Dextran sodium sulfate-induced colitis reveals nicotinic modulation of ion transport via iNOS-derived NO

Christina L. Green,1 Winnie Ho,2 Keith A. Sharkey,2 and Derek M. McKay1

1Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario L8N 3Z5; and 2Gastrointestinal Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1

Submitted 15 February 2004 ; accepted in final form 8 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In normal colon, ACh elicits a luminally directed Cl efflux from enterocytes via activation of muscarinic receptors. In contrast, in the murine model of dextran sodium sulfate (DSS)-induced colitis, an inhibitory cholinergic ion transport event due to nicotinic receptor activation has been identified. The absence of nicotinic receptors on enteric epithelia and the ability of nitric oxide (NO) to modulate ion transport led us to hypothesize that NO mediated the cholinergic nicotinic receptor-induced changes in ion transport. Midportions of colon from control and DSS-treated mice were examined for inducible NO synthase (iNOS) expression by RT-PCR and immunofluorescence or mounted in Ussing chambers for assessment of cholinergic-evoked changes in ion transport (i.e., short-circuit current) with or without pretreatment with pharmacological inhibitors of NO production. iNOS mRNA and protein levels were increased throughout the tissue from DSS-treated mice and, notably, in the myenteric plexus, where the majority of iNOS immunoreactivity colocalized with the enteric glial cell marker glial fibrillary acidic protein. The drop in short-circuit current evoked by the cholinomimetic carbachol in tissue from DSS-treated mice was prevented by selective inhibitors of iNOS activity {N6-(1-iminoethyl)-lysine HCl and N-[3-(aminomethyl)benzyl]acetamidine} or an NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or by removal of the myenteric plexus. Thus, in this model of colitis, a "switch" occurs from muscarinic to nicotinic receptor-dominated control of cholinergic ion transport. The data indicate a novel pathway involving activation of nicotinic receptors on myenteric neurons, resulting in release of NO from neurons or enteric glia and, ultimately, a dampening of stimulated epithelial Cl secretion that would reduce secretory diarrhea.

short-circuit current; chloride; inflammatory bowel disease


WATER MOVEMENT between the gut lumen and the interstitium is critically important for hydrating the surface of the enteric epithelium, providing the medium for contact digestion and nutrient absorption, and reducing the contact of pathogens and potentially noxious bacterial or environmental toxins with the enterocytes. This key function of the epithelium is accomplished by the coordinated activity of ion channels, cotransporters, and energy-dependent ion pumps that are asymmetrically arranged in the cell membrane of the polarized epithelium: this allows for vectorial ion transport, creating the driving forces for directed water movements (2). Many intrinsic and extrinsic agents influence epithelial ion transport, including neurotransmitters (e.g., ACh), biogenic amines, nutrients, toxins, and medications (2). The importance of this primary role of the epithelium is aptly illustrated by the fact that ion transport and, thus, water balance are characteristically perturbed in inflammatory enteropathies and can manifest as a severe dehydrating diarrhea.

Cholinergic nerves are a principal regulator of enteric epithelial ion transport, which can be conveniently gauged in tissue that has been mounted in an Ussing chamber by recording changes in short-circuit current (Isc) (38). Recently, we showed that colonic tissues from mice with dextran sodium sulfate (DSS)-induced colitis had not only diminished ion transport responses to the prosecretory cholinomimetic carbachol (CCh) but had, in fact, a net current response opposite to that observed in segments of normal murine colon (38). Pharmacological analyses have indicated that the Isc response to CCh in normal tissue is predominantly due to activation of muscarinic (M3) cholinergic receptors on the epithelium to produce a luminally directed Cl efflux (8, 38). In colonic tissue from mice with DSS-induced colitis, however, the effect of CCh was sensitive to nerve blockade with tetrodotoxin, occurred via a nicotinic cholinergic receptor that was sensitive to hexamethonium, and was dependent on the movement of Cl and HCO3 (38).

The DSS model of colitis shares some similarities with human ulcerative colitis (27), and in this context, it is intriguing that nicotine is of therapeutic benefit to a cohort of patients with ulcerative colitis (15, 35). Indeed, a preliminary study suggests that DSS-induced colitis is more severe in mice deficient in nicotinic receptors (28). Although part of the beneficial effect of nicotine is presumably due to its immunosuppressive properties (41, 47), cholinergic events that are tilted in favor of absorption, rather than secretion, would be expected to alleviate some of the diarrhea experienced by patients with ulcerative colitis. The present study was designed to further define the mechanism of altered ion transport in the colon of mice with DSS-induced colitis and has revealed a novel pathway involving nicotinic-nitrigeric communication and, possibly, participation by enteric glial cells in the cholinergic control of ion transport.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Induction of colitis. Male Balb/c mice [8–12 wk old; Harlan Animal Suppliers (Indianapolis, IN) or Charles River Laboratories] were given a 4% (wt/vol) DSS (40 kDa; ICN Biomedicals, Aurora, OH) drinking water solution for 5 days followed by 3 days of normal drinking water. On day 3 after DSS removal (i.e., 8 days after the start of DSS treatment), mice were killed and segments of colon were excised. Colitis was assessed by three parameters: clinical score (weight loss, 0–2; colon length, 0–2; diarrhea, 0–2; and evidence of bleeding, 0 or 1: maximum score = 7), a histological damage score based on hematoxylin-and-eosin-stained sections of midcolon (loss of tissue architecture, 0–3; cellular infiltrate, 0–3; goblet cell depletion, 0–1; presence of ulcers, 0–1; edema, 0–1; muscle thickening, 0–2; crypt abscess, 0–1: maximum score = 12), and myeloperoxidase activity in the terminal 30% of the colon (7). All procedures were approved by the McMaster University and University of Calgary Animal Care Committees and were performed according to the guidelines of the Canadian Council of Animal Care.

Assessment of ion transport. Two nonmacroscopically ulcerated whole-thickness segments of middistal colon per mouse were opened along the mesenteric border and mounted in Ussing chambers (0.6-cm2 opening) (24). [It is likely that tissue from DSS-treated mice would show microscopic evidence of inflammation (image in Fig. 5, bottom left, reveals a small ulcer).] Briefly, tissues were bathed in oxygenated Krebs buffer containing 10 mM glucose (serosal side) or 10 mM mannitol (luminal side) at 37°C, and net active ion transport across the epithelium was measured via an Isc (expressed in µA) injected through the tissue under voltage-clamp conditions. After a 15-min equilibration period, baseline Isc and potential difference (in mV) were recorded and ion conductance was calculated (in mS/cm2). The maximum change in Isc within 10 min of the addition of various pharmacological agents to the serosal buffer was also recorded. Each tissue was challenged with 100 µM CCh (Sigma, St. Louis, MO) with or without a 10- to 15-min pretreatment with the inhibitors of inducible nitric oxide (NO) synthase (iNOS), N6-(1-iminoethyl)-lysine HCl (L-NIL, 3 µM; Sigma) or N-(3-(aminomethyl)benzyl)acetamidine (1400w, 5 µM; AG Scientific, San Diego, CA), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 100 µM; Calbiochem, San Diego, CA), or the muscarinic antagonist atropine (ATR, 1 µM; Sigma). At the end of each experiment, all tissues were challenged with the cAMP-dependent secretagogue forskolin (10 µM; Sigma) to test for viability and to ensure that the tissue had been mounted in the correct orientation in the Ussing chamber.



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Fig. 5. Myenteric plexus is essential for nicotinic cholinergic Isc response elicited by carbachol (CCh). Colonic tissues from mice treated with 4% DSS for 5 days and normal drinking water for 3 days, from which external muscle layers and attendant myenteric plexus have been removed, do not display a downward deflection of Isc in response to 100 µM CCh. Values are means ± SE; n = 5. *P < 0.05 vs. full-thickness intact tissue. Insets: hematoxylin-and-eosin-stained sections of full-thickness (intact) and muscle-stripped segments of midcolon from DSS-treated mice.

 
In some experiments, tissues (colon or cecum) were challenged with the selective muscarinic agonist bethanechol (BCh, 100 µM; Sigma) with or without ATR. For analysis of putative involvement of the myenteric plexus in mediating the Isc events, the outer muscle layers, including the myenteric plexus of one colonic segment from each animal, were removed by blunt dissection. Removal of the muscle layers was confirmed by histological assessment. These studies were performed on tissue from DSS-treated animals only. [Trials with control tissue were unsuccessful, such that attempts to remove the muscle resulted in significant tissue damage and conductance values consistently >100 mS/cm2, and this may also indicate the presence of edema in the more readily stripped tissue from DSS-treated mice.]

RT-PCR for iNOS. RNA was extracted from colonic tissue using the TRIzol extraction method (Invitrogen, Burlington, ON, Canada), and cDNA was reverse transcribed from 2 µg of total RNA. cDNA was incubated in a reaction mixture that included platinum Taq polymerase (Invitrogen), 2 mM MgCl2, and nucleotide primers as follows: 5'-AGA CCT CAA CAG AGC CCT CA-3' (forward primer) and 5'-GCA GCC TCT TGT CTT TGA CC-3' (reverse primer) for iNOS gene product (305 bp, 0.3 µM final concentration) and 5'-CCA GAG CAA GAG AGG TAT CC-3' (forward primer) and 5'-CTG TGG TGG TGA AGC TGT AG-3' (reverse primer) for {beta}-actin gene product (436 bp, 0.06 µM final concentration). cDNA was amplified for 35 cycles on a Techne PHC-3 thermal cycler (Mandel Scientific, Guelph, ON, Canada), with denaturing, annealing, and extending temperatures of 94°C, 57°C, and 72°C, respectively. The final PCR product was run on a 2% agarose gel, and the amplified bands were viewed via DNA binding to ethidium bromide under ultraviolet light. In addition to colonic iNOS mRNA in control and DSS-treated mice (i.e., 5 days of DSS + 3 days of water), tissue was excised and processed from animals exposed to 4% DSS for 3 and 5 days, without any switch to normal water (n = 3–4). Separate RT-PCR experiments were performed using 1 µg of RNA isolated from muscle-myenteric plexus preparations that had been stripped via blunt dissection from the mucosa/submucosa of colonic segments immediately after death (n = 3).

Immunohistochemistry. Cryostat sections (10 µm) of whole-thickness tissue and whole-mount preparations were made of the longitudinal muscle-myenteric plexus and the submucosal plexus as previously described (22, 24). Tissues were fixed in Zamboni's fixative overnight and then washed in PBS (3 times for 10 min each). Whole-mount preparations of colonic submucosal and myenteric plexus were incubated with primary antibodies for 48 h at 4°C, washed in PBS (3 times for 10 min each), and incubated with the appropriate secondary antibody at room temperature for 1 h. Primary antibodies were rabbit anti-choline acetyltransferase (ChAT, 1:500; code P3YEB, a generous gift from Dr. M. Schemann, Technical University of Munich, to K. A. Sharkey), rabbit anti-iNOS (1:500; catalog no. N-32030, Transduction Laboratories, Lexington, KY), mouse antineuronal NOS (bNOS, 1:500; catalog no. N-2280, Sigma), and rabbit anti-glial fibrillary acidic protein (GFAP, 1:250; catalog no. BT575, Biomedical Technologies, Stoughton, MA). Secondary antibodies were donkey anti-rabbit Cy3 (1:100; catalog no. 711-165-152, Biocan Scientific, Mississauga, ON, Canada) and rabbit anti-mouse FITC (1:50; catalog no. 315-095-045, Biocan Scientific). For the double-labeling studies, primary antibodies were added sequentially with the appropriate secondary antibodies. Tissues were washed in PBS (3 times for 10 min each) and mounted in bicarbonate-buffered glycerol (pH 8.6). Sections were examined using a Zeiss Axioplan fluorescence microscope and photographed with a Sensys digital camera (Photometrics, Tucson, AZ) using V for Windows software (version 3.5, Digital Optics, Auckland, New Zealand). Montages were created in CorelDraw 11.

Data presentation and statistical analysis. Values are means ± SE, and n represents the number of mice used. Data were compared using Student's t-test (unpaired or paired) or, for multiple group comparisons, one-way ANOVA followed by post hoc statistics with the Newman-Keuls test as appropriate. A level of statistically significant difference was accepted at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
DSS-induced colitis causes loss of muscarinic cholinergic regulation of epithelial ion transport and increased expression of ChAT. Consistent with previous reports, mice given 4% DSS-water for 5 days developed an acute colitis (7). Table 1 shows the extent of the inflammatory disease when mice were killed 3 days after DSS-water was replaced with regular water. Complementing our earlier study, Fig. 1 shows that the perturbed Isc response to CCh in tissues from mice with DSS-induced colitis lasts for ≥12 days after withdrawal of the DSS but returns to normal by 3 wk after treatment. Baseline Isc and conductance values were not significantly different between controls and DSS-treated mice (data not shown) (38). Moreover, tissues from DSS-treated mice were virtually unresponsive to stimulation with the selective muscarinic cholinergic agonist BCh at 10–4 M ({Delta}Isc = –3.3 ± 1.8 µA/cm2, n = 9), in contrast to colonic segments from control mice, which displayed a 88.4 ± 17.8 µA/cm2 (n = 3) increase in Isc that was blocked by pretreatment with the muscarinic antagonist ATR: {Delta}Isc = 5.9 ± 5.9 µA/cm2 (n = 3). Moreover, although cecums of mice exposed to DSS become inflamed, as indicated by elevated myeloperoxidase activity [0.03 ± 0.02 and 0.81 ± 0.39 U/mg tissue in control and DSS mice, respectively, n = 3, consistent with our earlier study (38)], this tissue was still responsive to BCh: {Delta}Isc = 30.1 ± 3.9 (n = 3) compared with 27.4 ± 5.9 (n = 7) µA/cm2 in control cecum. These data, combined with those from our previous report (38), indicate a defect in muscarinic receptor-mediated control of ion transport in DSS-treated mice that is restricted to the colon and not evident in inflamed cecum.


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Table 1. Assessment of colitis after exposure to 4% DSS water for 5 days followed by 3 days of normal drinking water

 


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Fig. 1. Change in short-circuit current (Isc) in colonic segments from control mice or in tissues excised at various times after 5 days of treatment with 4% (wt/vol) dextran sodium sulfate (DSS). Values are means ± SE; n = 12 for control and 3–4 for other groups. *P < 0.05 vs. control (by ANOVA).

 
Immunolocalization studies on cryostat and whole-mount preparations revealed abundant ChAT immunoreactivity in nerve fibers and cell bodies in colonic segments from control (36) and DSS-treated mice. However, interestingly and seemingly paradoxically, our studies indicated an increase in the intensity of ChAT expression and the number of immunoreactive cell bodies in the myenteric plexuses of colonic segments from DSS-treated mice compared with controls (Fig. 2).



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Fig. 2. Fluorescent micrographs illustrating increase in choline acetyltransferase (ChAT) immunoreactivity in myenteric plexus of DSS-treated mice (4% DSS for 5 days + 3 days normal drinking water (B) compared with control mice (A). Arrows, immunopositive cell bodies. Tissues from 4 mice in each group were examined. Scale bar, 50 µm.

 
DSS colitis is accompanied by an increase in expression of iNOS in enteric glia. RT-PCR of whole tissue extracts revealed a time-dependent increase in iNOS mRNA. Tissues obtained from mice after 5 days of exposure to DSS and those from mice 3 days after withdrawal of DSS-water showed a significant increase in iNOS mRNA expression compared with control mice and mice that received DSS for 3 days (Fig. 3A). Increased iNOS mRNA expression was also observed in muscle layer-myenteric plexus preparations from mice exposed to DSS-water compared with those from control mice (Fig. 3C).



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Fig. 3. A: representative gel electrophoresis images after RT-PCR for inducible nitric oxide synthase (iNOS) and the housekeeping gene {beta}-actin in colonic segments from control and DSS-treated mice [4% for 3 (3d DSS) or 5 days (5d DSS) and 4% DSS for 5 days and normal drinking water for 3 days (5d DSS + 3d H2O)]. *, Negative control reaction conducted with omission of cDNA. B: densitometry analysis of iNOS RT-PCR product normalized against {beta}-actin in mice treated with DSS for 5 days and normal drinking water for 3 days compared with controls. Values are means ± SE; n = 3–4. *P < 0.05. C: representative gel electrophoresis images after RT-PCR for iNOS and {beta}-actin in outer muscle layer-myenteric plexus preparations of colonic segments from control and DSS-treated mice.

 
We used immunohistochemistry to assess the localization of iNOS protein in the wall of the colon in animals with DSS-induced colitis. Immunohistochemical studies revealed, as expected and as reported in other colitis models (17, 49), increased iNOS protein expression in patches of epithelium and submucosal/mucosal cells (presumably resident and infiltrated immune cells) in tissue from the DSS-treated mice (data not shown). Faint iNOS immunoreactivity was also observed in the myenteric plexus and scattered submucosal nerve fibers in control tissue (46) (data not shown), consistent with the expression of the PCR product (Fig. 3). iNOS positivity in submucosal plexuses in colon from DSS-treated mice (Fig. 4) was similar to that in control tissue; however, there was a striking and substantial increase in iNOS expression in the myenteric plexus in animals treated with DSS (Fig. 4). Subsequent colocalization studies confirmed that the increased iNOS expression observed in the myenteric plexus was not found in neurons expressing constitutive bNOS, the expression of which was not altered in colitis, nor did it obviously overlap with expression of the generalized neuronal marker PGP 9.5 (data not shown). Use of the specific glial cell marker GFAP revealed that the increased iNOS expression in the myenteric plexus of colons from DSS-treated mice was predominantly in the enteric glial cells (Fig. 4). Levels of iNOS expression in control tissue, although detectable, were too low for unequivocal characterization of neuronal or glial colocalization.



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Fig. 4. Fluorescence micrographs of iNOS and glial fibrillatory acidic protein (GFAP) immunoreactivity in myenteric plexus (A and B) and submucosal plexus (C) and iNOS and antineuronal NOS (bNOS) immunoreactivity in myenteric plexus (D and E). A and D are from controls, and B, C, and E are from mice treated with 4% DSS for 5 days and normal drinking water for 3 days. In control mice, there was low basal level of iNOS immunoreactivity in myenteric plexus (A and D) and submucosal plexus (not shown). GFAP immunoreactivity was distributed in enteric glia. In colon from DSS-treated mice, iNOS expression was increased in myenteric plexus and to a far lesser extent in submucosal plexus. Increased iNOS in myenteric plexus appeared to be mostly glial in origin, colocalizing with GFAP (B). bNOS immunoreactivity was found in enteric neurons and nerve fibers as previously reported (17). bNOS-immunoreactive neurons appeared unchanged by inflammation, although there was a reduction in immunoreactive fiber labeling. There was no obvious overlap between iNOS and bNOS in control or inflamed animals. Scale bar, 50 µm.

 
CCh modulation of ion transport via nicotinic receptors in DSS tissue requires the myenteric plexus and involves NO. Earlier studies showed that the CCh-induced reduction in Isc in tissues from mice with DSS-induced colitis was tetrodotoxin and hexamethonium sensitive, indicating mediation via neuronal nicotinic receptors (38). CCh applied to the serosal side of tissue from mice with DSS-induced colitis in which the external muscle layers containing the myenteric plexus had been stripped away by blunt dissection did not elicit this characteristic drop in Isc (Fig. 5).

Pharmacological studies with whole-thickness colonic segments (i.e., with an intact myenteric plexus) showed that the CCh-induced drop in Isc evoked via nicotinic cholinergic receptors in the myenteric plexus was also dependent on iNOS-derived NO, because pretreatment with either inhibitor of iNOS activity, L-NIL or 1400w, blocked this ion transport event (Fig. 6A). Previously, we showed that the Isc response to CCh in tissue from mice with DSS-induced colitis could be reproduced in normal tissue from control mice by blocking muscarinic receptors with ATR before CCh challenge (38). Here we show that the CCh-induced drop in colonic Isc from control mice where muscarinic receptors had first been blocked by the addition of ATR (i.e., ATR + CCh) was inhibited by pretreatment with L-NIL or 1400w (Fig. 6B). Finally, serosal addition of the NO scavenger cPTIO to Ussing chamber-mounted colonic tissue from mice with DSS-induced colitis 10 min before challenge with CCh significantly inhibited the Isc response (Fig. 6C).



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Fig. 6. iNOS-derived NO mediates Isc response to CCh in DSS-treated tissue. A: pretreatment of DSS colonic tissue with 3 µM N6-(1-iminoethyl)-lysine HCl (L-NIL) or 5 µM N-(3-(aminomethyl)benzyl)acetamidine (1400w) prevents decrease in Isc evoked by 100 µM CCh (n = 6). B: tissue from control mice pretreated with 1 µM atropine (ATR) to block cholinergic muscarinic receptors does not respond to CCh with a decrease in Isc when pretreated with L-NIL or 1400w (n = 5). Insets: sample current traces in which 1 colonic segment received 1400w and CCh, and the other segment received only CCh. C: pharmacological scavenging of NO with 100 µM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) also prevents decrease in Isc after CCh application to tissue from mice treated with 4% DSS for 5 days and normal drinking water for 3 days (n = 9). Values are means ± SE; *P < 0.05 vs. CCh or ATR + CCh.

 
In all experiments, tissues were challenged with forskolin, which consistently resulted in a sustained increase in Isc as a result of luminally directed active Cl secretion: the magnitude of this event was significantly reduced in tissues from mice with DSS-induced colitis: 238 ± 19 (n = 16) vs. 26 ± 6 µA/cm2 (n = 6). In agreement with results from other investigators (1), pretreatment with the iNOS inhibitor 1400w partially prevented the reduction in magnitude of the {Delta}Isc induced by forskolin in tissue from mice with DSS-induced colitis: 86 ± 28 µA/cm2 (n = 7, P < 0.05 compared with control and DSS tissue without 1400w).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Of the many factors that influence ion transport, the neurotransmitter ACh is of particular importance. In normal mammalian colon, the ion transport response to ACh or to synthetic derivatives (e.g., CCh) is dominated by activation of epithelial M3 cholinergic receptors, the ligation of which causes a Cl efflux from the cell (8, 38). We previously showed that cholinergic control of ion transport is perturbed in tissue from mice with DSS-induced colitis (38), complementing observations from other studies (21, 34). However, the DSS model is intriguing, because CCh produced a sustained reduction in Isc, rather than a diminished transient increase in Isc observed in other models, such as those evoked by 2,4,6-trinitrobenzenesulfonic acid, 2,4-dinitrobenzenesulfonic acid (21, 34), or oxazolone (unpublished observation). Underlying differences in the models may contribute to this discrepancy; for example, 2,4,6-trinitrobenzenesulfonic acid and 2,4-dinitrobenzenesulfonic acid elicit a polarized Th1-type inflammatory response and oxazolone produces a Th2-type response, whereas DSS-induced colitis involves a Th1- and a Th2-type inflammatory profile (reviewed in Ref. 3). Further analyses attributed this altered Isc response in colonic tissue from DSS-treated mice to the activation of neuronal nicotinic cholinergic receptors (38). Here we extend these observations by demonstrating a role for iNOS-derived NO in the modulation of cholinergic ion transport that is unmasked during DSS-induced colitis.

There is evidence of nonneuronal expression of nicotinic receptor mRNA or protein on immune cells and airway epithelial cells (37, 48). However, the response to CCh in DSS-treated animals was tetrodotoxin and hexamethonium sensitive (38), suggesting that the cholinergic receptor involved in this response was expressed on enteric neurons. This postulate is supported by the inability of nicotine to elicit any direct change in Isc in the T84 and HT-29 human colonic epithelial cell lines (unpublished observation). We reasoned that an intermediate molecule was mobilized to affect the epithelium subsequent to CCh challenge; previous pharmacological studies with this model ruled out the involvement of norepinephrine and opiates (38). NO was hypothesized as the intermediate factor, because 1) it can modulate ion transport, both directly and indirectly (18, 20, 24); 2) it acts as an intermediate in other cholinergic events (16, 42); and 3) it colocalizes with ChAT in the mouse colon (36).

Initial studies revealed, as expected, increased iNOS mRNA transcripts in colonic tissues from DSS-treated mice (38), a finding supported by the immunohistochemical expression of iNOS. Consistent with analyses of inflamed human colon and other rodent models of colitis (22, 40, 49), patches of epithelium and cells in the mucosa and submucosa displayed iNOS immunoreactivity in the colon of DSS-treated mice. A similar pattern of iNOS immunoreactivity was not found in control tissue but has been observed in other studies with the DSS model of colitis (17). Moreover, there was an obvious increase in iNOS expression, both mRNA and protein, in the myenteric plexuses of colons from DSS-treated mice, again, a finding that is not unprecedented (25, 44). Colocalization studies confirmed that the immunoreactivity detected in the myenteric plexus of tissue from DSS-treated and control mice was, in fact, iNOS, which was revealed by double labeling to be distinct from constitutively expressed bNOS. iNOS expression in control tissue may at first appear unusual, but it is consistent with the status of "physiological inflammation" that exists in the colon, which is constantly exposed to an immense microbial flora (9). Additional immunohistochemical studies revealed that the increased iNOS expression in the myenteric plexus of DSS-treated mice was predominantly found in enteric glial cells, a cell type that can be an active participant in neuroimmunophysiological events (4, 32). Enteric glia are of neural crest lineage and are similar to central nervous system (CNS) astrocytes (33). Astrocytes have been identified as a source of NO when stimulated with proinflammatory cytokines or in inflammatory pathologies of the CNS (26). The expression of iNOS in enteric glia in intestinal inflammation suggests that their response to inflammation is similar to that of astrocytes in the CNS. Astrocytes express nicotinic receptors in the CNS, and the expression profile of the subunits that compose the functional receptors differs between the astrocytes and the neurons (13). There is no evidence that enteric glia express cholinergic nicotinic receptors (50), and indeed our analyses indicated only neural expression of nicotinic receptors in the colon of control and DSS-treated mice (unpublished observation).

Use of two inhibitors of iNOS activity and a pharmacological scavenger of NO revealed that iNOS-derived NO was required for the drop in Isc after cholinergic stimulation of colonic tissue from DSS-treated mice. Furthermore, the drop in Isc evoked by CCh in control tissues after blockade of muscarinic receptors by ATR was also prevented by use of either iNOS inhibitor. However, neither iNOS nor NO inhibition restored a normal secretory response to CCh, suggesting that NO was not blocking epithelial muscarinic receptors. These data suggest that NO plays a physiological role in modulating nicotinic receptor-mediated cholinergic ion transport in the mouse colon and that this role becomes more prominent during inflammation associated with dysfunctional epithelial muscarinic cholinergic receptors. Such a possibility is supported by evidence showing that NO suppresses electrically evoked ion transport mediated by submucosal neurons in the guinea pig colon (31).

Colonic tissues from DSS-treated mice that had been stripped of the external muscle layers and attendant myenteric plexus did not display a CCh-induced drop in Isc. This clearly indicates that the nicotinic receptors that initiate the Isc response reside in the myenteric plexus and suggests that iNOS-positive neurons or glial cells are the source of the NO. Should enteric glia prove to be a significant source of the iNOS-derived NO, as our immunohistochemical data suggest, then the lack of demonstrable nicotinic receptors on the enteric glia indicate neuron-to-glial communication in the modulation of colonic ion transport, an unprecedented observation.

NO produced in the myenteric plexus is unlikely to reach and directly affect the epithelium and so must mobilize an intermediate mediator to influence epithelial ion transport. We can only speculate as to the nature of this mediator [e.g., neuropeptide Y (23), somatostatin (6), prostaglandin D2 (30)], but it is apparent that cholinergic control of colonic ion transport is more complex than simple interaction of ACh with epithelial M3 receptors. Perturbation of the cholinergic system is not uncommon in inflammatory conditions (12, 29, 39), and it is clear that the DSS-induced colitis has resulted in dysfunctional cholinergic muscarinic receptors on the epithelium. This could be the result of reduced receptor expression, altered receptor affinity, or failure of the intracellular signaling pathway. Evidence from other model systems can be advanced in support of each scenario (10, 19, 43). Although this study has focused on the cholinergic nicotinic-NO pathway of controlling epithelial ion transport, the failure of the muscarinic system is an issue that needs to be investigated. Our preliminary data indicate no obvious changes in the density of M3 receptor expression in colonic tissue from DSS-treated mice.

As noted, enteric inflammation can be associated with altered cholinergic responses, and in accordance with this study, others have reported increased ChAT levels in inflamed gut tissue (5). This presents the seemingly paradoxical situation in which tissues from mice with DSS-induced colitis have the ability to make increased amounts of ACh while simultaneously having dysfunctional muscarinic receptors. However, increased availability of ACh to bind to nicotinic receptors may represent a braking strategy to counter the prosecretory effects of the cholinergic muscarinic system that would contribute to diarrhea. Should the findings in the DSS model system extrapolate to human colonic inflammation, then nicotinic receptor-mediated NO regulation of epithelial ion transport represents another facet of the putative therapeutic action of nicotine, along with nicotine-NO-mediated muscle relaxation (14), direct immunosuppression (45), and stimulation of mucin synthesis (11).

Collectively, the data reveal in the colon, but not the cecum, a shift in the balance of cholinergic control from predominantly muscarinic to nicotinic receptor-driven ion transport events during DSS-induced colitis and likely involves enteric glia. The results fit a model where cholinergic stimulation activates nicotinic receptors expressed on myenteric plexus neurons, resulting in NO production from iNOS in enteric glia or neurons. The NO then activates or inhibits other neurons or stromal cells, which signal the epithelium to alter its ion transport properties in a manner that counters a hypersecretory state (Fig. 7). The ability to recreate the ion transport events observed in tissue from DSS-treated mice in control tissue using pharmacological agonists and antagonists suggests that this pathway is present in normal tissue but has not been detected in studies that report net changes in Isc induced by cholinomimetics, where M3 events will dominate. Thus the unique attributes of the DSS model of colitis have revealed a novel pathway of cholinergic regulation of epithelial electrolyte transport that may help define targets for therapeutic interventions to relieve the inflammation and symptoms associated with human inflammatory bowel diseases.



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Fig. 7. Model illustrating pathways and cellular mediators of responses to CCh observed in DSS-induced colitis. CCh activates nicotinic receptor (NicR) on myenteric neurons, which liberate a mediator that initiates NO release from surrounding enteroglia or neurons that express iNOS. NO can then act on stromal cells (I), directly at the enterocyte (II), or at the level of the submucosal plexus (III) to inhibit secretion. –ve, Negative signal or inhibition.

 

    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was funded by operating grants from the Crohn's and Colitis Foundation of Canada (to D. M. McKay) and the Canadian Institutes of Health Research (to D. M. McKay and K. A. Sharkey). C. L. Green is a recipient of a Research and Development Scholarship from the Canadian Institutes of Health Research, and K. A. Sharkey is a recipient of a Medical Scientist award from the Alberta Heritage Foundation for Medical Research.


    ACKNOWLEDGMENTS
 
Technical assistance from Jun Lu (McMaster University) is greatly appreciated.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. M. McKay, IDRP, HSC-3N5E, McMaster Univ., 1200 Main St. West, Hamilton, ON, Canada L8N 3Z5 (E-mail: mckayd{at}mcmaster.ca)

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Asfaha S, Bell CJ, Wallace JL, and MacNaughton WK. Prolonged colonic epithelial hyporesponsiveness after colitis: role of inducible nitric oxide synthase. Am J Physiol Gastrointest Liver Physiol 276: G703–G710, 1999.[Abstract/Free Full Text]
  2. Barrett KE and Keeley SJ. Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects. Annu Rev Physiol 62: 535–572, 2000.[CrossRef][ISI][Medline]
  3. Bouma G and Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 3: 521–533, 2003.[CrossRef][ISI][Medline]
  4. Cornet A, Savidge TC, Cabarrocas J, Deng WL, Colombel JF, Lassmann H, Desreumaux P, and Liblau RS. Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn's disease? Proc Natl Acad Sci USA 98: 13306–13311, 2001.[Abstract/Free Full Text]
  5. Davis KA, Masella J, and Blennerhassett MG. Acetylcholine metabolism in the inflamed rat intestine. Exp Neurol 152: 251–258, 1998.[CrossRef][ISI][Medline]
  6. Dharmsathaphorn K, Racusen L, and Dobbins JW. Effect of somatostatin on ion transport in the rat colon. J Clin Invest 66: 813–820, 1980.[ISI][Medline]
  7. Diaz-Granados N, Howe K, Lu J, and McKay DM. Dextran sulfate sodium-induced colonic histopathology, but not altered epithelial ion transport, is reduced by inhibition of phosphodiesterase activity. Am J Pathol 156: 2169–2177, 2000.[Abstract/Free Full Text]
  8. Dickinson KEJ, Frizzell RA, and Chandra SM. Activation of T84 cell chloride channels by carbachol involves a phosphoinositide-coupled muscarinic M3 receptor. Eur J Pharmacol 225: 291–298, 1992.[CrossRef][Medline]
  9. Elson CO, Cong Y, Iqbal N, and Weaver CT. Immuno-bacterial homeostasis in the gut: new insights into an old enigma. Semin Immunol 13: 187–194, 2001.[CrossRef][ISI][Medline]
  10. Emala CW, Clancy J, and Hirshman CA. Glucocorticoid treatment decreases muscarinic receptor expression in canine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 272: L745–L751, 1997.[Abstract/Free Full Text]
  11. Finnie IA, Campbell BJ, Taylor BA, Milton JD, Sadek SK, Yu LG, and Rhodes JM. Stimulation of colonic mucin synthesis by corticosteroids and nicotine. Clin Sci (Lond) 91: 359–364, 1996.[Medline]
  12. Galeazzi F, Haapala EM, Van Rooijen N, Vallance BA, and Collins SM. Inflammation-induced impairment of enteric nerve function in nematode-infected mice is macrophage dependent. Am J Physiol Gastrointest Liver Physiol 278: G259–G265, 2000.[Abstract/Free Full Text]
  13. Graham AJ, Ray MA, Perry E, Jaros E, Perry RH, Volsen SG, Bose S, Evans N, Lindstrom J, and Court JA. Differential nicotinic acetylcholine receptor subunit expression in the human hippocampus. J Chem Neuroanat 25: 97–113, 2003.[CrossRef][ISI][Medline]
  14. Green JT, Richardson C, Marshall RW, Rhodes J, McKirdy HC, Thomas GAO, and Williams GT. Nitric oxide mediates a therapeutic effect of nicotine in ulcerative colitis. Aliment Pharmacol Ther 14: 1429–1434, 2000.[CrossRef][ISI][Medline]
  15. Guslandi M and Tittobello A. Outcome of ulcerative colitis after treatment with transdermal nicotine. Eur J Gastroenterol Hepatol 10: 513–515, 1998.[ISI][Medline]
  16. Hebeiss K and Kilbinger H. Cholinergic and GABAergic regulation of nitric oxide synthesis in the guinea pig ileum. Am J Physiol Gastrointest Liver Physiol 276: G862–G866, 1999.[Abstract/Free Full Text]
  17. Hokari R, Kato S, Matsuzaki K, Kuroki M, Iwai A, Kawaguchi A, Nagao S, Miyahara T, Itoh K, Sekizuka E, Nagata H, Ishii H, and Miura S. Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis. Free Radic Biol Med 31: 153–163, 2001.[CrossRef][ISI][Medline]
  18. Izzo AA, Mascolo N, and Capasso F. Nitric oxide as a modulator of intestinal water and electrolyte transport. Dig Dis Sci 43: 1605–1620, 1998.[CrossRef][ISI][Medline]
  19. Jacoby DB and Fryer AD. Interaction of viral infections with muscarinic receptors. Clin Exp Allergy 29: 59–64, 1999.[CrossRef][ISI][Medline]
  20. MacNaughton WK. Nitric oxide-donating compounds stimulate electrolyte transport in the guinea pig intestine in vitro. Life Sci 53: 585–593, 1993.[CrossRef][ISI][Medline]
  21. MacNaughton WK, Lowe SS, and Cushing K. Role of nitric oxide in inflammation-induced suppression of secretion in a mouse model of acute colitis. Am J Physiol Gastrointest Liver Physiol 275: G1353–G1360, 1998.[Abstract/Free Full Text]
  22. McCafferty DM, Miampamba M, Sihota E, Sharkey KA, and 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]
  23. McKay DM, Berin MC, Fondacaro JD, and Perdue MH. Effects of neuropeptide Y and substance P on antigen-induced ion secretion in rat jejunum. Am J Physiol Gastrointest Liver Physiol 271: G987–G992, 1996.[Abstract/Free Full Text]
  24. McKay DM, Lu J, Jedrzkiewicz S, Ho W, and Sharkey KA. Nitric oxide participates in the recovery of normal jejunal epithelial ion transport following exposure to the superantigen, Staphylococcus aureus enterotoxin B. J Immunol 163: 4519–4526, 1999.[Abstract/Free Full Text]
  25. Miampamba M and Sharkey KA. Temporal distribution of neuronal and inducible nitric oxide synthase and nitrotyrosine during colitis in rats. Neurogastroenterol Motil 11: 193–206, 1999.[CrossRef][ISI][Medline]
  26. Murphy S. Production of nitric oxide by glial cells: regulation and potential roles in the CNS. Glia 29: 1–13, 2000.[CrossRef][ISI][Medline]
  27. Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, and Nakaya R. A novel method in induction of reliable experimental acute and chronic colitis in mice. Gastroenterology 98: 694–702, 1990.[ISI][Medline]
  28. Orr-Urtreger A, Kedmi M, Karmeli F, Yaron Y, and Rachmilewitz E. The severity of experimental colitis in mice is dependent on the presence of the {alpha}5 neuronal nicotinic acetylcholine receptor (nAChR) subunit (Abstract). Am J Hum Genet 67: 184, 2000.
  29. Palmer JM and Koch TR. Altered neuropeptide content and cholinergic enzymatic activity in the inflamed guinea pig jejunum during parasitism. Neuropeptides 28: 287–297, 1995.[ISI][Medline]
  30. Rangachari PK, Betti PA, Prior ET, and Roberts LJ II. Effects of a selective DP receptor agonist (BW 245C) and antagonist (BW A868C) on the canine colonic epithelium: an argument for a different DP receptor? J Pharmacol Exp Ther 275: 611–617, 1995.[Abstract]
  31. Reddix RA, Liu X, Miller MJ, Niu X, and Powell A. Constitutive nitric oxide release modulates neurally-evoked chloride secretion in guinea pig colon. Auto Neurosci 86: 47–57, 2000.[CrossRef][ISI]
  32. Rühl A, Franzke S, Collins SM, and Stremmel W. Interleukin-6 expression and regulation in rat enteric glial cells. Am J Physiol Gastrointest Liver Physiol 280: G1163–G1171, 2001.[Abstract/Free Full Text]
  33. Rühl A and Sharkey KA. Enteric glia. Neurogastroenterol Motil 16 Suppl 1: 44–49, 2004.
  34. Sánchez de Medina F, Pérez R, Martínez-Augustin O, González R, Lorente MD, Gálvez J, and Zarzuelo A. Disturbances of colonic ion secretion in inflammation: role of the enteric nervous system and cAMP. Pflügers Arch 444: 378–388, 2002.[ISI][Medline]
  35. Sandborn WJ. Nicotine therapy for ulcerative colitis: a review of rationale, mechanisms, pharmacology, and clinical results. Am J Gastroenterol 94: 1161–1171, 1999.[CrossRef][ISI][Medline]
  36. Sang Q and Young HM. The identification and chemical coding of cholinergic neurones in the small and large intestine of the mouse. Anat Rec 251: 185–199, 1998.[CrossRef][ISI][Medline]
  37. Sato KZ, Fujii T, Watanabe Y, Yamada S, Ando T, Kazuko F, and Kawashima K. Diversity of mRNA expression for muscarinic acetylcholine receptor subtypes and neuronal nicotinic acetylcholine receptor subunits in human mononuclear leukocytes and leukemic cell lines. Neurosci Lett 266: 17–20, 1999.[CrossRef][ISI][Medline]
  38. Sayer B, Lu J, Green CL, Söderholm JD, Akhtar M, and McKay DM. Dextran sodium sulphate-induced colitis perturbs muscarinic cholinergic control of colonic epithelial ion transport. Br J Pharmacol 135: 1794–1800, 2002.[Abstract/Free Full Text]
  39. Shi XZ and Sarna SK. Inflammatory modulation of muscarinic receptor activation in canine ileal circular muscle cells. Gastroenterology 112: 864–874, 1997.[ISI][Medline]
  40. Singer II, Kawka DW, Scott S, Weidner JR, Mumford RA, Riehl TE, and Stenson WF. Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease. Gastroenterology 111: 871–885, 1996.[ISI][Medline]
  41. Sykes AP, Brampton C, Klee S, Chander CL, Whelan C, and Parsons ME. An investigation into the effect and mechanisms of action of nicotine in inflammatory bowel disease. Inflamm Res 49: 311–319, 2000.[CrossRef][ISI][Medline]
  42. Toda N, Toda M, Ayajiki K, and Okamura T. Cholinergic nerve function in monkey ciliary arteries innervated by nitroxidergic nerve. Am J Physiol Heart Circ Physiol 274: H1582–H1589, 1998.[Abstract/Free Full Text]
  43. Vajanaphanich M, Schultz C, Rudolf MT, Wasserman M, Enyedi P, Craxton A, Shears SB, Tsien RY, Barrett KE, and Traynor-Kaplan A. Long-term uncoupling of chloride secretion from intracellular calcium levels by Ins(3,4,5,6)P4. Nature 371: 711–714, 1994.[CrossRef][ISI][Medline]
  44. Valentine JF, Tannahill CL, Stevenot SA, Sallustio JE, Nick HS, and Eaker EY. Colitis and interleukin 1{beta} up-regulate inducible nitric oxide synthase and superoxide dismutase in rat myenteric neurons. Gastroenterology 111: 56–64, 1996.[ISI][Medline]
  45. Van Dijk JP, Madretsma GS, Keuskamp ZJ, and Zijlstra FJ. Nicotine inhibits cytokine synthesis by mouse colonic mucosa. Eur J Pharmacol 278: R11–R12, 1995.[CrossRef][ISI][Medline]
  46. Vannucchi MG, Corsani L, Bani D, and Faussone-Pellegrini M-S. Myenteric neurons and interstitial cells of Cajal of mouse colon express several nitric oxide synthase isoforms. Neurosci Lett 326: 191–195, 2002.[CrossRef][ISI][Medline]
  47. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Hua LJ, Wang H, Yang H, Ulloa L, Al-Abed Y, Czura CJ, and Tracey KJ. Nicotinic acetylcholine receptor {alpha}7 subunit is an essential regulator of inflammation. Nature 421: 384–388, 2003.[ISI][Medline]
  48. Wessler IK and Kirkpatrick CJ. The non-neuronal cholinergic system: an emerging drug target in the airways. Pulmon Pharmacol Ther 14: 423–434, 2001.[CrossRef][ISI][Medline]
  49. Yue G, Lai PS, Yin K, Sun FF, Nagele RG, Liu X, Linask KK, Wang C, Lin KT, and Wong PYK. Colon epithelial cell death in 2,4,6-trinitrobenzenesulfonic acid-induced colitis is associated with increased inducible nitric-oxide synthase expression and peroxynitrite production. J Pharmacol Exp Ther 297: 915–925, 2001.[Abstract/Free Full Text]
  50. Zhou X, Ren J, Brown E, Schneider D, Caraballo-Lopez Y, and Galligan JJ. Pharmacological properties of nicotinic acetylcholine receptors expressed by guinea pig small intestinal myenteric neurons. J Pharmacol Exp Ther 302: 889–897, 2002.[Abstract/Free Full Text]




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