Nitric oxide synthase stimulates prostaglandin synthesis and barrier function in C. parvum-infected porcine ileum
Jody L. Gookin,1
Laurel L. Duckett,1
Martha U. Armstrong,1
Stephen H. Stauffer,1
Colleen P. Finnegan,2
Michael P. Murtaugh,2 and
Robert A. Argenzio1
1Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606; and 2Department of Veterinary and Biomedical Science, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108
Submitted 23 September 2003
; accepted in final form 13 May 2004
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ABSTRACT
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Cell culture models implicate increased nitric oxide (NO) synthesis as a cause of mucosal hyperpermeability in intestinal epithelial infection. NO may also mediate a multitude of subepithelial events, including activation of cyclooxygenases. We examined whether NO promotes barrier function via prostaglandin synthesis using Cryptosporidium parvum-infected ileal epithelium in residence with an intact submucosa. Expression of NO synthase (NOS) isoforms was examined by real-time RT-PCR of ileal mucosa from control and C. parvum-infected piglets. The isoforms mediating and mechanism of NO action on barrier function were assessed by measuring transepithelial resistance (TER) and eicosanoid synthesis by ileal mucosa mounted in Ussing chambers in the presence of selective and nonselective NOS inhibitors and after rescue with exogenous prostaglandins. C. parvum infection results in induction of mucosal inducible NOS (iNOS), increased synthesis of NO and PGE2, and increased mucosal permeability. Nonselective inhibition of NOS (NG-nitro-L-arginine methyl ester) inhibited prostaglandin synthesis, resulting in further increases in paracellular permeability. Baseline permeability was restored in the absence of NO by exogenous PGE2. Selective inhibition of iNOS [L-N6-(1-iminoethyl)-L-lysine] accounted for
50% of NOS-dependent PGE2 synthesis and TER. Using an entire intestinal mucosa, we have demonstrated for the first time that NO serves as a proximal mediator of PGE2 synthesis and barrier function in C. parvum infection. Expression of iNOS by infected mucosa was without detriment to overall barrier function and may serve to promote clearance of infected enterocytes.
Cryptosporidium parvum; permeability
THE SINGLE LAYER OF EPITHELIUM lining the small intestine provides a defensive barrier that restricts ingress of luminal aggressive factors while selectively absorbing the majority of nutrients, electrolytes, and water that sustain life. These barrier and absorptive functions are particularly vulnerable to microbial pathogens with tropism for intestinal epithelium, such as rotavirus, enteropathogenic Escherichia coli, and Cryptosporidium parvum. Although these infections hasten the loss of villous enterocytes, epithelial continuity is frequently preserved by hyperplastic crypts that provide a continuous source of replacement enterocytes to the villi. In contrast, a persistent source of barrier dysfunction in intestinal infection appears to arise from selective increases in epithelial paracellular permeability. Transmucosal hyperpermeability has been consistently demonstrated in infants and human immunodeficiency virus patients with diarrhea caused by these agents (12, 14, 48), in animal models of intestinal infection (4, 19), and after infection of epithelial monolayers in culture (1, 13, 18, 38). Increases in paracellular permeability may contribute to fluid losses and facilitate translocation of luminal factors in the subepithelium that perpetuate mucosal inflammation. Although much has been learned regarding mediators and mechanisms of disruption of paracellular permeability, less is understood regarding the local mechanisms responsible for sustaining barrier function during intestinal infection.
Nitric oxide (NO) is consistently elevated in patients with infectious diarrhea (27) and mediates demonstrable but paradoxical effects on paracellular permeability in a variety of experimental models. Cell culture models suggest that NO would mediate barrier disruption in intestinal epithelial infection because both normal and infected intestinal epithelia subjected to induction of NO synthesis or exogenous NO undergo increases in paracellular permeability (7, 34, 40, 42, 44). In contrast, in vivo studies performed with normal intestine (22, 23, 24) or during the acute phase of intestinal injury (25, 37) support the view that NO preserves barrier function by mechanisms related to subepithelial events, such as maintenance of mucosal blood flow (36), inhibition of leukocyte adhesion (26), and modulation of mast cell reactivity (22). However, in the presence of mucosal inflammation, the role of NO-mediated effects is less clear, with NO promoting barrier function in some studies (33) and mediating barrier disruption in others (45). Studies have yet to define the local effect of NO on paracellular permeability of an entire intestinal mucosa in the presence of both epithelial infection and the resulting subepithelial inflammatory infiltrate.
In the presence of inflammation, NO can combine with superoxide to generate potent free radicals, such as peroxynitrite, that exacerbate epithelial injury (35, 39, 43, 47). On the other hand, reactive nitrogen species have also been demonstrated to activate cyclooxygenase (COX) in enzymatic studies and cell culture models (15). We have previously shown that synthesis of prostaglandins (PG) by ileal mucosa from C. parvum-infected piglets is increased and mediates diarrhea via inhibition of NaCl absorption and stimulation of anion (Cl or HCO3) secretion (2). Treatment of infected or inflamed mucosa with a PG synthesis inhibitor [e.g., indomethacin (Indo)] will restore normal electrolyte transport but simultaneously increases intestinal permeability and villous damage (3, 20).
Using a well-established experimental model of neonatal porcine C. parvum infection, we have demonstrated for the first time that NO serves as a proximal mediator of PGE2 synthesis and barrier function in C. parvum infection. Inducible NOS (iNOS) is expressed by the infected mucosa and gives rise to an increase in PGE2 synthesis compared with uninfected mucosa. iNOS activity was without detriment to barrier function and may serve to promote clearance of C. parvum-infected enterocytes.
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MATERIALS AND METHODS
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Animals.
Experimental animals were 1-day-old crossbred piglets obtained from the College of Agriculture. Piglets were placed in infected and control isolation units and fed a liquid diet by an automatic delivery system. An inoculum of 108 C. parvum oocysts was given to piglets by orogastric tube on day 3 of life. Control and infected piglets were studied on days 35 after inoculation, a time shown previously to be at the peak of intestinal infection (4). Piglets were killed by intravenous pentobarbital sodium, and sections of ileum, beginning 10 cm above the ileocecal junction, were taken sequentially for histology, RNA extraction, and in vitro studies. Sections of ileum were fixed in formalin, paraffin-embedded, sectioned at 7 µm, and stained with hematoxylin and eosin (H&E) for examination by light microscopy. All infected piglets used in the study showed evidence of villous atrophy and organisms adherent to villus enterocytes, whereas control piglets showed normal villous architecture with no evidence of infection. All studies were approved by the Institutional Animal Care and Use Committee.
RNA isolation and real-time RT-PCR.
Mucosal scrapings from control and infected piglets (n = 5 each) were stored in >10 vol RNAlater (Qiagen, Valencia, CA) at 20°C. Samples (30 mg) were homogenized (QIAshredder; Qiagen), and total RNA was extracted using an RNeasy Mini Kit (Qiagen) with on-column DNase digestion (RNA-free DNase; Qiagen). Concentration of RNA was determined by absorbance at 260 nm (Biophotometer; Eppendorf, Westbury, NY) and evaluated for RNA integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA (4 µg) was reverse transcribed with 200 µg random hexamers in a 40-µl total volume using an Invitrogen RT kit with Superscript II RT (Invitrogen, Carlsbad, CA).
Primers for PCR amplification of cDNA were designed using Primer 3 Software (Whitehead Institute, Cambridge, MA) to span introns. Specific primer sequences and sources are listed in Table 1. RNA integrity and cDNA production were verified by amplification of cyclophilin.
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Table 1. Specific primer sequences and sources used in real-time RT-PCR and their resulting amplification product sizes and melting temperatures
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Real-time PCR was performed in a 15-µl reaction volume with 50 nM of each primer, 100 ng cDNA, and 2x SYBR Green Mastermix Kit (Applied Biosystems, Foster City, CA) using an ABI Prism 7900 thermocycler for 50 cycles at 95°C for 15 s and 60°C for 1 min after an initial incubation for 10 min at 95°C. PCR products also were analyzed on a 1.5% agarose gel stained with ethidium bromide. Comparative threshold cycle analysis was used as described in the ABI Prism 7700 Sequence Detection System User Bulletin no. 2 to calculate relative degree of changes.
Barrier function studies.
Methods used in this laboratory for in vitro Ussing chamber studies have been described in detail (17). Briefly, a 20-cm segment of ileum was opened along the anti-mesenteric border in an oxygenated Ringer solution, and the seromuscular layers were removed. Mucosal sheets were mounted in 1.13-cm2 aperture Ussing chambers and bathed on both surfaces with a Ringer solution containing glucose (10 mM serosal) and mannitol (10 mM mucosal). Solutions were oxygenated and circulated by gas lift (95% O2-5% CO2) and maintained at 37°C by water-jacketed reservoirs. The spontaneous potential difference (PD) was measured using Ringer-agar bridges connected to calomel electrodes, and the PD was short-circuited through Ag-AgCl electrodes using a voltage clamp that corrected for fluid resistance. If the spontaneous PD was between 1.0 and 1.0 mV, tissues were current clamped at ±100 µA for 5 s, and the PD was recorded. Transepithelial electrical resistance (TER;
·cm2) was calculated from the spontaneous PD and short-circuit current, which were recorded at 15- to 30-min intervals over a 315-min period.
TER is ordinarily expressed on the basis of serosal surface area (i.e., the aperture of the Ussing chamber). As a consequence of villous atrophy, control and C. parvum-infected mucosa differ markedly in their mucosal surface area and, accordingly, the surface area of paracellular pathway available per square centimeter of serosa for ion permeation. Thus TER values were calculated as described by Collett et al. (11) on the basis of total mucosal surface area of paracellular pathway available in each square centimeter of serosal area as approximated using Marcial et al.'s (32) measurements of 0.2180 µm paracellular pathway/µm2 surface area of villus and 0.7680 µm paracellular pathway/µm2 surface area of crypt (32) and morphometric analysis of representative histological sections disclosing a 2.75-fold difference in the mucosal-to-serosal surface area ratio of muscle-stripped and mounted ileal mucosa from control and C. parvum-infected piglets (6). A detailed application of surface area effects on TER of porcine ileal mucosa has been published (16).
Isotopic mannitol and Na+ flux studies.
Isotopic flux studies of mucosal permeability were performed using [3H]mannitol (0.2 µCi/ml in 16.6 µmol/ml mannitol) and the isotope 22Na+ (0.2 µCi/ml in 154.2 meq/ml Na+) under short-circuit conditions (tissues clamped to 0 mV). Isotope was added to the mucosal ([3H]mannitol) or serosal (22Na+) reservoir 15 min after mounting the mucosa on the chamber. For [3H]mannitol, one 60-min flux period (from 255 to 315 min) was performed by taking paired samples from the serosal reservoir. For 22Na+, one 30-min flux period (from 255 to 285 min) was performed by taking paired samples from the mucosal reservoir. Samples were counted for 3H or 22Na+ in a scintillation counter. Flux of mannitol from mucosa to serosa and Na+ from serosa to mucosa was calculated using standard equations.
Transmission electron microscopy.
To examine the ultrastructural effect of NO or paracellular anatomy of ileal epithelium from C. parvum-infected piglets, mucosal sheets were incubated for 300 min in the Ussing chamber in the presence or absence of 10 mM NG-nitro-L-arginine methyl ester (L-NAME) on the serosal side. Tissues were removed from the Ussing chamber and placed in Trump's 4F:1G fixative at 4°C. Samples were processed for transmission electron microscopy using standard techniques.
NO and eicosanoid analyses.
Total NO2 + NO3 concentration was measured in urine and serum samples taken from infected and control piglets after conversion of NO3 to NO2 by nitrate reductase with detection of NO2 using a commercial kit (Griess Assay; Cayman Chemical, Ann Arbor, MI). For eicosanoid analyses, paired samples were taken from the serosal chamber solution, gassed with N2, and frozen in liquid N2. Samples were stored at 20°C before assay. Samples were analyzed for concentrations of PGE2, 6-keto-PGF1
(the stable metabolite of PGI2), and thromboxane (TX) B2 (the stable metabolite of TXA2) using commercial ELISA kits according to the manufacturer's instructions (Biomedical Technologies, Stoughton, MA). Because of baseline differences in eicosanoid synthesis by control and infected mucosa and the variable influence of stripping and mounting of mucosa on initial eicosanoid synthesis, baseline samples were collected 30 min after tissue mounting for eicosanoid assay. Baseline eicosanoid concentrations were subtracted from concentrations measured after 300 min of exposure to each treatment.
Assessment of NOS effects on barrier function.
All treatments were added to the Ussing chamber 15 min after mounting of the mucosa (acclimation period) and allowed to remain in contact with the mucosa for 300 min. The following treatments were evaluated in this study: the nonselective NOS inhibitors L-NAME (10 mM serosal) and L-NG-monomethyl arginine (L-NMMA; 1 mM serosal); the selective iNOS inhibitors L-N6-(1-iminoethyl)-L-lysine (L-NIL; 30 and 100 µM serosal) and aminoguanidine (5 mM serosal); the reportedly selective neuronal NOS (nNOS) inhibitors 7-nitroindazole (7-NI), N
-propyl-L-arginine (NPA), and 1-(2-trifluoro-methyl-phenyl)imidazole (TRIM; each 11,000 µM serosal); and the nonselective COX inhibitor Indo (5 x 106 M serosal and mucosal). Exogenous prostaglandins were provided by addition of 16,16-dimethyl-PGE2 (106 M), the PGI2 analog carbacyclin (106 M), or the stable TXA2 analog carbocyclic TXA2 (106 or 5 x 108 M; each given serosal at 15 and 195 min).
Assessment of NOS effects on restitution.
Restitution rate of the infected epithelium in the presence and absence of L-NAME was quantified using an established Ussing chamber model (16, 17). Briefly, the villous epithelium was removed by transient exposure of the chambered mucosa to deoxycholate (1.5 mM for 15 min; Sigma, St. Louis, MO). Deoxycholate was then replaced by Ringer solution. Paired tissues from each piglet were allowed to restitute for 3 h in the presence and absence of L-NAME (10 mM serosal). Tissues were removed from the Ussing chamber, fixed in formalin, paraffin embedded, sectioned at 5 µm, and stained with H&E for examination by light microscopy. With use of an ocular micrometer, the linear length of villus perimeter and linear length of denuded villus were measured for five-well-oriented villi and used to calculate the percent villus reepithelialization of each tissue. All measurements were performed without knowledge of treatment group.
Data analysis.
Data are reported as means ± SE. For all analyses, P
0.05 was considered significant. One-way ANOVA and a post hoc Tukey's test, two-way repeated-measures ANOVA, or Student's paired t-test was used to compare differences between treatment and control tissues (SigmaStat; Jandel Scientific, San Rafael, CA). Number of pigs receiving treatment = n.
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RESULTS
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Infection of neonatal piglets with C. parvum results in villus atrophy and diarrhea.
Piglets were killed on days 35 postinfection. This time period corresponds to peak epithelial infection with C. parvum and liquid diarrhea (4, 5). Ileal mucosa from time-matched control piglets was comprised of elongated villi lined by vacuolated epithelium. In C. parvum infection, sporozoites parasitized enterocytes along the villus tips, resulting in enhanced epithelial cell loss and marked villus atrophy (villus height = 638 ± 39 µm control, 121 ± 7 µm infected; P < 0.001 1-way ANOVA).
NO synthesis is increased in piglets with C. parvum infection.
To determine whether endogenous NO synthesis is increased in piglets with C. parvum infection, the oxidative metabolites of NO (NO2 and NO3) were measured in serum and urine samples taken from control and infected animals on days 35 postinfection (Fig. 1). The concentration of NO metabolites in serum was not significantly different between control and infected animals. However, greater synthesis of NO by infected animals was demonstrated by a significantly larger concentration of NO metabolites excreted in urine.

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Fig. 1. Concentration of serum and urine metabolites of nitric oxide (NOx; NO2 + NO3) as determined for control and Cryptosporidium parvum-infected piglets on days 35 postinfection. Urinary excretion of NOx was significantly greater in infected piglets (*P < 0.05, 1-way ANOVA; n = no. of piglets).
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NO contributes to barrier function in intestinal C. parvum infection.
To assess the effect of NO on intestinal barrier function in the infection, sheets of ileal mucosa from control and infected piglets were incubated in Ussing chambers. In infected mucosa, baseline TER was significantly lower and the passive serosal-to-mucosal flux of 22Na+ was significantly higher than for control mucosa (Figs. 2 and 3). In the absence of treatment, both infected and control mucosa maintained stable TER values for over 315 min of incubation. After addition of the nonselective NOS inhibitor L-NAME to the serosal bath of control tissue, TER and serosal-to-mucosal flux of 22Na+ were unaffected. In contrast, TER of infected tissue progressively deteriorated, and there was a significant increase in the serosal-to-mucosal flux of 22Na+ (Figs. 2 and 3). Mucosal-to-serosal flux of [3H]mannitol was likewise increased by the addition of L-NAME to infected tissue [µmol·cm2·h = 0.28 ± 0.04 no treatment (no Tx); 0.31 ± 0.03 L-NAME, n = 8 each; P < 0.01, Student's paired t-test], and significant inhibition of TER was also obtained with the nonselective NOS inhibitor L-NMMA [
·cm2 mucosa after 300 min = 25 ± 1.1 no Tx; 19 ± 0.9 L-NMMA (1 mM), n = 8 each; P < 0.01, Student's paired t-test]. Incubation of control or infected mucosa with D-NAME, an inactive isomer of L-NAME, was without effect on TER (Fig. 2, inset).

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Fig. 2. Transepithelial electrical resistance (TER) of control and C. parvum-infected ileal mucosa (days 35 postinfection) mounted in Ussing chambers. After a 15-min acclimation period, treated tissues were exposed serosally to the nonselective NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 mM) for 300 min. In infected mucosa, L-NAME resulted in significant reduction in TER (***P < 0.001, 2-way repeated-measures ANOVA). The inactive D-isomer of NAME had no effect on TER (inset); n = no. of piglets. No Tx, no treatment.
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Fig. 3. Passive flux of 22Na+ from serosa to mucosa (Jsm) of control and C. parvum-infected ileal mucosa mounted in Ussing chambers. Fluxes were performed after 4 h of incubation in the absence (No Tx) or presence of the nonselective NO synthase (NOS) inhibitor L-NAME (10 mM applied to the serosal reservoir). Flux of 22Na+ was significantly greater in infected mucosa compared with control (***P < 0.001, Student's t-test). Flux of 22Na+ was significantly increased in infected mucosa after treatment with L-NAME (*P < 0.05, Student's t-test); n = no. of piglets. NS, not significant.
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NO promotes paracellular integrity in C. parvum infection.
Decline in barrier function of infected mucosa treated with L-NAME could result from either arrest of epithelial replacement (restitution) or loss of paracellular integrity. To determine which mechanism was responsible, infected and control tissues were incubated in the presence or absence of L-NAME and removed from the chamber after 300 min for examination by light microscopy. In the absence of treatment, control and infected epithelia were indistinguishable in appearance from their freshly obtained counterparts and were lined by a continuous, well-apposed layer of enterocytes (Fig. 4). In contrast, after incubation with L-NAME, infected mucosa showed dilation of intercellular space and expansion of the lamina propria. Only a minor amount of intercellular space dilation and expansion of lamina propria was visible in L-NAME-treated, uninfected mucosa. To more closely examine the nature of the barrier defect resulting from blockade of NO, transmission electron microscopy was performed on infected epithelium incubated in the presence and absence of L-NAME. In untreated epithelium, there was close apposition of the lateral membranes, whereas L-NAME-treated epithelium showed marked dilation of the lateral intercellular space below the tight junction (Fig. 5).

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Fig. 4. Light microscopic appearance of infected (A and B) and control mucosa (C and D) after incubation in Ussing chambers for 300 min in the absence (A and C) or presence (B and D) of L-NAME (10 mM). In the presence of L-NAME, infected epithelia showed dilated paracellular pathways and expansion of lamina propria without loss of confluence. Dilation of paracellular space was evident to a lesser degree in identically treated control mucosa. Bar = 100 µm (A and B) and 50 µm (C and D).
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Fig. 5. Transmission electron micrographs of C. parvum-infected villus epithelium after incubation in Ussing chambers for 300 min in the absence (A) or presence (B) of L-NAME (10 mM). An organism is present among the apical membrane microvilli in A and B. Dramatic dilation of the paracellular pathway was evident in mucosa treated with L-NAME.
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Infected mucosa treated with L-NAME remained covered by a confluent layer of epithelium, suggesting that ongoing restitution was not interrupted by blockade of NOS. To ensure that arrest of restitution would be demonstrable over the time period of our studies, restitution by infected mucosa was blocked by incubation with cytochalasin D (3 x 105 M), an inhibitor of actin polymerization. Such treatment resulted in demonstrable increases in exposure of villous basement membrane over the 300-min duration of incubation. We additionally quantified the underlying restitution rate of the infected epithelium in the presence and absence of L-NAME by measuring the degree of villous reepithelialization achieved after denudation by a low concentration of deoxycholate (1.5 mM for 15 min). The rate of restitution of infected mucosa was not impaired by the presence of L-NAME [%reepithelialization = 76 ± 9 no Tx; 86 ± 7 L-NAME (10 mM); n = 6 each; Fig. 6].

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Fig. 6. Restitution of C. parvum-infected porcine ileal mucosa after exfoliation of villous epithelium with low-dose deoxycholate. Infected mucosa before treatment with deoxycholate (A), immediately after a 15-min mucosal exposure to 1.5 mM deoxycholate (B), and after 180 min of restitution in the absence (C) and presence (D) of 10 mM L-NAME in the serosal reservoir. There was no significant difference in restitution rate of mucosa treated or not treated with L-NAME (data reported in text). Bar = 50 µm.
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NO promotes barrier function via PG synthesis in C. parvum infection.
Akin to our observations on the effects of L-NAME, we have previously demonstrated that TER of C. parvum-infected and not control mucosa is significantly inhibited by incubation with the nonselective COX inhibitor Indo (3). Furthermore, the barrier-maintaining properties of PG are attributed to paracellular effects related to stimulation of Cl secretion that promotes withdrawal of Na+ and water from the paracellular spaces of the crypt and inhibition of neutral NaCl absorption, resulting in deceased paracellular water absorption by the villus (3, 8, 16). We therefore hypothesized that NO promotes barrier function in C. parvum infection by mediating the synthesis of endogenous PG. Thus we measured PGE2, 6-keto-PGF1
(the stable metabolite of PGI2), and TXB2 (the stable metabolite of TXA2) production by control and infected mucosa after incubation in Ussing chambers in the presence of L-NAME, Indo, or both (Table 2). Production of PGE2 was significantly greater in infected mucosa. In the presence of L-NAME, synthesis of all three eicosanoids by both control and infected mucosa was inhibited significantly. To determine whether PG depletion was mediating the effect of L-NAME on barrier function in the infection, the ability of mucosa to maintain TER was determined during incubation with Indo alone (5 x 106 M) or in combination with L-NAME (10 mM). Indo inhibited TER and dilated paracellular pathways of infected mucosa in a manner analogous to that of L-NAME. Neither inhibitor achieved a significant reduction in TER of control mucosa [
·cm2 at 315 min (no. of piglets); control = 47 ± 3.6 (n = 12), control + L-NAME = 40 ± 2.3 (n = 12), control + Indo = 47 ± 3 (n = 12), control + L-NAME + Indo = 41 ± 3 (n = 11), infected = 22 ± 0.9 (n = 8), infected + L-NAME = 16 ± 0.9 (n = 8) (P < 0.001, 1-way ANOVA), infected + Indo = 17 ± 0.8 (n = 8) (P < 0.01, 1-way ANOVA), infected + L-NAME + Indo = 15 ± 1.0 (n = 8) (P < 0.001, 1-way ANOVA); Fig. 7].
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Table 2. Concentration of PGE2, 6-keto-PGF1 (the stable metabolite of PGI2), and TXB2 (the stable metabolite of TXA2) in the serosal bath of control and Cyrptosporidium parvum-infected porcine ileal mucosa as measured by ELISA
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Fig. 7. Light microscopic appearance of infected mucosa after incubation in Ussing chambers for 300 min in the presence of indomethacin (Indo; 5 x 106 M; A), L-NAME (10 mM; B), and L-NAME + exogenous PGE2 (C). Indo and L-NAME had identical effects on dilation of paracellular pathway of infected mucosa. Addition of exogenous PGE2 reversed the effect of L-NAME on paracellular pathway dilation. Bar = 50 µm.
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We next examined the ability of each eicosanoid to abrogate the inhibitory effect of L-NAME on barrier function of infected mucosa (Fig. 8). In the absence of L-NAME, exogenous PG had no effect on barrier function. However, the inhibitory effect of L-NAME on TER was significantly abrogated by exogenous PGE2, but not by carbacyclin (an analog of PGI2; 106 M) or carbocyclic TXA2 (5 x 108 or 106 M). Light microscopic examination of L-NAME-treated mucosa rescued with PGE2 demonstrated closure of the paracellular pathway (Fig. 7). Thus, although L-NAME inhibited synthesis of each eicosanoid (PGE2, PGI2, and TXA2), PGE2 was the primary mediator of NO effects on barrier function and closure of paracellular space in infected mucosa.

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Fig. 8. TER of C. parvum-infected ileal mucosa after 300 min of incubation in Ussing chambers in the presence of no treatment, L-NAME (10 mM), 16,16-dimethyl-PGE2, the PGI2 analog carbacyclin, or the stable thromboxane (TX)A2 analog carbocyclic TXA2 (each 106 M serosal). *P < 0.05, 1-way ANOVA. P = 0.01, L-NAME vs. L-NAME + PGE2, Student's paired t-test; n = no. of piglets.
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Expression of NOS isoforms by ileal mucosa in C. parvum infection.
In an effort to identify which NOS isoforms were responsible for mediation of PG synthesis and maintenance of barrier function in C. parvum infection, we quantified mRNA expression of each isoform in mucosa from control and infected piglets harvested at the time of our functional studies using real-time RT-PCR (n = 5 piglets each; days 35 postinfection). Expression of endothelial NOS (eNOS) and nNOS mRNA was not altered by C. parvum infection, whereas iNOS mRNA was significantly increased (Fig. 9).

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Fig. 9. Effect of C. parvum infection on expression of inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) NOS mRNAs in ileal mucosa. Data represent the degree of difference in mRNA levels adjusted for differences in housekeeping cyclophilin expression among samples. *P < 0.05 vs. control, 1-way ANOVA; n = no. of piglets.
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Constitutive NOS and iNOS promote PG synthesis in C. parvum infection.
To determine the contribution of constitutive NOS (cNOS) and iNOS to PG synthesis and barrier function in C. parvum infection, sheets of ileal mucosa were incubated in Ussing chambers with NOS isoform-specific inhibitors, and the effect on TER and PG synthesis was measured. As previously shown, inhibition of both iNOS and cNOS activity by L-NAME significantly inhibited PGE2 synthesis by both infected and control mucosa (Table 2). Greater than 80% of PGE2 synthesis was NOS dependent in control and infected mucosa. Incubation with L-NIL (30 µM), a selective and irreversible inhibitor of iNOS, had no effect on synthesis of PGE2 or PGI2 by control mucosa, as would be expected in the absence of this isoform. In infected mucosa, L-NIL inhibited 45 ± 15% of NOS-dependent PGE2 synthesis (Fig. 10A) and synthesis of PGI2 [pg/ml
PGI2 (no. of piglets); infected = 7,013 ± 1,045 (n = 7), infected + L-NIL = 3,874 ± 617 (n = 8) (P < 0.05, 1-way ANOVA)]. In terms of barrier function, inhibition of total NOS or iNOS activity had no effect on TER of control mucosa. In infected mucosa, L-NIL inhibited 50% of NOS-dependent TER, although alone the effect of L-NIL on TER was not statistically significant, even in the presence of high doses of the inhibitor (L-NIL; 1001,000 µM; Fig. 10B). These data suggest that the constitutive and inducible isoforms of NOS have an additive effect on PGE2 synthesis and that inhibition of both isoforms is necessary for a significant decline in barrier function.

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Fig. 10. Change in concentration of PGE2 in the serosal bath (A) and TER (B) of control and C. parvum-infected porcine ileal mucosa incubated in Ussing chambers in the presence of the iNOS-selective inhibitor L-N6-(1-iminoethyl)-L-lysine (L-NIL) or the nonselective NOS inhibitor L-NAME. Samples for PGE2 ELISA were obtained 30 min after each tissue was mounted in an Ussing chamber and subtracted from values obtained after incubation for 300 min in the presence of each treatment. For PGE2 data: *P < 0.05, **P < 0.01, and ***P < 0.001, 1-way ANOVA compared with no treatment. For TER data: **P < 0.01 and *P = 0.053, Student's t-test compared with no treatment at 315 min; n = no. of piglets.
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We attempted to define which constitutive isoform, nNOS or eNOS, contributed to barrier function and PG synthesis by incubating infected mucosa with several reportedly selective inhibitors of nNOS (7-NI, NPA, or TRIM). A significant drawback to this approach is the overlapping specificity of available cNOS inhibitors, their undetermined optimal dose for studies in intact tissue, and lack of means to separately quantify nNOS vs. eNOS activity. Thus we examined only conservative doses of each inhibitor (1, 100, and 1,000 µM; n
5 piglets each). Incubation of infected mucosa for 300 min in the presence of the nNOS inhibitors 7-NI, NPA, or TRIM had no significant effect on barrier function (data not shown). We additionally measured PG synthesis by infected mucosa in the presence of TRIM (100 µM) and observed no significant effect on PGE2 or PGI2 synthesis [pg/ml
PGE2 (no. of piglets); control = 1,456 ± 346 (n = 8), control + TRIM = 2,825 ± 570 (n = 8), infected = 2,789 ± 491 (n = 7), infected + TRIM = 4,394 ± 1,085 (n = 8):
PGI2 (no. of piglets); control = 5,443 ± 647 (n = 8), control + TRIM = 7,242 ± 671 (n = 8), infected = 7,013 ± 1,045 (n = 7), infected + TRIM = 6,551 ± 2,420 (n = 8)]. These results suggest that eNOS and not nNOS is likely the constitutive isoform contributing to PGE2 synthesis and barrier function in the infection.
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DISCUSSION
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The present study has demonstrated that NO, elaborated by cNOS and iNOS, serves as a proximal mediator of PGE2 synthesis and barrier function in C. parvum infection (Fig. 11). This conclusion derives from several observations. First, incubation of ileal mucosa with the nonselective NOS inhibitor L-NAME inhibited synthesis of PGE2 in a manner analogous to that of Indo, whereas selective blockade of iNOS using L-NIL blocked
50% of NOS-dependent (L-NAME-inhibitable) PGE2 synthesis. After blockade of iNOS activity, PGE2 synthesis was reduced to levels seen in uninfected tissue. Second, nonselective inhibition of NOS inhibited TER in a manner equivalent to that of Indo, whereas selective inhibition of iNOS attenuated TER by 50% but alone was insufficient to significantly decrease barrier function. Both nNOS and eNOS can contribute to constitutive NO synthesis. Inhibitors of nNOS had no effect on PGE2 synthesis or barrier function. Because of inadequate means to selectively block eNOS activity, we could not discern if eNOS inhibition alone could account for the entirety of L-NAME effects on barrier function. However, given the equal contribution of cNOS and iNOS activity to PGE2 synthesis in the present study and the intermediate effect of L-NIL on TER reduction (Fig. 10), an additive contribution of eNOS and iNOS to barrier function is plausible. Finally, the loss of barrier function resulting from L-NAME was recovered by exogenous addition of 16,16-dimethyl-PGE2, but not by the synthetic analogs of PGI2 or TXA2.

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Fig. 11. Hypothetical schema for the action of iNOS and constitutive NOS (cNOS) on PGE2 synthesis with relative contributions to barrier function of C. parvum-infected porcine ileal mucosa.
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Increases in transepithelial permeability associated with C. parvum are well documented in cell monolayers, in vivo experimental models, and naturally occurring infection (1, 4, 12, 14, 18, 19, 48). In the present study, baseline barrier function, as determined by TER and passive serosal-to-mucosal flux of 22Na+, was significantly impaired by C. parvum infection and worsened further by the absence of NO-mediated elaboration of PGE2. NO and reactive nitrogen metabolites, such as peroxynitrite (formed by the reaction of NO with superoxide), have been demonstrated in a variety of experimental models to stimulate COX enzyme activity and PG synthesis (15, 42, 46). We have previously shown that mucosal PG synthesis is increased in C. parvum-infected piglets and calves and contributes to barrier function (3, 10). It is likely that the interplay between NOS and COX in the present study involves specific sites of NO production and cellular interactions, insofar as bathing infected mucosa with NO donors fails to recapitulate the effect of endogenous NOS activity on barrier function. Both endogenous NO activity and peroxynitrite formation are diffusion limited, and their effect on PG synthesis has been shown in some studies to depend critically on the identity of the COX isoform with which they interact (9). Additionally, broad application of NO has been demonstrated to result in a variety of effects that diminish barrier function of normal epithelial cells in culture (34, 42). The source of elevated PG in C. parvum infection has not been definitively established but may be the result of infiltrating polymorponuclear neutrophils and macrophages, the products of which have been shown to strongly induce PG synthesis by mesenchymal cells in the lamina propria (24, 21). Additionally, C. parvum has been shown to directly activate COX-2 expression and PGE2 synthesis by cultured human intestinal epithelial cells (28).
The mechanism by which PGE2 promotes barrier function of C. parvum-infected mucosa remains to be fully elucidated but appears to involve maintenance of paracellular space closure rather than an effect on epithelial restitution. In mucosa treated with Indo or L-NAME, decline in barrier function was associated with distension of paracellular pathways and expansion of lamina propria consistent with absorption, whereas these effects were reversed with exogenous PGE2. Prior studies of C. parvum infection have demonstrated that PG induce anion (Cl or HCO3) secretion, which promotes withdrawal of Na+ and water from the paracellular spaces of the crypts and inhibit neutral NaCl absorption, resulting in decreased paracellular water absorption by the villus (3). These alterations are attributed to direct effects of PGE2 on the epithelium and indirect effects via PGI2 activation of the enteric nervous system (2). Our observation that PGI2 did not contribute to barrier function is supported by previous studies in C. parvum-infected piglets demonstrating that inhibition of PGI2-activated neurons (using clonidine and somatostatin) does not significantly decrease TER (2).
In the present study, both L-NAME and Indo inhibited TER of infected and not control mucosa. This observation is consistent with prior studies demonstrating that Indo has no effect on TER of uninjured mucosal epithelium but significantly impairs barrier function of leaky epithelium arising as a result of C. parvum infection (3), ischemia (8), or bile salt injury (16). Each of these injuries is associated with a significant increase in mucosal PGE2 synthesis. Although iNOS inhibition appeared to abolish the rise in PGE2 synthesis resulting from C. parvum infection, a reduction in PGE2 synthesis below that seen in uninfected tissues was necessary to significantly impair barrier function. We speculate that basal amounts of PG are not necessary to maintain intercellular space closure in the absence of epithelial injury and have shown that Indo has no effect on NaCl transport or resistance of uninfected tissue (3). In contrast, with ongoing epithelial injury and accelerated restitution, PG effects on NaCl transport may contribute to reestablishment of paracellular space closure and resistance, as has been shown after ischemia and bile salt injury (8, 16).
Results of the present study demonstrate that C. parvum infection leads to induction of iNOS and increased synthesis of NO and PGE2 by parasitized intestinal mucosa. Studies in mice have shown that C. parvum infection is associated with an increase in iNOS expression by the epithelium (31). Although we did not specifically investigate the long-term consequences of iNOS expression in C. parvum infection, prior observations have shown that exposure to high concentrations of NO promotes cytotoxicity and permeability of cultured epithelia (7, 34, 41, 43, 46). These observations suggest a role for iNOS in purging the intestine of the infection. More specifically, NO donors have been shown in vitro to reduce viability of C. parvum sporozoites (30), and iNOS knockout mice or mice treated with NOS inhibitors have more severe intestinal infection and delayed parasite clearance (30, 31). Nevertheless, the role of epithelial vs. subepithelial iNOS in mediating these effects remains unclear. Although iNOS may promote elimination of infected enterocytes, overall epithelial barrier function appears to be promoted by the paracellular effects of NO-mediated PGE2 synthesis.
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GRANTS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02868 (to J. L. Gookin).
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ACKNOWLEDGMENTS
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We thank Sophia Chiang, Jessica Allen, and Philip Ruckart for excellent technical assistance; the Laboratory for Advanced Electron and Light Optical Methods, North Carolina State University, College of Veterinary Medicine, for assistance with transmission electron microscopy; and the National Institutes of Health-Center for Gastrointestinal Biology and Disease Immunotechnology Core for performance of eicosanoid assays.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. L. Gookin, College of Veterinary Medicine, North Carolina State Univ., 4700 Hillsborough St., Raleigh, North Carolina, 27606 (E-mail: Jody_Gookin{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.
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