expression of inducible nitric oxide synthase mrna in rat digestive tissues after endotoxin and its...

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 224, 703–708 (1996)ARTICLE NO. 1087

Expression of Inducible Nitric Oxide Synthase mRNA in Rat DigestiveTissues after Endotoxin and Its Role in Intestinal Mucosal Injury

Kai Chen,1 Masahiro Inoue, and Akira Okada

Department of Pediatric Surgery, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan.

Received June 14, 1996

Nitric oxide (NO) production is increased in the intestine and may contribute to intestinal injury in sepsis.However, the tissue expression of inducible NO synthase (iNOS) mRNA throughout the digestive tract andits relation with the mucosal damage after endotoxin challenge remain unknown. We therefore measuredtissue expression of mRNA encoding iNOS by Northern blot analysis and reverse transcription PCR. TheiNOS mRNA was detectable at 1 h, peaked at 4 h, and remained faint at 24 h after endotoxin injection inesophagus, duodenum, jejunum, ileum, and colon, but not in the stomach. Pre-treatment with dexamethasoneattenuated the rise of iNOS mRNA. Both dexamethasone and NOS inhibitor, L-NAME, ameliorated theendotoxin-induced increase in intestinal mucosal permeability. Our results indicate that there is tissue-specific expression of iNOS mRNA in the digestive tract. The manipulations that decrease NO productionmay have therapeutic potential in preserving intestinal mucosal integrity in sepsis. q 1996 Academic Press, Inc.

Increased nitric oxide (NO) formation via the expression of an endotoxin-inducible NOsynthase (iNOS) is thought to be responsible for the pathophysiology of septic shock such asthe cardiovascular collapse. This is supported by the findings that endotoxin induces iNOSactivity and gene expression in various tissues including lung, liver, spleen, kidney and skeletalmuscle(1, 2). The inhibition of NO formation by inhibitors has showed the therapeutic value inthe treatment of septic shock(3, 4).

Recently, the gastrointestinal tract is increasingly being recognized as an important organduring sepsis and stress, as these stressed conditions are often associated with mucosal barrierdysfunction(5). The latter is of major concern inasmuch as breakdown of the mucosal barriercan lead to bacterial translocation, systemic accumulation of toxic factors leading to multipleorgan failure. Furthermore, an increased activity of iNOS has been detected in the rat intestinein sepsis and experimental inflammatory bowel disease(6). Overproduction of NO appears tobe an important mechanism involved in the intestinal injury. However, the iNOS mRNAexpression and the effect of glucocorticoids in the digestive tissues remains unknown.

In view of this, we have now investigated the tissue localization and time course of iNOSexpression in rat digestive tract after administration of endotoxin. In addition, the effect ofNOS inhibitors, L-NAME and dexamethasone, on intestinal mucosal barrier function wasstudied, in an attempt to understand the mechanism of mucosal injury in endotoxemia and thebeneficial effect of NOS inhibitors.

1 To whom correspondence should be addressed. Fax: (06) 879-3759. E-mail: [email protected]: NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; GAPDH,

glyceraldehyde 3-phosphate dehydrogenase; PCR, polymerase chain reaction; i.p., intraperitoneal; bp, base pairs;kb, kilo-bases; ET, endotoxin; Dex, dexamethasone; L-NAME, NG-nitro-L-arginine methyl ester; FITC, fluoresceinisothiocyanate.

0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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Vol. 224, No. 3, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODSAnimals. Male Sprague-Dawley rats (Oriental Yeast Co., Osaka, Japan), weighing 250 to 280 g were obtained and

allowed to acclimate for 5 days before start of the experiment. All experimental procedures were conducted inaccordance with Osaka University Medical School Guideline for the Care and Use of Laboratory Animals. The ratswere injected intraperitoneally with endotoxin (E.Coli lipopolysaccharide serotype 0127:B8, Sigma) at the dose of 10mg/kg. At 0, 1, 4, 8 and 24 h after challenge, rats were killed under the anesthesia. For rats in endotoxin plusdexamethasone group, dexamethasone (3 mg/kg, i.p.) was injected 45 minutes prior to the administration of endotoxin.The rats in this group were sacrificed 4 h after endotoxin injection. Samples of esophagus, stomach, duodenum,jejunum, ileum and colon were removed and immediately frozen in liquid nitrogen. Tissues were stored at 0807C.

RNA extraction and reverse transcription polymerase chain reaction. Total RNA from tissue samples was extractedusing a commercial kit (Isogene). The amount of extracted RNA was calculated from optical density measurementsat 260 nm ( 1 OD260Ç40mg/ml). RNA (1.0 mg) was used to generate first-strand complementary DNA by usingreverse transcriptase (Gibco BRL, Gaithersburg, MD) following the manufacturer’s recommended procedures. Then,PCR was performed for iNOS and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the same complementaryDNA samples by using a Takara PCR Thermal Cycler. Oligonucleotide primers for iNOS and GAPDH were: 5* ATGGCT TGC CCT TGG AAG TTT CTC 3*, 5* TCC AGG CCA TCT TGG TGG CA AGA 3* and 5* TCC CTC AAGATT GTC AGC AA 3*, 5* AGA TCC ACA ACG GAT ACA TT 3*, which correspond to the rat iNOS and ratGAPDH cDNA respectively(7, 8). Amplification was initiated by 5 min of denaturation at 957C for 1 cycle followedby 25 cycled at 957C for 30 s, 607C for 45 s, and 727C for 1 min. After the last cycle of amplification, the sampleswere incubated for 10 min at 727C. The PCR products -- a 574 bp iNOS fragment and a 309 bp GAPDH fragment -were then visualized by UV illumination after electrophoresis through 1.5% agarose gels containing 0.5 mg/ml ethidiumbromide. The gel photographs were scanned with a computerized densitometer. Semiquantitative analysis of iNOSwas performed by compared to the housekeeping gene GAPDH.

Nucleotide sequencing. To confirm that the PCR-amplified bands correspond to the original sequence, the PCRamplified iNOS was sequenced by a subcloning, sequencing procedure. The PCR product was subcloned into thevector pGEM-T. The nucleotide sequence was determined by Applied Biosystems 373A DNA sequencer with oligonu-cleotide primers and a Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CA).

Northern blot analysis. Northern Blot analysis was performed with 10mg of total RNA per lane on a denaturing1.0% agarose gel. RNA was blotted onto a nylon membrane (Hybond N, Amersham) followed by hybridization. BothiNOS and GAPDH cDNA were labelled with 32P-dCTP by the random-primer method (Amersham). Prehybridizationand hybridization temperatures were 507C and 427C, respectively. The membranes were washed once in 2XSSC, 0.1%SDS for 5 minutes at room temperature, twice in 1XSSC, 0.1% SDS for 30 minutes at 657C, once in 0.2X SSC, 0.1%SDS for 30 minutes at 657C. Then, the membranes were exposed to X-ray film at 0807C with intensifying screens.

Intestinal permeability test. In a separate experiment, 20 rats were divided into 4 groups: Control group (injectedwith saline), endotoxin (ET) group, endotoxin plus dexamethasone group (ET/Dex, dexamethasone was injected 45min prior to endotoxin) and endotoxin plus NG-nitro-L-arginine methyl ester group (ET/NAME, 3 mg/kg L-NAMEwas subcutaneously injected 3 hours after endotoxin. Four hours after saline or endotoxin injection, rats were anesthe-tized (sodium pentobarbital, 40 mg/kg, i.p.) and intestinal permeability was measured by a modification of the methoddescribed by Otamiri et al(9). Briefly, 1 ml of permeability test solution containing 25 mg of fluorescein isothiocyanate(FITC)-dextran (Mw 4000) was instilled into the lumen of a 20 cm-ligated segment of ileum. After 30 minutes, bloodsample was taken by puncture of the portal vein. Plasma was analyzed for the FITC-dextran concentration byfluorescence spectrometry (Hitachi F-3000, Japan) at an excitation wavelength of 480 nm and an emission wavelengthof 520 nm.

Statistical methods. Data are expressed as mean{standard deviation (SD). Statistical comparisons between control,endotoxin and dexamethasone groups were made using one-way analysis of variance (ANOVA) with Student-Newman-Keuls multiple comparisons test. Values of põ0.05 were considered to be statistically significant.

RESULTSSequencing of iNOS PCR Product

The PCR product amplified using iNOS specific primers showed clear bands at the predictedsizes of 574 bp. The sequence of PCR-amplified iNOS cDNA corresponded exactly with thecloned rat hepatocyte iNOS cDNA sequence and showed a 92% homology with the correspond-ing sequence of the cloned macrophage iNOS cDNA.

Time Course of iNOS mRNA Expression after Endotoxin

We show that there is no detectable iNOS gene expression in the digestive tissues underthe normal condition (Fig. 1). The expression of iNOS mRNA was detectable at 1 h, peaked

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FIG. 1. The time-course of iNOS mRNA expression after endotoxin challenge in ileum and colon. (A) Gelphotograph of PCR-amplified iNOS and GAPDH cDNA derived from iNOS and GAPDH mRNA. The PCR productfragments of iNOS and GAPDH mRNA are 574 and 309 bp, respectively. (B) Bar graph showing the relative amountof iNOS mRNA quantified by densitometry and expressed as mean iNOS mRNA:GAPDH mRNA ratios. Error barsrepresent SD, nÅ5 for each group. * põ0.05 relative to all other time-points, † põ0.05 relative to the 24 hr’s.

at 4h, remained elevated at 8h and reduced to be faint at 24 h in both ileum and colon afterendotoxin challenge, using RT-PCR method. There were no significant differences in GAPDHmRNA expression at any time-point.

Expression of iNOS mRNA in the Digestive Tract and the Effect of Pre-treatment withDexamethasone

Four hours after endotoxin injection, Northern blot analysis demonstrated the induction ofa 4.4 kb iNOS mRNA in the tissues from esophagus, duodenum, jejunum, ileum and colon(Fig. 2). However, the iNOS signal was absent in RNAs from stomach. The additional RT-PCR was performed using stomach tissue, which indicated the identical result with Northernblot analysis (data not shown). Treatment with dexamethasone prior to endotoxin resulted ina significant reduction in iNOS mRNA expression in the jejunum, ileum and colon, while induodenum there was a non-significant trend for reduced expression. Dexamethasone did notaffect expression of GAPDH mRNA within the tissues.

The Effect of NO Production Inhibitors on Intestinal Mucosal Permeability

Administration of endotoxin led to a significant increase in ileal mucosal permeability ascompared with the control (Fig. 3) Both dexamethasone and L-NAME treatments reduced thesubsequent increase in the mucosal permeability.

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FIG. 2. Representative quantitative Northern blot analysis showing the expression of iNOS mRNA in the digestivetissues at 4 hours after endotoxin injection and the effect of dexamethasone pre-treatment upon iNOS mRNA expres-sion. Abbreviations: N, normal control; E, endotoxin treatment; D, dexamethasone pre-treatment 45 min prior toendotoxin injection.

DISCUSSION

NO is thought to play a key role in the mortality caused by sepsis. High-dose endotoxinadministration causes endotoxemia and sepsis, while inhibitors of NOS can reverse or preventthe hypotension induced in animals by endotoxin. It is known that null mutant iNOS mice(iNOS-/-) are resistant to the hypotension and death caused by endotoxin(10). Those data furtherestablished that iNOS has a crucial role in endotoxin-induced death.

The epithelium of the intestine serves as a barrier to the entry of intraluminal microorganismsand toxins. The loss of this barrier integrity under various pathophysiological conditions maybe a consequence of mucosal inflammation due to the over-production of NO by the inducibleisoform of NO synthase(11). Knowledge of the distribution of iNOS is a prerequisite to under-standing its diverse biological functions. The widespread expression of iNOS mRNA has beenwell studied(2), but little is known in the digestive tissues. This study demonstrated for the firsttime that the iNOS mRNA expression was increased in various tissues of digestive tract exceptthe stomach. Since iNOS is transcriptionally regulated(12), increases in iNOS mRNA levels aretherefore indicative of increased NO levels. In addition, there are tissue-specific differencesamong the tissues in expression of iNOS mRNA in response to endotoxin challenge.

Both small intestine and colon showed significantly remarkable expression in comparedwith the upper digestive tissues. This is consistent with the fact that NO over-productionconsists of one of the main pathophysiology of inflammation occurred in the intestine(13).

FIG. 3. Intestinal permeability to FITC-dextran (MW 4000). The portal blood FITC-dextran concentration in ETgroup was significantly higher than that in the control group. Both dexamethasone and L-NAME treatments significantlydecreased the intestinal permeability in compared with the ET group. DataÅmean{SD, nÅ5 for each group.

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Moreover, dexamethasone treatment caused a significant suppression of the endotoxin-inducediNOS mRNA expression, which may be one of the mechanisms underlying the anti-inflamma-tory action of glucocorticoids.

For the assessment of barrier function of intestinal mucosa, a permeability test can be asuitable method. The marker FITC-dextran (MW 4000) used in this study is considered topenetrate through a paracellular route via the tight junctions according to its size(14). As thisparacellular pathway is thought to be the same route of permeation from the intestinal lumentoward plasma used in patients, our result may nicely fit into the clinical circumstances(15).Although dexamethasone and L-NAME are considered to affect NO production by inhibitingthe mRNA expression and NO synthase respectively, the subsequent inhibition of nitric oxidesynthesis may contribute to the amelioration of increase intestinal permeability by endotoxin.Therefore, this study has provided direct evidence for the involvement of NO in the changesof intestinal mucosal integrity.

It is not known from this study in which mucosal cell types the increased NO productiontakes place. Previous reports suggest that the enterocyte can produce NO after exposure toendotoxin in vitro. It is possible, however, that macrophages and perhaps other mucosal cellsas well release increased NO. Further experiments, including in situ hybridization, will berequired to define the specific cells that produce NO in the intestinal mucosa during endo-toxemia.

A beneficial role of endogenous NO, synthetised by the constitutive NO synthase enzyme(cNOS), in the maintenance of microvascuar integrity has been observed, since administrationof NO inhibitor, L-NAME, concurrently with endotoxin aggravated endotoxin-induced micro-vascular damage in the gut(16, 17). The beneficial effect of NO inhibitor in this study may bedue to the delayed administration of L-NAME, until the time of significant expression of theiNOS mRNA. Clarification of the defensive role of constitutive NOS, as well as the pathologicalrole of inducible NOS appear to be important. The inhibition of iNOS may represent a newtreatment strategy for sepsis; however, the use of NOS inhibitors in the treatment of sepis canbe problematic in the light of their non-selective inhibition on both iNOS and cNOS. Theselective inhibitor directly toward inducible NOS would offer obvious potential therapeuticbenefits in the management of endotoxemia and septic shock.

ACKNOWLEDGMENTS

We gratefully acknowledge Hiroshi Takemori, M.S., and Ling Fu, M.D.( Osaka University Medical School, Osaka,Japan ), for their excellent technical assistance.

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expression of inducible nitric oxide synthase mrna in rat digestive tissues after endotoxin and its...

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