Q J Med 2003; 96: 57-65
© 2003 Association of Physicians
Effect of smoking and transdermal nicotine on colonic nicotinic acetylcholine receptors in ulcerative colitis
From the Departments of 1 Gastroenterology and 2 Pathology, University Hospital of Wales, Cardiff, UK, 3 Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, USA, 4 Department of Biology and Biochemistry, University of Bath, Bath, and 5 Department of Human Morphology, Medical School, University of Southampton, Southampton, UK
Received 4 January 2002 and in revised form 4 November 2002
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Background: Ulcerative colitis (UC) is a disease largely of non-smokers, in which nicotine is of therapeutic value. The mode of action is unknown, but may involve nicotinic acetylcholine receptors (nAChRs) in the bowel wall.
Aim: To investigate the presence of nAChRs in rectal mucosa, and the effect of smoking and nicotine on their expression.
Design: Prospective case-control study.
Methods: In situ hybridization (ISH) and immunocytochemistry (ICC) were used to show 
3 nAChRs in colonic mucosa. Rectal mucosa was examined from controls (n=55) and patients with inactive UC (n=62), both smokers and non-smokers, by ICC, using two antibodies to show the density and distribution of receptors in the mucosa. Non-smokers with UC (n=43) were given transdermal nicotine or placebo patches for 6 months, and rectal biopsies, taken before and after treatment, were examined by ICC to show nAChRs.
Results: In normal colon, ISH and ICC showed 3 subunit in a wide variety of cells, including mucosal epithelium. In rectal biopsies, neither smoking nor nicotine influenced the expression of 3 immunoreactivity in epithelium, either in controls or UC. However, controls had a significantly greater density of immunodetectable mucosal epithelium 3 subunit, compared with UC patients.
Discussion: The presence of nAChRs in colonic epithelium may be pertinent to the beneficial effect of nicotine in UC, but since neither smoking nor nicotine treatment is associated with any change in the expression of epithelial 3 nAChRs, the effect may be due to functional changes in the receptor. The decreased number of 3 nAChRs in UC compared with controls may be related to an increased cell turnover in UC.
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Introduction |
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Ulcerative colitis (UC) was first identified 20 years ago as largely a disease of non-smokers,1 and numerous studies have since confirmed the association.2 Non-smoking is currently the most consistent epidemiological finding associated with UC;3 current smokers with UC appear to remain in remission,4 while intermittent smokers often find their symptoms actually improve with smoking.5 Nicotine has been examined as a possible therapeutic agent to account for the associations, and two randomized, placebo-controlled trials have shown significant clinical benefit from transdermal nicotine,6,7 with the remission rate doubled after 6 weeks treatment.. Various mechanisms have been considered to explain this benefit, including effects on the epithelial mucus, gut motility, humoral and cellular immunity.8–11 While the mechanisms responsible for this therapeutic effect remain elusive, we have recently demonstrated nicotinic acetylcholine receptors (nAChRs) in the mucosal epithelium, enteric ganglia, nerves and lymphoid tissue of normal small bowel.12 The presence of similar nAChRs in the colon would open another possible route to explore mechanisms which may mediate the effects of nicotine in UC.
The nicotinic acetylcholine receptor gene family consists of ten (1 to 10) four ß (ß1 to ß4) and one 
, 
and 
subunits.13,14 Functional nAChRs are composed of five subunits arranged around a central ion channel like the staves of a barrel.15,16 Although many combinations of subunits are possible, a few predominate. In the peripheral nervous system, the 3 subunit, in combination with one or more other types of subunit, is the major contributor to functional nAChRs.17 In human small bowel, nAChRs containing the 3 subunit were identified in the mucosal epithelium.12 While their functional significance is not known, both skin keratinocytes and bronchial epithelial cells also express this subunit, and when cultured in the presence of nicotine, are stimulated to proliferate and mature.18,19
Since the mucosa is predominantly affected in UC, it is tempting to speculate that the beneficial effect of nicotine may be mediated by its binding to epithelial nAChRs. While the effect may be a purely functional one, it may also be associated with a measurable change in their number and distribution. Chronic nicotine exposure upregulates numbers of 3 nAChRs in the bronchial epithelium of smokers compared to non-smokers,19 and in the human neuroblastoma cell line SH-SY5Y.20,21 Furthermore, in the central nervous system, the exposure of nAChRs to nicotine produces a variety of effects which depend on the nAChR subtype and the duration of exposure: acute exposure produces receptor activation, longer exposure leads to desensitization and prolonged exposure to permanent inactivation, as well as upregulation.22,23
The present study was done in three parts. The first used in situ hybridization (ISH) and immunocytochemistry (ICC), with tissue from normal distal large bowel, to confirm that 3 subunits are expressed with a similar distribution to that described in human small bowel.12 The second used ICC on rectal mucosal biopsies from controls and patients with inactive UC to investigate the effect of smoking status on the density and distribution of immunodetectable 3 nAChR subunit in the mucosal epithelium. In the third study, patients with inactive UC were treated with transdermal nicotine or placebo for 6 months to examine the effect on the density and distribution of the nAChR subunit in the mucosal epithelium.
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Part I: colonic tissue
Histologically normal human colon was obtained from the colonic resections of five males, median age 74, range 32–83 years. The tissue, which comprised full bowel wall thickness, had been taken from the resection margins of surgical specimens for carcinoma of the colon, and was formalin-fixed and embedded in paraffin. All procedures were elective, with cases of bowel obstruction or perforation excluded. ISH and ICC were performed on sections taken from these tissue blocks.
Part II: rectal biopsies
Biopsies of rectal mucosa, taken at the time of rigid sigmoidoscopy, were examined in the following subjects.
- Normal rectal biopsies from controls were obtained from 20 smokers (8 male) and 35 non-smokers (20 male) with irritable bowel syndrome and/or exit rectal bleeding. The mean age of smokers and non-smokers was 45.6±12.0 (mean±SD) and 52.5±17.1 years respectively (p=0.1).
- Biopsies from patients with UC in remission—19 smokers (8 male) and 43 non-smokers (21 male)—were also examined. The mean ages of the smokers and non-smokers were 46.3±8.0 and 39.8±10.2 years, respectively; smokers were significantly older (p=0.02).
The smokers with inactive colitis have been described previously,4 and the non-smokers with colitis had taken part in a previous study of transdermal nicotine in UC.24
Part III
In the third part, non-smokers with inactive colitis were given either a 15 mg nicotine patch (20 patients) or a placebo patch (23 patients) daily for 26 weeks, and rectal mucosal biopsies taken both before and on completion of the trial, were examined. Serum cotinine levels were measured in these patients during the study, to reflect the level of nicotine exposure.
In-situ hybridization
In-situ hybridization (ISH) was performed on five normal full-thickness colonic tissues. Sections were cut at 4 µm thickness, from routine formalin-fixed, paraffin-embedded tissue.
For each tissue section, ISH was performed on two slides, using oligonucleotide probe cocktails directed against the 3 nAChR subunit on one slide and the kappa (
) immunoglobulin light chain on the other. Both cocktails contained eight oligonucleotide probes, 30 bases in length (with the exception of one oligonucleotide probe directed against the 3 nAChR subunit that was 24 bases long), and were used at a total concentration of 2 ng/ml. As such, the anti- probe cocktail provided a balanced positive control for the anti-3 nAChR subunit probe. The probes and technique used have been described previously.12
Immunocytochemistry
Immunocytochemistry was performed on the five normal full-thickness colonic tissues described above and on the various rectal biopsy specimens. All samples were routinely formalin-fixed, paraffin-wax-embedded, and sectioned at 4 µm thickness. Immunocytochemistry was performed on each specimen using two antibodies, mAb 210 and mAb 35, at concentrations of 0.25 µg/ml and 0.05 µg/ml, respectively; control experiments in which no primary antibody was applied also being performed. These monoclonal antibodies bind to the main immunogenic region of 1, 3 and 5 nAChR subunits.13,25 The technique used has been described previously.12 In addition, each biopsy was also stained with haematoxylin and eosin to assess the grade of colitis activity.
Assessment of slides
Full-thickness specimens
Two experienced histopathologists (GT and BJ) carried out a joint assessment of the slides and agreed on the presence of staining with the two ISH probes and two monoclonal antibodies. Staining was assessed in epithelium, enteric ganglia (both submucosal and myenteric ganglia), smooth muscle and lymphoid tissue (if present). The intensity of staining was not assessed.
Rectal biopsy specimens
The mucosal biopsies were examined by the two histopathologists, who carried out a blind joint assessment, of the epithelial staining obtained with the monoclonal antibodies. The intensity of staining was scored as: 0, negative; 1, mild; 2, moderate; and 3, strong.
Early in the work, it was noted that the antibodies did not produce uniform staining of the mucosal epithelium—the intensity often differed between epithelium lining the colonic lumen and the crypts. In view of this, separate scores were given to the epithelium at these two sites. Since these were mucosal biopsies, smooth muscle and enteric plexus tissue were not consistently present and were therefore not scored.
Colitis activity was assessed using a modification of the system described by Truelove and Richards in 1956.26
Comparisons
Rectal biopsies were available from smokers and non-smokers for controls and patients with ulcerative colitis, which made it possible to compare the intensity of staining in the following: (i) ulcerative colitis was compared with controls in smokers and non-smokers; and (ii) smokers were compared with non-smokers in controls and patients with ulcerative colitis.
The effect of nicotine in patients with inactive UC was investigated by comparison of rectal biopsies taken before and at the end of treatment with either transdermal nicotine or a placebo patch.24
Statistical analysis
Results were analysed statistically using the non-parametric Wilcoxon and Mann-Whitney tests, on SPSS for Windows version 9.
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Results |
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Part I: normal colon—ISH and ICC
ISH showed a widespread distribution of
3 nAChR subunit mRNA in all the sections examined; the 3 nAChR probe stained mucosal epithelium, enteric ganglia, smooth muscle and follicular lymphoid tissue (Figure 1
). The kappa light chain probe produced the expected strong staining of plasma cells throughout the lamina propria, with a weak staining of lymphoid tissue, but did not stain the enteric ganglia, smooth muscle or epithelium (Figure 1). The results from ICC were supportive of those obtained with ISH. Both antibodies produced staining of the same tissue structures as the 3 nAChR probe: the mucosal epithelium, enteric ganglia, smooth muscle and lymphoid tissue. The intensity of staining was consistently greater for mAb 210 than mAb 35. The two complementary techniques were considered to have identified 3 nAChR mRNA and the subunit protein itself.
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The presence of positive staining within colonic epithelium provided the basis for the investigations which followed, with rectal biopsies to examine the effect of smoking and/or nicotine on mucosal epithelial nAChRs, using the simpler technique of ICC alone.
Part II: the effect of smoking on antibody staining of the mucosal epithelium of rectal biopsies
The mucosal epithelial staining of the rectal biopsies with mAb 210 and 35 was identical to that observed in the full thickness colonic sections described above; the intensity of staining was also consistently greater with mAb 210 than with mAb 35.
In Figure 2, each histogram represents a particular subject group investigated (e.g. control smokers), with one of the monoclonal antibodies, and compares the intensity of staining of the surface (luminal) epithelium with that of the crypt epithelium. The intensity and distribution of staining was similar in all the groups of subjects investigated—controls, inactive colitis, smokers and non-smokers (Figure 2). It can be seen that in a majority of cases the intensity of staining in the luminal epithelium was greater than in the crypt epithelium—and on five occasions this difference was statistically significant, as exemplified by control non-smokers (Figures 2 and 3).
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Comparison of ulcerative colitis and controls
Smokers
Twenty of the controls and 19 UC patients were smokers; their mean ages were not significantly different. The colitis was inactive in most patients, with 14/19 recording histological grade 0.
The intensity and distribution of epithelial staining was similar to that seen in non-smokers (Figure 2
). In Figure 4, the histograms of row a show results from both luminal and crypt epithelial regions with both monoclonal antibodies, and compare the percentage of biopsies with an epithelial score of 0, 1, 2 or 3, in controls and patients with UC. For both of the epithelial areas assessed, luminal and crypt, the staining intensity was greater in the controls compared with inactive UC and reached statistical significance for mAb 210 (p<0.01) but not for mAb 35 (Figure 4). Similar results were obtained when the analysis was repeated using only the UC patients with a histological activity score of 0; the staining intensity remained greater in the controls and achieved statistical significance for mAb 210 (p=0.03) but not for mAb 35 (p=0.09).
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Non-smokers
Thirty-five of the controls and 43 with UC were non-smokers. The colitis was inactive in most patients, with 40 of the 43 recording a histological grade 0.
For both areas of the epithelium assessed the staining intensity was greater in the controls compared with ulcerative colitis for both antibodies (p<0.05) (Figure 4, row b). As before, similar results were obtained when the analysis was repeated using only the UC patients with a histological activity score of 0; the staining intensity remained greater in the controls and achieved statistical significance with both antibodies (p<0.05).
Comparison of smokers and non-smokers
Controls
Of the controls, 20 were smokers and 35 non-smokers. There was no significant difference in the intensity or distribution of antibody staining between smokers and non-smokers in either of the epithelial regions assessed for either of the antibodies (Figure 4).
Ulcerative colitis subjects
Of the patients with ulcerative colitis, 19 were smokers and 43 non-smokers. Again, there was no significant difference in the intensity or distribution of mucosal epithelial staining as assessed by either antibody (Figure 4).
Part III: effect of transdermal nicotine in ulcerative colitis
Twenty of the subjects given nicotine and 23 given placebo were investigated from the study of Thomas et al 1995.24 The mean±SD ages of the nicotine and placebo groups were 42.9±9.4 and 37.2±10.2 years, respectively (p=0.06). On entry to the study, most patients had inactive disease—graded 0 in 18 of the 20 given the nicotine patch and in 22 of the 23 given the placebo. The serum cotinine concentration varied considerably in patients given nicotine, but repeated measurements in individual patients were more constant. Before treatment, the serum cotinine concentration was 1.0±0.3 ng/ml (mean±SEM), range 0.1–6.2 ng/ml. During treatment, mean values were 77.7±11.3 ng/ml, range 0.5–175 ng/ml; these concentrations are lower than those seen in habitual smokers.27
Subjects given transdermal nicotine showed no significant change in the intensity or distribution of epithelial staining (p>0.05, Figure 4)—likewise with the placebo patch. However, a significant difference in the intensity of mAb 210 epithelial staining was observed before and after treatment with the placebo patch: luminal staining was significantly greater than crypt (p=0.007). For the nicotine patch, no such difference was seen either before or after treatment (Figure 2). The reason for this difference is not clear and could not be explained on the grounds of differences in histological disease activity. In the clinical study, transdermal nicotine was no more effective than placebo in maintenance of remission, despite its efficacy in the treatment of active disease.24
The effect of disease activity in ulcerative colitis
In a final analysis of all the UC rectal biopsies, from smokers and non-smokers, before and after the nicotine or placebo patch, with a disease activity score 
1 (n=11) were combined and compared to all those with a disease activity score of 0 (n=86). For both epithelial regions, and with both antibodies used, there was greater epithelial staining in the group with a disease activity of 0; but statistical significance was only achieved for the mAb 35 staining of the luminal epithelium (p=0.02).
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Discussion |
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This is the first demonstration of nicotinic acetylcholine receptors in the colon. Both ISH and ICC show a wide distribution of
3 nAChR subunit mRNA and protein, respectively, in normal human colon—in mucosal epithelium, submucosal and myenteric plexuses, smooth muscle and lymphoid tissue. On the basis of these findings, the monoclonal antibodies were then used to examine the effects of smoking and transdermal nicotine on the distribution and density of immunodetectable epithelial nAChRs. Smokers and non-smokers did not differ in either controls or patients with UC, nor did transdermal nicotine affect the results in the non-smoking UC group. However, the antibody staining intensity was greater in both the luminal and crypt epithelium of controls compared to UC, for both smokers and non-smokers. We have interpreted this as indicative of a greater nAChR density in the mucosal epithelium of controls compared to UC patients. The validity of our ISH and ICC findings is based on previous observations with the same oligonucleotide probe cocktail and monoclonal antibodies used with small bowel tissue.12 Our non-smoking group with UC are probably representative of the total UC out-patients, since they were chosen randomly on the basis of clinical remission and willingness to take part in the nicotine study. The number of smokers with UC in clinical remission was limited because there are few of them: in our original survey, only 8% of UC patients smoked cigarettes.1 To identify our UC smokers involved a special survey of out-patients. The UC numbers were matched approximately with similar numbers of controls, both smokers and non-smokers. Furthermore, the work with transdermal nicotine was based on paired biopsies from each of the 43 non-smoking patients. Every effort was made to include only patients with clinically inactive colitis, which on the basis of histological assessment, was largely achieved. The numbers of subjects used to examine the effects of smoking and nicotine are in line with numbers used in other studies to show the effect of a pharmacological agent on a disease process. Whether the conclusions can be applied more generally to all patients with UC is somewhat limited by the relatively small numbers in the groups. An analysis of the possible impact of age and gender on the results is similarly limited by small numbers, but we were unable to identify such an effect.
The increased staining in luminal epithelium suggests that expression of nAChRs is related to terminal differentiation in colonocytes. Goblet cells also stain, and in both types of epithelial cell, the nAChR protein appears to be cytoplasmic as well as membranous. In keratinocytes in cell culture, nAChRs were localized to the plasma membrane, and patch clamp studies indicated similar ion gating and pharmacological properties to 3 nAChR characterized in neurons.18 Similar physiological properties and plasma membrane localisation were demonstrated in the a3 nAChRs of the bronchial epithelium.19
The work confirms our previous findings on small bowel with ISH and ICC.12 These are the only descriptions of nAChR subunit expression in the gut epithelium, although nAChRs have previously been described in both keratinocytes and bronchial epithelium in man.18,28,29 When this programme of work first began, we performed autoradiography with tritiated epibatidine, a nicotinic agonist that has proven especially useful for the study of 3 nAChRs,30,31 on specimens of freshly resected human colon from four subjects with carcinoma, one with UC and one with colonic Crohn's disease.32 In the specimens, there was clear labelling in the cytoplasm of the ganglion cells in the nerve plexuses, but the mucosal epithelium was not convincingly labelled.32 It may be that this technique detected a high density of nAChRs in nerve plexus ganglia, but was not sufficiently sensitive to detect labelling elsewhere in the colonic tissues. Alternatively, since epibatidine binds to the whole nAChR, its affinity is dependent on all of the subunits present, not just the 3; the nAChR subunit composition of non-neuronal tissues may have conferred a lower affinity than neuronal tissues.
Since smoking and nicotine appear to have no effect on either the density or distribution of epithelial 3 subunits in the colon, if an action of these 3 nAChRs is pertinent to the effect of smoking and nicotine in UC, then some functional change in the receptors may be responsible. Functional effects mediated through nAChRs in other epidermal tissues have been described in man; keratinocyte and bronchial epithelium maturation, differentiation and cellular adhesion is influenced by 3 nAChR stimulation, an effect mediated by a calcium influx.18,28,29 Alternatively, since nicotine influences gut smooth muscle largely through nitric oxide (NO)-dependent mechanisms,9 one could speculate on a NO effect on epithelial cell function following nicotinic stimulation. Whatever the potential mechanism, an effect of nicotine on colonic epithelial stability would be of considerable interest in the pathogenesis of UC, but must be considered only speculative at present. The decreased expression of 3 nAChRs in the epithelium of patients with ulcerative colitis, a finding seen in both smokers and non-smokers, is of some interest and could be related to the increased cell turnover present, even in ‘quiescent’ colitis.33,34 The observation of a decreased epithelial staining in cases of more active UC is compatible with cell turnover influencing the expression of 3 nAChRs; unfortunately the small number of cases in the ‘active’ group prevents firm conclusions from being drawn. A number of metabolic abnormalities have been described in quiescent colitis, including reduced butyrate oxidation, phenol sulphation and mucin glycosylation.35 The reduced expression of nAChRs in UC may be detrimental to epithelial cell function, and nicotinic stimulation might also have a beneficial effect in this situation.
In conclusion, we have demonstrated the presence of nAChRs in the human colon using ISH and ICC with a substantial density of receptors in the luminal epithelium. Smoking and transdermal nicotine have no effect on either the density or distribution of 3 nAChRs in the mucosal epithelium of the rectum. A greater density of 3 nAChRs in the mucosal epithelium of controls compared with inactive UC may relate to an increased cell turnover in UC that decreases the time available for 3 nAChR subunit maturation. The presence of nAChRs in colonic epithelium may be pertinent to the association between smoking, nicotine and ulcerative colitis.
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Acknowledgments |
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We are grateful for statistical advice to Dr Robert G. Newcombe, Reader in Medical Statistics, Department of Epidemiology, Statistics And Public Health, College of Medicine, Heath Park, Cardiff.
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Address correspondence to Professor John Rhodes, Department of Gastroenterology, University Hospital of Wales, Heath Park, Cardiff CF14 4XW
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References |
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1. Harries AD, Baird A, Rhodes J. Non smoking: a feature of ulcerative colitis. Br Med J 1982; 284:706.
2. Lidberg E, Tysk C, Andersson K, Jarnerot G. Smoking and inflammatory bowel disease: a case-control study. Gut 1988; 29:352–7.
3. Calkins BM. A meta-analysis of the role of smoking in inflammatory bowel disease. Dig Dis Sci 1989; 34:1841–54.[CrossRef][Web of Science][Medline]
4. Green JT, Rhodes J, Ragunath K, Thomas GAO, Williams GT, Mani V, Feyerabend C, Russell MAH. Clinical studies of ulcerative colitis in patients who smoke. Am J Gastroenterol 1998; 93:1463–7.[CrossRef][Web of Science][Medline]
5. Rudra T, Motley R, Rhodes J. Does smoking improve colitis? Scand J Gastroenterol 1989; 170(Suppl.):61–3.
6. Pullan RD, Rhodes J, Ganesh S, Mani V, Morris JS, Williams GT, Newcombe RG, Russell MAH, Feyerabend G, Thomas GAO, Sawe U. Transdermal nicotine for active ulcerative colitis. N Engl J Med 1994; 330:811–15.
7. Sandborn WJ, Tremaine W, Offord KP, et al. Transdermal nicotine for mildly to moderately active ulcerative colitis: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 1997; 126:364–71.
8. Finnie IA, Campbell BJ, Taylor BA, Milton JD, Sadek JD, Yu LG, Rhodes JM. Stimulation of colonic mucin synthesis by corticosteroids and nicotine. Clin Sci 1996; 91:359–64.[Medline]
9. Green JT, Richardson C, Marshall RW, Rhodes J, McKirdy H, Thomas GA, Williams GT. Nitric oxide mediates a therapeutic effect of nicotine in ulcerative colitis. Aliment Pharmacol Ther 2000; 14:1429–34.[CrossRef][Web of Science][Medline]
10. Holt PG. Immune and inflammatory function in cigarette smokers. Thorax 1987; 8:381–91.
11. Miller LG, Goldstein G, Murphy M, Ginns LC. Reversible alterations in immunoregulatory T cells in smoking. Chest 1982; 5:527–9.
12. Richardson CE, Morgan JM, Jasani B, Green JT, Rhodes J, Williams GT, Lindstrom J, Wonnacott S, Thomas GAO, Smith V. Megacystis-Microcolon-Intestinal Hypoperistalsis Syndrome (MMIHS) is associated with absence of the 3 nicotinic acetylcholine receptor subunit. Gastroenterology 2001; 121:350–7.[CrossRef][Web of Science][Medline]
13. Lindstrom J. Acetylcholine receptors and myasthenia. Muscle Nerve 2000; 23:453–71.[CrossRef][Web of Science][Medline]
14. Lustig LR, Peng H, Hiel H, Yamamoto T, Fuchs PA. Molecular cloning and mapping of the human nicotinic acetylcholine receptor alpha10 (CHRNA 10). Genomics 2001; 73:272–83.[CrossRef][Web of Science][Medline]
15. Unwin N. Nicotinic acetylcholine receptor at 9A resolution. J Mol Biol 1993; 229:1101–24.[CrossRef][Web of Science][Medline]
16. Unwin N. Acetylcholine receptor channel imaged in the open state. Nature 1995; 373:37–43.[CrossRef][Medline]
17. Wang F, Gerzanich V, Wells GB, Anand R, Peng X, Keyser K, Lindstrom J. Assembly of human neuronal nicotinic receptor 5 subunits with 3, ß2 and ß4 subunits. J Biol Chem 1996; 271:17656–65.
18. Grando SA, Horton RM, Pereira EF, Diethelm-Okita BM, George PM, Albuquerque EX, Conti-Fine BM. A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes. J Invest Dermatol 1995; 105:774–81.[CrossRef][Web of Science][Medline]
19. Zia S, Ndoye A, Nguyen VT, Grando SA. Nicotine enhances expression of the 3, 4, 5 and 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells. Res Commun Mol Pathol Pharmacol 1997; 97:243–62.[Web of Science][Medline]
20. Peng X, Gerzanich V, Anand R, Wang F, Lindstrom J, et al. Chronic nicotine treatment up-regulates 3 AChRs and 7 AChRs expressed by the human neuroblastoma cell line SH-SY5Y. Mol Pharmacol 1997; 45:546–54.
21. Ridley DL, Rogers A, Wonnacott S. Differential effects of chronic drug treatment on 3 and 7 nicotinic receptor binding sites, in hippocampal neurones and SH-SY5Y cells. Br J Pharmacol 2001; 133:1286–95.[CrossRef][Web of Science][Medline]
22. Peng X, Anand R, Whiting P, Lindstrom J. Nicotine induced upregulation of neuronal nicotinic receptors results from a decrease in the rate of turnover. Mol Pharmacol 1994; 46:523–30.[Abstract]
23. Olale F, Gerzanich V, Kuryatov A, Wang F, Lindstrom J. Chronic nicotine exposure differentially affects the function of human 3, 5 and 7 neuronal nicotinic receptor subtypes. J Pharmacol Exp Ther 1997; 283:675–83.
24. Thomas GAO, Rhodes J, Mani V, Williams GT, Newcombe RG, Russell MAH, Feyerabend C. Transdermal nicotine as maintenance therapy for ulcerative colitis. N Engl J Med 1995; 332:988–92.
25. Lindstrom J. Monoclonal antibodies to nicotinic acetylcholine receptors. Neurotransmissions 1996; 12:1–9.
26. Truelove SC, Richards WCD. Biopsy studies in ulcerative colitis. Br Med J 1956; 2:1315–21.
27. Benowitz NL, Kuyt F, Jacob P III, Jones RT, Osmann AL. Cotinine disposition and effects. Clin Pharmacol Ther 1983; 34:604–6.[Web of Science][Medline]
28. Grando SA, Crosby AM, Zelickson BD, Dahl MV. Agarose gel keratinocyte outgrowth system as a model of skin re-epithelization: requirement of endogenous acetylcholine for outgrowth migration. J Invest Dermatol 1993; 101:804–10.[CrossRef][Web of Science][Medline]
29. Grando SA, Horton RM, Mauro TM, Kist DA, Lee TX, Dahl MV. Activation of keratinocyte nicotinic cholinergic receptors stimulates calcium influx and enhances cell differentiation. J Invest Dermatol 1996; 107:412–18.[CrossRef][Web of Science][Medline]
30. Badio B, Daly J. Epibatidine, a potent analgesic and nicotinic agonist. Mol Pharmacol 1994; 45:563–569.[Abstract]
31. Gerzanich V, Peng X, Wang F, Wells G. Anand R, Fletcher S, Lindstrom J. Comparative pharmacology of epibatidine: a potent agonist for neuronal nicotinic acetylcholine receptors. Mol Pharmacol 1995; 48:774–82.[Abstract]
32. Green JT, Peel S, Wonnacott S, Jasani B, Williams GT, Thomas GAO, Rhodes J. Demonstration of nicotinic acetylcholine receptors in human colon. Digestion 1998; 59:696.[CrossRef][Web of Science][Medline]
33. Allan A, Bristol JB, Williamson RN. Crypt cell production rate in ulcerative procto-colitis differential increments in remission and relapse. Gut 1985; 26:999–1003.
34. Serafini EP, Kirk AP, Chambers TJ. Rate and pattern of epithelial cell proliferation in ulcerative colitis. Gut 1981; 22:648–52.
35. Gibson PR, Barkla DH. Mucosal metabolism and proliferation. In: Allan RN, Rhodes JM, Hanauer SB, eds. Inflammatory Bowel Diseases. Churchill Livingstone, 1997.
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