The small intestine is the principal site of nutrient and water absorption. These functions are served by a single layer of epithelial cells that are generated in intestinal crypts and migrate up mucosal villi, where they express digestive enzymes and nutrient transporters. The protozoal parasite Cryptosporidium parvum replicates within villus epithelial cells resulting in accelerated loss of enterocytes, villous atrophy, nutrient malabsorption, and severe diarrhea. Infection by Cryptosporidium is responsible for 6% of all diarrheal disease and 37.7% of recreational water–associated and 8.5% of drinking water–associated outbreaks of gastroenteritis of known and suspected infectious etiology (1,2). The infection is responsible for 24% of cases of chronic diarrhea in people with human immunodeficiency virus–acquired immune deficiency syndrome worldwide (1,3,4) and up to 26% of cases of chronic diarrhea in children in developing countries (5).
There are no consistently effective antimicrobial treatments or vaccines for Cryptosporidium infection. Recovery from infection depends upon innate and specific epithelial defense mechanisms that remain poorly understood and culminates in repopulation of the villi by functionally mature intestinal epithelium. These reparative mechanisms must outpace the life-threatening consequences of diarrheal dehydration and starvation. Accordingly, therapeutic approaches that promote epithelial defense and repair are likely to have a significant impact on the morbidity and mortality of C parvum infection.
The amino acid L-arginine has been demonstrated in several models of intestinal injury to promote epithelial defense and repair (6–10). These effects are attributed to metabolism of L-arginine to nitric oxide by nitric oxide synthase (NOS) and conversion of L-arginine by arginase to L-ornithine, the precursor of polyamines. We and others have shown that in vivo, C parvum infection results in epithelial induction of nitric oxide synthase II (iNOS) (11,12). Knockout or pharmacological inhibition of iNOS activity results in significant increases in epithelial parasitism and oocyst excretion (11–13). These effects occur in the absence of alterations in epithelial turnover or secretion, suggesting that nitric oxide exerts direct inhibitory effects on the parasite (11). Accordingly, exogenous nitric oxide has been shown in vitro to inhibit excystation of C parvum sporozoites and reduce their viability (13). Nitric oxide is synthesized exclusively from L-arginine. In the neonate, milk is a poor source of L-arginine (14,15) and the majority of L-arginine must be synthesized endogenously by the intestinal epithelium (16,17). It is also presumed that severe villous atrophy, a key feature of C parvum infection, culminates in an immature, crypt-like epithelium that is incapable of nutrient transport. We therefore surmised that L-arginine deficiency may contribute to poor epithelial defense of the newborn against C parvum infection. Whether the intestinal epithelium is able to transport L-arginine in C parvum infection has important clinical implications for use of oral L-arginine to promote epithelial defense. Four kinetically defined systems are reportedly capable of L-arginine transport: y+, B0,+, b0,+, and y+L. Genes encoding the proteins responsible for the activity of system y+ and B0,+ have been cloned and designated as the cationic amino acid–specific transporters (CAT-1–4) (18–22) and ATB0,+(23), respectively. In a variety of cell and tissue types, L-arginine transporters are coordinately induced with iNOS enabling extracellular L-arginine to be used preferentially for nitric oxide synthesis (24–27). We are not aware of any studies examining the ability of infected intestinal epithelium to transport luminal L-arginine, the specific transport system activities and transporter proteins responsible for L-arginine uptake by infected gut, or the influence of iNOS activity on epithelial transport of L-arginine.
The C parvum–infected neonatal piglet is a unique experimental model that fully recapitulates human cryptosporidiosis, the pathophysiology of which is not reproduced in traditional animal models. In the present study, we used intact mucosa from C parvum–infected neonatal piglets to demonstrate that infected ileum transports L-arginine at rates equivalent to that of uninfected epithelium despite the presence of profound villous atrophy. This could be attributed to enhanced uptake of L-arginine by individual epithelial cells in the infection. Unexpectedly, there were no differences in L-arginine transport system activities (y+ and B0,+) or level of transporter gene expression (CAT-1, CAT-2A, and ATB0,+) between uninfected and C parvum–infected epithelial cells. However, infected epithelia had induced expression of the L-arginine hydrolytic enzyme arginase II and lower concentrations of L-arginine. Furthermore, transport of L-arginine by the infected epithelium was significantly inhibited by pharmacological blockade of arginase activity. These results suggest that intracellular catabolism of L-arginine by arginase II facilitates the uptake of L-arginine through transport systems that do not differ from that of uninfected cells. Induction of arginase II has not been described previously for the intestinal epithelium and may limit nitric oxide synthesis by competing with iNOS for utilization of L-arginine or promote the use of L-arginine for the synthesis of reparative polyamines.
MATERIALS AND METHODS
Experimental animals were 1-day-old crossbred piglets obtained from the College of Agriculture and Life Sciences. Piglets were placed into infected or control isolation facilities and fed a liquid diet (Advance Liqui-Wean, Milk Specialties, Dundee, IL; 8.52 g/kg dry matter L-arginine) hourly by an automated delivery system. An inoculum of 108C parvum oocysts (Bunch Grass Farm, Troy, ID) was given to piglets by orogastric tube at 3 days of age. Piglets were studied on days 3 to 5 of infection, a time period shown to correspond to peak epithelial infection (28). Piglets were anesthetized with ketamine (15 mg/kg) and xylazine (0.5 mg/kg) given intramuscularly, then euthanized with an intracardiac dosage of sodium pentobarbital. Sections of ileum, beginning 5 cm above the ileocecal junction, were then immediately taken for ex vivo studies. For each piglet, a section of ileum was fixed in formalin, paraffin-embedded, sectioned at 7 μm, and stained with hematoxylin and eosin for examination by light microscopy. All of the studies were approved by the North Carolina State University Institutional Animal Care and Use Committee.
High Performance Liquid Chromatography
Mucosal samples were obtained by opening the ileum lengthwise, rinsing out the luminal content with cold Ringer solution, scraping the mucosa off the seromuscular layers over ice using a glass microscope slide, and freezing at −20°C. Blood was obtained from anesthetized piglets by cardiocentesis. After centrifugation at 2000g for 5 minutes, serum was frozen at −20°C. Mucosal scrapings and serum samples were shipped on dry ice to the laboratory of Dr Guoyao Wu at Texas A&M University for analysis of L-arginine content by high performance liquid chromatography.
14C-labeled L-Arginine Uptake Studies in Intact Ileal Mucosa
A 20-cm segment of ileum from each piglet was removed from the abdomen and opened along the antimesenteric border in an oxygenated Ringer solution, then the seromuscular layers were removed. Mucosal sheets were mounted in each of six 1.13-cm2 aperture Ussing chambers and bathed on both surfaces with a Ringer solution containing glucose (10 mmol/L serosal) and mannitol (10 mmol/L mucosal). Solutions were oxygenated and circulated by gas-lift (95% O2 and 5% CO2) and maintained at 37°C by water-jacketed reservoirs. After a 60-minute incubation period, 14C-L-arginine (5 mmol/L; 0.2 μCi/mL) was added to the mucosal reservoir of 5 chambers while the sixth served as a negative control (or “blank”). At 3, 5, 10, 15, and 30 minutes after addition of 14C-arginine, a chamber was removed and flushed with cold Ringer solution, and the epithelium and lamina propria were scraped into 1 mL of ice-cold 1N NaOH. Acetic acid was added to normalize the pH of each sample and radioactivity was counted in a liquid scintillation counter. Counts per minute per picomole L-arginine was determined for each chamber by counting the radioactivity of a sample withdrawn from the mucosal reservoir. The concentration of L-arginine was chosen on the basis of its physiological effects on migration of razor-wounded porcine intestinal epithelial cells and bile salt–injured porcine ileal mucosa (29,30), and its comparability to high physiological concentrations of L-arginine measured in the intestinal lumen of neonatal piglets (31).
14C-labeled L-Arginine Uptake Studies in Epithelial Cells
Sheets of ileal mucosa (2.5 cm2) were obtained from control and infected piglets at peak infection (days 3–5) and denuded of villous epithelium by immersion and agitation in an oxygenated citrate-phosphate buffer containing (mmol/L): NaCl 137, KCl 2.7, Na2HPO4 8.0, KH2PO4 1.5, EDTA 30, and glucose 2.5 for 30 minutes at 37°C (32). Exfoliated cells were filtered (100-μm nylon cell strainer), pelleted at 400g, resuspended, and separated from the intraepithelial lymphocytes by centrifugation at 600g for 20 minutes through a discontinuous 25%–40% Percoll gradient as described for the selective recovery of intestinal epithelial cells by a number of investigators (33–35). Cell viability was assessed by trypan blue exclusion. Epithelial cells at a concentration of 1 × 106 live cells per milliliter were incubated at 37°C for 30 minutes in each assay solution to deplete intracellular arginase (ARG) and trans-stimulation of ARG transport (36–39). Cells were then treated with defined concentrations of L-arginine (0.1, 0.5, 2.5, or 12.5 mmol/L) (2 μCi 14C-labeled ARG/reaction; Amersham Life Science, Birmingham, UK). At 30 seconds and 1, 2, and 3 minutes after addition of ARG, duplicate samples were removed and transport activity terminated by washing 3 times in ice-cold phosphate-buffered saline (PBS). After the final wash, cells were resuspended in 1 N NaOH to lyse cells and release isotope. Acetic acid was added to normalize the pH of each sample and radioactivity was counted in a liquid scintillation counter. Transport rate of 14C-arginine was determined by linear regression analysis and expressed as picomoles of amino acid per 106 cells per minute. Prior studies have demonstrated that 3H-arginine uptake is linear and metabolism of arginine is negligible during the chosen time period (36–39).
Identity of L-Arginine Transport System Activities
To determine the identity of the cationic amino acid transport system(s) present on luminal epithelium of the ileum, control and infected mucosae were incubated in Ussing chambers for 60 minutes in the presence of transport system–selective inhibitors followed by addition of 250 μmol/L ARG (2 μCi 14C-ARG) to the mucosal reservoir for 10 minutes. Preliminary studies determined that L-arginine uptake remains linear during this period of time. Sample processing and counts were performed as aforementioned. For these studies, the relative contributions of each carrier system were distinguished by each of the following uptake assay conditions: normal Ringer solution, Na+-free Ringer solution (mmol/L: d-glucamine Cl 130.1, KCl 3.4, CaCl2 1.2, MgCl2 0.7, K2SO4 1.3, KH2PO4 0.3, choline HCO3 24), normal Ringer solution and L-leucine (10 mmol/L), and Na+-free Ringer solution and L-alanine (10 mmol/L). Table 1 indicates the method used to estimate the relative contribution of each transport activity.
Expression Analysis CAT-1, CAT-2A, CAT-2B, and ATB0,+ mRNA
Ileal epithelial cells were exfoliated from the mucosa of control and C parvum–infected piglets (days 3–5 of infection), filtered (100-μm nylon cell strainer), pelleted at 400g, resuspended, and separated from the intraepithelial lymphocytes by centrifugation at 600g for 20 minutes through a discontinuous 25%–40% Percoll gradient, and frozen at −20°C in RLT buffer. Approximately 30 mg of each sample was homogenized (QIAshredder; Qiagen, Hilden, Germany) and total RNA extracted using an RNeasy Mini Kit (Qiagen) with on-column DNAse digestion (RNA-free DNase; Qiagen). Total RNA was reverse transcribed with random hexamers (Applied Biosystems, Foster City, CA) using an Invitrogen RT kit with Superscript II reverse transcriptase. RNA integrity and cDNA production were verified by electrophoresis of RNA and polymerase chain reaction (PCR) of the housekeeping gene cyclophilin (AY008846). Real-time PCR was performed using a BioRad iCycler in a 15-μL reaction volume with 50 nmol/L of each primer, 60 ng cDNA, and 2X SYBR Green Mastermix Kit (Applied Biosystems). To ensure amplification of only cDNA and not genomic sequences, primers were designed to amplify across intron-exon junctions as predicted by comparison of mRNA and gene sequence data (Table 2). PCR products were analyzed by gel electrophoresis with ethidium bromide and their identity confirmed by size, melting temperature, and gene sequencing (University of North Carolina Automated Sequencing Facility). Positive controls included normal porcine liver (CAT-2A) and ConA and IL-2–stimulated porcine peripheral blood T lymphocytes (CAT-2B) (41). Negative controls included porcine genomic DNA, no template, and RNA only (no reverse transcription [RT]).
Immunoblotting of Intestinal Epithelial Protein for Arginase I and Arginase II
For demonstration of arginase I and arginase II protein expression, ileal epithelial cells were exfoliated from the mucosa of control and C parvum–infected piglets (days 3–5 of infection) as previously described (11), frozen in liquid nitrogen, and stored at −80°C. Samples were thawed on ice in RIPA buffer (0.15 mol/L NaCl, 50 mmol/L Tris [pH 7.2], 1% deoxycholic acid, 1% Triton X-100, 0.1% sodium dodecyl sulfate) containing bestatin, leupeptin, aprotinin, sodium orthovanadate, and phenyl-methyl sulfonyl fluoride. This mixture was sonicated and centrifuged at 10,000g for 10 minutes at 4°C. The supernatants were saved and their protein concentration determined (Dc protein assay, Bio-Rad, Hercules, CA). Samples of equal protein concentration were suspended in 2X–sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer, boiled for 4 minutes, and loaded in sodium dodecyl sulfate–polyacrylamide gels. Equal protein loading was confirmed by Coomassie blue staining of gels after electrophoretic separation of proteins. Electrophoresis was carried out and proteins were transferred to a nitrocellulose membrane (Hybond ECL, Amersham) using an electroblotting minitransfer apparatus. Membranes were blocked overnight in Tris-buffered saline plus 0.05% Tween-20 (TBST) and 5% powdered milk, bovine serum albumin, or StartingBlock (Pierce, Rockford, IL). Membranes were incubated in primary antibody (mouse anti-arginase I 1:1000 [BD Pharmingen, San Diego, CA] or rabbit anti-arginase II 1:200 [Santa Cruz Biotechnology, Santa Cruz, CA]). After 3 washes each with TBST, the membranes were incubated for 45 minutes with horseradish peroxidase conjugated secondary antibody (1:5,000 to 1:10,000; Santa Cruz Biotechnology). After 3 additional washes for 5 minutes each with TBST, the membranes were developed for visualization of protein by addition of enhanced chemiluminescence reagent. Positive controls for arginase I and II expression included protein lysates of porcine liver and kidney, respectively.
Samples of ileal mucosa were embedded in optimal cutting temperature medium, frozen in liquid N2, and sectioned at 5-μm thickness. Sections were fixed in 100% ethanol at −20°C for 30 minutes and rinsed with PBS containing 1.2 mmol/L CaCl2 at pH 7.4 (PBS+). Sections were treated with blocking buffer (consisting of PBS+, 1% bovine serum albumin, and 2% goat serum) for 1 hour, followed by incubation with polyclonal rabbit anti-human arginase II antibody (1:100) or isotype control antibody in blocking buffer for 1 to 2 hours at RT. Sections were rinsed in PBS+ (4 times for 5 minutes each) followed by incubation with goat anti-rabbit immunoglobulin G secondary antibody-Cy3 conjugate (1:100 in blocking buffer) for 30 minutes at RT in the dark. Sections were rinsed in PBS+ (4 times for 5 minutes each), coverslipped with mounting media containing DAPI (Vectashield; Vector Laboratories, Burlingame, CA), and imaged using an epifluorescence microscope.
Effect of Nitric Oxide Synthase and Arginase Inhibitors on Uptake of 14C-arginine
To determine the effect of NOS and arginase activity on intestinal epithelial uptake rate of L-arginine, control and infected ileal mucosae were incubated in Ussing chambers for 60 minutes in the presence of serosally applied L-NGω-nitro-arginine methyl ester (L-NAME) (10 mmol/L) or di-fluoro-methyl-ornithine (DFMO) (10 mmol/L) followed by addition of 2.5 mmol/L ARG (2 μCi 14C-ARG) to the mucosal reservoir. Ten minutes after addition of 14C-arginine, the chamber was removed and flushed with cold Ringer solution, and the epithelium and lamina propria scraped into 1 mL of ice-cold 1N NaOH. Acetic acid was added to normalize the pH of each sample and radioactivity was counted in a liquid scintillation counter.
Data are reported as mean ± standard error (SE). For all of the analyses, P ≤ 0.05 was considered significant. All of the data were tested for normality and equal variance using a statistical software package (SigmaStat, Jandel Scientific, San Rafael, CA). Normally distributed data were analyzed using an analysis of variance or Student t test and nonparametric data were analyzed using a Kruskal-Wallis analysis of variance on ranks or Mann-Whitney rank sum test when appropriate.
Uptake of L-Arginine From the Lumen of the Ileum Is Preserved in C parvum Infection
To determine the ability of C parvum–infected intestinal epithelium to transport L-arginine when presented to the lumen of the gut, sheets of ileal mucosa from uninfected control and C parvum–infected piglets were incubated in Ussing chambers in the presence of 5 mmol/L L-arginine (0.2 μCi 14C-labeled-L-arginine per milliliter) added to the luminal reservoir for durations of 3, 5, 10, 15, and 30 minutes. On the basis of serosal surface area (ie, the aperture of the Ussing chamber) uptake of 14C-labeled L-arginine by control and C parvum–infected mucosa was nearly identical (Fig. 1). All of the infected piglets used in the study had severe villous atrophy and organisms present in villous enterocytes, whereas control piglets had normal villous architecture with no evidence of infection. Equivalent rates of uptake were observed despite a substantial difference in mucosal surface area between control and infected intestines (Fig. 2).
Epithelial Cells Isolated From C Parvum–infected Piglets Have Increased Transport Rate of L-Arginine
To determine whether equivalent uptake of L-arginine by control and atrophic C parvum–infected epithelium could be attributed to enhanced transport by individual epithelial cells, uptake of 14C-labeled L-arginine by epithelial cells isolated from the ileum of control and infected piglets was measured under initial rate conditions. Transport rate of L-arginine was significantly greater in C parvum–infected epithelial cells compared with uninfected control epithelial cells (Fig. 3).
Luminal Uptake of L-Arginine From the Ileum of Control and C parvum–infected Piglets Is Mediated by Transport Systems y+ and B0,+
Cellular transport of L-arginine can be mediated by the kinetically defined systems y+, y+L, b0,+, and B0,+. To determine the identity of the amino acid transport activities present in control and C parvum–infected epithelium in vivo, in situ uptake of 14C-arginine by mucosal epithelium was performed in Ussing chambers in the presence of transport system-selective inhibitors as outlined in Table 1. These studies were performed with a low dose of L-arginine (250 μmol/L; 0.2 μCi/mL) to ensure sufficient inhibitory effects of competing amino acids. The identity and percent contribution of L-arginine transport system activities present were not significantly different between uninfected control and C parvum–infected epithelium, and were attributed predominantly to systems y+ and B0,+ (Fig. 4).
C parvum–infected Mucosa Express CAT-1, CAT-2A, Scant CAT-2B, and ATB0,+ Transporter Transcripts
To determine whether an increase in the expression of genes encoding the proteins responsible for activity of system y+ or B0,+ were responsible for increased L-arginine uptake by intestinal epithelium of C parvum–infected piglets, we designed primers for the specific amplification of system y+ (CAT-1, CAT-2A, CAT-2B) and system B0,+ (ATB0,+) transporter protein genes. Levels of mRNA expression were compared between control and infected piglets using real-time RT-PCR. There were no differences in expression of cationic amino acid–specific or ATB0,+ transporters between control and C parvum–infected piglets. The ubiquitously expressed transporter CAT-1 and ATB0,+ were present in greatest abundance. CAT-2A mRNA was detected in lesser amounts, whereas CAT-2B was detected only at low levels (Table 3). RT-PCR products for CAT-1, CAT-2B, CAT-2A, and ATB0,+ shared 100% sequence identity with corresponding mRNA sequences in Genbank (Table 2). Therefore, increased L-arginine transport by C parvum–infected epithelium could not be attributed to an increase in expression of genes responsible for y+ or B0,+ transport activities.
C parvum Infection Is Associated With Cellular L-Arginine Deficiency
An enhanced uptake of L-arginine, in the absence of changes in transporter gene expression, has been demonstrated in alveolar macrophages to be mediated by intracellular consumption of L-arginine by NOS (42). We therefore surmised that in C parvum–infected epithelium, intracellular catabolism of L-arginine may promote an enhanced uptake of L-arginine. To determine whether cellular concentrations of L-arginine were consistent with consumption of L-arginine in the infection, we examined the L-arginine content of ileal mucosa from control and C parvum–infected piglets. In infected animals, ileal mucosal L-arginine concentrations were significantly lower than in uninfected animals (nmol/g = 1440 ± 72 uninfected control; 951 ± 31 C parvum; n = 5 each; P < 0.001, 1-way analysis of variance). Furthermore, reduced cellular concentrations of L-arginine in the infection could not be attributed to serum deficiency because there were no significant differences in serum concentration of L-arginine between uninfected control and C parvum–infected piglets (μmol/L = 75 ± 18 uninfected control; 94 ± 18 C parvum; n = 5 each).
C parvum Infection Induces Expression of Arginase II by Intestinal Epithelium In Vivo
Key enzymes mediating catabolism of L-arginine are NOS and arginase. We have previously shown that iNOS is expressed intensely by C parvum–infected epithelium in vivo (11,43), but arginase expression has not been explored previously. Expression of arginase is reportedly negligible in intestinal epithelium of neonates (31,44). To determine whether expression of arginase by the intestinal epithelium could be induced by C parvum infection and thereby contribute to L-arginine catabolism, intestinal epithelial protein lysates from control and C parvum–infected piglets were immunoblotted for arginase I and II expression. Arginase I was not expressed by intestinal epithelial cells from either uninfected or infected piglets. Arginase II expression was similarly negligible in uninfected piglets but strongly induced by intestinal epithelium from piglets infected with C parvum. To determine the location of arginase II expression along the crypt–villus axis in C parvum infection, immunofluorescence was performed to localize arginase II expression. In C parvum–infected ileum, arginase II expression was observed in both villous and crypt epithelium (Fig. 5).
Enhanced Uptake of L-Arginine by the Ileum of C parvum–infected Piglets Is Mediated by Arginase
To determine the contribution of NOS and arginase to uptake of L-arginine from the lumen of the intestine, sheets of ileal mucosa from uninfected control and C parvum–infected piglets were incubated in Ussing chambers with a NOS inhibitor (L-NAME, 10 mmol/L) or arginase inhibitor (DFMO, 10 mmol/L) for 1 hour before determination of L-arginine uptake rate (2.5 mmol/L; 0.2 μCi 14C-labeled-L-arginine per milliliter). Inhibition of NOS had no effect on the uptake rate of L-arginine by either control or infected ileum. In contrast, inhibition of arginase significantly decreased the uptake of L-arginine by C parvum–infected but not uninfected ileum, which is consistent with arginase II induction by infected and not control epithelium (Fig. 6).
The ability of the neonatal gut to transport L-arginine from the lumen of the intestine has important implications for the efficacy of enteral L-arginine to correct deficiency states and/or promote intestinal epithelial defense and repair. In the present studies, we used intact sheets of intestinal mucosa mounted in Ussing chambers and exposed to luminal L-arginine to identify transport systems y+ and B0,+ as mediators of L-arginine uptake by neonatal ileum. Furthermore, these same transport activities were preserved in C parvum–infected ileum despite the presence of severe villous atrophy. Most reports on the identity of transport system activities in intestinal epithelium are consistent with system y+ transport by the apical membrane (45), although findings vary considerably depending on species, location along the length of the gastrointestinal tract, and location between the apical and basolateral membranes. Likewise, system B0,+ has been identified as a luminal transport mechanism of intestinal epithelium in a number of species (46–49), although previously reported to be absent in weaned pigs (50). In contrast, the presence of system B0,+ activity in neonatal piglet intestinal epithelium, as described here, may reflect age-dependent modulation of system B0,+ activity, as has been demonstrated in response to dietary and growth factors (45,51). Indeed, in the present study, fully half of L-arginine transport by neonatal piglet epithelium was dependent on the presence of Na+, a characteristic uniquely ascribed to transport of L-arginine by system B0,+.
Recently, genes encoding the proteins responsible for the activity of system y+ and B0,+ have been cloned and designated as the cationic amino acid–specific transporters (CAT-1, -2A, -2B, -3, and -4) (18–22) and ATB0,+(23), respectively. CAT-3 and -4 have not been described for intestinal epithelium. In the present study, we identified expression of the system y+ and B0,+ transporter genes CAT-1, CAT-2A, and ATB0,+ at comparable levels in intestinal epithelium from both control and C parvum–infected piglets. CAT-1 is a ubiquitously expressed transporter (except in liver) (52) for which transcripts have been previously identified in murine intestine and CaCo-2 cells (53,54). CAT-2A is primarily expressed in liver and skeletal muscle (18) and was reportedly absent in studies of murine intestine and CaCo-2 cells (53,54). Our studies are the first, to our knowledge, to demonstrate expression of CAT-2A by intestinal epithelium. Finally, although expression of CAT-2B is co-induced with iNOS in a variety of nonepithelial cell and tissue types (24,26,27,38), only scant CAT-2B transcripts were amplified from intestinal epithelium in the present study.
Preservation of L-arginine transporter expression by the infected epithelium was somewhat unexpected, given the severity of villous atrophy caused by C parvum. However, other laboratories have demonstrated normal or increased expression of ASC (55) and PepT1 transporters (56) in cryptosporidiosis, suggesting that the infected epithelium remains capable of sustained expression of nutrient transporters despite a high rate of cellular turnover. This may be attributed in part to compensatory induction of transporter expression by crypt epithelium as has been described for ASC (55). A lack of specific antibodies for identification of L-arginine transport proteins precluded studies to locate their expression along the crypt–villus axis in the present study.
In addition to preserving the expression of L-arginine transporter genes, individual epithelial cells isolated from the infected mucosa exhibited an increased uptake rate of L-arginine. We did not perform detailed studies of specific transport system activities in epithelial cells after their isolation from the mucosa because the length required of these studies adversely affected cell survival and precluded study of luminal transport mechanisms by disrupting epithelial cell polarity. Our observation that C parvum–infected mucosa had significantly lower cellular concentrations of L-arginine despite normal serum concentrations led us to surmise that intracellular consumption of L-arginine may promote facilitated transport by the infected epithelium. The 2 major pathways for intracellular consumption of L-arginine are conversion by NOS to nitric oxide and L-citrulline and hydrolyzation by arginase to urea and L-ornithine. We and others have shown that C parvum infection results in epithelial induction iNOS (11,12), and a significant effect of iNOS activity on transport rate of L-arginine has been shown in some studies (42), but not in others (57). However, arginase is normally absent in intestinal epithelial cells before weaning (31), at which time cortisol-induced expression of arginase becomes a major pathway for L-arginine degradation (44). The studies reported here are the first to demonstrate induction of arginase II by intestinal epithelium in response to infection in vivo. Induction of arginase could not be attributed to early weaning alone (58) because arginase expression was not observed in identically weaned, formula-fed littermates who were not infected with C parvum. A definitive role of arginase in promoting increased uptake of L-arginine by the infected epithelium was demonstrated by pharmacological studies in which the uptake of L-arginine was significantly inhibited by treatment with the L-ornithine analog DFMO but not the NOS inhibitor L-NAME. Although arginase activity promoted uptake of L-arginine by C parvum–infected epithelium, its contribution to total absorption was only modest, accounting for ∼25% of cellular L-arginine uptake (Fig. 6). DFMO was chosen for use in these studies because of its lack of effects on iNOS activity and potent inhibitory effects on cellular arginase activity at concentrations that do not interfere with L-arginine uptake mechanisms (59). Although DFMO is also an inhibitor of ornithine decarboxylase activity, and therefore polyamine synthesis, the time required for depletion of intracellular polyamines (up to 6 days) (60) greatly exceeds the short duration of use in these studies (1 hour).
Co-induction of arginase II and iNOS by intestinal epithelium has not been reported previously. At low concentrations of L-arginine, arginase and iNOS compete at similar rates for consumption of the amino acid (61). Therefore, sufficient quantities of arginase can limit the availability of L-arginine for nitric oxide synthesis (62,63). We and others have shown that nitric oxide decreases epithelial parasitism in vivo and inhibits excystation and viability of C parvum sporozoites in vitro (11–13). In models of infection caused by Giardia lamblia, Helicobacter pylori, and Trypanosoma brucei, consumption of L-arginine by pathogen-derived arginase or arginase induced within the host inhibits nitric oxide synthesis in favor of pathogen survival (64–66). Arginase may also protect host epithelium from the deleterious effects of sustained overproduction of nitric oxide. In addition, arginase mediates the conversion of L-arginine to L-ornithine, a key substrate for synthesis of polyamines. Polyamines play key reparative roles in promoting epithelial cell proliferation and restitution (60,67). In mice infected with Citrobacter rodentium, induction of arginase I ameliorates colitis by enhancing the synthesis of polyamines (68). In the present study, apparently greater expression of arginase II within hyperplastic crypts, rather than villous epithelium (where parasites and iNOS expression predominate), suggests that arginase II may promote polyamine synthesis in direct support of enhanced epithelial cell replacement and restitution, rather than serving as a primary competitor for nitric oxide synthesis. However, whether arginase II expression by intestinal epithelium in C parvum infection limits nitric oxide synthesis or confers a survival advantage to the host or parasite is unclear. These questions will need to be answered with in vivo studies, in which the effect of arginase on synthesis of nitric oxide and polyamines, epithelial repair and barrier function, and parasite and host survival can be ascertained (11).
The authors thank Martha U. Armstrong for excellent technical assistance.
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