L. GRASA, M.P. ARRUEBO, M.A. PLAZA, M.D. MURILLO*

A DOWNREGULATION OF nNOS IS ASSOCIATED TO DYSMOTILITYEVOKED BY LIPOPOLYSACCHARIDE IN RABBIT DUODENUM.

Department of Pharmacology and Physiology, Faculty of Veterinary Medicine, University of Zaragoza, Spain.

Alterations in gastrointestinal motility have been reported in response to endotoxin.The effects of lipopolysaccharide (LPS) on motility have been attributed to severalsubstances, including prostaglandins and nitric oxide. The aim of this study was toinvestigate the expression and the contribution of NOS and COX enzymes to thelocal effect of LPS on ACh-evoked contractions in rabbit duodenum. The AChevoked contractions were inhibited by LPS in longitudinal and circular muscles ofduodenum. L-NNA, aminoguanidine, ODQ, indomethacin, and NS-398 but notNPLA antagonized the inhibitory effect of LPS. Western blot analysis showedprotein bands of 155, 130, 70 and 72 kDa for nNOS, iNOS, COX-1 and COX-2respectively in rabbit duodenum. All of these isoforms were expressed constitutivelyand only the nNOS was reduced in the presence of LPS. Expression of nNOS, iNOS,COX-1 and COX-2 was detected by inmunohistochemistry in the smooth musclelayers and in the neurons of the myenteric ganglia of rabbit duodenum. Inconclusion, LPS locally administered reduces the contractility of rabbit duodenumand a downregulation of nNOS is associated to this effect. The iNOS, COX-1 andCOX-2 were expressed constitutively but their expression was not modified by LPS.

K e y w o r d s : cyclooxygenase, lipopolysaccharide, motility, nitric oxide synthase, smallintestine

INTRODUCTION

Inflammatory reactions can cause various clinical manifestations frequentlyassociated with abnormal motility of the gastrointestinal tract, such as nausea,vomiting, ileus, or diarrhea (1). The mechanisms underlying the altered motorfunction are not fully understood but they may reflect alterations in smooth

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2008, 59, 3, 511�524

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muscle and/or nerve activity (2). Lipopolysaccharide (LPS), an endotoxin ofGram-negative bacteria, is a known causative agent of inflammation. Alterationsin gastrointestinal motility have been widely reported in response to the systemicadministration of the endotoxin (3 - 7). In contrast, the local effect of LPS onintestinal motility has scarcely been studied (8). Previous studies by our group (6)have reported a local inhibitory effect of LPS on the ACh-evoked contractionswhen rabbit intestinal segments (duodenum, jejunum or ileum) were incubatedwith LPS in vitro. We have used in this work a model of dysmotility evoked bythe local effect of LPS in rabbit duodenum to study the role of COX and NOSproteins in this effect. It has been reported that rabbit (6) is more sensitive to LPSthan other animal models such us rats (3, 5) and in this sense it is closer to thehuman model (9).

The effects of LPS on motility have been attributed to several activesubstances, including prostaglandins and nitric oxide (6, 10, 11). Nitric oxideis a nonadrenergic, noncholinergic neurotransmitter that causes inhibition ofintestinal motility (12). NO is largely produced by three isoforms of nitricoxide synthase (NOS) (13). The neuronal (nNOS) and endothelial (eNOS)NOS isoforms that are constitutively expressed are widely distributed andsynthesize NO at low concentrations which regulates numerous physiologicalfunctions. The third isoform is named inducible NOS (iNOS) as its expressionrequires transcriptional activation. LPS induces iNOS in the rat small intestine(3, 5).

Prostaglandins play a significant role in the physiology and pathophysiologyof the digestive system, affecting water and electrolyte transport, mucoussecretion, blood flow and motility (14, 15). Prostaglandins are locally actinghormones derived from the metabolism of arachidonic acid by cyclooxygenase(COX), which exists as two isoenzymes, COX-1 and COX-2. COX-1 is aconstitutive enzyme found in the gastrointestinal tract, and expression of COX-2is induced by a variety of stimuli, including LPS (1). LPS and other inflammatorycytokines are able to induce both iNOS and COX-2 (16).

Pharmacological handling of NO has yielded inconsistent and contradictoryresults. It has been suggested that some of these inconsistencies may be attributedto differential effects depending on dose, species, tissue, the nature of theinflammatory state and the activities of the three NOS isoforms (16). The role ofNO in disturbed motor activity related to inflammatory bowel diseases hastraditionally focused on the iNOS. However, it is now known whether theterminology "constitutive" versus "inducible" is an oversimplification, as it hasbeen stated that the constitutive isoforms may also contribute to the inflammation(17) and, conversely, that iNOS may function as a "constitutive" enzyme in somecells in physiological situations (18). In the same way, a constitutive expressionof COX-2 has been found in gastrointestinal tissues (19). On the other hand,recent research has demonstrated that there is a correlation between the release ofnitric oxide and the production of prostaglandins (8, 20, 21). Therefore, the aims

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of this study were 1) to investigate the contribution of nNOS, iNOS, COX-1 andCOX-2 enzymes to the local effect of LPS on the ACh-evoked contractions inlongitudinal and circular smooth muscle of rabbit duodenum in vitro; 2) toevaluate the protein expression of the constitutive and inducible isoforms of bothNOS and COX using western blotting in both control and LPS incubatedsegments of rabbit duodenum; and 3) to localize NOS and COX proteins usingimmunohistochemistry. To our knowledge, this is the first study that evaluates theexpression of both the NOS and COX isoform proteins on the local effect of LPSin small intestine.

MATERIALS AND METHODS

The handling, equipment used and sacrifice of animals complied with European Councillegislation 86/609/EEC concerning experimental animal protection. Male New Zealand rabbitsweighing 2 - 2.5 kg were kept at a constant room temperature (22 °C) with free access to water andstandard rabbit fodder. All experimental protocols were approved by the Ethics Committee of theUniversity of Zaragoza (Spain).

Functional studies: Muscle contractility

After 24 h of fasting, the animals were humanely killed by a blow to the head. Pieces of rabbitduodenum, were removed, washed, freed from mesenteric attachment, and cut into smallersegments. Whole thickness segments (10 mm long and 5 mm wide) were suspended in the directionof longitudinal or circular smooth muscle fibers in a thermostatically controlled (37 °C) organ bath(10 ml capacity) containing Krebs solution, and maintained continuously gassed with 95% O2 and5% CO2. Each segment was connected to an isometric force transducer (Pioden UF1, Graham BellHouse, Canterbury, UK) and stretched passively to an initial tension of 20 mN. Signal output of themechanical activity was amplified (The Mac Lab Bridge Amp, AD Instruments Inc., Milford, MA,U.S.A.) with a range of 2 mV, recorded on a computer for later analysis using the Mac LabSystem/8e computer program (AD Instruments Inc., Milford, MA, U.S.A.) and digitized at twosamples per second per channel. Before testing, segments were allowed to equilibrate in Krebssolution for 60 min. During that time, the nutrient solution was changed every 20 min.

Each experimental protocol was systematically performed on eight segments of duodenum (4longitudinal and 4 circular muscle) taken from the same rabbit, and repeated in three or fourdifferent animals. Thus, each preparation served as its own control. Segments that did not showspontaneous activity were discarded.

In order to investigate the local effect of LPS on the longitudinal and circular smooth muscle ofrabbit duodenum we added ACh 100 µM to the bath after the equilibration period. ACh 100 µMevoked contractile responses that were recorded and considered as the control. The duodenumsegments were then incubated for 90 min with Krebs or LPS (0.3 µg ml-1) and afterwards ACh 100µM was added to the bath. This second ACh response was compared with the control and expressedas a percentage. We examined the role of NO and prostaglandins in the LPS-induced effects bymeans of L-NNA (10 µM, a non selective NOS inhibitor), aminoguanidine (AG, 10 µM, a selectiveiNOS inhibitor), Nω-propyl-L-arginine (NPLA, 10 µM, a selective nNOS inhibitor), ODQ (0.1-1µM, a guanylate cyclase inhibitor), indomethacin (1 µM, a non selective COX inhibitor) and NS-398 (0.1 µM, a selective COX-2 inhibitor). These agents were added to the bath 15 min beforeincubation for 90 min with Krebs or LPS (0.3 µg ml-1).

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Western blot analysis

Western blot analysis was performed on protein extracted from three different samples ofduodenum taken from the same rabbit. One of them was taken immediately after the sacrifice ofthe animal, with no incubation in the organ bath. The other samples were obtained 90 min afterincubation in the organ bath with Krebs or LPS (0.3 µg ml-1). We obtained samples from threedifferent rabbits and performed two western blots for each rabbit. Thus, six western blots wereused to detect each isoform. Duodenum segments were opened on the longitudinal axis andmucosa was scraped off with a clean glass and separated from the muscular layers. Samplescontaining the mucosa layer or muscular layer were homogenized with a Polytron homogenizer(DI 25 Basic, IKA-WERKE, Germany) in cold RIPA buffer containing 0.1% PMSF (100 mM),0.3% aprotinin and 0.1% sodium orthovanadate (1 M). The homogenates were centrifuged at15000 g for 20 min at 4 °C. The supernatant containing proteins was stored at -80 °C until use.Protein concentration was determined by the Bradford method (Biorad, Germany). Samples weresolubilized in 2 x sodium dodecyl sulfate (SDS) sample buffer and then boiled at 100 °C during5 min.

Equal amounts of protein (20 µg/lane) were loaded and separated by electrophoresis on 5 or 8%SDS-polyacrilamide gel. The membranes were blocked with 5% non-fat dry milk in 0.1% Tween20/phosphate buffered saline (TPBS) for 6 hours at 4 °C. Mouse antibody anti-nNOS (against N-terminus, 1:500) or anti-iNOS (1:50) and goat antibody anti-COX-1 (1:500), anti-COX-2 (1:200) oranti-actin (1:500) were applied overnight at 4 °C. Peroxidase-conjugated anti-mouse (1:2000) oranti-goat (1:5000) antibody were applied at room temperature for 2 hours. All the antibodies werepurchased from Santa Cruz Biotechnology, CA, U.S.A. (SC-5302, SC-7271, SC-1754, SC-1747,SC-1616, SC-2005, SC-2354). Visualization was achieved by enhanced chemiluminiscencetechnique (Amersham Biosciences, UK). In control experiments, primary antibodies were omittedas negative controls and different tissues were used as positive controls.

Immunohistochemistry

Duodenum segments incubated in the organ bath for 90 min with Krebs or LPS (0.3 µg ml-1)from six different animals, were fixed in 10% formalin and embedded in paraffin. Deparaffinedduodenum 4 µm sections were incubated in 0.9% H2O2/methanol for 30 min and then microwavedin 10 mM sodium citrate buffer twice for 5 min. After blocking with 30% normal donkey serum,sections were incubated overnight at 4 °C with either mouse antibody anti-nNOS (against N-terminus, 1:50) or anti-iNOS (1:50), goat antibody anti-COX-1 (1:300) or anti-COX-2 (1:500)diluted in 5% normal donkey serum. These antibodies were the same as those used in western blotanalysis. Sections were incubated with a biotin-conjugated donkey, anti-mouse or anti-goatantibody 1:100 for 30 min at room temperature. The avidin-biotin method was used as described inthe ABC elite kit (Vector Laboratories, CA, U.S.A.). Antibody localizations on the specimens werevisualized with a diaminobenzidine kit (Vector Laboratories, CA, U.S.A.). Sections werecounterstained with Mayer´s hemalum solution and finally mounted in DPX. In controlexperiments, the primary antibody was omitted.

Data analysis and statistics

All of the intestinal segments included in the analyses showed spontaneous contractions. TheACh motor responses (MR) were measured as integrated mechanical activity (IMA) per second,expressed as mN s-1 (6) and normalised per square millimeter of cross-sectional area (CSA, mm2)as follows: MR = A1 - A0, where A is the integrated area per second per mm2 during either the first3 min of response to ACh (A1) or the spontaneous motility, 3 min before adding ACh (A0). The

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integrated area was calculated using a baseline of 0 mN. CSA was determined for each muscle stripusing the equation CSA (mm2) = mass (mg)[length (mm)·density (mg·mm-3)]-1, where rabbitintestinal muscle density was assumed to be 1.05 mg·mm-3; and length and mass (wet weight) ofeach segment were noted on completion of experiments. Results were expressed as a percentage ofthe ACh control values (100%).

Protein expression was measured by densitometry using appropriate software and the data wasexpressed as arbitrary units relative to the samples incubated with Krebs 90 min (100%).

Results are expressed as mean ± s.e.m. Comparisons between means were made using one-wayanalysis of variance (ANOVA) tests and P-values were determined using the Scheffé F test.Differences with P-values <0.05 were considered statistically significant.

Drugs and solutions

The composition of the normal Krebs solution in mM was as follows: NaCl 120, KCl 4.7, CaCl2

2.4, MgSO4 1.2, NaHCO3 24.5, KH2PO4 1.0, and glucose 5.6, pH 7.4. Acetylcholine (ACh), Nω-nitro-L-arginine (L-NNA), aminoguanidine, indomethacin and lipopolysaccharide (LPS, fromEscherichia coli serotype 0111-B4) were purchased from Sigma (Madrid, Spain). NS-398, 1H-[1,2, and 4] Oxadiazolo [4, 3-α] quinoxalin-1-one (ODQ) and Nω-propyl-L-arginine (NPLA) wereacquired from Tocris (Madrid, Spain). Drug solutions were prepared in milliQ water, except forindomethacin, which was dissolved in 5% sodium bicarbonate.

RESULTS

Functional studies: Muscle contractility

Effect of LPS on ACh-evoked contractions: ACh (100 µM) evokedcontractions in the longitudinal and circular smooth muscle of duodenum thatwere not significantly modified after the incubation with Krebs for 90 min (Fig.1a, b). These ACh-evoked contractions were inhibited in the presence of LPS (0.3µg ml-1, 90 min) compared with Krebs (90 min) in longitudinal and circularmuscles (Fig. 1a, b).

Effects of NOS and COX inhibitors on ACh-evoked contractions: Someduodenum segments were incubated for 90 min with the NOS and COXinhibitors to check that they have not any effect "per se" on ACh-evokedcontractions. The incubation with L-NNA (10 µM, 105 min), aminoguanidine(AG, 10 µM, 105 min), NPLA (10 µM, 105 min), ODQ (0.1 µM in longitudinaland 1 µM in circular muscle, 105 min), indomethacin (1 µM, 105 min) or NS-398 (0.1 µM, 105 min) did not modify significantly the ACh-evokedcontractions respect to Krebs in both longitudinal (L-NNA: 76.7 ± 16.1, AG:97.7 ± 14.8, NPLA: 90.5 ± 9.5, ODQ: 93.9 ±15.3, indomethacin: 92.5 ± 23.7,NS-398: 78.5 ± 7.0 vs. Krebs: 84.4 ± 7.6) and circular (L-NNA: 92.1 ± 14.7, AG:94.2 ± 7.7, NPLA: 90.8 ± 12.5, ODQ: 86.1 ±13.4, indomethacin: 110 ± 15.7, NS-398: 107.8 ± 20.7 vs. Krebs: 103 ± 10.3) muscle of duodenum (n = 4-6 segmentstaken from 4 different rabbits).

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Role of NO in the inhibitory effect of LPS: L-NNA (10 µM, 105 min) andaminoguanidine (AG, 10 µM, 105 min) antagonized the inhibitory effect of LPSon the ACh contractions in both longitudinal and circular muscle. In contrast,NPLA (10 µM, 105 min) did not block the inhibitory effects induced by LPS.Finally, ODQ (105 min) at the dose of 0.1 µM blocked the effect of LPS inlongitudinal but not in circular muscle. It was necessary a dose of 1 µM of ODQto block the effect of LPS in the circular muscle (Fig. 1a, b).

Role of prostaglandins in the inhibitory effect of LPS: Indomethacin (1 µM,105 min) and NS-398 (0.1 µM, 105 min), reversed the inhibitory effect of LPS onthe ACh contractions in both longitudinal and circular muscle (Fig. 1a, b).

Western blot analysis

Distinct proteins of apparently different molecular masses were identified byanti-NOS and anti-COX antibodies. We observed single protein bands ofapproximately 130, 70 and 72 kDa for iNOS, COX-1 and COX-2 respectively inboth muscular and mucosa layers of rabbit duodenum. A protein band of 155

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Fig. 1. Effects of LPS and inhibitors of NOS and COX on contractility. Effects of 90 min ofincubation with Krebs (control) and LPS (0.3 µg ml-1) on contractions evoked by ACh (100 µM) inlongitudinal (a) and circular (b) smooth muscle of rabbit duodenum. Influence of L-NNA (10 µM),aminoguanidine (AG, 10 µM), NPLA (10 µM), ODQ (0.1 µM in longitudinal and 1 µM in circular),indomethacin (1 µM) or NS-398 (0.1 µM) added 15 min before the LPS (0.3 µg ml-1). Data areexpressed as percentage of the response to ACh control values (100%). Columns are mean valuesand vertical bars indicate s.e.m, n = 8 segments. *P<0.05, **P<0.01, ***P<0.001 vs Krebs.

kDa for nNOS was observed in the muscular layer but not in the mucosa layerof rabbit duodenum (Fig. 2). The specificity of the bands was controlled byomitting the primary antibodies. In addition, rat brain for nNOS, and RAW 264.7+ LPS/PMA cell lysate for iNOS, COX-1 and COX-2 (3, 22) were used aspositive controls (Fig. 2).

In nonincubated tissues from rabbit duodenum, the iNOS, COX-1 and COX-2 proteins were expressed constitutively in the muscular and the mucosa layers.Expression of these proteins was not modified significantly by the incubationwith LPS (0.3 µg ml-1) for 90 min in comparison with Krebs in the muscular ormucosa layers (Fig. 2 and 3). The nNOS protein was only expressedconstitutively in the muscular layer. This expression was higher in tissues afterincubation with Krebs for 90 min than after incubation for 90 min with LPS (0.3µg ml-1) or in nonincubated tissues (Fig. 2 and 3).

Immunohistochemistry

The expression of NOS and COX isoforms was examined in the rabbitduodenum incubated with Krebs or LPS (0.3 µg ml-1) for 90 min. A moderateexpression of nNOS was detected only in the neurons of the myenteric plexus ofrabbit duodenum (Fig. 4a, d). High expression of iNOS, COX-1 and COX-2 was

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Fig. 2. Expression of NOS andCOX isoforms. Western blotanalysis shows single proteinbands of approximately 130 kDa,70 kDa and 72 kDa for iNOS,COX-1 and COX-2 respectivelyin muscular and mucosa layers ofrabbit duodenum. A protein bandof 155 kDa for nNOS wasobserved in the muscular layerbut not in the mucosa layer ofrabbit duodenum. Samples weretaken from tissues withoutincubation (K 0) or after 90 minof incubation with either Krebs(K 90) or LPS (0.3 µg ml-1, LPS90). The specificity of the bandswas controlled by omitting theprimary antibodies. In addition,rat brain for nNOS and RAW264.7 + LPS/PMA cell lysate foriNOS, COX-1 and COX-2 wereused as positive controls.Representative western blotsfrom 3 different animals.

found in the circular and longitudinal smooth muscle layers and in the neurons ofthe myenteric ganglia (Fig. 4b, e, g-j). The proteins COX-1 and COX-2 werestrongly expressed in neurons of the submucosal ganglia and blood vesselsthroughout the duodenal wall (Fig. 4g-j). On the contrary, weak labeling of bloodvessels was observed by staining with anti-iNOS antibody (Fig. 4e).

The epithelial cells of the duodenal villi were stained strongly by anti-COX-1and anti-COX-2 antibodies. The COX-1 isoform was detected at a particularlyhigh level in the goblet cells, and the COX-2 isoform in the intestinal brushborder membrane (Fig. 4g-j). Conversely, only a moderate expression of iNOSwas observed in these cells (Fig. 4b, e). In the submucosa layer, expression ofCOX-1 and COX-2 was found in the muscularis mucosae (Fig. 4g-j).

No differences between the immunoreactivity of NOS and COX proteins werefound in the segments incubated with LPS and those incubated with Krebs, exceptfor nNOS, which was expressed at a lower level in the neurons of the myentericplexus of duodenal segments incubated with LPS (Fig. 4a, d).

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Fig. 3. Densitometry analysis of NOS and COX isoforms. Densitometry analysis of nNOS, iNOS,COX-1 and COX-2 protein expression in muscular (a) and mucosa (b) layers of nonincubatedsegments (K 0) or after 90 min of incubation with either Krebs (K 90) or LPS (0.3 µg ml-1, LPS 90)from rabbit duodenum. The data was expressed as arbitrary units ± s.e.m. relative to Krebs 90 min(100 %), n = 6 western blots from 3 different animals in duplicate. ***P<0.001 vs Krebs 90 min.

The specificity of the staining obtained was controlled by omitting theprimary antibody in some sections, with a clear suppression of staining beingobserved (Fig. 4c, f).

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Fig. 4. Localization of NOS and COX isoforms in rabbit duodenum. Localization of nNOS (a, d),iNOS (b, e), COX-1 (g, i) and COX-2 (h, j) proteins in rabbit duodenum after incubation withKrebs (K 90) or LPS 0.3 µg ml-1 (LPS 90) for 90 min using the avidin-biotin method anddiaminobenzidine. Panels c and f show sections of duodenum in which the primary antibody wasomitted to control the specificity of the labelling obtained. Representative sections from 6different animals. Bars = 50 µm. Magnification x200 (a, d, c, f, h-j), x400 (b, e, g). V: epithelialcells of intestinal villi; SM: submucosa; L and C: Longitudinal (L) and circular (C) smoothmuscle layers; large arrows: neurons of the myenteric ganglia; small arrows: neurons of thesubmucosal ganglia.

DISCUSSION

Previous studies by our group (6) have reported a local inhibitory effect ofLPS on the ACh-evoked contractions when rabbit intestinal segments(duodenum, jejunum or ileum) were incubated with LPS in vitro. The mainfinding of the present study is that a downregulation of nNOS is associated to thislocal effect of LPS. The isoforms iNOS, COX-1 and COX-2 were expressedconstitutively in different layers of rabbit duodenum wall and their expressionwas not modified by LPS.

The nNOS produces basal levels of NO to maintain the physiological tonicinhibition of the intestine (23). In our study, we observed a single protein band of155 kDa in the rabbit duodenum by staining with the antibody anti-nNOS (againstN-terminus). A similar size for this protein has been reported in rat gastric mucosa(24, 25). We found expression of this constitutive isoform in the neurons of themyenteric plexus of rabbit duodenum. Similar results were observed in themyenteric plexus of rat intestine (26, 27). Although nNOS is constitutivelyexpressed, increasing evidence indicates that nNOS expression can bedynamically regulated in various physiological and pathological conditions in thegastrointestinal tract. In fact, in our study nNOS expression was increased by theincubation of duodenum in Krebs for 90 min when compared with nonincubatedspecimen. The nNOS mRNA up-regulation seems to represent a general responseof neuronal cells to stress induced by a large array of physical agents such as heat,light exposure or mechanical lesions (22). In our experiments, the intestinalsegments were cut and exposed to light in the organ bath and this could explainour results. This study also shows a reduction in the expression of the nNOSprotein induced by endotoxin in the neurons of the myenteric plexus of rabbitduodenum. Similarly, a downregulation of the nNOS was found in the rat stomachassociated to a delay of gastric emptying induced by endotoxin (28), in a model ofLPS-induced intestinal injury in neonatal rats (29) and in a model of inflammatorybowel disease induced by indomethacin in rat intestine (30). On the contrary, otherauthors have reported an upregulation of nNOS and iNOS in jejunal and ilealsmooth muscle of rat during endotoxemia (31). It has been speculated that down-regulation of nNOS could be due either to an inhibition of gene transcription bycytokines, or to an increase in mRNA breakdown, or to a negative feedbackexerted by NO itself (32). One likely hypothesis is that intracellular calcium, aspreviously reported for nNOS in neurons and cardiac myocytes, may modulatemRNA expression via the CREB family members (32, 33).

Similarities between nitric oxide synthase and cyclooxygenase are apparent.Both enzymes are present in a constitutive form under basal conditions, and in thepresence of certain stimuli, such as LPS or cytokines, the induced form of theenzyme will begin to express itself. In our study, we detected protein bands of 130kDa and 72 kDa for iNOS and COX-2 respectively in the rabbit duodenum.Similar sizes for these proteins have been reported in other gastrointestinal

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segments for iNOS (3, 25) and for COX-2 (19). In this study both isoforms weredetected constitutively in the smooth muscle layers and in the neurons of themyenteric and submucosal ganglia of rabbit duodenum. Similar locations in ratileum and mouse colon were reported for iNOS and COX-2 proteins by otherauthors (8, 19, 34). Moreover, the location of COX-1 and COX-2 isoforms agreeswith the distribution of prostaglandin receptors (35). Other authors have alsodetected a constitutive expression of iNOS and COX-2 in stomach, smallintestine and colonic samples from control animals (8, 36, 37). The functionalsignificance of this expression is unknown. However, since NO is known to betoxic to certain microorganisms, a role for continuous expression of iNOS inmucosal integrity could be postulated (3). Porcher et al. (19) have reported thatcolonic intersticial cells of Cajal may be involved in enteric motorneurotransmission and production of NO, as well as in the synthesis ofprostaglandins and contribute to paracrine regulation of colonic motility.

It has been widely reported that LPS is able to induce both iNOS and COX-2in the gastrointestinal tract (8, 16). In our study no changes in iNOS or COX-2expression were induced by endotoxin in rabbit duodenum. Although theexpression of iNOS and COX-2 proteins was not modified, our functional studiessuggest that these constitutively expressed isoforms may produce NO andprostaglandins in sufficient amounts to play a role in the effect of LPS on ACh-evoked contractions. In our study, L-NNA, a non selective NOS inhibitor, andaminoguanidine, a selective iNOS inhibitor, antagonized the LPS effect on theACh contractions, indicating that NO derived from iNOS could play a role in thiseffect. In fact, we have previously showed that sodium nitroprusside (SNP), anagent that releases NO, reduces both the spontaneous motility and thecontractions induced by ACh in rabbit intestine (38). Similarly, L-NNA andaminoguanidine reversed the inhibitory effect of LPS in ileal muscle (3, 8). Inaddition, in our model, NPLA, a selective nNOS inhibitor, did not block the localinhibitory effect of LPS, indicating that LPS does not evoke the release of NOderived from nNOS. Growing experimental evidence has led to the developmentof a theory on a cross talk between constitutive NOS and iNOS. It has beensuggested that NO derived from nNOS activity keeps iNOS suppressed undernormal conditions, whereas in the course of intestinal inflammation,downregulation of nNOS is a necessary condition to facilitate the expression ofiNOS and the release of large amounts of NO (39). In fact, a cyclical oscillationin the expression of iNOS with a sustained downregulation of nNOS has beenreported in a model of inflammatory bowel disease (30). Thus, we hypothesizethat the inhibitory effect of LPS in our model is associated to a downregulationof nNOS that seems to be necessary for increasing the expression of iNOS,although the expression of this isoform was not increased after 90 min ofincubation with LPS. It would probably be necessary a longer time of incubationwith LPS to detect a higher expression of iNOS in rabbit duodenum. Protein geneexpression, synthesis of the protein and functional activity of the protein

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including dimerization of the iNOS has been shown to be a slow process (40).Hori et al. (8) found that in rat ileum COX-2 and iNOS mRNAs began to increasein LPS-treated tissue at 30 and 90 min and reached their peak at 60 and 240 min,respectively. Recently, it has been reported that a systemic administration of lowdoses of endotoxin (µg/Kg) in rats is associated with rapid changes ingastrointestinal motor function and these appear to be mediated by an increase inNO synthesis in the brainstem, as well as in the gastric myenteric plexus thirtyminutes after endotoxin administration. This synthesis is due to constitutive nitricoxide synthase and occurs before induction of NOS takes place (18).

In our study, ODQ, a guanylate cyclase inhibitor, reversed the effect of LPS,indicating that NO-induced inhibition is mediated by cyclic GMP. Ingastrointestinal smooth muscles, the NO-induced relaxation is mediated by theguanylyl cyclase-cyclic GMP pathway and also by membrane hyperpolarization(12). In rabbit small intestine, the relaxations evoked by SNP on spontaneousmotility were eliminated in the presence of ODQ, indicating that the relaxationsevoked by this NO donor are completely dependent on guanylate cyclaseactivation (38).

Our results indicate that synthesis of prostaglandins triggered by endotoxinseems to be mediated by the COX-2 isoenzyme because the COX-2 selectiveinhibitor, NS-398, significantly prevented the effect of endotoxin in a similarmanner to that observed in indomethacin-incubated duodenum. The LPS effect onACh-induced contractions was also antagonized by indomethacin in rabbit smallintestine (6). It has also been reported that indomethacin and NS-398 preventedthe delay in gastric emptying induced by endotoxin in rats (29).

In conclusion, LPS locally administered reduces the contractility of rabbitduodenum and a downregulation of nNOS is associated to this effect. The iNOS,COX-1 and COX-2 isoforms were expressed constitutively in the muscle layersand the neurons of the myenteric ganglia of rabbit duodenum and their expressionwas not modified by LPS.

Acknowledgments: We would like to thank Dr José Emilio Mesonero for his technicalassistance in the western blotting study. This work was funded by the Spanish Ministry of Scienceand Technology (Dirección General de Investigación AGL2003-03291, AGL2006-04317 andERDF) and the Regional Government of Aragon (A-24/2005 and B61/2006). A personal grant toLaura Grasa was provided by the Government of Aragon and the European Social Fund(B010/2003, Spain).

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R e c e i v e d : March 6, 2008A c c e p t e d : July 30, 2008

Author�s address: M. Divina Murillo, Department of Pharmacology and Physiology, Facultyof Veterinary Medicine, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza (Spain).Tel: 34-976-761652, Fax: 34-976-761612; e-mail: dmurillo@unizar.es

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