Department of Pediatrics, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island 02903
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 wasRNA 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.
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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.
|
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.
|
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.
|
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).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ajizian, SJ,
English BK,
and
Meals EA.
Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-.
J Infect Dis
179:
939-944,
1999[ISI][Medline].
2.
Angele, MK,
Knoferl MW,
Schwacha MG,
Ayala A,
Cioffi WG,
Bland KI,
and
Chaudry IH.
Sex steroids regulate pro- and anti-inflammatory cytokine release by macrophages after trauma-hemorrhage.
Am J Physiol Cell Physiol
277:
C35-C42,
1999
3.
Boughton-Smith, NK,
Evans SM,
Laszlo F,
Whittle BJ,
and
Moncada S.
The induction of nitric oxide synthase and intestinal vascular permeability by endotoxin in the rat.
Br J Pharmacol
110:
1189-1195,
1993[Abstract].
4.
Brown, JF,
and
Tepperman BL.
Ontogeny of nitric oxide synthase activity and endotoxin-mediated damage in the neonatal rat colon.
Pediatr Res
41:
635-640,
1997[Abstract].
5.
Bustamante, SA,
Pang Y,
Romero S,
Pierce MR,
Voelker CA,
Thompson JH,
Sandoval M,
Liu X,
and
Miller MJ.
Inducible nitric oxide synthase and the regulation of central vessel caliber in the fetal rat.
Circulation
94:
1948-1953,
1996
6.
Chamulitrat, W,
Skrepnik NV,
and
Spitzer JJ.
Endotoxin-induced oxidative stress in the rat small intestine: role of nitric oxide.
Shock
5:
217-222,
1996[ISI][Medline].
7.
Chavez, AM,
Menconi MJ,
Hodin RA,
and
Fink MP.
Cytokine-induced intestinal hyperpermeability: role of nitric oxide.
Crit Care Med
27:
2246-2251,
1999[ISI][Medline].
8.
Chavez, AM,
Morin MJ,
Unno N,
Fink MP,
and
Hodin RA.
Acquired interferon--responsiveness during enterocytic differentiation: effects on iNOS gene expression.
Gut
44:
659-665,
1999
9.
Chen, CC,
and
Wang JK.
p38 but not p44/42 mitogen-activated protein kinase is required for nitric oxide synthase induction mediated by lipopolysaccharide in RAW 264.7 macrophages.
Mol Pharmacol
55:
481-488,
1999
10.
Chirgwin, JM,
Pryzbla AE,
MacDonald RJ,
and
Rutter WJ.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:
5294-5299,
1979[ISI][Medline].
11.
Cook, HT,
Bune AJ,
Jansen AS,
Taylor GM,
Loi RK,
and
Cattell V.
Cellular localization of inducible nitric oxide synthase in experimental endotoxic shock in the rat.
Clin Sci (Colch)
87:
179-186,
1994[ISI][Medline].
12.
Crissinger, KD,
and
Granger DN.
Mucosal injury induced by ischemia and reperfusion in the piglet intestine: influences of age and feeding.
Gastroenterology
97:
920-926,
1989[ISI][Medline].
13.
Curran, RD,
Ferrari FK,
Kispert PH,
Stadler J,
Stueher DJ,
Simmons RL,
and
Billiar TR.
Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis.
FASEB J
5:
2085-2092,
1991
14.
Da Silva, J,
Pierrat B,
Mary J,
and
Lesslauer W.
Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric oxide synthase expression in mouse astrocytes.
J Biol Chem
272:
28373-28380,
1997
15.
Deitch, EA.
The role of intestinal barrier failure and bacterial translocation in the development of systemic infection and multiple organ failure.
Arch Surg
125:
403-404,
1990[Abstract].
16.
Dijkstra, G,
Moshage H,
and
van Dullenmen HM.
Expression of nitric oxide synthase and formation of nitrotyrosine and reactive oxygen species in inflammatory bowel disease.
J Pathol
186:
416-421,
1998[ISI][Medline].
17.
Enslen, H,
Raingeaud J,
and
Davis RJ.
Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6.
J Biol Chem
273:
1741-1748,
1998
18.
Fink, MP.
Gastrointestinal mucosal injury in experimental models of shock, trauma and sepsis.
Crit Care Med
19:
627-641,
1991[ISI][Medline].
19.
Ford, H,
Watkins S,
Reblock K,
and
Rowe M.
The role of inflammatory cytokines and nitric oxide in the pathogenesis of necrotizing enterocolitis.
J Pediatr Surg
32:
275-282,
1997[ISI][Medline].
20.
Goplerud, JM,
and
Delivoria-Papadopoulos M.
Principles in cellular oxygenation: fetal and neonatal intestines.
Clin Perinatol
13:
191-196,
1986[ISI][Medline].
21.
Gosche, JR,
Harris PD,
and
Garrison RN.
Age-related differences in intestinal microvascular responses to low-flow states in adult and suckling rats.
Am J Physiol Gastrointest Liver Physiol
264:
G447-G453,
1993
22.
Grisham, MB,
Hernandez LA,
and
Granger DN.
Xanthine oxidase and neutrophil infiltration in intestinal ischemia.
Am J Physiol Gastrointest Liver Physiol
251:
G567-G574,
1986
23.
Hale, KK,
Trollinger D,
Rihanek M,
and
Manthey CL.
Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta, gamma and delta in inflammatory cell lineages.
J Immunol
162:
4246-4252,
1999
24.
Han, J,
Lee JD,
Bibbs L,
and
Ulevitch RJ.
A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells.
Science
265:
808-811,
1994[ISI][Medline].
25.
Henning, SJ.
Ontogeny of enzymes in the small intestine.
Annu Rev Physiol
47:
231-245,
1985[ISI][Medline].
26.
Hoffman, RA,
Zhang G,
Nussler NC,
Gleixner SL,
Ford HR,
Simmons RL,
and
Watkins SC.
Constitutive expression of inducible nitric oxide synthase in the mouse ileal mucosa.
Am J Physiol Gastrointest Liver Physiol
272:
G383-G392,
1997
27.
Jiang, Y,
Gram H,
Zhao M,
New L,
Gu J,
Fend L,
Di Padova F,
Ulevitch RJ,
and
Han J.
Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38delta.
J Biol Chem
272:
30122-30128,
1997
28.
Keesler, GA,
Bray J,
Hunt J,
Johnson DA,
Gleason T,
Yao Z,
Wang SW,
Parker C,
Yaman H,
Cole C,
and
Lichtenstein HS.
Purification and activation of recombinant p38 isoforms alpha, beta, gamma and delta.
Protein Expr Purif
14:
221-228,
1998[ISI][Medline].
29.
Klemm, K,
and
Moody FG.
Regional intestinal blood flow and nitric oxide synthase inhibition during sepsis in the rat.
Ann Surg
227:
126-133,
1998[ISI][Medline].
30.
Knowles, RG,
and
Monecada S.
Nitric oxide synthases in mammals.
Biochem J
298:
249-258,
1998.
31.
Laubach, VE,
Foley PL,
Shockey KS,
Tribble CG,
and
Kron IL.
Protective votes of nitric oxide and testosterone in endotoxemia: evidence from NOS-2 deficient mice.
Am J Physiol Heart Circ Physiol
275:
H2211-H2218,
1998
32.
Lee, JC,
Kassis S,
Kumar S,
Badger A,
and
Adams JL.
p38 mitogen-activated protein kinase inhibitorsmechanisms and therapeutic potentials.
Pharmacol Ther
82:
389-397,
1999[ISI][Medline].
33.
Lizasoain, I,
Weiner CP,
Knowles RG,
and
Moncada S.
The ontogeny of cerebral and cerebellar nitric oxide synthase in the guinea pig and rat.
Pediatr Res
39:
779-783,
1996[Abstract].
34.
Losonczy, G,
Kriston T,
Szabo A,
Muller V,
Harvey J,
Hamar P,
Heemann U,
and
Baylis C.
Male gender predisposes to development of endotoxic shock in the rat.
Cardiovasc Res
47:
183-191,
2000[ISI][Medline].
35.
Marletta, MA.
Nitric oxide synthase structure and mechanism.
J Biol Chem
268:
12231-12234,
1993
36.
Mercer, DW,
Smith GS,
Cross JM,
Russell DH,
Chang L,
and
Cacioppo J.
Effects of lipopolysaccharide on intestinal injury: potential role of nitric oxide and lipid peroxidation.
J Surg Res
63:
185-192,
1996[ISI][Medline].
37.
Miller, MJ,
Thompson JH,
Zhang X,
Sadowska-Krowicka H,
Kakkis JL,
Munshi U,
Sandoval M,
Rossi JL,
Eloby-Childress S,
Beckman JS,
Ye YZ,
Rodi CP,
Manning PT,
Currie MG,
and
Clark DA.
Role of inducible nitric oxide synthase expression and peroxynitrite formation in guinea pig ileitis.
Gastroenterology
109:
1475-1483,
1995[ISI][Medline].
38.
Moncada, S,
Palmer RMJ,
and
Higgs EA.
Nitric oxide: physiology, pathophysiology and pharmacology.
Pharmacol Rev
43:
109-142,
1991[ISI][Medline].
39.
Morin, MJ,
Unno N,
Hodin RA,
and
Fink MP.
Differential expression of inducible nitric oxide synthase messenger RNA along the longitudinal and crypt-villus axes of the intestine in endotoxemic rats.
Crit Care Med
26:
1258-1264,
1997[ISI].
40.
Musemeche, CA,
Henning SJ,
Baker JL,
and
Pizzini RP.
Inflammatory enzyme composition of the neonatal rat intestine: implications for susceptibility to ischemia.
J Pediatr Surg
28:
788-791,
1993[ISI][Medline].
41.
Nathan, C.
Nitric oxide as a secretory product of mammalian cells.
FASEB J
6:
3051-3064,
1992
42.
Nathan, C,
and
Xie QW.
Nitric oxide synthases: roles, tolls and controls.
Cell
78:
915-918,
1994[ISI][Medline].
43.
Neudling, S,
Kahlert S,
Loebbert K,
Doevendans PA,
Meyer R,
Vetter H,
and
Grohe C.
17-estradiol stimulates expression of endothelial and inducible NO synthase in rat myocardium in-vitro and in-vivo.
Cardiovasc Res
43:
666-674,
1999[ISI][Medline].
44.
Neugarten, J,
Ding Q,
Friedman A,
Lei J,
and
Silbiger S.
Sex hormones and renal nitric oxide synthases.
J Am Soc Nephrol
8:
1240-1246,
1997[Abstract].
45.
North, AJ,
Star RA,
Brannon TS,
Ujiie K,
Wells LB,
Lowenstein CJ,
Snyder SH,
and
Shaul PW.
Nitric oxide synthase type I and type III gene expression are developmentally regulated in rat lung.
Am J Physiol Lung Cell Mol Physiol
266:
L635-L641,
1994
46.
Rairigh, RL,
Le Cras TD,
Ivy DD,
Kinsella JP,
Richter G,
Horan MP,
Fan ID,
and
Abman SH.
Role of inducible nitric oxide synthase in regulation of pulmonary vascular tone in the late gestation ovine fetus.
J Clin Invest
101:
15-21,
1998
47.
Rouse, J,
Cohen P,
Trigon S,
Morange M,
Alonso-Llamazares A,
Zamanillo D,
Hunt T,
and
Nebreda AR.
A novel kinase cascade triggered by stress and heat shock protein that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins.
Cell
78:
1027-1037,
1994[ISI][Medline].
48.
Schumann, RR,
Pfeil D,
Lamping N,
Kirschning C,
Scherzinger G,
Schlag P,
Karawajew L,
and
Herrmann F.
Lipopolysaccharide induces the rapid tyrosine phosphorylation of the mitogen-activated protein kinases erk-1 and p38 in cultured human vascular endothelial cells requiring the presence of soluble CD14.
Blood
87:
2805-2814,
1996
49.
Tepperman, BL,
Brown JF,
and
Whittle BJ.
Nitric oxide synthase induction and intestinal epithelial cell viability in rats.
Am J Physiol Gastrointest Liver Physiol
265:
G214-G218,
1993
50.
Unno, N,
Wang H,
Menconi MJ,
Tytgat SHAJ,
Larkin V,
Smith V,
Morin MJ,
Hodin RA,
and
Fink MP.
Inhibition of inducible nitric oxide synthase ameliorates lipopolysaccharide-induced gut mucosal barrier dysfunction in rats.
Gastroenterology
113:
1246-1257,
1997[ISI][Medline].
51.
Wang, XS,
Diener K,
Manthey CL,
Wang S,
Rosenzweig B,
Bray J,
Delaney J,
Cole CN,
Chan-Hui PY,
Mantlo N,
Lichenstein HS,
Zukowski M,
and
Yao Z.
Molecular cloning and characterization of a novel p38 mitogen-activated protein kinase.
J Biol Chem
272:
23668-23674,
1997
52.
Whittle, BJ.
Nitric oxidea mediator of inflammation or mucosal defense.
Eur J Gastroenterol Hepatol
9:
1026-1032,
1997[ISI][Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |