Am J Physiol Gastrointest Liver Physiol
281: G645–G653, 2001.
Glutamine transporter in crypts compensates for loss
of villus absorption in bovine cryptosporidiosis
ANTHONY BLIKSLAGER,1 ELAINE HUNT,2 RICHARD GUERRANT,3
MARC RHOADS,4 AND ROBERT ARGENZIO5
Departments of 5Anatomy, Physiological Sciences, and Radiology, 1Clinical Sciences and
2
Food Animal Health and Resource Management, College of Veterinary Medicine, North Carolina
State University, Raleigh 27606, 4Department of Pediatrics, School of Medicine, University of
North Carolina, Chapel Hill, North Carolina 27599; and 3Division of Geographic Medicine,
Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908
Received 16 January 2001; accepted in final form 9 May 2001
THE USE OF ORAL REHYDRATION solutions (ORS) containing
glucose has become one of the most important therapeutic advances in the last century for the treatment of
diarrheal disease. These ORS became well established
in the treatment of cholera and other enterotoxigenic
bacterial diarrheas that resulted in toxin-mediated increases in intestinal epithelial cAMP or cGMP but
produced no structural damage. The effect of raising
these intracellular second messengers was to inhibit
an electrically neutral NaCl absorptive mechanism
and to elicit anion secretion; however, the glucoselinked Na⫹ absorption on the small intestinal villus
remained intact. This physiological principle provided
the rationale for the use of oral glucose-containing
electrolyte solutions, which were capable of stimulating NaCl and water absorption, thereby counteracting
the second messenger-mediated events (37). Nevertheless, there are several potential difficulties with this
concept in diarrheal illnesses in which there is structural damage to the intestinal mucosa. For example,
the Na⫹-glucose cotransporter (SGLT1) has been localized to the oldest cells in the terminal villus (18). In
most enteric viral diseases and those caused by invasive or effacing organisms, including Cryptosporidium,
these are the first cells lost and last to be replaced in
the damage-repair cycle of the villus. Furthermore,
there is emerging evidence that SGLT1 may be specifically inhibited in certain viral infections (15, 21), even
if these are not accompanied by significant structural
damage (15).
Previous studies of cryptosporidiosis (3) or rotavirus
(21, 35) in piglet models have shown that indeed, the
Na⫹-glucose driven absorption is severely attenuated.
However, in both models (4, 35) it was shown that
luminally administered glutamine was capable of fully
restoring Na⫹ transport despite the villous atrophy.
Thus glutamine, a preferred fuel of the small intestine,
was able to powerfully stimulate Na⫹ absorption in
both these infections even though the absorptive epithelium on the villus was comprised exclusively of
juvenile cells. In the rotavirus model, alanine was
similarly as effective, suggesting that the effect of
glutamine was not simply the provision of a superior
metabolic substrate but might involve a regulation or
distribution of amino acid transport systems that differ
from the Na⫹-glucose transporter in infected tissue.
Na⫹-dependent amino acid transport systems are
complex and overlapping. For example, system ASC,
Address for reprint requests and other correspondence: R. Argenzio, Dept. of Anatomy, Physiological Sciences, and Radiology, College
of Veterinary Medicine, North Carolina State Univ., 4700 Hillsborough St. at William Moore Dr., Raleigh, NC 27606 (E-mail:
[email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
sodium absorption; sodium/hydrogen exchanger; villous atrophy; crypt hyperplasia; prostaglandin E2
http://www.ajpgi.org
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society
G645
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 14, 2017
Blikslager, Anthony, Elaine Hunt, Richard Guerrant, Marc Rhoads, and Robert Argenzio. Glutamine
transporter in crypts compensates for loss of villus absorption in bovine cryptosporidiosis. Am J Physiol Gastrointest
Liver Physiol 281: G645–G653, 2001.—Cryptosporidium parvum infection represents a significant cause of diarrhea in
humans and animals. We studied the effect of luminally
applied glutamine and the PG synthesis inhibitor indomethacin on NaCl absorption from infected calf ileum in Ussing
chambers. Infected ileum displayed a decrease in both mucosal surface area and NaCl absorption. Indomethacin and
glutamine or its stable derivative alanyl-glutamine increased
the net absorption of Na⫹ in infected tissue in an additive
manner and to a greater degree than in controls. Immunohistochemical and Western blot studies showed that in control animals neutral amino acid transport system ASC was
present in villus and crypts, whereas in infected animals,
ASC was strongly present only on the apical border of crypts.
These results are consistent with PGs mediating the altered
NaCl and water absorption in this infection. Our findings
further illustrate that the combined use of a PG synthesis
inhibitor and glutamine can fully stimulate Na⫹ and Cl⫺
absorption despite the severe villous atrophy, an effect associated with increased expression of a Na⫹-dependent amino
acid transporter in infected crypts.
G646
LOSS OF VILLUS ABSORPTION IN BOVINE CRYPTOSPORIDIOSIS
MATERIALS AND METHODS
Experimental animals were 1-day-old Holstein calves obtained from a local farm and housed in isolation facilities at
the College of Veterinary Medicine. All procedures were
approved by the University Animal Care and Use Committee. Calves were evaluated for colostral antibody status and
fed an antibiotic-free synthetic diet twice daily at a rate of
10% body wt/day (Purina kid milk replacer). Rectal swabs
were taken daily from all calves and examined for the presence of Cryptosporidium. All calves infected with Cryptosporidium were positive for the organism, and control calves
were always negative.
Purified Cryptosporidium parvum oocysts were obtained
from Pleasant Hill Farm (Troy, ID). An inoculum of 108
oocysts was given orally to the calves on day 7 of life. Control
and infected calves were killed on day 4 postinfection, a time
determined previously to be at the peak of diarrhea (unpublished observations). Calves were given a lethal overdose of
intravenous pentobarbital sodium, and sections of ileum beginning 10 cm proximal to the ileocecal valve were taken for
in vitro studies.
Tissue preparation. The section of ileum was opened along
the antimesenteric border in a dissection tray and bathed in
an oxygenated Ringer solution. The outer muscular layers
AJP-Gastrointest Liver Physiol • VOL
were removed by blunt dissection, and part of the tissue was
fixed in formalin for light microscopy and immunohistochemistry or snap frozen in liquid nitrogen for Western blot analysis.
Morphometric analysis. Formalin-fixed tissues were embedded in paraffin, cut in slices 5 ␮m thick, and stained with
hematoxylin and eosin for light microscopy. Four to five
well-oriented villi from eight infected and eight control calves
were measured to determine mean villus height and diameter. Crypt depth was also measured. Villus measurements
were converted to a three-dimensional parameter using
equations for a cylinder to yield villus surface area, as described previously (3).
Gel electrophoresis and Western blotting. Tissues were
stored at ⫺70°C before preparation for SDS-PAGE, at which
time they were thawed at 4°C. Tissue portions (1 g) were
added to 3 ml of chilled RIPA buffer (0.15M NaCl, 50 mM
Tris, pH 7.2, 1% deoxycholic acid, 1% Triton X-100, and 0.1%
SDS), including protease inhibitors. This mixture was homogenized on ice and centrifuged at 4°C, and the supernatant was saved. Protein analysis of extract aliquots was
performed (DC protein assay, Bio-Rad, Hercules, CA). Tissue
extracts (amounts equalized by protein concentration) were
mixed with an equal volume of 2⫻ SDS-PAGE sample buffer
and boiled for 4 min. Lysates were loaded on a 10% SDSpolyacrylamide gel, and electrophoresis was carried out according to standard protocols. Proteins were transferred to a
nitrocellulose membrane (Hybond ECL, Amersham Life Science, Birmingham, UK) using an electroblotting minitransfer apparatus according to the manufacturer’s protocol.
Membranes were blocked at room temperature for 60 min in
Tris-buffered saline plus 0.05% Tween 20 (TBST) and 5% dry
powdered milk. Membranes were washed twice with TBST
and incubated for 1 h in primary antibody (anti-ASCT-1,
Chemicon, Temecula, CA). After being washed three times
for 10 min each with TBST, the membranes were incubated
for 45 min with horseradish peroxidase-conjugated secondary antibody. After being washed three additional times for
10 min each with TBST, the membranes were developed for
visualization of protein by addition of enhanced chemiluminescence reagent according to the manufacturer’s instructions (Amersham, Princeton, NJ). Densitometry was performed on select blots using appropriate software.
Immunohistochemistry. Tissues were fixed in 10% neutral
buffered formalin, routinely processed for paraffin embedding, and cut into 5-␮m sections. After placement on slides,
sections were deparaffinized and rehydrated. Slides were
subsequently incubated in 3% H2O2, washed, and subjected
to Pronase digestion for 10 min. Slides were washed in PBS
and incubated with normal goat serum (Biogenex, San
Ramon, CA) for 20 min and then incubated for 1 h with rabbit
anti-human ASCT-1 polyclonal antibody (Chemicon). This
step was not performed on negative control slides. Slides
were washed four times in PBS between 20-min incubations
with biotinylated goat anti-rabbit antibody and streptavadinlabeled peroxidase (Biogenex, San Ramon, CA). Slides were
then placed in 3-amino-9-ethylcarbazole, washed in distilled
water, counterstained with 0.5% methyl green for 30 s, and
mounted.
Ussing chamber studies. Methods used in this laboratory
for in vitro studies have been described in detail previously
(2). Briefly, tissues stripped of the muscularis were mounted
in Ussing chambers and bathed on both surfaces with 10 ml
Ringer solution. Serosal glucose (10 mM) was osmotically
balanced with mucosal mannitol (10 mM). In some experiments, tissues were stripped and bathed in indomethacin (1
␮mol/l). In other experiments, 10 mM glutamine was added
281 • SEPTEMBER 2001 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 14, 2017
an Na⫹-dependent system for neutral amino acids, is
typically assigned to the basolateral membrane; however, studies of membrane vesicles from guinea pig
ileum have identified the ASC system as the major
Na⫹-dependent carrier for neutral amino acids in the
brush border (16). In addition, a recent reexamination
(31) of amino acid uptake across the mucosal membranes of intact rabbit ileum has shown that a lowaffinity system for glutamate uptake, previously
thought to represent system B, actually is the equivalent of system ASC. System ASC has also been shown
(32) to be responsible for part of the glutamine uptake
across the apical border of pig ileum. Theoretically, the
presence of ASC on both apical and basolateral membranes would allow the major fuel glutamine to be
taken up from either lumen or blood during digestive
or interdigestive periods, respectively, whereas vectorial transport could be accomplished by Na⫹-independent systems on the basolateral membranes.
Preliminary studies with isolated cells recently
showed that system ASC, which was credited with 50%
of the glutamine uptake, was not only present on villus
cells but also mechanistically expressed in isolated
crypt cells of the guinea pig small intestine (12). Similarly, studies (29) with the rat intestinal crypt cell line
IEC-17 have shown a low-affinity, Na⫹-dependent system resembling ASC to be present in these cells. Although cells in these latter studies do not display
polarized membranes, it is possible that an apically
located ASC transporter in the crypt of intact tissue
could compensate for diminished absorption on the
atrophic villus. The present study examines this hypothesis in a calf model of Cryptosporidium infection.
The calf was selected for study because it is the principal natural host for Cryptosporidium and exhibits a
profuse, watery diarrhea resembling the cholera-like
diarrhea in immunodeficient humans infected with
Cryptosporidium.
G647
LOSS OF VILLUS ABSORPTION IN BOVINE CRYPTOSPORIDIOSIS
fluxes from mucosa to serosa (Jm3s) and from serosa to
mucosa (Js3m) were calculated using standard equations. Gt
was calculated from the spontaneous PD and short-circuit
current (Isc) or by clamping the tissue at ⫾100 ␮A for 5 s and
recording the PD.
Statistical analysis. Data were analyzed using a paired
t-test for paired treatments or a one-way ANOVA for multiple comparisons followed by a Tukey’s test to determine
differences among treatments (Sigma Stat, Jandel Scientific,
San Rafael, CA).
RESULTS
to the mucosal side of the tissues, and this was balanced
with 10 mM mannitol added to the serosal solution. PGE2
(10⫺6 M) or bumetanide (10⫺4 M) was added to the serosal
solution in some experiments. Solutions were oxygenated
and circulated with 95% O2-5% CO2 in water-jacketed reservoirs maintained at 39°C.
The spontaneous potential difference (PD) was measured
with Ringer-agar bridges connected to matched calomel electrodes, and the PD was short-circuited through Ag-AgCl
electrodes, using an automatic voltage clamp that corrected
for fluid resistance. Tissues were given 30 min to equilibrate,
then the isotopes 22Na and 36Cl were added to the mucosal or
serosal reservoirs bathing paired tissues, which were
matched according to their conductance (Gt); if this differed
by ⬎25%, the tissues were discarded from analysis. After
allowing 20 min for isotopic equilibration, standards and
time 0 samples were taken from the reservoirs. Samples were
removed after 30 min (flux period 1), received treatment
(such as glutamine or PGE2 addition), and then underwent a
20-min equilibration period and a second 30-min flux period.
Samples were counted for 22Na in a crystal scintillation
counter and for 36Cl in a liquid scintillation counter. The
contribution of 22Na counts to 36Cl counts was determined
and subtracted from the total. Unidirectional Na⫹ and Cl⫺
Table 1. Effect of indomethacin on Na⫹ and Cl⫺ transport in control and infected calves
Na⫹
Control
Infected
Control ⫹ Indo
Infected ⫹ Indo
Cl⫺
n
Jm3s
Js3m
Jnet
Jm3s
Js3m
Jnet
ISC
Gt
18
9
15
7
10.6 ⫾ 0.5*
6.6 ⫾ 0.4†
10.8 ⫾ 0.5*
8.3 ⫾ 0.4‡
9.6 ⫾ 0.5*
6.2 ⫾ 0.3†
10.2 ⫾ 0.3*
6.3 ⫾ 0.4†
1.0 ⫾ 0.3*
0.5 ⫾ 0.2*
0.7 ⫾ 0.3*
2.0 ⫾ 0.2†
7.8 ⫾ 0.5*
4.8 ⫾ 0.4†
8.4 ⫾ 0.4*
6.2 ⫾ 0.4‡
6.1 ⫾ 0.4*
4.3 ⫾ 0.3†
6.9 ⫾ 0.3*
5.3 ⫾ 0.4†
1.7 ⫾ 0.3*
0.5 ⫾ 0.2†
1.5 ⫾ 0.2*
0.9 ⫾ 0.3*†
⫺0.2 ⫾ 0.1*
0.6 ⫾ 0.1†
⫺0.2 ⫾ 0.1*
0.5 ⫾ 0.1†
24.7 ⫾ 1.8*
12.8 ⫾ 0.6†
27.6 ⫾ 1.3*
14.8 ⫾ 0.8†
Values are means ⫾ SE; n ⫽ no. of animals. Fluxes and short-circuit current (Isc) are given in ␮eq 䡠 cm⫺2 䡠 h⫺1 and tissue conductance (Gt)
in mS/cm2. Indomethacin (Indo)-treated tissues were stripped and bathed in 10⫺6 M Indo. A 1-way ANOVA was used to compare treatment
means, followed by a post hoc Tukey’s test. Jm3s, mucosal-to-serosal flux; Js3m, serosal-to-mucosal flux; Jnet, net flux. Means with different
superscripts are significantly different from each other (P ⬍ 0.05).
AJP-Gastrointest Liver Physiol • VOL
281 • SEPTEMBER 2001 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 14, 2017
Fig. 1. Villus height and crypt depth of control and infected mucosa
on postinfection day 4. A 1-way ANOVA yielded significant differences in both villus height and crypt depth between control and
infected mucosa.
Cryptosporidium infection induces villous atrophy
and crypt hyperplasia. As shown in Fig. 1, ileal mucosa
from infected calves examined at the peak of diarrhea
(postinfection day 4) displayed shortened villi and
deepened crypts, which is characteristic of the entire
small intestine during Cryptosporidium infection in
these animals (17). Surface area calculations, based on
a three-dimensional construct of the villus, showed a
similarly reduced villus absorptive surface area (P ⬍
0.05).
Cryptosporidium infection alters ion transport and
Gt in calf ileum. Unidirectional and net ion fluxes,
together with the Isc and Gt for control and infected calf
ileum, are shown in Table 1. The infection reduced net
Cl⫺ absorption and increased Isc. Unidirectional fluxes
of Na⫹ and Cl⫺ were reduced in both directions, and
this was paralleled by a significant reduction in Gt. Net
Na⫹ absorption was reduced by one-half; however, this
effect was not statistically significant.
Because control tissue demonstrated no significant
basal Isc, whereas infected tissue showed a positive Isc,
we next treated the serosal side of infected tissue with
bumetanide (10⫺4 M), a specific inhibitor of the Na⫹K⫹-2Cl⫺ uptake process on the serosal membranes.
Bumetanide, at a dose shown to fully inhibit PG-stimulated Cl⫺ secretion in piglet ileum (4), reduced Isc
from 12 ⫾ 2 to 4 ⫾ 1 ␮A/cm2 (n ⫽ 3), illustrating that
the majority of Isc in infected tissue represented Cl⫺
secretion.
Indomethacin enhances ion absorption in infected
ileum. Previous studies with infected piglet ileum had
shown that elevated levels of tissue PGs inhibited Na⫹
and Cl⫺ absorption and induced anion (Cl⫺ and HCO3⫺)
secretion in that tissue (2). Therefore, the PG synthesis
inhibitor indomethacin was added to calf ileum to determine if endogenous PGs similarly altered the trans-
G648
LOSS OF VILLUS ABSORPTION IN BOVINE CRYPTOSPORIDIOSIS
Table 2. Effect of PGE2 on Na⫹ and Cl⫺ transport in control ileum
Na⫹
Control
⫹PGE2
P
Cl⫺
Jm3s
Js3m
Jnet
Jm3s
Js3m
Jnet
Isc
Gt
9.8 ⫾ 0.7
8.0 ⫾ 0.8
⬍0.01
8.9 ⫾ 0.8
8.3 ⫾ 0.8
NS
1.0 ⫾ 0.2
⫺0.3 ⫾ 0.2
⬍0.01
7.5 ⫾ 0.8
4.3 ⫾ 0.6
⬍0.001
5.6 ⫾ 0.6
4.6 ⫾ 0.3
NS
1.9 ⫾ 0.4
⫺0.3 ⫾ 0.6
⬍0.005
0.1 ⫾ 0.1
0.7 ⫾ 0.1
⬍0.001
21.3 ⫾ 2.4
17.1 ⫾ 1.5
NS
Values are means ⫾ SE of n ⫽ 10 animals. Fluxes and Isc are given in ␮eq 䡠 cm⫺2 䡠 h⫺1 and Gt in mS/cm2. A paired t-test was used to compare
treatment means. PGE2 (10⫺6 M) was added to the serosal side of the tissue for the second flux period. NS, not significant.
AJP-Gastrointest Liver Physiol • VOL
Similar to control tissue, both glutamine and alanylglutamine increased net Na⫹ transport and Isc with
equal potency in the infected tissue. Indomethacin
increased net Na⫹ transport without affecting Isc. AdDownloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 14, 2017
port of these ions. The effect of indomethacin addition
to control and infected ileum is shown in Table 1. In
control tissue, ion fluxes or electrical parameters were
unaltered by indomethacin. In contrast, indomethacin
added to infected tissue increased both net Na⫹ and
Cl⫺ to values significantly greater (Na⫹) or equal (Cl⫺)
to control values. These increases in net absorption of
Na⫹ and Cl⫺ were the result of significant increases in
the Jm3s of these ions; flux in the opposite direction
was not significantly affected. However, indomethacin
had no effect on the altered Isc or Gt induced by the
infection.
Exogenous PGs inhibit ion absorption in control ileum. Because the addition of indomethacin had no
significant effect in control tissue, we added PGE2 to
control tissue to determine if this maneuver would
mimic the altered transport of the infection. As shown
in Table 2, PG addition abolished net Na⫹ and Cl⫺
absorption. This effect was the result of significant
decreases in Jm3s of these ions; Js3m was not significantly affected. PG addition also increased Isc; Gt was
not significantly affected. Thus the PGs induced an
impairment in Na⫹ and Cl⫺ absorption in control ileum that was qualitatively similar to the altered transport of the infection.
Glutamine enhances net Na⫹ absorption in infected
ileum. Previous studies (4) with the infected piglet had
shown that glutamine was more effective than glucose
in stimulating Na⫹ absorption. Because glutamine is
reported to be unstable in solutions at room temperature, we also compared the effect of alanyl-glutamine; a
stable dipeptide reported to be extensively hydrolyzed
by mucosal enzymes in the intestine (26). Figure 2
shows the effect of equimolar glutamine and alanylglutamine on net Na⫹ and Cl⫺ transport and Isc in
control and infected tissue. The effect of glutamine
combined with indomethacin was also examined. In
control tissue (Fig. 2), both glutamine and the dipeptide increased net Na⫹ absorption by ⬃1 ␮eq䡠cm⫺2 䡠h⫺1.
A significant increase in Isc was also observed with
both compounds, which was equivalent to the net increase in Na⫹ absorption (1 ␮eq 䡠 cm⫺2 䡠 h⫺1). Cl⫺ transport was not significantly affected. As shown before
(Table 1), indomethacin alone had no effect on NaCl
fluxes or Isc. The addition of indomethacin and glutamine together produced no further stimulation of
Na⫹ or Cl⫺ absorption than glutamine alone and even
appeared to reduce glutamine-stimulated Na⫹ absorption and significantly reduced glutamine-induced Isc
(P ⬍ 0.001).
Fig. 2. Effect of glutamine (Gln), alanyl-Gln (Ala-Gln), indomethacin,
and Gln plus indomethacin on net NaCl fluxes (J), short-circuit current
(Isc), and conductance (Gt) in control and infected calf ileum. A control
flux period with normal Ringer (NR control) or indomethacin Ringer (IR
control) was followed by a second flux period in which 10 mM Gln or
Ala-Gln were added to the mucosal side of the tissues. Significant
changes in net transport were accompanied by significant changes in
mucosal-to-serosal fluxes, whereas changes in the opposite direction
were not significantly affected (data not shown). A 1-way ANOVA was
used to compare treatments followed by a post hoc Tukey’s test. Data
are means ⫾ SE; n ⫽ no. of animals; NS, not significant.
281 • SEPTEMBER 2001 •
www.ajpgi.org
LOSS OF VILLUS ABSORPTION IN BOVINE CRYPTOSPORIDIOSIS
Fig. 4. Western blot of ASCT-1 transport protein in control and
infected calf ileum. A dense band in the region of 60–65 kDa was
apparent in all tissue extracts. Densitometric analysis of n ⫽ 3 blots
using tissues from different animals showed that the density of the
ASCT-1 band in control tissue stripped and bathed in indomethacin
was significantly less than in tissues bathed in Ringer (* P ⬍ 0.05).
transporter in control and infected tissue. The commercial antibody used is raised against a neutral amino
acid transporter, ASCT-1, part of a family of structurally related glutamate transporters. As shown in Fig.
4, densitometric analysis of the Western blots showed
similar transporter protein expression in control and
infected tissue after equal loading of tissue protein
(n ⫽ 3). As also shown in Fig. 4, indomethacin significantly reduced the expression of the ASC protein in
control tissue (P ⬍ 0.05), but had no such effect in
infected tissue.
As shown for control tissue in Fig. 5, D and G, the
ASC system was expressed on both basal and apical
borders of villus epithelia. The protein was also detected in the crypts, but with diminished intensity (Fig.
5E). Intense staining for ASC in infected tissue was
seen on the apical border of the crypt epithelium (Fig.
5F). Very little staining could be seen on the infected
villus epithelium, although it could be detected on
some cells. Tissues stained with secondary antibody
alone (negative controls) showed no evidence of stain
uptake (Fig. 5, A–C).
DISCUSSION
Fig. 3. Effect of PGE2 replacement on basal Isc and Gln-stimulated
Isc in indomethacin-treated tissues. After tissues bathed in normal or
indomethacin Ringer were mounted, PGE2 at the concentrations
shown was added to the serosal side of indomethacin-treated tissues
and after a 20-min equilibration period, Isc was averaged for a
30-min period (period 1). Gln (10 mM) was then added to the mucosal
side of the tissues, and, after a 20-min equilibration, Isc was averaged
over the next 30 min (period 2). The Gln stimulation of Isc seen in
normal Ringer was inhibited in indomethacin Ringer; however, addition of PGE2 reversed the inhibition, and, at concentrations of 10⫺6
M, the Gln-stimulated Isc was fully restored (P ⬍ 0.05, n ⫽ 4).
AJP-Gastrointest Liver Physiol • VOL
Cryptosporidiosis is a zoonotic disease and a major
cause of diarrhea in calves and humans (13). Presently,
there is no effective antimicrobial treatment, and therapy is restricted to oral or intravenous rehydration.
The pathophysiology of the diarrhea is complex. Detailed studies of the piglet model of cryptosporidiosis
have identified an impairment in Na⫹-coupled glucose
absorption, presumed due to villous atrophy (3). In
addition to this structural pathogenesis, increased tissue PG levels were shown to mediate inhibition of an
electrically neutral NaCl absorptive process and to
elicit an electrogenic Cl⫺ and HCO3⫺ secretory process
(2). Thus net Na⫹ absorption was strongly inhibited.
281 • SEPTEMBER 2001 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 14, 2017
dition of both indomethacin and glutamine further
stimulated net Na⫹ absorption to a value seven times
greater than the basal level and to a value now even
significantly greater than the similarly treated control
(P ⬍ 0.01). In further contrast to control tissue, the
glutamine-induced Isc was unaffected by indomethacin.
Neither glutamine nor alanyl-glutamine by themselves
significantly altered net Cl⫺ transport; however, the
further addition of indomethacin induced a significant
increase in net Cl⫺ absorption compared with glutamine alone. Thus these present figures indicate that
the combined glutamine and indomethacin-induced increase in total net ion absorption in infected tissue was
3.5 ⫾ 0.09 vs. 0.42 ⫾ 1.2 ␮eq 䡠 cm⫺2 䡠 h⫺1 in the control
(P ⬍ 0.05).
PGE2 reverses indomethacin-induced inhibition of
control Isc. The above studies showed that indomethacin paradoxically inhibited the glutamine-induced Isc
in control tissue but had no such effect in the infected
ileum. To determine if the inhibitory effect of indomethacin observed in control ileum was due to a nonspecific effect of the inhibitor, tissue bathed in indomethacin was pretreated with varying concentrations
of PGE2 before addition of glutamine. As shown in Fig.
3, PGE2 dose dependently reversed the indomethacininduced inhibition of Isc.
Western blot and immunohistochemistry for ASC system. In an attempt to account for the augmented stimulation of Na⫹ transport induced by glutamine in the
infected atrophic tissue, we performed Western blot
analyses and immunohistochemistry for the ASC
G649
G650
LOSS OF VILLUS ABSORPTION IN BOVINE CRYPTOSPORIDIOSIS
As shown for several species, including humans,
pigs, and rabbits, the neutral NaCl absorptive process
represents two apically located independent Na⫹/H⫹
and Cl⫺/HCO3⫺ exchange mechanisms, whose degree of
coupling is dependent on the intracellular pH and
whose action in the absence of substrate-coupled Na⫹
absorption accounts for most, if not all, of the net Na⫹
and Cl⫺ absorption in the ileum of these species (9).
PGE2 and other cAMP agonists are capable of inhibiting the action of these neutral exchanges, possibly by
action involving protein kinase A-mediated phosphorylation of the Na⫹/H⫹ exchanger (27). In contrast,
cAMP elevations in the crypt generally induce Cl⫺
secretion by phosphorylating the apical Cl⫺ channel; in
some instances, HCO3⫺ secretion is also activated, but
by a poorly understood mechanism (9). In piglet ileum,
both the PG-mediated anion secretory processes of Cl⫺
AJP-Gastrointest Liver Physiol • VOL
and HCO3⫺ are electrogenic, and in the absence of
Na⫹-coupled substrate absorption, were shown to fully
account for Isc (4).
The source of the elevated tissue PGs in Cryptosporidium infections has not been definitively established
but may be the result of infiltrating macrophages and
polymorphonuclear neutrophils (3, 20), whose products
have been shown to induce PG synthesis from mesenchymal cells in the lamina propria (6). Furthermore,
recent studies of human intestinal epithelial cell lines
showed that C. parvum infection directly activates
PGH synthase 2 expression and PG production by
these cells (23). Thus PGs could act on villus epithelium in an autocrine or paracrine manner. The present
studies in the calf are consistent with a PG-mediated
inhibition of neutral NaCl absorption. Thus indomethacin treatment of infected tissue restored net rates of
281 • SEPTEMBER 2001 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 14, 2017
Fig. 5. Immunohistochemical analysis of control and infected calf ileum for ASCT-1 protein. Control tissue showed
stain uptake of upper (D) and lower villus and crypt (E) and on both apical and basal membranes (G). Infected
ileum showed intense staining that can clearly be seen only on apical border of crypt epithelium (F). Negative
controls showed no stain uptake (A–C). Scale bar for A–E ⫽ 100 ␮m; scale bar for G ⫽ 50 ␮m.
LOSS OF VILLUS ABSORPTION IN BOVINE CRYPTOSPORIDIOSIS
AJP-Gastrointest Liver Physiol • VOL
calf and the full stimulation of Na⫹-coupled glutamine
absorption in the calf despite a clear loss of cells on the
villus. One explanation for these differing results may
be related to the enterocyte life span. For example,
studies with tritiated thymidine showed that the age of
an enterocyte at the time of sloughing at the villus tip
is 9 days in the neonatal pig, whereas it is shortened to
3 days in the adult (28). Thus the relatively older
epithelium on the neonatal villus may be capable of
expressing the transporter at a more proximal site on
the crypt-villus axis. Indeed, autoradiographic studies
(38) have shown that enterocytes all along the cryptvillus axis take up alanine in the neonatal pig, whereas
in the adult rabbit alanine uptake was confined to the
villus tip. Although the present immunohistochemical
studies in the control calf are consistent with this
possibility, one would expect a compensatory increase
in crypt cell production and a more rapid migration of
crypt cells onto the villus in a repairing epithelium
(39), which might offset the normally longer life span of
the neonatal enterocyte.
An alternative explanation for our results is a sitespecific increase in the expression of the Na⫹ transporters in the infection. Such region-specific upregulation has been demonstrated for NHE3 in villus
epithelium of dexamethasone-treated rats (11). In
these studies (11), glucocorticoids increased the levels
of mRNA transcript for NHE3, which correlated with
increased Na⫹ uptake into apical membrane vesicles
(effects that were restricted to ileum and proximal
colon). In studies of the streptozocin-diabetic rat, Na⫹dependent glutamine transport in brush-border membrane vesicles was augmented by a mechanism increasing the maximal rate of specific uptake with no
change in the affinity constant (42). A similar adaptive
upregulation of glutamine transport by brush-border
vesicles was demonstrated in the short bowel syndrome in human patients treated with human growth
hormone (19). Thus evidence exists for hormonal regulation of both the Na⫹/H⫹ exchanger and Na⫹-coupled glutamine transporter.
Based on the present immunohistochemical results,
we propose that at least part of the effective stimulation of Na⫹ absorption lies in an increased expression
of Na⫹-dependent transporters in the crypts of the
infected intestine. The detection of ASC immunostaining in the crypts of control tissue suggests that system
ASC is normally expressed to some extent in the
crypts. However, in contrast to control tissue, infected
calf ileum showed a marked increase in the intensity of
the stain on the luminal border of crypt epithelium,
whereas very little signal was present on the infected
villus. We are uncertain as to why there is an absence
of stain on the infected villus, but it may be related to
the apical membrane invagination of villus cells during
parasite attachment (25). We have not seen parasites
infecting crypt cells in the ileum of calves or piglets,
whereas quantitative analysis of piglet ileum showed
that 71% of the villus cells were infected (4). Western
blotting also showed that ASC protein was present in
similar concentrations in homogenized control or in281 • SEPTEMBER 2001 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 14, 2017
NaCl absorption to normal, whereas addition of PG to
control tissue inhibited the net absorption of these
ions. Interestingly, in contrast to the pig model (4),
Na⫹ and Cl⫺ transport processes appear loosely coupled because indomethacin strongly increased neutral
Na⫹ absorption, whereas its effect on net Cl⫺ absorption was less dramatic and not consistently significant.
Furthermore, indomethacin failed to abolish the bumetanide-sensitive Isc in infected calf ileum, suggesting that as-yet-unidentified, noncyclooxygenase products contribute to Cl⫺ secretion in this tissue.
The current studies of infected tissue also showed
that luminal glutamine plus indomethacin stimulated
Na⫹ absorption in an additive manner and emphasize
the finding that despite the villous atrophy, maximal
rates of NaCl absorption can be established. Similar
results were obtained with glutamine in the Cryptosporidium-infected piglet (4); however, the mechanism of
the glutamine-stimulated events appears to differ in
the two species. In the piglet models of both rotavirus
(35) and Cryptosporidium (4), the primary effect of
glutamine was to strongly stimulate electroneutral
NaCl absorption. For example in indomethacintreated, Cryptosporidium-infected tissue, glutamine
stimulated net Na⫹ absorption by 4.3 ⫾ 0.7 ␮eq 䡠 cm⫺2 䡠
h⫺1, whereas the increase in Isc was only 0.8 ⫾ 0.1
␮eq 䡠 cm⫺2 䡠 h⫺1 (4). In contrast, in the present study of
the calf, the net increase in Na⫹ absorption induced by
glutamine was numerically equal to the glutaminestimulated Isc, and no significant net change in Cl⫺
absorption was demonstrable.
Recently, Abely et al. (1) calculated that the relative
magnitude of the two Na⫹ absorptive processes stimulated by glutamine can vary from 0.10 to 3.5 (neutral/
electrogenic) according to animal species, age, or
pathological condition. For example, in rabbit ileum
infected with an attaching, effacing strain of Escherichia coli (RDEC-1), glutamine stimulation of Na⫹
absorption was entirely electrogenic (33). However, in
contrast to the present results, the magnitude of this
process (33) was greatly reduced from the control and
there also was loss of neutral NaCl absorption as well
as the Na⫹-dependent glucose absorptive processes in
these adult rabbits. These results with infected rabbit
ileum (33) are consistent with the location of the Na⫹
absorptive mechanisms on the crypt-villus axis in rabbit intestine. Recent in situ hybridization and immune
complex studies (7, 14, 18) of adult rodent and rabbit
intestine have shown that the messages for both
SGLT1 and Na⫹/H⫹ exchanger 3 (NHE3), the Na⫹/H⫹
exchange isoform thought to mediate vectorial Na⫹
transport, are found only in villus cells with expression
of the proteins in the most mature cells at the tip of the
villus. These results support the paradigm of nutrient
and Na⫹ absorptive processes residing on the most
mature cells and could explain the impaired Na⫹ absorption in infections associated with villus cell loss
and replacement with immature crypt-like cells.
Nevertheless, this hypothesis does not explain the
completely intact neutral Na⫹ absorption unmasked by
indomethacin in the Cryptosporidium-infected pig and
G651
G652
LOSS OF VILLUS ABSORPTION IN BOVINE CRYPTOSPORIDIOSIS
AJP-Gastrointest Liver Physiol • VOL
cially available. It is also noteworthy that glutamine
and alanyl-glutamine have both been shown to enhance epithelial repair in several models of intestinal
injury (5, 22, 24, 40) and to stimulate epithelial proliferation or reduce apoptosis in cell culture (8, 34). Thus
increased uptake of oral glutamine in the crypts not
only could promote a compensatory increase in Na⫹
absorption but also would place this nutrient in the
ideal location to promote crypt cell production and
restoration of the villus architecture.
This work was supported by United States Department of Agriculture Grant 2000-02092 and by a grant from the North Carolina
State University College of Veterinary Medicine.
REFERENCES
1. Abely M, Dallet P, Boisset M, and Desjeux JF. Effect of
cholera toxin on glutamine metabolism and transport in rabbit
ileum. Am J Physiol Gastrointest Liver Physiol 278: G789–G796,
2000.
2. Argenzio RA, Lecce J, and Powell DW. Prostanoids inhibit
intestinal NaCl absorption in experimental porcine cryptosporidiosis. Gastroenterology 104: 440–447, 1993.
3. Argenzio RA, Liacos JA, Levy ML, Meuten DJ, Lecce JG,
and Powell DW. Villous atrophy, crypt hyperplasia, cellular
infiltration, and impaired glucose-Na⫹ absorption in enteric
cryptosporidiosis of pigs. Gastroenterology 70: 1156–1160, 1990.
4. Argenzio RA, Rhoads JM, Armstrong M, and Gomez G.
Glutamine stimulates prostaglandin-sensitive Na⫹-H⫹ exchange in experimental porcine cryptosporidiosis. Gastroenterology 106: 1418–1428, 1994.
5. Blikslager AT, Rhoads JM, Bristol DG, Roberts MC, and
Argenzio RA. Glutamine and transforming growth factor-␣
stimulate extracellular regulated kinases and enhance recovery
of villous surface area in porcine ischemic-injured intestine.
Surgery 125: 186–194, 1999.
6. Bern MJ, Sturbaum CW, Karayalcin SS, Berschneider H,
Wachsman JT, and Powell DW. Immune system control or rat
and rabbit colonic electrolyte transport. Role of prostaglandins
and the enteric nervous system. J Clin Invest 83: 1810–1820,
1989.
7. Bookstein C, DePaoli AM, Xie Y, Niu P, Musch MW, Rao
MC, and Chang EB. Na/H exchangers, NHE-1 and NHE-3, of
rat intestine. Expression and localization. J Clin Invest 93:
106–113, 1994.
8. Brito GC, Alcantara CS, and Guerrant RL. Stable glutamine
derivatives reduce apoptosis in intestinal epithelial cells and
inhibit cholera toxin-induced secretion. Am J Trop Med Hyg 62:
367–368, 2000.
9. Chang EB and Rao MC. Intestinal water and electrolyte transport. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited
by Johnson LR. New York: Raven, 1994, p. 2027–2081.
10. Cheeseman CI. Upregulation of SGLT1 transport activity in
rat jejunum induced by GLP-2 infusion in vivo. Am J Physiol
Regulatory Integrative Comp Physiol 273: R1965–R1971, 1997.
11. Cho JH, Musch MW, DePaoli AM, Bookstein CM, Xie Y,
Burant CF, Rao MC, and Chang EB. Glucocorticoids regulate
Na⫹/H⫹ exchange expression and activity in region- and tissuespecific manner. Am J Physiol Cell Physiol 267: C796–C803,
1994.
12. Del Castillo JR, Sulbaran MC, and Burguillos LO. Identification of a novel Na⫹-dependent transport mechanism, highly
specific for glutamine, in isolated intestinal villus and crypt cells
(Abstract). Gastroenterology 118: A70, 2000.
13. Fayer R and Ungar BLP. Cryptosporidium spp. and cryptosporidiosis. Microbiol Rev 50: 458–483, 1986.
14. Ferraris RP, Villenas SA, Hirayama BA, and Diamond J.
Effect of diet on glucose transporter site density along the cryptvillus axis. Am J Physiol Gastrointest Liver Physiol 262: G1060–
G1068, 1992.
15. Halaihel N, Lievin V, Alvarado F, and Vasseur M. Rotavirus
infection impairs intestinal brush-border membrane Na⫹-solute
281 • SEPTEMBER 2001 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 14, 2017
fected mucosa despite the loss of one-half of the villus
surface area. Together, these findings strongly suggest
an adaptive increase in expression of ASC protein and
glutamine transport by the hyperplastic crypts of infected calf intestine. Whether a similar explanation
applies to the Na⫹/H⫹ exchanger requires further examination; however, the strong stimulation of neutral
Na⫹ absorption by indomethacin to levels even greater
than in control tissue (Table 1 and Fig. 2), suggests
that electroneutral Na⫹ transporters are upregulated
during infection even though their action is suppressed
by the elevated PGs.
An interesting, but as yet unresolved finding in this
study was the inhibitory effect of indomethacin on the
glutamine transporter in control villus, whereas no
such inhibition was seen for the glutamine transporter
in the infected crypts. These inhibitory effects appeared due to the absence of basal PG production based
on the PGE2 replacement studies. There may be at
least two plausible explanations for this phenomenon.
First, it is possible that elimination of the housekeeping PGs renders transporters on the villus more susceptible to a luminal injury by indomethacin than the
better-protected transporters in the crypt (41). Alternatively, PGs may regulate trafficking or action of the
ASC transporter. For example, there is evidence that
PGE2 and other cAMP agonists can acutely upregulate
SGLT1 expression on the villus by a mechanism that
may be related to increased trafficking of SGLT1 from
an intracellular pool to the brush-border membrane
(10, 36). cAMP has also been shown (30) to stimulate
system ASC in rat hepatocytes, but by a mechanism
involving hyperpolarization of the membrane potential. The failure of indomethacin to attenuate Na⫹glutamine cotransport in the infected calf tissue could
be explained by the presence of alternative cAMP stimulants in the infected intestine, a possibility consistent
with the failure of indomethacin to abolish the Cl⫺
secretory process. Obviously, further study is needed to
distinguish among these possibilities.
In summary, the present study has shown that maximal rates of NaCl absorption can be stimulated in
infected calf intestine with the combined use of a PG
synthesis inhibitor and luminally administered glutamine. This maneuver allowed uninhibited operation
of the neutral Na⫹ absorptive mechanism as well as
maximal stimulation of the electrogenic process. The
addition of a nonselective cyclooxygenase inhibitor to
ORS may be contraindicated because of the potential
deleterious effects on the mucosal barrier. The use of
selective cyclooxygenase inhibitors, however, is worthy
of further examination because it may be possible to
preserve PG-mediated cytoprotection while eliminating their inhibitory effect on the neutral NaCl absorptive mechanisms. Similarly, potential problems with
the use of glutamine exist, because glutamine is relatively unstable in solutions kept at room temperature.
However, the stable dipeptide alanyl-glutamine was
shown to be as effective as glutamine in stimulating
Na⫹ absorption. Thus the dipeptide may be advantageous for use in ORS as soon as it becomes commer-
LOSS OF VILLUS ABSORPTION IN BOVINE CRYPTOSPORIDIOSIS
16.
17.
18.
19.
20.
22.
23.
24.
25.
26.
27.
28.
AJP-Gastrointest Liver Physiol • VOL
29. Mordrelle A, Huneau JF, and Tome D. Sodium-dependent
and -independent transport of L-glutamate in the rat intestinal
crypt-like cell line IEC-17. Biochem Biophys Res Commun 233:
244–247, 1997.
30. Moule SK, Bradford NM, and McGivan JD. Short-term stimulation of Na⫹-dependent amino acid transport by dibutyryl
cyclic AMP in hepatocytes. Characteristics and partial mechanism. Biochem J 241: 737–743, 1987.
31. Munck BG and Munck LK. Effects of pH changes on systems
ASC and B in rabbit ileum. Am J Physiol Gastrointest Liver
Physiol 276: G173–G184, 1999.
32. Munck LK, Grondahl ML, Thorboll JE, Skadhauge E, and
Munck BG. Transport of neutral, cationic and anionic amino
acids by systems B, bo,⫹, XAG, and ASC in swine small intestine.
Comp Biochem Physiol A Physiol 126: 527–537, 2000.
33. Nath SK, Dechelotte P, Darmaun D, Gotteland M, Rongier
M, and Desjeux JF. [15N] and [14C]glutamine fluxes across
rabbit ileum in experimental bacterial diarrhea. Am J Physiol
Gastrointest Liver Physiol 262: G312–G318, 1992.
34. Rhoads JM, Argenzio RA, Chen W, Rippe RA, Westwick
JK, Cox AD, Berschneider HM, and Brenner DA. L-glutamine stimulates intestinal cell proliferation and activates mitogen-activated protein kinases. Am J Physiol Gastrointest Liver
Physiol 272: G943–G953, 1997.
35. Rhoads JM, Keku EO, Quinn J, Woosely J, and Lecce JG.
L-glutamine stimulates jejunal sodium and chloride absorption
in pig rotavirus enteritis. Gastroenterology 100: 683–691, 1991.
36. Scholtka B, Stumpel F, and Jungermann K. Acute increase,
stimulated by prostaglandin E2, in glucose absorption via the
sodium dependent glucose transporter-1 in rat intestine. Gut 44:
490–496, 1999.
37. Schultz SG. Sodium-coupled solute transport by the small intestine: a status report. Am J Physiol Endocrinol Metab Gastrointest Physiol 233: E249–E254, 1977.
38. Smith MW. Autoradiographic analysis of alanine uptake by
newborn pig intestine. Experientia 37: 868–870, 1981.
39. Thake DC, Moon HW, and Lambert G. Epithelial cell dynamics in transmissible gastroenteritis of neonatal pigs. Vet Pathol
10: 330–341, 1973.
40. Tremel H, Kienie B, Weilemann LS, Stehle P, and Furst P.
Glutamine dipeptide-supplemented parenteral nutrition maintains intestinal function in the critically ill. Gastroenterology
107: 1595–1601, 1994.
41. Uribe A, Alam M, and Soderman C. Cell kinetic events in
early indomethacin-induced gastrointestinal ulcerations in the
rat. Eur J Gastroenterol Hepatol 9: 267–273, 1997.
42. Van Voorhis K, Said HM, Abumrad N, and Ghishan FK.
Effect of chemically-induced diabetes mellitus on glutamine
transport in rat intestine. Gastroenterology 98: 862–866, 1990.
281 • SEPTEMBER 2001 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 14, 2017
21.
cotransport activity in young rabbits. Am J Physiol Gastrointest
Liver Physiol 279: G587–G596, 2000.
Hayashi K, Dojo S, Nakashima K, Nishio E, Kurushima H,
Saeki M, Amioka H, Hirata Y, Ohtani H, and Hiraoka M.
Analysis of neutral amino acid transport systems in the small
intestine: a study of brush border membrane vesicles. Gastroenterol Jpn 26: 287–293, 1991.
Heine J, Pohlenz FL, Moon HW, and Woode GN. Enteric
lesions and diarrhea in gnotobiotic calves monoinfected with
Cryptosporidium species. J Infect Dis 150: 768–775, 1984.
Hwang E-S, Hirayama BA, and Wright EM. Distribution of
the SGLT-1 Na⫹/glucose cotransporter and mRNA along the
crypt-villus axis of rabbit small intestine. Biochem Biophys Res
Commun 181: 1208–1217, 1991.
Iannoli P, Miller JH, Ryan CK, Gu LH, Ziegler TR, and Sax
HC. Human growth hormone induces system B transport in
short bowel syndrome. J Surg Res 69: 150–158, 1997.
Kandil HM, Berschneider HM, and Argenzio RA. Tumour
necrosis factor-␣ changes porcine intestinal ion transport
through a paracrine mechanism involving prostaglandins. Gut
35: 934–940, 1994.
Keljo DJ, MacLeod RJ, Perdue MH, Butler DG, and Hamilton JR. D-glucose transport in piglet jejunal brush-border
membranes: insights from a disease model. Am J Physiol Gastrointest Liver Physiol 249: G751–G760, 1985.
Klimberg VS, Salloum RM, Kasper M, Plumley DA, Dolson
DJ, and Hautamaki D. Oral glutamine accelerates healing of
the small intestine and improves outcome after whole body
irradiation. Arch Surg 125: 1040–1045, 1990.
Laurent F, Kagnoff MF, Savidge TC, Naciri M, and Eckmann L. Human intestinal epithelial cells respond to Cryptosporidium parvum infection with increased prostaglandin H
synthase 2 expression and prostaglandin E2 and F2 ␣ production.
Infect Immun 66: 1787–1790, 1998.
Lima AAM, Brito GAC, Carvalho GHP, Macdonald TL, and
Guerrant RL. Glutamine and derivatives stimulate electrolyte
transport, mucosal barrier repair, cell proliferation and reduce
apoptosis. The Fourth Oxford Glutamine Workshop Programme
and Abstracts. Oxford: St. Catharine’s College, 2000, p. 1–8.
Marcial M and Madara JL. Cryptosporidium: cellular location, structural analysis of absorptive cell-parasite membranemembrane interactions in guinea pigs, and suggestion of protozoan transport by M cells. Gastroenterology 90: 583–594, 1986.
Minami H, Morse EL, and Adibi SA. Characteristics and
mechanism of glutamine-dipeptide absorption in human intestine. Gastroenterology 103: 3–11, 1992.
Moe OW, Amemiya M, and Yamaji Y. Activation of protein
kinase A acutely inhibits and phosphorylates Na⫹/H⫹ exchanger
NHE-3. J Clin Invest 96: 2187–2194, 1995.
Moon HW. Epithelial cell migration in the alimentary mucosa of
the suckling pig. Proc Soc Exp Biol Med 137: 151–154, 1971.
G653