AJP - GI Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 293: G1281-G1287, 2007. First published October 18, 2007; doi:10.1152/ajpgi.00357.2007
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/G1281    most recent
00357.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Petersson, J.
Right arrow Articles by Holm, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Petersson, J.
Right arrow Articles by Holm, L.

INFLAMMATION/IMMUNITY/MEDIATORS

eNOS involved in colitis-induced mucosal blood flow increase

Joel Petersson,1,* Olof Schreiber,1,* Andreas Steege,2 Andreas Patzak,2 Anna Hellsten,1 Mia Phillipson,1 and Lena Holm1

1Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden; and 2Institute of Vegetative Physiology, Charité-Universitätsmedizin Berlin, Berlin, Germany

Submitted 8 August 2007 ; accepted in final form 17 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The role of NO in inflammatory bowel disease is controversial. Studies indicate that endothelial nitric oxide synthase (eNOS) might be involved in protecting the mucosa against colonic inflammation. The aim of this study was to investigate the involvement of nitric oxide (NO) in regulating colonic mucosal blood flow in two different colitis models in rats. In anesthetized control and colitic rats, the distal colon was exteriorized and the mucosa visualized. Blood flow (laser-Doppler flowmetry) and arterial blood pressure were continuously monitored throughout the experiments, and vascular resistance was calculated. Trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS) was used to induce colitis. All groups were given the NOS inhibitor N{omega}-nitro-L-arginine (L-NNA) or the inducible NOS (iNOS) inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL). iNOS, eNOS, and neuronal NOS (nNOS) mRNA in colonic samples were investigated with real-time RT-PCR. Before NOS inhibition, colonic mucosal blood flow, expressed as perfusion units, was higher in both colitis models compared with the controls. The blood flow was reduced in the TNBS- and DSS-treated rats during L-NNA administration but was not altered in the control group. Vascular resistance increased more in the TNBS- and DSS-treated rats than in the control rats, indicating a higher level of vasodilating NO in the colitis models. L-NIL did not alter blood pressure or blood flow in any of the groups. iNOS and eNOS mRNA increased in both colitis models, whereas nNOS remained at the control level. TNBS- and DSS-induced colitis results in increased colonic mucosal blood flow, most probably due to increased eNOS activity.

Trinitrobenzene sulfonic acid; dextran sulfate sodium; inducible nitric oxide synthase; neuronal nitric oxide synthase

NITRIC OXIDE (NO) HAS BEEN shown to be involved in the pathogenesis of the inflammatory bowel diseases (IBD) ulcerative colitis and Crohn's disease (18, 24, 55). However, the role of NO in IBD is still controversial, since some studies suggest that NO has protective influence, whereas others indicate a pathogenic role [reviewed by Kolios and colleagues (18)]. Recently, high levels of luminal NO have been found during intestinal inflammation (28–31, 44, 50), highlighting the association of NO with IBD.

The involvement of NO in blood flow regulation in the upper gastrointestinal tract and the importance of gastric mucosal blood flow in the defense against injury to the gastric mucosa have been well studied (1, 17). The regulation of colonic mucosal blood flow is poorly understood, and its importance during colonic injury is still unclear. Studies in recent years have yielded contradictory data, showing either an increased (8, 11, 14, 27) or a decreased (7, 11, 21, 22, 38) blood flow in different animal models of colitis, using a variety of measuring techniques. The few blood flow studies conducted in humans with IBD have indicated that the mucosal blood flow is increased in inflamed regions (3, 15).

A well-regulated colonic mucosal blood flow is of utmost importance for sustained homeostasis. Epithelial integrity is dependent on mucosal blood flow for delivering oxygen, nutrients, and leukocytes when required and for removing reactive oxygen species and intruding bacteria (32). During periods of ischemia, the colonic barrier immediately breaks down, a condition which, if left untreated, will lead to sepsis.

In the colon, mucosal capillaries normally form a regular honeycomb-like plexus around the opening of the mucosal glands (53), and this colonic microvasculature becomes dilated during colitis (10, 34, 45, 48). NO formed in endothelial cells plays an important part in the regulation of the regional blood flow. Endothelial nitric oxide synthase (eNOS) produces NO that dilates the submucosal arterioles, leading to increased blood flow in the mucosal capillaries (55). In mice deficient in eNOS, the inflammation induced in different models of colitis is more severe, indicating that eNOS has an important protective role against mucosal inflammation (51, 56).

Although NO is involved in many physiological functions, it also contributes to tissue injury during inflammatory processes. When produced in small amounts, NO generally exerts positive effects in the gastrointestinal tract (55). However, under inflammatory colonic conditions, greatly increased NO levels have been reported, for example, in human IBD (47, 49) and in animal models of colitis (26). Under these conditions, NO is produced in large amounts mainly by inducible NOS (iNOS), and it has been proposed that this high NO level causes injury through formation of peroxynitrite (24). Expression of iNOS is induced during inflammation, and hence iNOS is highly upregulated in inflamed colonic mucosa (4, 33, 45). iNOS is primarily expressed in macrophages and neutrophils (55), but during colitis it is also expressed in large amounts by epithelial cells (35, 45). Interestingly, induction of experimental colitis in iNOS-deficient mice results in less severe inflammation (4, 20, 59) in contrast to the findings in mice with eNOS deficiency.

Neuronal NOS (nNOS) in the colonic myenteric plexus has been shown to play an important part in the regulation of colonic peristalsis and transit (54), and there are data indicating that loss of nNOS during colonic inflammation results in dysfunction of colonic peristalsis (36).

The aim of this study was to measure colonic mucosal blood flow both in untreated rats and in rats with colitis induced by 2.4.6-trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS). Furthermore, since large amounts of NO are present in the colon during colitis (28, 29, 31, 44, 50), a further purpose was to determine whether NO is involved in the regulation of colonic mucosal blood flow in colitis and in control animals. An attempt was also made to ascertain which of the NOS isoforms is most important in blood flow regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. All experiments were approved by the regional ethics committee for animal experiments in Uppsala.

Male Sprague-Dawley rats (B&K, Sollentuna; Sverige and Mollegaard Breeding Center, Ejby, Denmark), weighing between 190 and 290 g (weight before treatment), were kept under standardized conditions at a temperature of 21–22°C and with 12 h light and 12 h dark per day. They were allowed to acclimatize for 1 wk before the experiments were started.

The rats were anesthetized with 120 mg/kg body wt of 5-ethyl-5-(1-methylpropyl)-2-thiobutabarbital sodium (Inactin; Sigma, St. Louis, MO), given intraperitoneally. The body temperature was maintained at 37–38°C, using a heating pad controlled by a rectal thermistor probe. To facilitate spontaneous breathing, a short PE-200 cannula was placed in the trachea. A PE-50 cannula containing heparin (Leo Pharma, Malmoe, Sweden; 12.5 IU/ml) dissolved in 0.9% saline was placed in the right femoral artery to monitor blood pressure, and the right femoral vein was catheterized to enable a continuous infusion of Ringer's solution (120 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, and 0.75 mM CaCl2) at a rate of 1 ml per h during the experiment. In some experiments the right femoral vein was also the route for administration of N{omega}-nitro-L-arginine (L-NNA, Sigma) or L-N6-(1-iminoethyl)-lysine (L-NIL, Sigma) in the Ringer's solution. The colon was exteriorized through a midline abdominal incision, and the descending colon was opened longitudinally 1–2 cm. The rat was placed on its right side on a Lucite microscope stage. The colon was everted and loosely draped over a truncated cone. A double-bottom mucosal chamber with 37°C circulating water and a hole in the bottom was fitted over the mucosa, exposing ~0.5 cm2 of the mucosal surface. The junction was sealed with silicon grease (Dow Corning high vacuum grease; Dow Corning, Weisbaden, Germany). The mucosal chamber was filled with ~5 ml 37°C saline to keep the tissue moist and warm. This model is further described by Atuma et al. (2, 13).

Colonic mucosal blood flow measurements. The colonic mucosal blood flow was measured continuously from the mucosal side by using laser Doppler flowmetry (PeriFlux 4001 Master and PeriFlux PF3; Perimed, Järfälla, Sweden). Red monochromatic laser light (wavelength 635 nm) is guided via an optic fiber down to the tissue. In the tissue the light is reflected against moving objects, and a shift in frequency, referred to as the Doppler Effect, will take place. The size of the shift is dependent on the velocity and number of red blood cells in the illuminated tissue. A fraction of the backscattered light returns via two equal-sized optic fibers. A laser probe fixed to a micromanipulator was placed in the saline 1–2 mm above the mucosa to monitor the mucosal blood flow. Blood flow in this study is expressed both in perfusion units (PU) and in relative terms as percentage of baseline values (% of control) over 10-min periods.

TNBS-induced colitis. To induce colitis, 30 mg TNBS (Fluka Chemie, Darmstadt, Germany) in 0.25 ml 50% ethanol (ethanol 99.5% Solveco Chemicals, Täby, Sweden) was administered rectally using an 8-cm-long plastic catheter in accordance with the model developed by Morris et al. (39). Before the administration of TNBS the rats were sedated with 0.5 mg midazolam (Dormicum, 5 mg/ml; Roche, Basel, Switzerland) given intraperitoneally. After TNBS administration, the rats were given 1 ml saline subcutaneously in the event of acute diarrhea or appreciable weight loss. The body weight of the rats was measured daily from the day of TNBS administration to the day of the experiment, and a weight loss of up to 15% was tolerated. The animal's condition and any diarrhea or bloody stool were also noted everyday. All experiments were performed on day 7 after the induction of colitis. The saline control rats were treated in the same manner as the rats with induced colitis, but instead of TNBS, the controls received 0.9% saline rectally.

DSS-induced colitis. Rats were given 5% (wt/vol) DSS (37–40 kilodaltons; TdB Consultancy, Uppsala, Sweden) in their drinking water for 9 days. Body weight, presence of fecal blood or gross rectal bleeding, and stool consistency were noted daily. The control rats for the DSS group were untreated.

Assessment of severity of colitis. After the experiment, the rats were killed with an intravenous injection of saturated potassium chloride solution, and the colon was removed, opened longitudinally, and rinsed with deionized water. The colon was inspected macroscopically, and its weight and length were measured in all animals. In the TNBS-treated animals the colonic damage was evaluated and assigned a macroscopic damage score as described by Morris et al. (39). This scoring takes into account the presence of diarrhea, adhesions, macroscopically visible damage, and bowel wall thickness. In the DSS-treated animals, the severity of the colitis was assessed on the basis of clinical parameters, such as stool consistency and fecal blood content. This severity, together with any weight loss was scored daily with the use of disease activity index (DAI), a scoring method described in detail by Cooper et al. (5). In both models, the animal weight was monitored daily from the induction of the inflammation to the day of the experiment.

Experimental protocol. After the preparation, the rats were given at least 1 h to stabilize before the blood flow measurements began. Systemic blood pressure and colonic blood flow had been at steady-state for at least 20 min before the experiment started. The rats were divided into four treatment groups: 1) saline intrarectally in a single dose 7 days prior to the experiment (TNBS control), 2) TNBS intrarectally in a single dose 7 days prior to the experiment, 3) untreated (DSS control), and 4) DSS given in the drinking water for 9 days.

The colonic mucosal blood flow was followed for 100 min in all four groups. In different sets of experiments, the NOS inhibitor L-NNA (10 mg/kg bolus in the volume of 1.0 ml followed by an infusion of 3 mg·kg–1·h–1 iv with the infusion rate of 1.0 ml/h) or the iNOS inhibitor L-NIL (10 mg/kg bolus in the volume of 1.0 ml followed by an infusion of 3 mg·kg–1·h–1 iv with the infusion rate of 1.0 ml/h) was given, starting 20 min after the start of the experiment, in all four groups. The recorded mean arterial blood pressure (expressed in mmHg) and mucosal blood flow (in PU) were used to calculate the vascular resistance during the experiments.

RNA purification. Another 12 rats (4 controls, 4 TNBS-treated, and 4 DSS-treated) were used for these experiments. The rats were anesthetized with 120 mg/kg body wt of 5-ethyl-5-(1-methylpropyl)-2-thiobutabarbital sodium, and the distal colon was excised and immediately frozen in liquid nitrogen. To investigate changes in eNOS, iNOS, and nNOS, whole colonic tissue RNA was isolated using an RNA isolation kit (Peqlab, Erlangen, Germany). The tissue samples were homogenized in a mortar in liquid nitrogen. For the isolation of total RNA, 20 µg of homogenized tissue was used in accordance with manufacturer's instructions.

mRNA quantification. The amounts of eNOS, iNOS, and nNOS mRNA were estimated by real-time RT-PCR analyses. Five hundred nanograms of the isolated RNA was reverse transcribed using SuperScriptII Reverse Transcriptase and random hexamers (Invitrogen, Karlsruhe, Germany) in accordance with the manufacturer's instructions. Quantitative PCR analysis was performed in triplicate using a GeneAmp 5700 (Applied Biosystems, Darmstadt, Germany) as instructed by the manufacturer. SYBR Green was used for the fluorescent detection of DNA amplified during the PCR. The PCR reaction took place in a total volume of 25 µl with 0.2 pmol/µl of each primer, and SYBR Green master mix (Bioline, Luckenwalde, Germany). One microliter cDNA corresponding to 20 ng RNA was used as template. Published sequences for rat eNOS (NM_021838 [GenBank] ), rat iNOS (NM_012611 [GenBank] ), rat nNOS (NM_052799 [GenBank] ), and rat β-actin (NM_031144 [GenBank] ) were used to design primers that bridge at least one intron. Primer sequences were as follows: eNOS sense 5'-CAG CAC CAG ACC ACA GCC CC-3' and antisense 5'-TCC TGC TGA GCC TGT GCA CT-3'; iNOS sense 5'-CAG GTG CTA TTC CCA GCC CAA CA-3' and antisense 5'-CAT TCT GTG CAG TCC CAG TGA GGA A-3'; nNOS sense 5'-ATC CAG GTG GAC AGA GAC CTC GAT G-3' and antisense 5'-CCG AGG TAG GGG ACT GTT CCT TCT CT-3'; β-actin sense 5'-GGT CCA CAC CCG CCA CCA GTT-3' and antisense 5'-ACC CAT ACC CAC CAT CAC ACC CTG-3'. Conditions were 50°C, 2 min; 95°C, 10 min; 40 cycles: 95°C, 30; 67°C, 1 min; 77°C, 30 s. The levels of expression of the eNOS, iNOS, and nNOS mRNA were normalized to the levels of β-actin using the delta CT method. Parallelism of standard curves of the test and control was confirmed.

Chemicals. NaCl was obtained from Fluka Chemie, and NaHCO3, KCl, and CaCl2 from Merck, Buchs, Switzerland. NaCl (20 ml ampule, 9 mg/ml) was from Braun Medical.

Statistical analysis. All data are expressed as means ± SE. For statistical analysis two- and one-way ANOVA repeated measurements with Tukey's multiple comparison test were performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA). Student's t-test was used for statistical evaluation of unpaired data. P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Blood pressure and mucosal blood flow remained stable during the entire experimental period in all three groups, controls, TNBS-treated, and DSS-treated (Fig. 1A). Since there were no differences in the blood flow values between the untreated animals (group 1) and the animals given saline rectally 7 days prior to the experiments (group 3), these animals were regarded as one control group. When we compared the colonic mucosal blood flow, expressed in PU, during the control period (the first 20 min) in all experiments (n = 75), we found that it was significantly higher in both the TNBS-treated (184 ± 21 PU, n = 17) and the DSS-treated animals (189 ± 21 PU, n = 22) than in the controls (117 ± 7 PU, n = 36) (Fig. 2). When taking all experiments in account, there were no differences in mean arterial blood pressure during the control period between the groups (control, 100 ± 2 mmHg n = 36; DSS-treated, 97 ± 2 mmHg n = 22; TNBS-treated, 99 ± 4 mmHg n = 17).


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 1. Dynamic changes in mean arterial pressure (MAP), colonic mucosal blood flow [presented as laser Doppler flowmetry (LDF) % of control] and vascular resistance (presented as % of control) in untreated (control), dextran sodium sulfate (DSS)-treated and 2.4.6.-trinitrobenzene sulfonic acid (TNBS)-treated animals in the control situation (A), after nitric oxide synthase (NOS) inhibition (B), and after inducible NOS (iNOS) inhibition (C). Control (untreated), n = 11; DSS-treated, n = 6; TNBS-treated, n = 5; NOS inhibition, n = 14/5/4; iNOS inhibition, n = 5/6/5. Values are expressed as means ± SE.

 

Figure 2
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 2. Colonic mucosal blood flow, expressed in perfusion units during, the first 20 min of the experiment in untreated control rats (n = 36) and in rats treated with DSS (n = 22) and TNBS (n = 17). Values are means ± SE. *P < 0.05 compared with control.

 
NOS and Colonic Mucosal Blood Flow

L-NNA administered to control animals resulted in a significant rise in blood pressure (by 50%), but no significant changes in mucosal blood flow were observed. The calculated vascular resistance consequently increased (by 54 ± 11%), indicating that NO might be involved in maintaining baseline blood flow. Another theoretical possibility is that the blood flow is regulated by myogenic (or metabolic) response to the increased blood pressure. In TNBS- and DSS-treated rats a significant reduction in blood flow was seen during treatment with L-NNA (by 33 ± 4% and by 33 ± 7%, respectively) (Fig. 1B). As in the control animals, a significant rise in blood pressure was also seen in these rats when treated with L-NNA.

The calculated vascular resistance increased to a significantly greater extent in the TNBS- and DSS-treated rats (by 102 ± 10% and by 118 ± 10%, respectively) than in the control animals during L-NNA administration, indicating a higher level of vasodilating NO in colitis.

iNOS and Colonic Mucosal Blood Flow

Blood pressure and mucosal blood flow remained unchanged when L-NIL was given to control animals. Similarly, L-NIL administration did not affect either blood flow or blood pressure in the TNBS- and DSS-treated rats (Fig. 1C), suggesting that iNOS-induced NO was not involved in the blood flow increase in the rats with colitis. The used dose and route of administration of L-NIL has earlier been shown to inhibit iNOS in vivo (46).

eNOS, iNOS, and nNOS mRNA Expression

The relative expression of eNOS mRNA was significantly increased in the TNBS- and DSS-treated animals compared with controls (Fig. 3). iNOS expression was also significantly higher in the colitic animals compared with the controls. There was, however, no difference in nNOS expression between the groups (Fig. 3).


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 3. RT-PCR relative amounts of endothelial NOS (eNOS), iNOS, and neuronal NOS (nNOS) in control animals and in animals treated with TNBS or DSS. Values are expressed as means ± SE. *P < 0.05 compared with control.

 
Severity of Colitis

TNBS. TNBS induced severe colitis with penetrating ulcers, a reduced colon length (control, 19.4 ± 0.4 cm; TNBS, 16.0 ± 0.4 cm), and thickening of the colonic wall (Fig. 4). All the animals in the TNBS-treated group had a damage score that was at least four times higher than the highest damage score in the control group. These colitic rats also had a higher colon weight per centimeter than the controls (TNBS, 0.16 ± 0.01 g/cm; controls, 0.09 ± 0.02 g/cm), and they lost body weight after the treatment with TNBS. The controls, on the other hand, gained weight.


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 4. Severity of the colitis in the different models. A: TNBS-induced colitis, macroscopic damage score. B: DSS-induced colitis, disease activity index values are expressed as means ± SE. *P < 0.05 compared with control.

 
DSS. DSS induced a more diffuse mucosal inflammation covering the entire colon and with a higher fecal blood content than in the TNBS-treated rats. In the DSS model, a shortening of the colon was observed (control, 19.4 ± 0.4 cm; DSS, 16.3 ± 1.1 cm), but no increase in thickness of the colonic wall or weight per centimeter was observed in these animals. The DSS-treated animals reached a DAI of 2.2 ± 0.1 at day 9 of DSS treatment, whereas none of the controls showed any signs of diarrhea, weight loss or bloody stool (Fig. 4).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study we used two common models of experimental colitis, the TNBS and the DSS model. TNBS induced severe colitis with penetrating ulcers, a reduced colon length, and thickening of the colon wall (41). This model shares some of the histopathological features of Crohn's disease (39). DSS is a model of ulcerative colitis, induced a more diffuse mucosal inflammation covering the entire colon, and resulted in diarrhea and gross rectal bleeding (5, 40, 43).

Colonic mucosal blood flow was measured by laser Doppler flowmetry, a well-established method for continuous measurements of mucosal blood flow (12, 25, 42). Our results show that irrespective of the disease model used, colitic rats have a higher colonic mucosal blood flow than control rats and that NO is involved in the regulation of the mucosal blood flow in the inflamed colon. Both iNOS and eNOS are upregulated in TNBS- as well as DSS-induced colitis, whereas nNOS remained unchanged. Inhibition of iNOS did not influence the colonic mucosal blood flow either in the control or in the colitic rats, whereas L-NNA, an unselective inhibitor of NOS, reduced the blood flow in the colitic rats, demonstrating the importance of constitutively expressed NOS in the blood flow increase seen during colitis.

Bacaner and Hultén et al. (3, 15) found an increased blood flow in inflamed regions of the bowel in patients with ulcerative colitis and Crohn's disease. IBD comprises a group of inflammatory conditions with greatly heterogeneous signs and symptoms in the affected patients. Owing to this variation, it is very difficult to study these conditions in humans. Blood flow measurements in patients with IBD are difficult due to problems such as determination of the phase of the inflammation, limitations of the blood flow techniques, and the influence of the patient's medication. It is therefore important to investigate the blood flow in animal models of colitis, where functional studies under controlled conditions can be performed.

Reports on the changes in mucosal blood flow in various experimental models of colitis provide conflicting findings. That acute inflammation involves hyperemia is not questioned, but in the chronic situation, both increased blood flow and reduced blood flow have been recorded.

Buhr and colleagues (7, 21–23), using intravital microscopy, have repeatedly observed an increased mucosal blood flow within 24 h after induction of colitis with TNBS and a reduced blood flow after 3 days. Deniz et al. (6) recorded decreased arterial mesenteric blood flow at early stages in TNBS-induced colitis in mice. The first days after TNBS administration, the induced colitis is still in an acute phase. In the present study, increased blood flow was noted 1 wk after TNBS administration, hence during a more manifest stage of the inflammation, which may account for the discrepancies seen. In accordance with our results, Hosoi et al. (14) found an increased mucosal blood flow (hydrogen gas clearance) 1 wk after induction of colitis with TNBS. The question whether the mucosal blood flow increases or decreases during TNBS-induced colitis might depend on where the measurements have been made. Most likely, there are regional differences in blood flow within the inflamed area, where deep penetrating ulcers are ischemic, whereas the surrounding mucosa is hyperemic. Our measurements were always performed about 2 cm proximal to the ulcerated area in the distal colon.

Inducing colitis with DSS renders a gradual disease progression, with a disease severity that increases with the time of DSS exposure. In the DSS model, a reduction in blood flow of small submucosal arterioles only was noted on days 4 and 6 in the mouse proximal colon (38). Another study, measuring blood flow in the proximal colon of mice, showed no blood flow changes during DSS treatment (52). However, a higher colonic blood flow was seen in the distal rat colon on days 6 and 7 with use of microspheres (8). This is in accordance with our findings of a higher colonic blood flow in the distal colon 9 days after the start of DSS administration. We and others (7, 9) have observed that the degree of inflammation in DSS-induced colitis tends to be greater in the distal than in the proximal colon, which might explain the differences in reported colonic blood flow.

Several studies have demonstrated that colitis is associated with changes in the colonic microvasculature. McLaren et al. (34) concluded that DSS-induced colitis in rats augments the capillary diameters and increases the vessel density in the colonic mucosa. Enlarged colonic vessels have also been found in biopsy samples from patients with Crohn's disease and with ulcerative colitis (45). Recently Ravnic et al. (48) showed that the diameter of the colonic mucosal capillaries increased during TNBS-induced colitis in mice. They also measured the velocity of the blood flow in the mucosal plexus and found that it was decreased. Thus despite the decrease in velocity, due to the dilatation, the net effect was an increased blood flow to the mucosa. In our studies we noticed that the small mucosal venules visible through the microscope were enlarged in both the DSS- and TNBS-induced colitis models, confirming the above described microvascular changes.

In the present study, a twofold upregulation of eNOS mRNA and a 20-fold upregulation of iNOS mRNA was detected both in DSS- and TNBS-induced colitis using real-time RT-PCR. nNOS mRNA was not significantly altered in either of the colitis models compared with the controls, which is in agreement with findings in a previous study of manifest DSS-induced colitis (4). In other studies, however, in which measurements were made only in the muscle layer of the colon, a reduced nNOS activity was observed in the DSS model, suggesting that this downregulation is responsible for impaired colonic peristalsis during colitis (19, 36). In line with this reduction, our study with measurement of the mRNA levels throughout the colonic wall, showed a slightly nonsignificant decrease in nNOS in the DSS-treated animals, which might have been significant if measurements had been conducted in the muscle layer alone.

The massive increase in iNOS mRNA we measure is not surprising, since iNOS is a hallmark of inflammation, and several earlier studies have demonstrated an upregulation of iNOS in mucosal samples both in DSS (4) and TNBS models (33), as well as in human patient data representing both Crohn's disease and ulcerative colitis (45). iNOS has been localized in infiltrating macrophages and neutrophils in the colonic mucosa and submucosa, surrounding the crypts, and in epithelial cells both in animal models (35) and in patients with Crohn's disease (58) and ulcerative colitis (45). The increase in iNOS in the tissue is correlated with the severity of the disease (57). The magnitude of increase in iNOS mRNA expression we detect during inflammation in our models indicates there may be production of a large amount of NO; however, specific inhibition of iNOS did not alter the blood flow or vascular resistance in our colitic rats. In addition, L-NNA, which is a poor inhibitor of iNOS but a potent eNOS and nNOS inhibitor (37), lead to a colonic blood flow reduction. These results are certainly surprising and difficult to explain. One possible explanation is that the NO produced by iNOS in the mucosa diffuses into the venules and is transported away from the arterioles in the submucosa without influencing the blood flow. eNOS, which is produced in the vessel wall, has a much greater influence of the vascular resistance than NO produced in mucosal cells.

The increase in eNOS mRNA observed in the present study during colitis confirms previous reports. Elevated levels of eNOS have been found both in colonic tissue from DSS-induced colitis in mice (4) and in mesenteric endothelial cells from patients with ulcerative colitis (16).

Recent results have indicated that eNOS might be involved in protecting the colon against mucosal inflammation, since the disease activity was increased in eNOS-deficient mice treated with DSS (51). Vallance et al. (56) confirmed these findings in the TNBS model, where the eNOS-deficient animals suffered from more severe colitis. In the present study we have found indications that eNOS plays an important role in regulating the colonic mucosal blood flow during inflammation. When the NOS activity was blocked with the nonselective inhibitor, L-NNA [potency; eNOS = nNOS > iNOS (37)], the vascular resistance increased more in the colitic rats than in the controls, pointing to a higher level of vasodilating NO in these rats. Since eNOS, and not nNOS, is upregulated in the colitis models and since eNOS is expressed mainly in endothelial cells in close proximity to arteriolar smooth muscles, we can conclude that the blood flow alterations seen in the colitis models most probably depend on the eNOS activity.

In conclusion, we have shown in this study that mucosal blood flow is increased in the distal colon in both the TNBS- and DSS-induced colitis models. This blood flow increase is most probably due to upregulation of eNOS and not to the high iNOS levels recorded. One protective action of eNOS may therefore be to increase the mucosal blood flow during inflammation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by grants from the Swedish Research Council (04X-08646).


   ACKNOWLEDGMENTS
 
We thank Annika Jägare and Ulrike Neumann for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Lena Holm, Dept. of Medical Cell Biology, Uppsala Univ., S-751 23 Uppsala, Sweden (e-mail: Lena.Holm{at}mcb.uu.se)

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.

* J. Petersson and O. Schreiber contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abdel-Salam OM, Czimmer J, Debreceni A, Szolcsanyi J, Mozsik G. Gastric mucosal integrity: gastric mucosal blood flow and microcirculation. An overview. J Physiol Paris 95: 105–127, 2001.[CrossRef][ISI][Medline]
  2. Atuma C, Strugala V, Allen A, Holm L. The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo. Am J Physiol Gastrointest Liver Physiol 280: G922–G929, 2001.[Abstract/Free Full Text]
  3. Bacaner MB. Quantitative measurement of regional colon blood flow in the normal and pathological human bowel. Gastroenterology 51: 764–777, 1966.[ISI][Medline]
  4. Beck PL, Xavier R, Wong J, Ezedi I, Mashimo H, Mizoguchi A, Mizoguchi E, Bhan AK, Podolsky DK. Paradoxical roles of different nitric oxide synthase isoforms in colonic injury. Am J Physiol Gastrointest Liver Physiol 286: G137–G147, 2004.[Abstract/Free Full Text]
  5. Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest 69: 238–249, 1993.[ISI][Medline]
  6. Deniz M, Cetinel S, Kurtel H. Blood flow alterations in TNBS-induced colitis: role of endothelin receptors. Inflamm Res 53: 329–336, 2004.[ISI][Medline]
  7. Foitzik T, Kruschewski M, Kroesen A, Buhr HJ. Does microcirculation play a role in the pathogenesis of inflammatory bowel diseases? Answers from intravital microscopic studies in animal models. Int J Colorectal Dis 14: 29–34, 1999.[CrossRef][ISI][Medline]
  8. Garrelds IM, Heiligers JP, Van Meeteren ME, Duncker DJ, Saxena PR, Meijssen MA, Zijlstra FJ. Intestinal blood flow in murine colitis induced with dextran sulfate sodium. Dig Dis Sci 47: 2231–2236, 2002.[CrossRef][ISI][Medline]
  9. Harris NR, Whatley JR, Carter PR, Specian RD. Venular constriction of submucosal arterioles induced by dextran sodium sulfate. Inflamm Bowel Dis 11: 806–813, 2005.[CrossRef][ISI][Medline]
  10. Hatoum OA, Binion DG. The vasculature and inflammatory bowel disease: contribution to pathogenesis and clinical pathology. Inflamm Bowel Dis 11: 304–313, 2005.[CrossRef][ISI][Medline]
  11. Hatoum OA, Binion DG, Gutterman DD. Paradox of simultaneous intestinal ischaemia and hyperaemia in inflammatory bowel disease. Eur J Clin Invest 35: 599–609, 2005.[CrossRef][ISI][Medline]
  12. Holm-Rutili L, Berglindh T. Pentagastrin and gastric mucosal blood flow. Am J Physiol Gastrointest Liver Physiol 250: G575–G580, 1986.[Abstract/Free Full Text]
  13. Holm-Rutili L, Obrink KJ. Rat gastric mucosal microcirculation in vivo. Am J Physiol Gastrointest Liver Physiol 248: G741–G746, 1985.[Abstract/Free Full Text]
  14. Hosoi T, Goto H, Arisawa T, Niwa Y, Okada N, Ohmiya N, Hayakawa T. Role of nitric oxide synthase inhibitor in experimental colitis induced by 2,4,6-trinitrobenzene sulphonic acid in rats. Clin Exp Pharmacol Physiol 28: 9–12, 2001.[CrossRef][ISI][Medline]
  15. Hulten L, Lindhagen J, Lundgren O, Fasth S, Ahren C. Regional intestinal blood flow in ulcerative colitis and Crohn's disease. Gastroenterology 72: 388–396, 1977.[ISI][Medline]
  16. Iwashita E, Miyahara T, Hino K, Tokunaga T, Wakisaka H, Sawazaki Y. High nitric oxide synthase activity in endothelial cells in ulcerative colitis. J Gastroenterol 30: 551–554, 1995.[CrossRef][ISI][Medline]
  17. Kawano S, Tsuji S. Role of mucosal blood flow: a conceptional review in gastric mucosal injury and protection. J Gastroenterol Hepatol 15 Suppl: D1–6, 2000.[ISI][Medline]
  18. Kolios G, Valatas V, Ward SG. Nitric oxide in inflammatory bowel disease: a universal messenger in an unsolved puzzle. Immunology 113: 427–437, 2004.[CrossRef][ISI][Medline]
  19. Kono T, Chisato N, Ebisawa Y, Asama T, Sugawara M, Ayabe T, Kohgo Y, Kasai S, Yoneda M, Takahashi T. Impaired nitric oxide production of the myenteric plexus in colitis detected by a new bioimaging system. J Surg Res 117: 329–338, 2004.[CrossRef][ISI][Medline]
  20. Krieglstein CF, Cerwinka WH, Laroux FS, Salter JW, Russell JM, Schuermann G, Grisham MB, Ross CR, Granger DN. Regulation of murine intestinal inflammation by reactive metabolites of oxygen and nitrogen: divergent roles of superoxide and nitric oxide. J Exp Med 194: 1207–1218, 2001.[Abstract/Free Full Text]
  21. Kruschewski M, Anderson T, Buhr HJ, Loddenkemper C. Selective COX-2 inhibition reduces leukocyte sticking and improves the microcirculation in TNBS colitis. Dig Dis Sci 51: 662–670, 2006.[CrossRef][ISI][Medline]
  22. Kruschewski M, Anderson T, Loddenkemper C, Buhr HJ. Endothelin-1 receptor antagonist (LU-135252) improves the microcirculation and course of TNBS colitis in rats. Dig Dis Sci 51: 1461–1470, 2006.[CrossRef][ISI][Medline]
  23. Kruschewski M, Foitzik T, Perez-Canto A, Hubotter A, Buhr HJ. Changes of colonic mucosal microcirculation and histology in two colitis models: an experimental study using intravital microscopy and a new histological scoring system. Dig Dis Sci 46: 2336–2343, 2001.[CrossRef][ISI][Medline]
  24. Kubes P, McCafferty DM. Nitric oxide and intestinal inflammation. Am J Med 109: 150–158, 2000.[CrossRef][ISI][Medline]
  25. Kvietys PR, Shepherd AP, Granger DN. Laser-Doppler, H2 clearance, and microsphere estimates of mucosal blood flow. Am J Physiol Gastrointest Liver Physiol 249: G221–G227, 1985.[Abstract/Free Full Text]
  26. Lamine F, Fioramonti J, Bueno L, Nepveu F, Cauquil E, Lobysheva I, Eutamene H, Theodorou V. Nitric oxide released by Lactobacillus farciminis improves TNBS-induced colitis in rats. Scand J Gastroenterol 39: 37–45, 2004.[CrossRef][ISI][Medline]
  27. Laroux FS, Grisham MB. Immunological basis of inflammatory bowel disease: role of the microcirculation. Microcirculation 8: 283–301, 2001.[CrossRef][ISI][Medline]
  28. Ljung T, Axelsson LG, Herulf M, Lundberg JO, Hellstrom PM. Early changes in rectal nitric oxide and mucosal inflammatory mediators in Crohn's colitis in response to infliximab treatment. Aliment Pharmacol Ther 25: 925–932, 2007.[ISI][Medline]
  29. Ljung T, Beijer E, Herulf M, Weitzberg E, Lundberg JO, Finkel Y, Hellstrom PM. Increased rectal nitric oxide in children with active inflammatory bowel disease. J Pediatr Gastroenterol Nutr 34: 302–306, 2002.[CrossRef][ISI][Medline]
  30. Ljung T, Herulf M, Beijer E, Jacobsson H, Lundberg J, Finkel Y, Hellstrom PM. Rectal nitric oxide assessment in children with Crohn's disease and ulcerative colitis. Indicator of ileocaecal and colorectal affection. Scand J Gastroenterol 36: 1073–1076, 2001.[CrossRef][ISI][Medline]
  31. Ljung T, Lundberg S, Varsanyi M, Johansson C, Schmidt PT, Herulf M, Lundberg JO, Hellstrom PM. Rectal nitric oxide as biomarker in the treatment of inflammatory bowel disease: responders versus nonresponders. World J Gastroenterol 12: 3386–3392, 2006.[ISI][Medline]
  32. Matheson PJ, Wilson MA, Garrison RN. Regulation of intestinal blood flow. J Surg Res 93: 182–196, 2000.[CrossRef][ISI][Medline]
  33. McCafferty DM, Miampamba M, Sihota E, Sharkey KA, Kubes P. Role of inducible nitric oxide synthase in trinitrobenzene sulphonic acid induced colitis in mice. Gut 45: 864–873, 1999.[Abstract/Free Full Text]
  34. McLaren WJ, Anikijenko P, Thomas SG, Delaney PM, King RG. In vivo detection of morphological and microvascular changes of the colon in association with colitis using fiberoptic confocal imaging (FOCI). Dig Dis Sci 47: 2424–2433, 2002.[CrossRef][ISI][Medline]
  35. Miller MJ, Thompson JH, Zhang XJ, Sadowska-Krowicka H, Kakkis JL, Munshi UK, Sandoval M, Rossi JL, Eloby-Childress S, Beckman JS, Ye YZ, Rodi CP, Manning PT, Currie MG, Clark DA. Role of inducible nitric oxide synthase expression and peroxynitrite formation in guinea pig ileitis. Gastroenterology 109: 1475–1483, 1995.[CrossRef][ISI][Medline]
  36. Mizuta Y, Isomoto H, Takahashi T. Impaired nitrergic innervation in rat colitis induced by dextran sulfate sodium. Gastroenterology 118: 714–723, 2000.[CrossRef][ISI][Medline]
  37. Moncada S, Higgs A, Furchgott R. International Union of Pharmacology Nomenclature in Nitric Oxide Research. Pharmacol Rev 49: 137–142, 1997.[Abstract/Free Full Text]
  38. Mori M, Stokes KY, Vowinkel T, Watanabe N, Elrod JW, Harris NR, Lefer DJ, Hibi T, Granger DN. Colonic blood flow responses in experimental colitis: time course and underlying mechanisms. Am J Physiol Gastrointest Liver Physiol 289: G1024–G1029, 2005.[Abstract/Free Full Text]
  39. Morris GP, Beck PL, Herridge MS, Depew WT, Szewczuk MR, Wallace JL. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96: 795–803, 1989.[ISI][Medline]
  40. Murthy SN, Cooper HS, Shim H, Shah RS, Ibrahim SA, Sedergran DJ. Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin. Dig Dis Sci 38: 1722–1734, 1993.[CrossRef][ISI][Medline]
  41. Neurath M, Fuss I, Strober W. TNBS-colitis. Int Rev Immunol 19: 51–62, 2000.[Medline]
  42. Nilsson GE, Tenland T, Oberg PA. Evaluation of a laser Doppler flowmeter for measurement of tissue blood flow. IEEE Trans Biomed Eng 27: 597–604, 1980.[ISI][Medline]
  43. Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 98: 694–702, 1990.[ISI][Medline]
  44. Olesen M, Middelveld R, Bohr J, Tysk C, Lundberg JO, Eriksson S, Alving K, Jarnerot G. Luminal nitric oxide and epithelial expression of inducible and endothelial nitric oxide synthase in collagenous and lymphocytic colitis. Scand J Gastroenterol 38: 66–72, 2003.[CrossRef][ISI][Medline]
  45. Palatka K, Serfozo Z, Vereb Z, Hargitay Z, Lontay B, Erdodi F, Banfalvi G, Nemes Z, Udvardy M, Altorjay I. Changes in the expression and distribution of the inducible and endothelial nitric oxide synthase in mucosal biopsy specimens of inflammatory bowel disease. Scand J Gastroenterol 40: 670–680, 2005.[ISI][Medline]
  46. Phillipson M, Henriksnas J, Holstad M, Sandler S, Holm L. Inducible nitric oxide synthase is involved in acid-induced gastric hyperemia in rats and mice. Am J Physiol Gastrointest Liver Physiol 285: G154–G162, 2003.[Abstract/Free Full Text]
  47. Rachmilewitz D, Stamler JS, Bachwich D, Karmeli F, Ackerman Z, Podolsky DK. Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease. Gut 36: 718–723, 1995.[Abstract/Free Full Text]
  48. Ravnic DJ, Konerding MA, Tsuda A, Huss HT, Wolloscheck T, Pratt JP, Mentzer SJ. Structural adaptations in the murine colon microcirculation associated with hapten-induced inflammation. Gut 56: 518–523, 2007.[Abstract/Free Full Text]
  49. Reinders CI, Hellstrom PM, Bjork J, Weitzberg E, Lundberg JO. Effect of intravenous L-NMMA on nitric oxide production in collagenous colitis. Scand J Gastroenterol 39: 32–36, 2004.[ISI][Medline]
  50. Reinders CI, Herulf M, Ljung T, Hollenberg J, Weitzberg E, Lundberg JO, Hellstrom PM. Rectal mucosal nitric oxide in differentiation of inflammatory bowel disease and irritable bowel syndrome. Clin Gastroenterol Hepatol 3: 777–783, 2005.[CrossRef][ISI][Medline]
  51. Sasaki M, Bharwani S, Jordan P, Elrod JW, Grisham MB, Jackson TH, Lefer DJ, Alexander JS. Increased disease activity in eNOS-deficient mice in experimental colitis. Free Radic Biol Med 35: 1679–1687, 2003.[CrossRef][ISI][Medline]
  52. Satoyoshi K, Akita Y, Nozu F, Yoshikawa N, Mitamura K. Hemodynamics in the colonic mucosa of rats with dextran sulfate-induced colitis in the early phase. J Gastroenterol 31: 512–517, 1996.[CrossRef][ISI][Medline]
  53. Skinner SA, O'Brien PE. The microvascular structure of the normal colon in rats and humans. J Surg Res 61: 482–490, 1996.[CrossRef][ISI][Medline]
  54. Takahashi T. Pathophysiological significance of neuronal nitric oxide synthase in the gastrointestinal tract. J Gastroenterol 38: 421–430, 2003.[CrossRef][ISI][Medline]
  55. Wallace JL, Miller MJ. Nitric oxide in mucosal defense: a little goes a long way. Gastroenterology 119: 512–520, 2000.[CrossRef][ISI][Medline]
  56. Vallance BA, Dijkstra G, Qiu B, van der Waaij LA, van Goor H, Jansen PL, Mashimo H, Collins SM. Relative contributions of NOS isoforms during experimental colitis: endothelial-derived NOS maintains mucosal integrity. Am J Physiol Gastrointest Liver Physiol 287: G865–G874, 2004.[Abstract/Free Full Text]
  57. Vento P, Kiviluoto T, Jarvinen HJ, Soinila S. Changes in distribution of three isoforms of nitric oxide synthase in ulcerative colitis. Scand J Gastroenterol 36: 180–189, 2001.[ISI][Medline]
  58. Zhang XJ, Thompson JH, Mannick EE, Correa P, Miller MJ. Localization of inducible nitric oxide synthase mRNA in inflamed gastrointestinal mucosa by in situ reverse transcriptase-polymerase chain reaction. Nitric Oxide 2: 187–192, 1998.[CrossRef][ISI][Medline]
  59. Zingarelli B, Szabo C, Salzman AL. Reduced oxidative and nitrosative damage in murine experimental colitis in the absence of inducible nitric oxide synthase. Gut 45: 199–209, 1999.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
W.-H. Kan, J.-T. Hsu, M. G. Schwacha, M. A. Choudhry, R. Raju, K. I. Bland, and I. H. Chaudry
Selective inhibition of iNOS attenuates trauma-hemorrhage/resuscitation-induced hepatic injury
J Appl Physiol, October 1, 2008; 105(4): 1076 - 1082.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/G1281    most recent
00357.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Petersson, J.
Right arrow Articles by Holm, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Petersson, J.
Right arrow Articles by Holm, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.