
n = 8). The food intake during this
postoperative period was not significantly different from that at the
preoperative level (preoperative = 19.1 ± 0.4, 2 wk = 17.6 ± 0.9, 8 wk = 17.8 ± 0.5 g/day). The water intake was markedly
increased after proctocolectomy (preoperative = 24 ± 3, 2 wk = 48 ± 4 ml/day; P < 0.05) and
sustained at this increased level until 8 wk after surgery (50 ± 4 ml/day). On the other hand, nonoperated 12-wk-old rats (age matched to
the proctocolectomized animals at the 4-wk time point) exhibited a
weight gain of 48 ± 2% during the 4-wk period, a food intake of
18.5 ± 1.6 g/day, and a water intake of 23 ± 1 ml/day
(n = 4). Thus the proctocolectomy did
not change the food intake but reduced the efficiency of the weight
gain per gram of food intake and increased the water intake.
Urine volume was slightly but significantly decreased after the
proctocolectomy (Fig.
1A).
Both urine Na+ and
K+ concentrations were decreased
considerably and showed little recovery by 8 wk after surgery (Fig.
1B).

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Fig. 1.
Changes in urine volume (A) and
changes in Na+ and
K+ concentrations in the urine
(B) after proctocolectomy. Control
values are those obtained before surgery. Values are means ± SE for
5 animals. Values with different letters are significantly different
from each other according to ANOVA.
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Fecal water content 2 wk after proctocolectomy was increased by
approximately threefold compared with that at preoperative levels (Fig.
2A). The
increased fecal water content was not significantly altered until 8 wk.
The concentrations of Na+ and
K+ in the fecal water were
increased considerably 2 wk after surgery (Fig.
2B). However, at 4 wk, the increase
in fecal Na+ concentration was
significantly attenuated, and, at 8 wk, the increase was not
statistically significant compared with that at preoperative levels. In
contrast, the K+ concentration in
the fecal water remained elevated until 8 wk after surgery.

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Fig. 2.
Changes in fecal water content (A)
and changes in Na+ and
K+ concentrations in the fecal
water (B) after proctocolectomy.
Control values are those obtained before surgery. Values are means ± SE for 5 animals. Values with different letters are significantly
different from each other according to ANOVA.
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Plasma corticosteroid level.
The plasma aldosterone level was elevated by 30-fold 1 wk after surgery
(Fig.
3A). It
continued rising thereafter, achieving a level 80-fold higher than that
at the preoperative level, 8 wk after surgery. In contrast, the plasma
corticosterone level had not changed after the surgery (Fig.
3B).

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Fig. 3.
Changes in plasma aldosterone (A)
and plasma corticosterone (B) levels
after proctocolectomy. Control values are those obtained from
age-matched (2-4 wk) normal animals. Values are means ± SE.
The number of animals is given in parenthesis. Values with different
letters are significantly different from each other according to ANOVA.
Plasma corticosterone levels are not significantly different from each
other.
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Isc measurements in the small intestine.
We examined whether an amiloride-sensitive, electrogenic
Na+ absorption was functionally
expressed in the small intestine after proctocolectomy by measuring the
Isc. Addition of
amiloride, an ENaC inhibitor, to the mucosal solution (0.1 mM) had no
effect on Isc in
the terminal ileum (1-3 cm from ileocecal junction) of normal
rats, whereas it clearly decreased
Isc in the
corresponding intestinal segment of proctocolectomized rats (Fig.
4), suggesting that an amiloride-sensitive,
electrogenic Na+ absorption was
functionally activated in the ileum after proctocolectomy. No
significant change in transmural
Gt could be
detected in association with the
Isc decreased by
amiloride (see Table 1 for the values of
the electrical parameters). The concentration dependency of the effect
of amiloride on
Isc showed an
IC50 of 0.45 µM (Fig. 5). The inhibitory effect of
amiloride on Isc
in the ileum of proctocolectomized rats was similar to that observed in
the distal colon of unoperated rats treated with a
low-Na+ diet (Fig. 5).

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Fig. 4.
Effects of mucosal amiloride and tetraethylammonium (TEA) on
short-circuit current
(Isc) in the
terminal ileum (1-3 cm from the end of ileum). Typical tracings
from a control rat (A) and a
proctocolectomized rat (B) are
shown. Arrows indicate when amiloride (0.1 mM) and TEA (2 mM) were
added to the mucosal solution.
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Table 1.
Basal Isc and transmural Gt and effects of
amiloride and TEA on Isc in the terminal ileum after
proctocolectomy
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Fig. 5.
Dose dependency of the inhibitory effect of amiloride on
Isc. , Data
determined in the terminal ileum of proctocolectomized rats 4 wk after
surgery. , Data determined in the distal colon of unoperated rats
treated with a low-Na+ diet for
7-10 days. Amiloride was added cumulatively to the mucosal side at
a concentration ranging from 0.1 to 100 µM. Normalized
amiloride-sensitive
Isc was
determined by assuming that 100 µM amiloride maximally inhibits the
amiloride-sensitive component. Values are means ± SE for 5 animals
in each group.
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When tetraethylammonium (TEA), a
K+ channel inhibitor, was added to
the mucosal side (2 mM, Cl
salt) after amiloride treatment,
Isc increased in
the terminal ileum of proctocolectomized, but not control, rats (Fig.
4). A change in
Gt was not
detected with the
Isc increase by
TEA (see Table 1 for the values of the electrical parameters). The
TEA-induced Isc
increase was completely abolished when the tissue was pretreated with
serosal bumetanide (0.1 mM), a
Na+-K+-2Cl
cotransport inhibitor (data not shown, 4 proctocolectomized rats). Bumetanide alone increased
Isc (17.1 ± 12.1 µA/cm2). These
results suggest that the bumetanide-inhibitable, TEA-sensitive, electrogenic K+ secretion
documented in the mammalian distal colon (7, 45) was also activated in
the terminal ileum of proctocolectomized rats.
Table 1 summarizes the time course of changes in electrical parameters
in the terminal ileum (1-3 cm from ileoanal anastomosis) of
proctocolectomized rats. The basal
Isc was slightly,
but not significantly, increased after proctocolectomy (the increase in basal Isc reached
a significant level in another series of experiments, as shown in Table
2). Amiloride-sensitive
Isc was induced after surgery. The values of
Isc were comparable among 2, 4, and 8 wk. Similarly, the
Isc increase
caused by the 2 mM TEA was not significantly altered during this
period. Noticeably, the
Gt was gradually
reduced after proctocolectomy.
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Table 2.
Effect of mucosal benzamil on unidirectional fluxes of
22Na+ in the terminal ileum of control
and proctocolectomized rats
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We next examined the distribution of the amiloride-sensitive
Isc and
TEA-sensitive Isc
in the ileum after proctocolectomy (Fig.
6). In the terminal ileum, the values of
the Isc decrease at 2 and 4 wk after proctocolectomy caused by the 0.1 mM amiloride treatment were not different. In contrast, in the region 14-16 cm
from the anastomosis, the value of the amiloride-sensitive Isc was rather
low at 2 wk, but it was significantly increased by 4 wk after
surgery. In the region 24-26 cm from the anastomosis (intestinal
segment located approximately at two-thirds from the ligament of Treitz
to the end of the ileum), an amiloride-sensitive Isc was not
observed at either 2 or 4 wk. A similar time-dependent change in
distribution pattern was also observed for the value of the
Isc increase
induced by 2 mM TEA (Fig. 6B). Thus
amiloride-sensitive Isc and
TEA-sensitive Isc
are activated apparently in parallel, and they emerge first in the
terminal ileum region and then spread to the more proximal part of the
ileum, thereby probably increasing their capacities.

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Fig. 6.
Increases in regional distribution of the
Isc component
inhibited by mucosal 0.1 mM amiloride
(A) and the
Isc component
inhibited by mucosal 2 mM TEA (B).
Intestinal segments 2 cm (1-3 cm), 15 cm (14-16 cm), and 25 cm (24-26 cm) from the ileoanal anastomosis were obtained from
proctocolectomized animals 2 and 4 wk after surgery.
Isc change with
amiloride was determined first, and then
Isc change with
TEA was determined as shown in Fig. 4. Values are means ± SE for 5 animals. NS, not significant.
* P < 0.05.
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A transport study on the more proximal segments of the small intestine
was impossible to accomplish because the integrity of these tissues
appeared to decline in Ussing chambers, as judged from the absence of
glucose-induced
Isc. This is in
contrast to the brisk and reproducible
Isc response to
luminal glucose application in the ileum; the reason for this differing
viability among the different intestinal segments is not known.
22Na+
flux measurement in the terminal ileum.
We determined bidirectional
22Na+
fluxes in the terminal ileum of control and proctocolectomized rats
using benzamil (10 µM), a more specific inhibitor for the ENaC than
amiloride (3, 20) (Table 2). Neither
22Na+
absorption nor
Isc was affected
by mucosal benzamil in control rats. In contrast, in proctocolectomized
rats,
22Na+
absorption (and
Isc) was
significantly decreased by mucosal benzamil, mainly due to a decrease
in Jm
s
without significant changes in
Js
m.
Therefore, the amiloride (benzamil)-sensitive, electrogenic Na+ absorption is actually
activated after proctocolectomy. A dosage of 10 µM benzamil is
probably the maximum necessary, since benzamil is more potent than
amiloride by a factor of 10 (20). Thus the amiloride
(benzamil)-sensitive
22Na+
absorption rate represents only one-third of the total
22Na+
absorption (Table 2). In addition, it was found that the
Js
m in
the proctocolectomized rats was significantly smaller (by 20-40%) than that in control rats, and this was apparently associated with a
decrease in Gt by
a similar degree (Table 2).
Expression of rENaC subunit mRNAs in the small intestine.
To determine the mechanism of induction of the amiloride-sensitive,
electrogenic Na+ absorption in the
small intestine, we examined mRNA expression of
-,
-, and
-subunits of rENaC by Northern blot analysis. As shown in Fig.
7, the expression of
-,
-, and
-subunits of mRNA was very low in the upper, middle, and lower third
of the small intestine of control rats. On the other hand, the
expression of mRNAs for all three rENaC subunits was abundant in the
lower third of the small intestine of proctocolectomized rats, although they remained at a low level in the upper and middle third of the small
intestine. In the colon of normal rats, expression of
-subunit mRNA
was high but that of
-subunit mRNA was very low, consistent with a
previous report (2, 11, 13, 34, 35, 39, 44). Figure
8 shows the time course of the increase in mRNA levels for each subunit in the lower third of the small intestine after proctocolectomy. Expressions of
-,
-, and
-subunit rENaC mRNAs were all increased in a parallel fashion and almost reached a
maximum at 4 wk after proctocolectomy. Thus these increases in rENaC
mRNA levels apparently corresponded well with the increase in the
capacity of electrogenic Na+
absorption.

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Fig. 7.
Expressions of -, -, and -subunits of rat epithelial
Na+ channel (rENaC) mRNA in the
intestinal tissues of normal and proctocolectomized rats. Intestinal
segment from the ligament of Treitz to the end of ileum was equally
divided into 3 parts. Note that each part was ~25 cm in length. Upper
(lane 1), middle
(lanes 2 and
3), and lower (lane
4) third of intestine from control rats and the upper
(lanes 5 and
6), middle (lane
7), and lower (lanes
8 and 9) third of
intestine from proctocolectomized rats (4 wk after surgery) and the
colon of normal rats (lane 10) were
used. Total RNA was fractionated by electrophoresis through a 1%
agarose gel (40 µg/lane), transferred onto nylon membrane, and
sequentially hybridized with rENaC - and -subunit cDNA
probes (A) or with a -subunit
cDNA probe (B) and then with a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. Sizes of
the main hybridization fragments, estimated from 28S and 18S RNA,
corresponded with the sizes expected from previous reports (8, 9, 34,
35).
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Fig. 8.
Time course of the increase in rENaC -, -, and -subunit mRNA
levels in the lower third of the small intestine after proctocolectomy.
A and
B: Northern blotting of control rat
(lane 1) and rats 1 wk
(lanes 2 and
3), 2 wk (lanes
4 and 5), 4 wk
(lanes 6 and
7) and 8 wk (lanes
8 and 9) after
proctocolectomy. C: signal of each
band was quantified by an image analyzer. Results (means of 2 animals) are expressed, after being normalized with GAPDH mRNA levels,
as the ratio to the mRNA level at 8 wk after proctocolectomy for
each subunit.
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DISCUSSION |
The present study investigated the regulation of
Na+ and
K+ transport in the small
intestine after proctocolectomy in rats. The results have shown de novo
induction of an amiloride-sensitive, electrogenic
Na+ absorption in the lower small
intestine after proctocolectomy in a time-dependent fashion,
demonstrated by both molecular and electrophysiological methods. The
increases in the amount of mRNAs of the
-,
- and
-rENaC
subunits and in the capacity of the amiloride-sensitive, electrogenic
Na+ absorption occurred in a
parallel fashion, indicating that electrogenic Na+ absorption can be activated in
the remnant ileum through the induction of the rENaC mRNA. The
significant recovery from the elevation of the fecal
Na+ concentration, particularly
during the period between 2 and 4 wk after proctocolectomy (Fig.
2B), is possibly explained by this molecular and functional regulation of
Na+ absorption in the terminal ileum.
The plasma aldosterone level, but not the corticosterone level,
markedly increased after proctocolectomy. The increased aldosterone levels observed in this study could be one of the factors responsible for the induction of rENaC mRNA and the activation of electrogenic Na+ absorption in the ileum after
proctocolectomy. The presence of a mineralocorticoid receptor (17, 18)
and 11
-hydroxysteroid dehydrogenase type II (38, 43) has been
demonstrated in the ileum, supporting the hypothesis that the lower
small intestine is a target organ for aldosterone. Actually, it has
been reported that an amiloride-sensitive, electrogenic
Na+ absorption can be observed in
the lower small intestine of rats and chickens when the animals are fed
a salt-depleted diet or when administered aldosterone (22, 48). The
plasma aldosterone level in these
Na+-depleted rats was reported to
be 13 ng/ml, which is comparable to the levels observed in the
proctocolectomized rats of the present study (48). We have recently
demonstrated in the rat ileum in vitro in Ussing chambers that the
application of aldosterone to the bathing solution can induce
amiloride-sensitive
Isc (31). It
remains to be demonstrated, however, whether aldosterone may also
upregulate all three rENaC subunits in the ileal mucosa. The
amiloride-sensitive
Isc was observed
only in the terminal ileum at 2 wk after proctocolectomy but still was
detected in further proximal parts of the ileum at 4 wk. The plasma
aldosterone level was nearly doubled during the period between 2 and 4 wk after proctocolectomy, suggesting that there is a distal-to-proximal gradient of aldosterone sensitivity for induction of electrogenic Na+ absorption in the ileum. A
similar distal-to-proximal gradient of aldosterone sensitivity has been
demonstrated in the distal colon (12).
Regulation of apical ENaC activity through induction of mRNA is one of
the mechanisms of activation of electrogenic
Na+ absorption in epithelial
tissues by aldosterone (3, 20, 40). We have demonstrated that all
-,
-, and
-ENaC subunit mRNAs increased in the remnant ileal mucosa
after proctocolectomy. In the rat colon, the
-subunit gene is
constitutively expressed, whereas preferential gene induction of
-
and
-subunits has been demonstrated under secondary
hyperaldosteronism (2, 13, 34, 39, 44). Aldosterone enhanced
-subunit gene expression in the kidney but had a minimal effect on
the expression of
- and
-subunits. Thus what occurs in the ileum
after proctocolectomy is not similar to what occurs in either the
kidney or the colon.
The excellent temporal correlation demonstrated in the present study
suggests that the activation of electrogenic
Na+ absorption in the ileum
results in the decrease of fecal
Na+ concentration and plays a
crucial role in the prevention of fecal Na+ loss after proctocolectomy.
Hill et al. (25) have also reported the major contribution of the
terminal ileum in reducing fecal Na+ concentration in patients who
underwent colectomy, but the underlying mechanism has not been
determined. It has been demonstrated previously that the electrogenic
Na+ absorption can decrease the
Na+ concentration of the
intestinal content in the small and large intestine (15, 16, 48). In
contrast to the dramatic change in the
Na+ concentration, the fecal water
volume was not markedly changed during the 2-8 wk after surgery
(Fig. 2). This suggests that the activated electrogenic
Na+ absorption had a minimal
effect on the fecal water volume, although several previous studies
have shown that water absorption in the small and large intestine
increased in association with the activation of electrogenic
Na+ absorption (15, 16, 48).
Several problems, however, remain to be solved before concluding that
the activation of electrogenic Na+
absorption in the ileum is mainly responsible for the recovery from the
elevation of fecal Na+
concentration after proctocolectomy. First, the electrogenic Na+ absorption probably
constitutes only a minor component of total Na+ absorption in the ileum of
proctocolectomized rats. In the Ussing chamber experiments,
benzamil-insensitive
22Na+
absorption contributed to more than half of the total
22Na+
absorption (Table 2). Unfortunately, it was not clear from the present
results whether the benzamil-insensitive
22Na+
absorption in the ileum was also regulated after proctocolectomy. In
addition, other types of Na+
absorption mechanisms including nutrient-coupled ones may participate in the in vivo Na+ absorption
(14). Second, regulation of
22Na+
transport in the more proximal part of the small intestine should also
be determined. We could not perform the electrophysiological and
22Na+
flux experiments because of the poor viability of proximal segments in
Ussing chambers. Finally, digestive and absorptive processes of foods
in the intestine may well have been disturbed after proctocolectomy. If
so, this may produce the alteration in the ion and water absorption by
the intestine. Consequently, many factors other than electrogenic Na+ absorption can influence the
Na+ and water content in the ileal
effluent. Thus further studies are required to determine the underlying
mechanism of time-dependent changes in ion and water composition of
ileal effluent after proctocolectomy.
This study has also shown that the TEA- and bumetanide-sensitive
Isc, which are
absent in normal intestine, were enhanced in the rat ileum after
proctocolectomy. This
Isc component
probably is a result of an electrogenic
K+ secretion, which has been
demonstrated in the distal colon but not in the small intestine (7,
45). In the colon, a bumetanide-sensitive Na+-K+-2Cl
cotransport has been shown to be the primary mechanism of
K+ uptake across the basolateral
membrane, although K+ uptake via
Na+-K+-ATPase
occurs when
Na+-K+-2Cl
cotransport is impaired (45). The electrogenic
K+ secretion in the distal colon
can be activated by aldosterone (4, 23, 45). It is therefore possible
that aldosterone is also responsible for the activation of electrogenic
K+ secretion in the ileum in our
study. The electrogenic K+
secretion in the ileum may be responsible, at least in part, for the
elevation of fecal K+
concentration after proctocolectomy (Fig. 2). It has been shown that in
patients who have undergone colectomy the
K+ concentration in the ileal
effluent is elevated in association with the increase in aldosterone
levels (19, 21, 30, 33, 42). The activation of
K+ secretion seems to be an
unfavorable response to proctocolectomy because it precipitates
K+ deficiency (Fig. 1). However,
the electrogenic K+ secretion
might conceivably provide an electrical driving force for electrogenic
Na+ absorption, thereby increasing
Na+-retaining activity indirectly.
In the present study, we used 2 mM TEA, which inhibits only 50% of
K+ secretion in the rat distal
colon (Suzuki, unpublished observations). The potency of the inhibitory
effect of mucosal TEA on ileal K+
secretion remains unknown. Therefore, the actual amount of
K+ secretion enhanced after
proctocolectomy cannot yet be determined. For the same reason, it is
also difficult to evaluate whether Isc components
other than electrogenic Na+
absorption and K+ secretion are
also changed after proctocolectomy.
Our results have shown that the
Gt as well as the
serosal-to-luminal
22Na+
flux significantly decreased in the ileum after proctocolectomy (Tables
1 and 2), suggesting that permeability of the paracellular pathway was
diminished. The previous studies on the regulation of epithelial
permeability by aldosterone have provided conflicting results: both the
increase (47, 49) and the decrease (4, 26) of
Gt and passive
Na+ permeability have been
demonstrated. Interestingly, it has been reported that
Gt and
serosal-to-luminal
22Na+
flux are smaller in ileal biopsies taken from patients with ileostomy than those in control ileal mucosa (24), consistent with the results of
our present rat model. The decrease in paracellular Na+ permeability of the lower
small intestine after proctocolectomy may contribute to the low
concentration of fecal Na+ by
reducing the amount of Na+ leakage
into the lumen.
In conclusion, the present results have characterized the possible
mechanisms by which the small intestine participates in long-term
regulation of electrolyte and water balance in the whole body. Using
our rat model, we have demonstrated that amiloride-sensitive, electrogenic Na+ absorption and
probably also TEA-sensitive, electrogenic
K+ secretion were induced in the
ileum after proctocolectomy. We also indicated that the induction of
the electrogenic Na+ absorption
depended on the increase in the amount of all three subunits of the
ENaC mRNAs (
,
, and
), probably due to activation at the
transcription level. Hyperaldosteronemia, which was observed after
surgery, may play an important role in the induction of ENaC and the
functional activation of electrogenic
Na+ absorption. Thus the present
results confirmed and extended the previous observations in
Na+-deficient or
aldosterone-treated animals. It should be noted, however, that rats in
the present proctocolectomy model are different from
Na+-deficient or
aldosterone-treated animals in several respects, such as in the absence
of the large intestine and possible changes in small intestinal transit
time of the contents, compared with normal rats. Thus future study
using the present rat model may provide unique and significant
information concerning the regulation of small intestinal functions.
One of the interesting questions to be addressed may be whether the
proximal or middle part of the ileum expresses more amiloride-sensitive
Isc when, in
addition to the colon, the distal part of the ileum is removed. The
results of such a study may be useful for clinicians to help determine if the distal part of the ileum should be conserved in colectomy patients to prevent excessive fecal
Na+ loss. It remains to be
investigated, however, whether similar regulation processes
(demonstrated here in rats) can be induced in the human ileum.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by Grant-in-Aid for Scientific Research
07457266 (to I. Sasaki) and by a Grant-in-Aid for Scientific Research
on Priority Areas of "Channel-Transporter Correlation" (to Y. Suzuki) from the Ministry of Education, Science, Sports and Culture of Japan.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. Sasaki, First
Dept. of Surgery, Tohoku Univ. School of Medicine, Sendai
980-0872, Japan.
Received 6 October 1998; accepted in final form 22 December 1998.
 |
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