Developmental variability in expression and regulation of inducible nitric oxide synthase in rat intestine

Melinda J. Morin, Shannon M. Karr, Ronald A. Faris, and Philip A. Gruppuso

Department of Pediatrics, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island 02903


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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Inducible nitric oxide synthase (iNOS) may be a key mediator of intestinal injury, which varies with developmental age. One member of the mitogen-activated protein kinase (MAPK) family, p38, is involved in the lipopolysaccharide (LPS)-mediated iNOS induction. The involvement of p38 MAPK in basal and LPS-induced iNOS expression was examined in the rat intestine at two developmental ages. Neonatal (4 days postnatal) and adolescent (15 days postnatal) rats were injected with LPS (5 µg/g ip), a selective p38 inhibitor (SB 203580), or both. Tissue was removed after 4 h and 6 h for mRNA and protein analysis. iNOS mRNA and protein were markedly upregulated in the adolescent female following LPS exposure, whereas males had an attenuated response. Neonates had a minimal response. SB 203580 suppressed LPS-induced iNOS mRNA and protein in the ileum, more so in females than in males. Adolescent ileal p38 activation was constitutively high and nonresponsive to LPS. Basal and post-LPS p38 phosphorylation was low in neonatal ileum. We conclude that ileal iNOS expression is developmentally regulated and influenced by gender and that p38 is permissive for LPS effect. The developmental regulation of p38 may contribute to age-dependent variations of intestinal injury.

lipopolysaccharide; p38 mitogen-activated protein kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) IS A SHORT-LIVED proinflammatory mediator produced by mammalian cells (35, 38, 41). There is accumulating evidence that NO is a pivotal mediator of gut pathophysiology (18). One of three NO synthase isoforms involved in NO synthesis, inducible NO synthase (iNOS), is characterized by its cytokine upregulation and production of high quantities of NO (30, 42).

NO is synthesized in the intestine during endotoxin-mediated septic shock and may be a key component of sepsis-induced intestinal damage. During inflammatory states, gut-derived NO appears to cause direct mucosal injury with disruption of barrier function and maldistribution of blood flow (3, 11). iNOS inhibitors have been shown to ameliorate intestinal hyperpermeability (50) and improve cell viability (7, 49) in lipopolysaccharide (LPS)-exposed animals.

In recent years, the mechanisms involved in neonatal intestinal injury have come under close scrutiny. There is compelling evidence that the neonatal gastrointestinal tract may be uniquely susceptible to NO-induced injury in animals exposed to LPS or cytokines (40). Previous work has demonstrated that iNOS mRNA expression is differentially regulated along the longitudinal axis of the intestine, being most prominent in the ileum (39). iNOS mRNA localizes to the more superficial population of cells in the intestinal mucosa, particularly differentiated villus cells (37, 39). Other in vitro data support this dependence of iNOS expression on intestinal epithelial differentiation (8). These data may be interpreted as suggesting a developmental variability in LPS-induced iNOS upregulation underlying age-dependent patterns of intestinal injury.

Mammalian development involves significant changes in sex hormones. Gender differences in NO production have recently been demonstrated with increased iNOS mRNA expression (34) and total NO production (44) in female rats. Although known to modulate immune function, the effects on proinflammatory and cytokine cascades have yet to be fully defined.

A number of studies have demonstrated that the mitogen-activated protein kinase (MAPK) cascade is targeted by cytokines and LPS in vitro (24, 47, 48). After binding to CD14, LPS has been shown to trigger the activation of several protein kinases, leading to the secretion of various immunomodulatory molecules, including NO. In particular, the MAPK family member p38 has been found to play an important role in LPS-induced iNOS expression in several cell lines (1, 9, 14). The signaling pathways involved in iNOS upregulation in vivo have not been elucidated. The aim of this study was to determine whether an ontogenic pattern of iNOS expression existed in the small intestine of endotoxic animals and to determine if age-dependent regulation of p38 contributes to this pattern.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal studies. The experiments described were approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital and were performed in accordance with the National Institutes of Health guidelines for the care and handling of animals (DHHS Publication no. 85-23, 1985). Pregnant Sprague-Dawley rats were purchased from Charles River Breeding Labs (Wilmington, MA). After arrival, cages were checked twice daily for pups. Pups between 0 and 21 days of age were reared with their mother, who was fed and allowed water ad libitum. Weaning in our laboratory was observed to occur at 21 days of age. Study animals were not weaned. All animals were kept in a temperature-controlled room and maintained on a 12:12-h light/dark cycle.

Pups were randomized to four experimental groups: 1) 4-day-old control (intraperitoneal sterile saline injection), 2) 4-day-old LPS (Escherichia coli serotype 0111:B4, Sigma Chemical, St. Louis, MO; 5 µg/g ip in sterile saline), 3) 15-day-old control (sterile saline), and 4) 15-day-old LPS (5 µg/g ip in sterile saline). The volume of agents administered was <= 10 µl/g body wt. Adolescent tissue was segregated according to animal gender. Tissue for mRNA analysis was obtained 4 h after LPS injection; that for protein analysis was obtained 6 h after LPS injection. All animals were treated so that the time of death would be between 1300 and 1500 h to eliminate any potential diurnal variation.

Another group of adolescent rats received the p38 inhibitor SB 203580 (Calbiochem, San Diego, CA) (32) as part of the experimental protocol. SB 203580 (10 µg/g ip in saline) was injected simultaneously with the LPS or saline administration. Tissue was segregated by gender. To examine p38 activation in response to LPS, some animals received a single dose of LPS (5 µg/g ip) or saline. Ileum and liver tissue were removed at time points 2-20 min later. Isoflurane was used for rapidity of anesthesia before tissue excision in these animals. In all other cases, rats were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg) before euthanasia. After satisfactory anesthesia was obtained, a midline laparotomy incision was performed. Approximately 3 cm of distal ileum, located ~1 cm proximal to the ileocecal valve, was removed. Ileal segments from 15-day-old pups were excised longitudinally and gently rinsed with saline. One lobe of the liver was also excised. Tissue was immediately placed into Bouin's fixative, iced extraction buffer containing protease inhibitors, or guanidinium thiocyanate.

RNA isolation and Northern blot analysis. Total RNA was extracted using the guanidinium thiocyanate method (10). Total cellular RNA (20 µg) was analyzed by 1% agarose formaldehyde gel electrophoresis, blotting onto nitrocellulose membranes by capillary action, and ultraviolet cross-linking. Blots were hybridized overnight at 42°C with a hybridizing solution containing 50% (vol/vol) formamide in 5× saline citrate buffer (SSC), 1% (wt/vol) SDS, and denatured cDNA probe. After completion of hybridization, the blots were washed three times for 15 min at room temperature in 2× SSC then twice for 15 min at 50°C in 2× SSC containing 0.1% SDS. Autoradiography was performed at -70°C. Relative concentrations of mRNA were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA.

cDNA probes were dCTP labeled (with 32P) by the random primer method to a specific activity of ~3 × 108 dpm/µg of DNA. The iNOS probe was a 1.2-kb (EcoR I/BamH I) fragment derived from a human iNOS cDNA clone generously provided by Dr. David Geller (University of Pittsburgh). The GAPDH probe was a 1.27-kb fragment derived from human GAPDH (Clontech, Palo Alto, CA).

Protein extraction and Western immunoblot analysis. Rat ileum and liver were homogenized in extraction buffer, 1 ml/0.1 g tissue (10 mM Tris, pH 8.0, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, and 100 µM sodium vanadate) containing the protease inhibitors leupeptin (1 µg/ml), aprotinin (1 µg/ml), and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (2 µg/ml). Samples were then centrifuged at 18,000 g for 20 min. The supernatant was removed, and protein concentrations were determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL). To increase iNOS antibody specificity, immunoprecipitation was carried out using protein A-Sepharose CL-4B beads to which the iNOS antibody had been covalently coupled using dimethylpemilimidate (20 mM in 0.1 M sodium borate, incubated for 30 min at room temperature). Western immunoblotting was performed using rabbit polyclonal iNOS antibody (1:200 dilution; Transduction Laboratories, San Diego, CA) and 2 mg of homogenate protein. Proteins were separated by 7.5% polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. Proteins were transferred to a polyvinylidene difluoride membrane and incubated for 1 h at room temperature in blocking solution (5% nonfat dried milk and PBS). The membrane was then washed twice in Tris-buffered saline solution with 0.2% Tween (TBST), followed by a 16-h incubation with the same rabbit polyclonal iNOS antibody (1:200 dilution in TBST), rabbit polyclonal phospho-specific p38 antibody (1:1,000 dilution in TBST; New England Biolabs, Beverly, MA), or rabbit polyclonal p38 antibody (1:1,000 dilution in TBST; New England Biolabs). After being washed with TBST, protein was detected using a horseradish peroxidase-conjugated goat-anti-rabbit immunoglobulin as secondary antibody (1:10,000 dilution at room temperature; Pharmingen, San Diego, CA). After 5 additional washes, the immune complexes were visualized by using enhanced chemiluminescence detection reagents (Amersham). The resulting autoradiograph was analyzed by computer-assisted densitometry. Results are reported as relative absorbance units.

Immunohistochemistry. Sections of paraffin-embedded rat ileum (4 µm thick) were deparaffinized with xylene and graded ethanol. Endogenous peroxidase was blocked with 2% hydrogen peroxide in 70% methanol for 10 min at room temperature. Nonspecific binding was reduced by incubation with normal goat serum for 30 min. Sections were rinsed with PBS and incubated for 2 h with rabbit polyclonal anti-human iNOS IgG (N32030; Transduction Laboratories) at a dilution of 1:100, followed by biotinylated goat anti-rabbit secondary antibody (Pierce) at a dilution of 1:100 in PBS, and avidin-biotin peroxidase (Vector, Burlingame, CA). Sections incubated without primary antibody were included as controls. Sections were counterstained with methylene green, dehydrated, and mounted.


    RESULTS
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INTRODUCTION
METHODS
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DISCUSSION
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iNOS mRNA expression following LPS exposure. To determine iNOS mRNA expression at the two developmental ages under investigation, ileal segments and liver from LPS or saline-exposed, neonatal or adolescent rats were analyzed by Northern blot hybridization (Fig. 1). Male and female tissues were pooled. Control animals from both developmental groups displayed no iNOS mRNA signal after saline injection. Four hours after endotoxin exposure, Northern blot analysis demonstrated the induction of a 4.4-kb iNOS mRNA in both the ileum and liver (Fig. 1, top). The size of the iNOS transcript was the same at both developmental ages. Time course experiments in both neonatal and adolescent animals revealed maximal iNOS mRNA upregulation 4 h after LPS exposure (results not shown). Gender segregation revealed no difference in iNOS mRNA expression in the neonatal animals. However, iNOS expression in the adolescent was gender specific and consistently upregulated beyond that in the neonatal ileum (see Fig. 5, top). Hepatic induction was similar in both age groups. These changes in iNOS mRNA were quantified by scanning densitometry of Northern blots from three different experiments (Fig. 1, bottom). Following LPS exposure, adolescent ileum demonstrated a 10-fold higher mean level of iNOS transcript compared with neonates.


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Fig. 1.   Ontogeny of inducible nitric oxide synthase (iNOS) mRNA expression in the ileum and liver following saline or lipopolysaccharide (LPS) injection. Adolescent male and female tissues were pooled. A: representative Northern blot analysis for iNOS mRNA (4.4 kb) 4 h after saline (-) or LPS (+) injection. N, neonatal tissue; A, adolescent tissue. The Northern blot was sequentially hybridized with iNOS and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA probes. B: quantification of iNOS mRNA by densitometry. C, control. Data are shown as means ± SD for 3 independent experiments.

iNOS protein expression after LPS injection. Samples of ileum and liver from LPS- or saline-injected rats at the two developmental ages were analyzed for the presence of iNOS protein by Western immunoblot analysis (Fig. 2, top). Male and female tissues were pooled. An equal amount of protein was added to each lane. Tissue harvested from rats that had been injected with 5 µg/g LPS 6 h previously showed a 130-kDa band in ileal as well as hepatic samples of LPS-injected rats. Noninjected animals and those injected with saline showed no expression of iNOS protein. iNOS protein levels in LPS-injected neonatal animals were markedly lower than those seen in the adolescent rats. Consistent with the mRNA data, there was no discernible gender specificity to neonatal iNOS protein expression. Adolescent female animals had an amplified ileal response to LPS compared with male animals (see Fig. 6, top). Importantly, minimal difference in iNOS protein production was seen in the neonatal vs. adolescent liver. iNOS protein levels were quantified by scanning densitometry of Western blots from four different experiments (Fig. 2, bottom). Adolescent ileum demonstrated an ~10-fold greater increase in protein content after LPS compared with neonatal ileum.


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Fig. 2.   Western immunoblot analysis of age-dependent iNOS protein induction by LPS in the ileum and liver. Male and female adolescent tissues were pooled. A: representative blot showing immunoreactive iNOS (130 kDa) 6 h after injection of saline (-) or LPS (+). B: quantification of iNOS protein content by densitometric units. Data are shown as means ± SD of 4 experiments.

Immunohistochemical localization of iNOS. To localize the LPS-induced expression of iNOS protein in the ileum, sections were stained with rabbit anti-human iNOS antibody (Fig. 3). Following endotoxin exposure, immunostaining for iNOS was found in the mucosal layer of the ileum from adolescent rats (Fig. 3, B and D). Staining in the crypts was negligible but became more intense along the length of the villus, with appreciable iNOS immunostaining in the epithelial cells at the villous tips. Minimal or no staining was seen in other cells. iNOS expression was not detected in the non-LPS treated animals (Fig. 3, A and C) or when the primary antibody was omitted (not shown). In marked contrast, epithelial cells from LPS-injected 4-day-old rats showed a minimal level of iNOS immunoreactivity (Fig. 3F). No crypt-to-villus iNOS gradient was apparent in these animals. As was the case for older rats, the noninjected 4-day-old rats showed no immunoreactivity (Fig. 3E). Similar results were obtained in three additional experiments.


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Fig. 3.   Age-dependent expression of iNOS upregulation in the ileum following LPS or saline injection. All sections were stained with anti-human iNOS antibody, as described in METHODS. A: 16-day-old saline-injected rat (×20). B: 16-day-old rat injected with LPS 6 h before death (×20). C: 16-day-old saline-injected rat (×40). D: 16-day-old rat injected with LPS 6 h before death (×40). E: 4-day-old saline-injected rat (×20). F: 4-day-old rat injected with LPS 6 h before death (×20).

p38 activation state in neonatal and adolescent ileum and liver. p38 phosphorylation, as detected in Western immunoblotting with phospho-specific p38 antibodies (Fig. 4), was used as an indirect indicator of p38 activation state. Following saline (time 0) or LPS injection, both ileum and liver from neonatal animals showed little phospho-p38. In contrast, phospho-p38 was easily detected in ileum and liver from saline- or LPS-injected adolescent animals. LPS administration had no apparent effect on phosphorylation of p38. Total p38 content was similar in the ileum at the two developmental ages. Similar results were obtained in two additional experiments.


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Fig. 4.   p38 phosphorylation before and after LPS injection. Representative Western blots are shown for ileum (A) and liver (B), demonstrating time-independent phosphorylation of p38 mitogen-activated protein kinase and total p38 protein following LPS treatment. Saline-injected neonates and adolescents were harvested at time 0. Proteins were quantified by scanning densitometry of Western blots from 3 different experiments.

Effects of inhibition of p38 MAPK activity. On the basis of our findings indicating that p38 is constitutively active in adolescent ileum and liver, we attempted to inhibit p38 pharmacologically in these older animals to assess its role in the regulation of iNOS expression. Because of some variability observed in our prior results, experiments were repeated and tissue was segregated by gender. Animals were injected with the specific p38 inhibitor SB 203580, LPS, or both. The LPS and kinase inhibitor injections were simultaneous. Results (Fig. 5, top) showed that administration of SB 203580 at the time of LPS injection attenuated the increase in ileal iNOS mRNA induction in each of three replicate experiments. Female animals had an amplified ileal response to LPS compared with male rats. Female animals also had an exaggerated attenuation of LPS-induced iNOS expression following SB 203580 exposure. This effect was also seen at the level of ileal protein content in three additional experiments (Fig. 6, top). An equal amount of protein was added to each lane. Equivalent loading of lanes was verified by immunoblotting with p38, a constitutively expressed protein (data not shown). Of note, SB 203580 had no effect on hepatic mRNA or protein levels in the male but did attenuate iNOS expression in the female. These changes in iNOS mRNA and protein were quantified by scanning densitometry of Northern and Western blots (Fig. 5, bottom, and Fig. 6, bottom).


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Fig. 5.   Effects of p38 inhibition on iNOS mRNA expression and induction in adolescent ileum and liver. A: representative Northern blot analysis for iNOS mRNA. An ethidium bromide stains for 18S and 28S ribosomal RNA are shown. Male and female samples were analyzed on the same Northern blot. Results were normalized to the LPS-injected male animals for densitometry. B: quantification of iNOS mRNA by densitometry. Data are shown as means ± SD for 3 independent experiments; p38i, the p38 inhibitor SB203580.



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Fig. 6.   Effects of p38 inhibition on iNOS protein expression and induction in adolescent ileum and liver. A: representative Western immunoblot analysis for iNOS protein content. Male and female samples were analyzed on the same Western blot. Results were normalized to the LPS-injected male animals for densitometry B: quantification of iNOS protein content by densitometry. Data are means ± SD of 3 experiments.


    DISCUSSION
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The mucosal epithelium represents the primary barrier against bacterial translocation in the intestine. Current evidence suggests a paradoxical role for iNOS, with early induction considered a protective response (26). Continued iNOS expression, however, appears to result in mucosal injury (52). Following LPS exposure, Mercer et al. (36) demonstrated intermittent morphological injury in the rat small intestine, including areas of epithelial lifting and frank necrosis. Miller et al. (37), in their model of guinea pig ileitis, demonstrated that iNOS expression was increased in areas of inflammation, with iNOS protein localizing to the most superficial layer of the mucosa.

Although not fully characterized, the effects of LPS on iNOS expression in the small intestine are complex. Previous studies have inferred that the ileum may be more susceptible to NO-dependent oxidative stress (6) and LPS-induced injury (15). We have previously demonstrated (39) that regional differences in iNOS immunoreactivity exist, although the LPS-induced morphological injury was similar in the ileum and jejunum (36). Whether NO production or NO toxicity is area specific remains undetermined (16); however, the greater magnitude of iNOS expression in this segment of the gut is consistent with its importance in sepsis-induced intestinal damage.

The transition from fetal to adult life involves profound alterations in the response to tissue injury and infection (12, 21, 22). Musemeche et al. (40) found an induction of several intestinal inflammatory enzymes, including xanthine oxidase, in the rat during the first 15 postnatal days. Digestive enzymatic function dramatically changes during the first two postnatal weeks in the rat (25). Other in vitro studies have shown similar ontogenic patterns with increasing NOS activity during the first 2 weeks of postnatal life for brain (33) and pulmonary neuronal NOS (45). Coincident with the development of other inflammatory and digestive enzyme activity, NOS function has been demonstrated to mature postnatally in the colon (4).

There is growing evidence that sexual dimorphism exists with regard to the immune response (2, 31). That females appear to have an improved immune response to LPS, trauma, or hemorrhage may reflect their increased iNOS production. Sex hormones are developmentally regulated factors that have been recently shown to have a significant effect on NO production (34, 43). On the basis of our findings that adolescent rats had a variable upregulation of iNOS following LPS exposure, we repeated our experiments with attention to animal gender. Although we have shown that ileal iNOS mRNA and protein expression are exaggerated in females compared with males, the specific role of female sex steroids in the iNOS signaling pathways remains unknown. Unfortunately, the majority of previous investigations on iNOS regulation used only male animals.

It has previously been shown that fetal rats are able to upregulate iNOS in the pulmonary (46) and cardiac (4) vascular beds following endotoxin exposure. We chose to investigate iNOS expression in the period following birth and in the preweaned animal, two periods of intense developmental change. The ileum was studied because of its high expression of iNOS on stimulation and susceptibility to hypoxia and reperfusion injury (29). Unlike regulation of iNOS in the colon (4), we found a significant difference in ileal iNOS expression at the two developmental ages studied. The in vivo regulation of ileal iNOS appears to be distinct from hepatic iNOS expression. Liver tissue was included in this study for comparison given the marked response to endotoxin and high mRNA content in the adult rat. Furthermore, the role of iNOS in the liver is fundamentally different from that in the gut, controlling hepatic protein synthesis (13) as well as mediating blood flow. As an embryologically distinct derivative of the gastrointestinal tract, it is not surprising that iNOS regulation is dissimilar.

The apparent downregulation of neonatal iNOS expression in the ileum coincides with the replacement of fetal cells with adult enterocytes. It is reasonable to postulate that the downregulation of neonatal iNOS expression may be a mechanism by which the metabolic demands of the rapidly proliferating ileal mucosa are met. This diminished iNOS expression may have several other physiological advantages. It may serve as a protective mechanism during the development of intraluminal bacterial colonization when a pronounced iNOS response could be deleterious. With the replacement of fetal cells, the risk of increased mucosal permeability escalates. A diminished iNOS response may be advantageous because the mucosal vascular supply changes during the transition from an environment of relative hypoxia.

Of interest is the finding that enterocytic iNOS expression is high in infants with necrotizing enterocolitis (19). Despite intense investigation, the pathogenesis of necrotizing enterocolitis remains obscure. The neonatal intestine has a diminished compensatory response to hemorrhage, hypothermia, and hypoxia. As the typical arteriolar vasodilation response is absent in the neonate, the neonatal intestine is more susceptible to the pathophysiological stresses of postnatal life (20, 21). The present finding of diminished iNOS induction may underlie this fixed vasomotor tone in the neonate. Perhaps the downregulation of the early induction of iNOS, with the loss of its protective effects, is involved in the pathophysiological process leading to necrotizing enterocolitis. Why this differential regulation of iNOS occurs only in the neonate remains undetermined and may indicate a variation in the signaling pathways controlling NOS induction. We therefore undertook studies on the role of a critical stress-activated MAPK, p38, in the ontogeny of intestinal iNOS regulation.

Although ileal and hepatic p38 content are similar at the two developmental ages studied, p38 activation state, as indicated by p38 phosphorylation, is not. In both ileum and liver, p38 phosphorylation is constitutively high in adolescent rats compared with neonatal animals. LPS did not further stimulate p38 phosphorylation in either tissue at either developmental age. However, inhibition of p38 abrogates LPS-mediated iNOS induction in ileum from adolescent rats of both sexes, although the effect in females was greater. In the liver, this inhibitory effect was seen only in female animals and was less marked than in the ileum. These findings demonstrate the exaggerated iNOS response in females as well as the functional differences in the two organs studied with regard to NO physiology. In the neonate, ileal iNOS mRNA is weakly induced by LPS, yet no phospho-p38 activity is seen following LPS stimulation. In contrast, hepatic iNOS expression is strongly induced by LPS, also in the absence of phospho-p38 activity. This suggests that p38 activity is necessary, but not sufficient, for iNOS induction in ileum. Thus we have demonstrated a developmental role for p38 in the stress-mediated regulation of intestinal iNOS.

Important differences exist among the members of the p38 isoforms with regard to activation and biological effect (23, 27, 28). Wang et al. (51) have detected expression of multiple p38 isoforms in human intestine. Phospho-specific p38 antibodies are likely to recognize all forms of activated p38, given the high level of conservation at the activating phosphorylation site. Although SB 203580 is not equipotent for all p38 isoforms (17, 32), administration of this inhibitor did modify regulation of intestinal iNOS expression. It is possible that the relatively diminished SB 203580 effect in liver compared with ileum is the result of differential p38 isoform expression.

In conclusion, we have demonstrated that iNOS gene expression and protein content are developmentally regulated in the rat ileum. Induction after exposure to LPS is upregulated beyond the neonatal period in a gender-specific manner. Expression of mRNA coincides with protein content, consistent with transcriptional and/or posttranscriptional regulation. p38 MAPK activation is also developmentally regulated and contributes to the LPS-mediated induction of iNOS. p38 activity appears to permit, rather than mediate, LPS induction of iNOS, particularly in the female. This developmental pattern of p38 activity in the ileum may contribute to the complex maintenance of intestinal mucosal integrity and underlie the susceptibility of the neonatal gut to sepsis-induced mucosal damage.


    FOOTNOTES

Address for reprint requests and other correspondence: M. J. Morin, Division of Pediatric Critical Care, Rhode Island Hospital, 593 Eddy St., Providence, RI 02903 (E-mail: mjmorin{at}lifespan.org).

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 12 September 2000; accepted in final form 14 March 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 281(2):G552-G559
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society




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