Departments of 5 Anatomy, Physiological Sciences, and Radiology, 1 Clinical Sciences and 2 Food Animal Health and Resource Management, College of Veterinary Medicine, North Carolina State University, Raleigh 27606, 4 Department of Pediatrics, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599; and 3 Division of Geographic Medicine, Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908
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ABSTRACT |
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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.
sodium absorption; sodium/hydrogen exchanger; villous atrophy; crypt hyperplasia; prostaglandin E2
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INTRODUCTION |
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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 glucose-linked 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, 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 low-affinity 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.
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MATERIALS AND METHODS |
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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 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 µm 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% SDS-polyacrylamide 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-µm 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 streptavadin-labeled 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 µmol/l). In other experiments, 10 mM glutamine was added to the
mucosal side of the tissues, and this was balanced with 10 mM
mannitol added to the serosal solution. PGE2
(106 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.
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).
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RESULTS |
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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).
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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.
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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
HCO
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 Jm
s 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
Jm
s of these ions;
Js
m 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.
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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 alanyl-glutamine 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
µeq · cm
2 · h
1. A
significant increase in Isc was also observed
with both compounds, which was equivalent to the net increase in
Na+ absorption (1 µeq · cm
2 · 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).
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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 indomethacin-induced inhibition of
Isc.
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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 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.
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DISCUSSION |
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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
HCO
As shown for several species, including humans, pigs, and rabbits, the
neutral NaCl absorptive process represents two apically located
independent Na+/H+ and
Cl/HCO
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, HCO
and HCO
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 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 indomethacin-treated, Cryptosporidium-infected tissue, glutamine stimulated net
Na+ absorption by 4.3 ± 0.7 µeq · cm2 · h
1, whereas
the increase in Isc was only 0.8 ± 0.1 µeq · cm
2 · 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 glutamine-stimulated
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 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 crypt-villus 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 site-specific 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 infected 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 commercially 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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: robert_argenzio{at}ncsu.edu).
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.
Received 16 January 2001; accepted in final form 9 May 2001.
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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[ISI][Medline].
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[ISI][Medline].
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[ISI][Medline].
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[ISI][Medline].
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[ISI][Medline].
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[ISI][Medline].
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 tissue-specific 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[ISI].
13.
Fayer, R,
and
Ungar BLP
Cryptosporidium spp. and cryptosporidiosis.
Microbiol Rev
50:
458-483,
1986[ISI].
14.
Ferraris, RP,
Villenas SA,
Hirayama BA,
and
Diamond J.
Effect of diet on glucose transporter site density along the crypt-villus 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 cotransport activity in young rabbits.
Am J Physiol Gastrointest Liver Physiol
279:
G587-G596,
2000
16.
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[Medline].
17.
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[ISI][Medline].
18.
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[ISI][Medline].
19.
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[ISI][Medline].
20.
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[Abstract].
21.
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[ISI][Medline].
22.
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[Abstract].
23.
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
24.
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.
In: The Fourth Oxford Glutamine Workshop Programme and Abstracts. Oxford: St. Catharine's College, 2000, p. 1-8.
25.
Marcial, M,
and
Madara JL.
Cryptosporidium: cellular location, structural analysis of absorptive cell-parasite membrane-membrane interactions in guinea pigs, and suggestion of protozoan transport by M cells.
Gastroenterology
90:
583-594,
1986[ISI][Medline].
26.
Minami, H,
Morse EL,
and
Adibi SA.
Characteristics and mechanism of glutamine-dipeptide absorption in human intestine.
Gastroenterology
103:
3-11,
1992[ISI][Medline].
27.
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[ISI][Medline].
28.
Moon, HW.
Epithelial cell migration in the alimentary mucosa of the suckling pig.
Proc Soc Exp Biol Med
137:
151-154,
1971.
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[ISI][Medline].
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[ISI][Medline].
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[ISI].
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[ISI][Medline].
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[ISI][Medline].
39.
Thake, DC,
Moon HW,
and
Lambert G.
Epithelial cell dynamics in transmissible gastroenteritis of neonatal pigs.
Vet Pathol
10:
330-341,
1973[ISI][Medline].
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[ISI][Medline].
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[ISI][Medline].
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[ISI][Medline].