Departments of 1 Internal Medicine and 2 Pathology, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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Dietary sodium depletion has multiple diverse effects on ion transport in the rat colon, including both the induction and inhibition of electroneutral NaCl absorption in proximal and distal colon of rat, respectively. To establish the mechanism of the differential regulation of Na+ absorption by sodium depletion, this study utilized 1) HOE-694, a dose-dependent inhibitor of Na+/H+ exchanger (NHE) isoforms, in studies of proton gradient-driven 22Na uptake (i.e., Na+/H+ exchange) by apical membrane vesicles (AMV); 2) Northern blot analyses of NHE isoform-specific mRNA abundance; and 3) Western blot analyses of NHE isoform-specific protein expression. HOE-694 inhibition studies establish that 25 µM HOE-694-sensitive (NHE2) and 25 µM HOE-694-insensitive (NHE3) Na+/H+ exchange activities are present in AMV of both proximal and distal colon of normal rats. In proximal colon, dietary sodium depletion enhanced both NHE2 and NHE3 isoform-specific Na+/H+ exchange activities, protein expression, and mRNA abundance. In contrast, in distal colon both NHE2 and NHE3 isoform-specific Na+/H+ exchange activities, protein expression, and mRNA abundance were inhibited by sodium depletion. NHE1 isoform-specific mRNA abundance in proximal or distal colon was not altered by sodium depletion. Differential effects by sodium depletion on Na+/H+ exchange in rat colon are tissue specific and isoform specific; sodium depletion both induces and inhibits apical Na+/H+ exchange at a pretranslational level.
aldosterone; pretranslational; Na+/H+ exchange; membrane vesicles; uptake
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INTRODUCTION |
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THE MECHANISMS OF ACTIVE NaCl transport differ
significantly in the proximal and distal segments of the rat large
intestine (4). HCO3-dependent
electroneutral NaCl absorption is the primary transport process in the
distal colon and is the consequence of the coupling of parallel ion
exchanges,
Na+/H+
and
Cl
/HCO
3
exchanges (3, 14, 16, 23, 24). Although electroneutral
Na+ absorption is also
Cl
dependent in the
proximal colon, net Cl
movement is absent (13), and
Na+/H+
exchange is the predominant mechanism of
Na+ absorption (13,
23).
Aldosterone, either as a consequence of dietary sodium
depletion or as a result of its continuous infusion via
minipumps,1
alters both Na+ and
Cl absorption in both
proximal and distal segments of the rat colon (15, 18). However, the
effect of aldosterone in these two segments differs substantially:
aldosterone inhibits electroneutral NaCl absorption in the distal
colon; in contrast, in the proximal segment aldosterone enhances
Na+ absorption while inducing
Cl
absorption (15,
18).
Five different
Na+/H+
exchange isoforms have been cloned to date, and three isoforms (NHE1,
NHE2, and NHE3) have been identified in intestinal epithelial cells
(12, 17, 21, 32). Recently, a
Cl-dependent
Na+/H+
exchange has been identified in apical membranes of crypt cells of rat
distal colon (25), and it is not known whether any of the existing NHE
isoforms are responsible for
Cl
-dependent
Na+/H+
exchange activity.
The molecular regulation of colonic Na+/H+ exchange by aldosterone has recently been reported by Cho et al. (7). These studies examined the effect of aldosterone administrated intraperitoneally for only 3 days and concluded that aldosterone did not alter any parameter of Na+/H+ exchange in the distal colon while stimulating NHE3, but not NHE2, isoform-specific message, protein, and transport function in the proximal colon (7). We had previously observed that 3 days of continuous subcutaneous infusion of aldosterone did not modify electroneutral NaCl absorption in the distal colon, whereas 7 days resulted in its complete inhibition (18, 30). Because intraperitoneal administration of aldosterone for 3 days resulted in serum aldosterone levels that are 24% less than those observed in dietary sodium depletion, a physiological model of aldosterone excess, the present study was designed to provide a comprehensive characterization of the effects of dietary sodium depletion1 on Na+/H+ exchange in both proximal and distal segments of the rat colon.
The present results establish that sodium depletion alters both NHE2 and NHE3 isoforms at a pretranslational level in both proximal and distal segments; sodium depletion substantially increases both NHE2 and NHE3 function, protein, and message in the proximal colon but reduces NHE2 and NHE3 function, protein, and message in distal colon. In contrast, sodium depletion does not alter NHE1 isoform-specific function or message. Thus the differential effects of sodium depletion on Na+/H+ exchange are both isoform specific and tissue specific.
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METHODS |
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Membrane Vesicle Preparation
Apical membrane vesicles (AMV) and basolateral membrane vesicles (BLMV) were isolated from proximal and distal segments of the large intestine of both normal and aldosterone-treated rats (male, Sprague Dawley, 200-250 g) by the method of Stieger et al. (29) and Biber et al. (1), respectively, as described previously (23, 26, 27). Normal rats were fed with Purina rat chow ad libitum, whereas aldosterone rats were produced by feeding Na+-free diet (20 g · ratUptake Studies
Uptake of 22Na was performed for 6 s by the rapid filtration techniques, as both proton gradient-driven and potential-dependent 22Na uptake were linear for at least up to 8 s (23, 26, 27). Uptake studies were performed in at least three different membrane preparations. Results presented represent means ± SE of triplicate assays of typical experiments.mRNA Isolation
Total RNA from colonic epithelial cells were isolated by the CsCl cushioning centrifugation method of Sambrook et al. (28). In brief, proximal and distal colonic segments obtained from anesthetized rats were washed with ice-cold saline containing 0.5 mM dithiothreitol (DTT). Sacs of colonic segments filled with buffer A (4 mM HEPES-Tris, pH 7.6, 30 mM NaCl, 2 mM EDTA, and 0.5 mM DTT) were incubated in buffer B (buffer A without DTT) on ice. After 20-30 min of incubation, colonocytes were squeezed directly into 15-ml GIT buffer (4 M guanidineisothiocyanate, 25 mM Na+-acetate, and 0.85%cDNA Probes
NHE isoform-specific cDNA probes (NHE1: Pst I fragment that encodes nt 1237-2611; NHE2: Pvu II fragment that encodes nt 849-1590; and NHE3: Pst I fragment that encodes nt 1241-2522) were digested from their respective full-length cDNAs, generously provided by Dr. Gary E. Shull, University of Cincinnati, Cincinnati, OH (22, 33). Digestion products were electrophoresed on 1% agarose-ethidium bromide gel. Expected size cDNA fragments extracted using cDNA gel extraction kit (Qiagen, Chatsworth, CA) were used as NHE isoform-specific probes for Northern blot analyses. Glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was prepared using rat GAPDH control amplifier set (Clontech, Palo Alto, CA).Northern Blot
Five micrograms mRNA in 50% formamide-15% formaldehyde were electrophoresed on a 1% agarose-formaldehyde gel. Electrophoresed RNA was transferred to nylon membrane (NEN, Boston, MA) by capillary action and linked to membrane using UV-Stratalinker 2400 (Stratagene, La Jolla, CA). Blots were exposed for 2 h in prehybridization solution (50% deionized formamide, 10% dextran sulfate, 1 M NaCl, and 1% SDS) at 42°C. cDNA probes were labeled using [32P]dCTP and random primer labeling kit (Boehringer-Mannheim). 32P-labeled cDNA probes were added and hybridized for 18 h at 42°C. The blots were washed with 0.1× saline-sodium citrate-0.5% SDS at 45°C for 30 min and exposed to Hyperfilm (Amersham, Chicago, IL) atWestern Blot
SDS-PAGE was performed by the standard protocol as described earlier (26). To avoid aggregation, the protein samples (30 mg) of rat colonic apical and basolateral membranes were warmed to 37°C for 20 min before loading. Proteins were electrophoretically transferred from SDS-PAGE to nitrocellulose (Biotrace; Gelman Sciences, Ann Arbor, MI) in 192 mM glycine, 25 mM Tris, pH 8.3, and 20% methanol overnight at 30 V. After nonspecific sites were blocked with TBST consisting of 20 mM Tris, 137 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk, pH 7.5, immunostaining was performed with NHE2 and NHE3 isoform-specific antibodies (2, 19) and horseradish peroxidase-conjugated anti-rabbit IgG (Amersham) and anti-mouse IgG (Amersham), respectively. Antibody-specific bands were visualized by Supersignal enhanced chemiluminescence (Pierce Chemicals, Rockford, IL). Mouse monoclonal antibody againstStatistics
Results are reported as means ± SE. Student's t-test was used to determine statistical significance between two groups. For more than two-group comparison, a one-way ANOVA was performed. A P value of <0.05 was considered significant. ![]() |
RESULTS |
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Proximal Colon
22Na uptake studies. Previous studies of transepithelial 22Na fluxes across intact tissue established that dietary sodium depletion and aldosterone enhanced active Na+ absorption in rat proximal colon (13, 31). Therefore, experiments were designed to examine the mechanisms of this induction of electroneutral Na+ absorption by sodium depletion in rat proximal colon. In the initial studies, electroneutral Na+/H+ exchange activity was measured as outward proton gradient-driven 22Na uptake in both AMV and BLMV isolated from proximal colon of both normal and sodium-depleted rats. As shown in Fig. 1, the initial rate of proton gradient-driven 22Na uptake in AMV prepared from normal proximal colon was almost identical to that of our earlier results (23). Proton gradient-driven 22Na uptake in AMV from proximal colon of experimental rats was substantially higher than that in AMV prepared from normal rat proximal colon. Figure 1A presents the results of 22Na uptake by AMV from normal and sodium-depleted rats and demonstrated a 2.6-fold enhancement in the experimental group. In contrast, the rate of proton gradient-driven 22Na uptake in BLMV was similar in the two groups (Fig. 1B). Proton gradient-driven 22Na uptake was inhibited by 5 µM 5-ethylisopropylamiloride (EIPA), an amiloride analog that inhibits Na+/H+ exchange, in AMV of normal (37.2 ± 6.5 vs. 0.3 ± 0.4 pmol · mg protein
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Kinetic studies were performed to establish whether the
increase in proton gradient-driven
22Na uptake in AMV from proximal
colon of sodium-depleted rats is due to change in affinity
(Km) for
Na+ and/or the maximal
rate (Vmax) of
uptake. As shown in Fig. 2, increasing
extravesicular Na+ concentration
both increased and saturated proton gradient-driven 22Na uptake in AMV from proximal
colon of both normal and experimental rats. Lineweaver-Burke plot
analyses of proton gradient-driven 22Na uptake in AMV from proximal
colon of normal and sodium-depleted rats yielded
Km values for
Na+ of 8.3 ± 0.3 and 9.7 ± 0.4 mM (not significant) and
Vmax values of
4.8 ± 0.2 and 17.4 ± 5.7 nmol · mg
protein1 · 6 s
1
(P < 0.05), respectively (Fig.
2B). Significant increase in
Vmax (3.6 fold)
but not Km for
Na+ of the proton gradient-driven
22Na uptake suggests that sodium
depletion might have either enhanced the turnover rate of the existing
Na+/H+
exchange or induced the synthesis of new
Na+/H+
exchange proteins.
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Studies to distinguish NHE2 and NHE3 isoforms
activities. Studies were designed to identify whether
NHE2 and/or NHE3 isoforms are responsible for the enhancement
of
Na+/H+
exchange activity by sodium depletion in AMV from proximal colon. In
these studies, HOE-694 (3-methylsulfonyl-4-piperidinobenzoyl guanidine), an amiloride analog that inhibits the functional expression of NHE isoforms in a dose-dependent manner (9,
35),2
was used to estimate the several components of proton gradient-driven 22Na uptake by specific NHE
isoforms. In these experiments the effect of HOE-694 on
Na+/H+
exchange was determined by assessing its ability to alter the EIPA-sensitive component of proton gradient-driven
22Na uptake. As shown in Fig.
3B, proton
gradient-driven 22Na uptake in
BLMV was inhibited by 1 µM HOE-694 in both normal and
sodium-depleted groups. This fraction represents the NHE1 isoform and
is ~96% of total
Na+/H+
exchange function in BLMV. In contrast, proton gradient-driven 22Na uptake in AMV was not
significantly inhibited by 1 µM HOE-694 in either group (Fig.
3A), confirming that NHE1 isoform is
localized to basolateral, not apical, membranes and that sodium
depletion does not alter the membrane localization of NHE1
isoform.
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Figure 4 presents 25 µM
HOE-694-sensitive and 25 µM HOE-694-insensitive proton
gradient-driven 22Na uptake in AMV
of both normal and sodium-depleted rats. These HOE-694-sensitive and
HOE-694-insensitive components presented represent EIPA-sensitive
proton gradient-driven 22Na
uptake. On the basis of the inhibition of proton gradient-driven 22Na uptake by HOE-694, NHE3 is
the predominant NHE isoform present in AMV. Sodium depletion induced
both NHE2 isoform and NHE3 isoform activities, although not
proportionately. As a result, NHE2 isoform represents 11% of total
EIPA-sensitive proton gradient-driven 22Na uptake in normal rats,
whereas in the experimental group NHE2 isoform represents 31% of
EIPA-sensitive proton gradient-driven 22Na uptake. Thus sodium depletion
increased the NHE2 isoform component of proton gradient-driven
22Na uptake by 7.6-fold, whereas
the NHE3 isoform component was increased by only 2-fold. These results
suggest that both NHE2 and NHE3 isoforms are responsible for the
increase in
Na+/H+
exchange function in sodium-depleted rats.
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Western blot analyses. Although
previous studies have established that NHE2 and NHE3 isoform-specific
proteins are localized to the apical membrane of proximal colon of
normal rats (5, 6, 8), only NHE3 isoform-specific protein expression
was increased by aldosterone (7). Because the functional
22Na uptake studies (Fig. 4)
revealed that both NHE2 and NHE3 activities were increased by sodium
depletion, Western blot analyses were performed with apical membranes
isolated from proximal colon of normal and sodium-depleted rats using a
polyclonal antibody specific to NHE2 isoform (19) and a monoclonal
antibody specific to NHE3 isoform (2). Figure
5A
demonstrates that both NHE2 and NHE3 isoform-specific proteins were
expressed in apical membranes of proximal colon of normal rats. The
expression of both NHE2 and NHE3 isoform-specific proteins was
substantially increased by approximately threefold and sevenfold,
respectively (Fig. 5B), in apical
membranes from proximal colon of sodium-depleted rats. In contrast,
neither NHE2 nor NHE3 isoform-specific proteins were expressed in
basolateral membranes of normal or experimental animals (data not
shown).
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Northern blot analyses. Our
observations establish that sodium depletion markedly increases both
NHE2 and NHE3 activities and protein expression. Therefore, to
determine whether these observations are due to transcriptional events,
NHE isoform-specific mRNA abundance was examined by Northern blot
analyses. The results shown in Fig.
6A
demonstrate that NHE1, NHE2, and NHE3 cDNA probes hybridize with 4.8-, 4.4-, and 5.6-kb messages, respectively, in mRNA isolated from normal
rat proximal colon and confirm previous studies of the presence of NHE
isoform mRNA in rat colon (22, 33). Although all three NHE
isoform-specific mRNAs are also expressed in proximal colon of
sodium-depleted rats, only NHE2 and NHE3 mRNA abundances were
substantially increased in the experimental animals (Fig.
6A). As shown in Fig.
6B, quantitation of NHE
isoform-specific mRNA abundance using GAPDH as an internal control
revealed that sodium depletion enhanced both NHE2 and NHE3
isoform-specific mRNA levels by 1.9- and 3.0-fold, respectively. In
contrast, NHE1 isoform-specific mRNA level was not altered in
sodium-depleted rats (Fig. 6B).
These results suggest that sodium depletion stimulates electroneutral
Na+ absorption by enhancing apical
(i.e., NHE2 and NHE3), but not basolateral (i.e., NHE1),
membrane-specific NHE isoform-specific mRNAs in rat proximal colon via
pretranslational regulation.
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Distal Colon
Previous studies established that the effect of sodium depletion and aldosterone1 on Na+ transport in the distal colon differs qualitatively from their effects in proximal colon (13, 15). Aldosterone inhibits both electroneutral Na+ absorption in intact tissue (18) and Na+/H+ exchange in AMV (26). As a result, the experiments in the distal colon examined the effects of sodium depletion on NHE isoforms by determining NHE isoform-specific 22Na uptake, protein, and message in normal and sodium depleted-animals.22Na uptake
studies. Previous studies of proton gradient-driven
22Na uptake have been performed in
AMV from normal and sodium-depleted rats (26). Proton gradient-driven
22Na uptake was identified in AMV
from distal colon of both groups but in the sodium-depleted animals was
inhibited by low-dose amiloride (26), suggesting that proton
gradient-driven 22Na uptake in the
experimental animals represented proton diffusion-coupled electrogenic
uptake (26). To confirm this possibility, proton gradient-driven
22Na uptake under nonvoltage
clamped condition was assessed in AMV prepared from distal colon of
normal and sodium-depleted rats. First, the effect of 1 µM EIPA on
proton gradient-driven 22Na uptake
was determined (Fig.
7A).
Because 80% of proton gradient-driven 22Na uptake in AMV from normal
animals was inhibited by 1 µM EIPA, Na+/H+
exchange is responsible for the major fraction of Na uptake across AMV
in control rats. The results shown in Fig.
7A with benzamil, an amiloride analog
that blocks Na+ channels but does
not inhibit NHE activity, confirm our previous findings (26) that
Na+ channels are not present in
apical membrane of normal rat colonocytes. The effect of both EIPA and
benzamil on proton gradient-driven 22Na uptake in AMV from
sodium-depleted rats differs substantially from that seen in normal
rats. Figure 7B demonstrates that
proton gradient-driven 22Na uptake
was inhibited by benzamil by 95% but is not inhibited by EIPA. These
results indicate that
Na+/H+
exchange is present in AMV from distal colon of normal but not of
sodium-depleted rats, whereas in the sodium-depleted animals proton
gradient-driven 22Na uptake occurs
via apical membrane Na+
channels.
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To determine the NHE isoform(s) responsible for Na+ uptake across apical and basolateral membrane of normal rats, the effect of 1 µM HOE-694 on proton gradient-driven 22Na uptake was also examined. Figure 7A presents results that are similar to those seen in proximal colon. Proton gradient-driven 22Na uptake in AMV was not inhibited by 1 µM HOE-694; in contrast, proton gradient-driven 22Na uptake in BLMV of both normal and sodium-depleted animals was effectively abolished (data not shown). Thus proton gradient-driven 22Na uptake in AMV from distal colon is not mediated by NHE1 isoform but by NHE2 and/or NHE3 isoforms. In contrast, the proton gradient-driven 22Na uptake in BLMV is regulated by NHE1 isoform.
Studies to distinguish NHE2 and NHE3 isoforms
activities. To establish whether NHE2 and/or
NHE3 isoform is responsible for Na+ movement across apical
membrane, the effect of 25 µM HOE-694 on proton gradient-driven
22Na uptake in AMV from normal
distal colon was also determined under voltage-clamped conditions.
Similar to our earlier observation (26), voltage clamping did not
affect proton gradient-driven 22Na
uptake in AMV from normal rat, but substantially inhibited in AMV from
the sodium-depleted animal (12.6 ± 1.3 vs. 0.0 ± 0.4 pmol · mg
protein1 · 6 s
1).
Figure 8 demonstrates that 25% of proton
gradient-driven 22Na uptake in AMV
of normal rats was inhibited by 25 µM HOE-694, indicating that NHE2
isoform accounts for ~25% of basal apical membrane
Na+/H+
exchange activity. In contrast, ~75% of total proton gradient-driven 22Na uptake is insensitive to 25 µM HOE-694 and represents NHE3 isoform. Sodium depletion virtually
abolished NHE3 isoform component (Fig.
8B) while substantially reducing the
NHE2 isoform component of proton gradient-driven
22Na uptake (Fig.
8A).
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Western blot analyses. Western blot
analyses confirmed the presence of NHE2 and NHE3 isoform-specific
proteins in apical membranes of distal colon in normal rats (Fig.
9A).
These studies also established that sodium depletion decreased NHE2
isoform-specific protein expression by 33%, while NHE3
isoform-specific protein expression was decreased by 75% (Fig.
9B). These observations in sodium
depletion parallel directly with the results of
22Na uptake
studies.
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Northern blot analyses. To determine
whether the decrease in both NHE2 and NHE3 activities (Figs. 7 and 8)
and protein expression (Fig. 9) by sodium depletion in distal colon
represents transcriptional regulation, NHE isoform-specific mRNA
abundance was examined by Northern blot analyses. Figure
10A
presents NHE isoform-specific mRNA levels and reveals that NHE1, NHE2,
and NHE3 isoform-specific mRNAs are present in distal colon of normal
rats. Figure 10 also reveals that sodium depletion markedly reduced
NHE2 isoform-specific mRNA levels and almost completely abolished NHE3
isoform-specific mRNA levels. Sodium depletion did not alter NHE1
isoform-specific mRNA levels. The results with NHE isoform-specific
message parallel the observations that were obtained with HOE-694
inhibition of proton gradient-driven
22Na uptake and NHE
isoform-specific protein. This result differs substantially from the
changes in NHE2 and NHE3 isoform-specific mRNA abundance seen in
proximal colon of sodium-depleted animals (Fig. 6). Sodium depletion
upregulates NHE2 and NHE3 isoform-specific mRNA levels in proximal
colon but downregulates NHE2 and NHE3 isoform-specific message levels
in distal colon. The changes in NHE isoforms observed in both proximal
and distal colon appear to represent pretranslational regulation of
Na+/H+
exchange by dietary sodium depletion.
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DISCUSSION |
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There are several significant differences in the
characteristics and regulation of electroneutral NaCl absorption in
proximal and distal colon (3, 4, 13, 18, 23, 24). First, electroneutral
Na+ absorption in rat distal colon
is Cl-dependent, and net
Na+ absorption is approximately
equivalent to net Cl
absorption (13). The demonstration of
Na+/H+
exchange and
Cl
/HCO
3
exchange in isolated AMV suggested that electroneutral NaCl absorption
in distal colon occurs via the coupling of these two dual-ion exchanges
by intracellular pH (23, 24). Second, in contrast in proximal colon,
although electroneutral Na+
absorption is also
Cl
-dependent, net
Cl
absorption is not
present (13). Third, either sodium depletion or aldosterone inhibits
both Na+ and
Cl
absorption in distal
colon when studied in vitro across isolated colonic mucosa under
voltage clamp conditions, while inducing both net
Na+ and
Cl
absorption in proximal
colon (13, 18). Recent studies in proximal colon indicated that
aldosterone enhanced NHE3 isoform, but not NHE2 isoform, at a
pretranslational level (7). These studies did not reveal any effects of
aldosterone on NHE function in the distal colon.
The effect of glucocorticoids and aldosterone on
Na+ and
Cl absorption differs
strikingly in proximal colon (4). Although glucocorticoids and
aldosterone both enhance electroneutral NaCl absorption in proximal
colon of the rat, their effects are distinct and appear mediated by
separate receptors. Aldosterone stimulates both electroneutral NaCl
absorption that is inhibited by spironolactone, a mineralocorticoid
receptor antagonist, and electrogenic
K+ secretion (31). In contrast,
RU-28362, a glucocorticoid receptor-specific agonist, stimulates
spironolactone-insensitive NaCl absorption and does not affect
K+ transport (31). These
observations are best explained by separate and distinct corticosteroid
receptors that regulate the effects of glucocorticoids and aldosterone
on
Na+/H+
exchange in the proximal colon.
These present studies demonstrate that sodium depletion upregulates both NHE2 and NHE3 isoform-specific mRNA, protein, and Na+/H+ exchange activity in proximal colon (Figs. 4-6). In contrast, prior studies revealed that dexamethasone increased NHE3, but not NHE2 isoform-specific mRNA and protein abundance (8). Similar observations with methylprednisolone, a glucocorticoid agonist, have recently been reported in rabbit ileum (35, 36). As a result, it would appear that although both aldosterone and glucocorticoids increase Na+/H+ exchange activity, their effects on NHE isoform-specific mRNAs are not identical. Aldosterone affects both NHE2 and NHE3 isoforms, whereas glucocorticoids regulate only the NHE3 isoform.
The present results indicate that the NHE3 isoform,
which represents 89% of the EIPA-sensitive
Na+/H+
exchange in AMV (Fig. 4), is the primary NHE isoform responsible for
electroneutral Na+ absorption in
the proximal colon of normal rats. Because sodium depletion increased
NHE2 isoform-specific
Na+/H+
exchange activity considerably more than NHE3 isoform-specific Na+/H+
exchange function, we suspect that NHE2 isoform, but not NHE3 isoform,
is primarily responsible for the stimulation of electroneutral Na+ absorption by sodium depletion
in proximal colon. This possibility is further supported by the recent
observation that aldosterone induced
Na+ absorption in avian colon by
selectively stimulating the NHE2 isoform (11). Our results, however,
differ from those of Cho et al (7), who demonstrated that NHE3 isoform,
but not NHE2 isoform, message abundance was increased by aldosterone in
proximal colon of rat. This discrepancy may be due to different
experimental maneuvers used in these two studies. Cho et al. (7)
produced the hyperaldosterone state by intraperitoneal injection of
aldosterone for 3 days, while in the present study increased plasma
aldosterone levels were the result of 7 days of dietary sodium
depletion. Although previous studies have established that dietary
sodium depletion for 7 days and continuous subcutaneous infusion of
aldosterone via minipumps for 7 days produce identical changes of
colonic Na+ and
Cl transport
(15),1 it should be noted that
intraperitoneal administration of aldosterone for 3 days resulted in
serum aldosterone levels that are 24% less than those seen both in
dietary sodium depletion for 7 days and in animals that had received
continuous subcutaneous administration of aldosterone for 3 days (7);
it is not known whether enhancement of NHE2 isoform-specific abundance
requires elevated aldosterone levels for more than 3 days.
Similar to observations in proximal colon (Fig. 4), both NHE2- and NHE3-specific Na+/H+ exchange activities are expressed in AMV of the distal colon of normal rats (Fig. 8). NHE3 activity represents almost 87%, while the remaining EIPA-sensitive Na+/H+ exchange activity represents NHE2 isoform in AMV of normal distal colon. In contrast to proximal colon, sodium depletion inhibited both NHE2 and NHE3 isoform-specific mRNA abundance, protein expression, and Na+/H+ exchange activities but not NHE1 isoform-specific mRNA abundance and Na+/H+ exchange activity (Figs. 8-10). The inhibition of NHE2 isoform protein by sodium depletion was substantially less than that of Na+/H+ exchange activity and would be compatible with a decrease in turnover rate of NHE2 isoform protein. Although Cho et al. (7) demonstrated enhanced NHE3 message by aldosterone in proximal colon, they also reported that aldosterone did not have any effect on NHE isoform message and protein abundance in the distal colon. It is likely that their failure to observe changes in NHE isoform message and protein may also be related to the different methods used to enhance serum aldosterone levels and the duration during which aldosterone levels were elevated. Although dietary sodium depletion and subcutaneous administration of aldosterone for 7 days resulted in identical inhibition of both Na+/H+ exchange in AMV and electroneutral NaCl absorption in the distal colon, electroneutral NaCl absorption was not inhibited by 3 days of subcutaneous administration of aldosterone (18). Thus it is likely that the differences in observations between these present results and those of Cho et al. (7) are likely due to the shorter (3 days) period that aldosterone was administered and/or the lower serum aldosterone levels achieved by the intraperitoneal route of its administration.
The present data indicate that NHE isoforms that are localized in colonic apical membrane are regulated by aldosterone at a pretranslational level because these isoform-specific mRNA abundances, protein expressions, and Na+/H+ exchange activities were altered in parallel. It is highly likely that the effect of sodium depletion to stimulate NHE3 isoform-specific message, protein, and Na+/H+ exchange activities in rat proximal colon (Figs. 4-6; Ref. 7) is not a consequence of aldosterone's activation of the glucocorticoid receptor (31). Because the stimulation of electroneutral Na+ absorption by aldosterone in the proximal colon is inhibited by spironolactone (31), a mineralocorticoid-receptor antagonist, we conclude that both NHE2 and NHE3 isoforms are regulated at a pretranslational level by aldosterone following its interaction with the mineralocorticoid receptor in rat colon. However, the mechanism of regulation of apical membrane NHE isoforms by aldosterone differs in a tissue-specific pattern, because NHE2 and NHE3 mRNA abundance, protein expression, and Na+/H+ exchange activities are upregulated in proximal (Figs. 4-6), but downregulated in distal, colon (Figs. 8-10). More than one explanation could account for this tissue-specific differential regulation of NHE mRNA and Na+/H+ exchange activities. First, identical NHE isoforms are present in both proximal and distal colon but are under the control of different promoters. Therefore, it is likely that these differential effects of aldosterone in the proximal and distal colon are the result of the tissue-specific localization of different aldosterone-responsive promoters in proximal and distal colon. Second, it is also possible that apical membrane NHE isoforms expressed in proximal and distal colon represent highly homologous but not identical isoforms similar to that recently shown for anion exchange isoforms with NH2-terminal variations (34). However, this hypothesis will require further identification and sequencing for substantiation.
In summary, both NHE2 and NHE3 isoform-specific Na+/H+ exchange activities are present in AMV, while NHE1 isoform is present in BLMV of proximal and distal colon of normal rats. Sodium depletion inhibits electroneutral Na+ absorption by reducing both NHE2 and NHE3 isoforms in distal colon while it enhances Na+ absorption in proximal colon by stimulating both NHE2 and NHE3 isoforms. In contrast, NHE1 isoform in proximal or distal colon was not altered by sodium depletion. Regulation of apical membrane-specific NHE isoforms by dietary sodium depletion occurs at a pretranslational level and is both tissue specific and isoform specific.
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ACKNOWLEDGEMENTS |
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Prof. R. Gregor generously provided HOE-694. We thank Dr. Sarah Kolla for advice about cDNA probe preparation and Andrea Mann for performing the Western blot analyses.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-14669-25.
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.
1 Previous studies demonstrated identical changes in Na+ transport in dietary Na+-depleted animals and rats given aldosterone subcutaneously via osmotic minipumps (15). Serum aldosterone levels were equivalent in these two experimental groups (15). As a result, the experimental groups used in this present study are referred to as either aldosterone-treated or Na+-depleted rats.
2 HOE-694 inhibits 22Na uptake in PS120 cells that had been individually transfected with NHE1, NHE2, and NHE3 isoform cDNAs with half-maximal inhibitory concentration of 0.16 mM, 5 µM, and 650 µM, respectively (9). As a result of these previous observations, the NHE1 isoform in these present studies of 22Na uptake by colonic AMV is defined as proton gradient-driven 22Na uptake that is inhibited by 1 µM HOE-694. NHE2 isoform is defined as proton gradient-driven 22Na uptake inhibited by 25 µM HOE-694, and proton gradient-driven 22Na uptake that is insensitive to 25 µM HOE-694 represents NHE3 isoform activity.
Address for reprint requests: V. M. Rajendran, Dept. of Internal Medicine, Yale Univ. School of Medicine, Box 208019, 333 Cedar St., New Haven, Connecticut 06520.
Received 31 August 1998; accepted in final form 26 October 1998.
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