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According to the adenoma-carcinoma sequence, colon cancer results from accumulating somatic gene mutations; environmental growth factors accelerate and augment this process. For example, diets rich in meat and fat increase fecal bile acids and colon cancer risk. In rodent cancer models, increased fecal bile acids promote colon dysplasia. Conversely, in rodents and in persons with inflammatory bowel disease, low-dose ursodeoxycholic acid treatment alters fecal bile acid composition and attenuates colon neoplasia. In the course of elucidating the mechanism underlying these actions, we discovered that bile acids interact functionally with intestinal muscarinic receptors. The present communication reviews muscarinic receptor expression in normal and neoplastic colon epithelium, the role of autocrine signaling following synthesis and release of acetylcholine from colon cancer cells, post-muscarinic receptor signaling including the role of transactivation of epidermal growth factor receptors and activation of the ERK and PI3K/AKT signaling pathways, the structural biology and metabolism of bile acids and evidence for functional interaction of bile acids with muscarinic receptors on human colon cancer cells. In murine colon cancer models, deficiency of subtype 3 muscarinic receptors attenuates intestinal neoplasia; a proof-of-concept supporting muscarinic receptor signaling as a therapeutic target for colon cancer.

Previous work suggests that vagus nerve disruption reduces hepatocyte and oval cell expansion after liver injury. The role of postneuronal receptor activation in response to liver injury has not been ascertained. We investigated the actions of scopolamine, a nonselective muscarinic receptor antagonist, and specific genetic ablation of a key cholinergic receptor, muscarinic subtype-3 (Chrm3), on azoxymethane (AOM)-induced liver injury in mice. Animal weights and survival were measured as was liver injury using both gross and microscopic examination. To assess hepatocyte proliferation and apoptosis, ductular hyperplasia, and oval cell expansion, we used morphometric analysis of 5-bromo-2'-deoxyuridine-, activated caspase-3-, hematoxylin and eosin-, cytokeratin-19-, and epithelial cell adhesion molecule-stained liver sections. Sirius red staining was used as a measure of collagen deposition and its association with oval cell reaction. In AOM-treated mice, both muscarinic receptor blockade with scopolamine and Chrm3 ablation attenuated hepatocyte proliferation and augmented gross liver nodularity, apoptosis, and fibrosis. Compared with control, scopolamine-treated and Chrm3(-/-) AOM-treated mice had augmented oval cell reaction with increased ductular hyperplasia and oval cell expansion. Oval cell reaction correlated robustly with liver fibrosis. No liver injury was observed in scopolamine-treated and Chrm3(-/-) mice that were not treated with AOM. Only AOM-treated Chrm3(-/-) mice developed ascites and had reduced survival compared with AOM-treated wild-type controls. In AOM-induced liver injury, inhibiting postneuronal cholinergic muscarinic receptor activation with either scopolamine treatment or Chrm3 gene ablation results in prominent oval cell reaction. We conclude that Chrm3 plays a critical role in the liver injury response by modulating hepatocyte proliferation and apoptosis.

Purpose of Review: Update on the role of gastrointestinal peptides in regulating gastric acid secretion., Recent Findings: A novel transgenic mouse that expresses the entire human gastrin gene locus in G-cells of gastrin-null mice will facilitate investigation of gastrin gene regulatory elements. Isolation of a highly homogeneous population of G-cells permits the elucidation of stimulatory and inhibitory ligands without the confounding presence of other neuroendocrine cells. The use of somatostatin receptor knockout mice demonstrated the plasticity of gastric acid regulatory mechanisms and compensation by upregulation of the galanin pathway which inhibits secretion by enterochromaffin-like cells. The importance of adenosine in regulating somatostatin release was shown using adenosine receptor knockout mice., Summary: The importance of gastrointestinal peptides for regulating gastric acid is evident. Ongoing investigations will characterize the mechanisms underlying actions of these agents on gastric acid secretion, particularly with regard to their combinatorial effects and interplay with other acid-regulating pathways.

The human apical sodium-dependent bile acid transporter (ASBT; SLC10A2) is the primary mechanism for intestinal bile acid reabsorption. In the colon, secondary bile acids increase the risk of cancer. Therefore, drugs that inhibit ASBT have the potential to increase the risk of colon cancer. The objectives of this study were to identify FDA-approved drugs that inhibit ASBT and to derive computational models for ASBT inhibition. Inhibition was evaluated using ASBT-MDCK monolayers and taurocholate as the model substrate. Computational modeling employed a HipHop qualitative approach, a Hypogen quantitative approach, and a modified Laplacian Bayesian modeling method using 2D descriptors. Initially, 30 compounds were screened for ASBT inhibition. A qualitative pharmacophore was developed using the most potent 11 compounds and applied to search a drug database, yielding 58 hits. Additional compounds were tested, and their K(i) values were measured. A 3D-QSAR and a Bayesian model were developed using 38 molecules. The quantitative pharmacophore consisted of one hydrogen bond acceptor, three hydrophobic features, and five excluded volumes. Each model was further validated with two external test sets of 30 and 19 molecules. Validation analysis showed both models exhibited good predictability in determining whether a drug is a potent or nonpotent ASBT inhibitor. The Bayesian model correctly ranked the most active compounds. In summary, using a combined in vitro and computational approach, we found that many FDA-approved drugs from diverse classes, such as the dihydropyridine calcium channel blockers and HMG CoA-reductase inhibitors, are ASBT inhibitors.

Previously, we showed that ACh-induced proliferation of human colon cancer cells is mediated by transactivation of epidermal growth factor (EGF) receptors (EGFRs). In the present study, we elucidate the molecular mechanism underlying this action. ACh-induced proliferation of H508 colon cancer cells, which express exclusively M3 muscarinic receptors (M3Rs), was attenuated by anti-EGFR ligand binding domain antibody, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, anti-MMP7 antibody, a diphtheria toxin analog that blocks release of an EGFR ligand [heparin-binding EGF-like growth factor (HBEGF)], and anti-HBEGF antibody. Conditioned media from ACh-treated H508 cells induced proliferation of SNU-C4 colon cancer cells that express EGFR but not M3R. These actions were attenuated by an EGFR inhibitor and by anti-EGFR and anti-HBEGF antibodies. In H508, but not SNU-C4, colon cancer cells, ACh caused a striking dose- and time-dependent increase in levels of MMP7 mRNA and MMP7 protein. Similarly, ACh induced robust MMP1 and MMP10 gene transcription. ACh-induced MMP1, MMP7, and MMP10 gene transcription was attenuated by atropine, anti-EGFR antibody, and chemical inhibitors of EGFR and ERK activation. In contrast, inhibitors of phosphatidylinositol 3-kinase and NF-kappaB activation did not alter MMP gene transcription. Collectively, these findings indicate that MMP7-catalyzed release of HBEGF mediates ACh-induced transactivation of EGFR and consequent proliferation of colon cancer cells. ACh-induced activation of EGFR and downstream ERK signaling also regulates transcriptional activation of MMP7, thereby identifying a novel feed-forward mechanism for neoplastic cell proliferation.

Most colon cancers overexpress M3 muscarinic receptors (M3R), and post-M3R signaling stimulates human colon cancer cell proliferation. Acetylcholine (ACh), a muscarinic receptor ligand traditionally regarded as a neurotransmitter, may be produced by nonneuronal cells. We hypothesized that ACh release by human colon cancer cells results in autocrine stimulation of proliferation. H508 human colon cancer cells, which have robust M3R expression, were used to examine effects of muscarinic receptor antagonists, acetylcholinesterase inhibitors, and choline transport inhibitors on cell proliferation. A nonselective muscarinic receptor antagonist (atropine), a selective M3R antagonist (p-fluorohexahydro-sila-difenidol hydrochloride), and a choline transport inhibitor (hemicholinum-3) all inhibited unstimulated H508 colon cancer cell proliferation by approximately 40% (P<0.005). In contrast, two acetylcholinesterase inhibitors (eserine-hemisulfate and bis-9-amino-1,2,3,4-tetrahydroacridine) increased proliferation by 2.5- and 2-fold, respectively (P<0.005). By using quantitative real-time PCR, expression of choline acetyltransferase (ChAT), a critical enzyme for ACh synthesis, was identified in H508, WiDr, and Caco-2 colon cancer cells. By using high-performance liquid chromatography-electrochemical detection, released ACh was detected in H508 and Caco-2 cell culture media. Immunohistochemistry in surgical specimens revealed weak or no cytoplasmic staining for ChAT in normal colon enterocytes (n=25) whereas half of colon cancer specimens (n=24) exhibited moderate to strong staining (P<0.005). We conclude that ACh is an autocrine growth factor in colon cancer. Mechanisms that regulate colon epithelial cell production and release of ACh warrant further investigation.

Colon epithelial cells express and most colon cancers overexpress M(3) muscarinic receptors (M(3)R). In human colon cancer cells, post-M(3)R signaling stimulates proliferation. To explore the importance of M(3)R expression in vivo, we used the azoxymethane-induced colon neoplasia model. Mice treated with weekly i.p. injection of saline [10 wild-type (WT) mice] or azoxymethane (22 WT and 16 M(3)R(-/-) mice) for 6 weeks were euthanized at 20 weeks. At week 20, azoxymethane-treated WT mice weighed approximately 16% more than M(3)R(-/-) mice (33.4 grams +/- 1.0 grams versus 27.9 grams +/- 0.5 grams; mean +/- SE, P < 0.001). In azoxymethane-treated M(3)R(-/-) mice, cell proliferation (BrdUrd staining) was reduced 43% compared with azoxymethane-treated WT mice (P < 0.05). Whereas control mice (both WT and M(3)R(-/-)) had no colon tumors, azoxymethane-treated WT mice had 5.3 +/- 0.5 tumors per animal. Strikingly, azoxymethane-treated M(3)R(-/-) mice had only 3.2 +/- 0.3 tumors per mouse (P < 0.05), a 40% reduction. Tumor volume in azoxymethane-treated M(3)R(-/-) mice was reduced 60% compared with azoxymethane-treated WT mice (8.1 mm(3) +/- 1.5 mm(3) versus 20.3 mm(3) +/- 4.1 mm(3); P < 0.05). Compared with WT, fewer M(3)R(-/-) mice had adenomas (6% versus 36%; P = 0.05), and M(3)R(-/-) mice had fewer adenocarcinomas per mouse (0.6 +/- 0.1 versus 1.7 +/- 0.4; P < 0.05). Eleven of 22 WT but no M(3)R(-/-) mice had multiple adenocarcinomas (P < 0.001). Compared with WT, azoxymethane-treated M(3)R-deficient mice have attenuated epithelial cell proliferation, tumor number, and size. M(3)R and post-M(3)R signaling are novel therapeutic targets for colon cancer.

Muscarinic cholinergic mechanisms play a key role in stimulating gastric pepsinogen secretion. Studies using antagonists suggested that the M3 receptor subtype (M3R) plays a prominent role in mediating pepsinogen secretion, but in situ hybridization indicated expression of M1 receptor (M1R) in rat chief cells. We used mice that were deficient in either the M1 (M1R-/-) or M3 (M3R-/-) receptor or that lacked both receptors (M(1/3)R-/-) to determine the role of M1R and M3R in mediating cholinergic agonist-induced pepsinogen secretion. Pepsinogen secretion from murine gastric glands was determined by adapting methods used for rabbit and rat stomach. In wild-type (WT) mice, maximal concentrations of carbachol and CCK caused a 3.0- and 2.5-fold increase in pepsinogen secretion, respectively. Maximal carbachol-induced secretion from M1R-/- mouse gastric glands was decreased by 25%. In contrast, there was only a slight decrease in carbachol potency and no change in efficacy when comparing M3R-/- with WT glands. To explore the possibility that both M1R and M3R are involved in carbachol-mediated pepsinogen secretion, we examined secretion from glands prepared from M(1/3)R-/- double-knockout mice. Strikingly, carbachol-induced pepsinogen secretion was nearly abolished in glands from M(1/3)R-/- mice, whereas CCK-induced secretion was not altered. In situ hybridization for murine M1R and M3R mRNA in gastric mucosa from WT mice revealed abundant signals for both receptor subtypes in the cytoplasm of chief cells. These data clearly indicate that, in gastric chief cells, a mixture of M1 and M3 receptors mediates cholinergic stimulation of pepsinogen secretion and that no other muscarinic receptor subtypes are involved in this activity. The development of a murine secretory model facilitates use of transgenic mice to investigate the regulation of pepsinogen secretion.

Emerging evidence indicates that some secondary bile acids interact functionally with muscarinic cholinergic receptors. Using thoracic aortic rings prepared from rats and mice, we examined the mechanism of deoxycholyltaurine-induced vasorelaxation. Increasing concentrations of both acetylcholine (1 nM to 0.1 mM) and deoxycholyltaurine (0.1 microM to 1 mM) stimulated relaxation of phenylephrine-constricted rings prepared from rat thoracic aortae. These effects were reduced by endothelial denudation and by treatment with an inhibitor of nitric oxide formation and with a synthetic acetylcholine:bile acid hybrid that acts as a muscarinic receptor antagonist. Likewise, both acetylcholine (1 nM to 0.1 mM) and deoxycholyltaurine (0.1 microM to 0.1 mM) stimulated relaxation of phenylephrine-constricted rings prepared from mouse thoracic aortae. These effects were reduced by endothelial denudation, addition of an inhibitor of nitric oxide formation, and by muscarinic M(3) receptor knockout. We conclude that the systemic vasodilatory actions of deoxycholyltaurine are mediated in part by a nitric oxide-, muscarinic M(3) receptor-dependent mechanism. In advanced liver disease, interaction of serum bile acids with endothelial muscarinic receptors may explain nitric oxide overproduction in the systemic circulation and resulting peripheral arterial vasodilation.

Background: Competence in gastrointestinal (GI) endoscopy correlates with the number of procedures performed by the endoscopist. For each GI endoscopic procedure, the American Society for Gastrointestinal Endoscopy (ASGE) guidelines recommend minimum numbers needed to assess competence., Methods: We conducted an anonymous mail survey to determine whether GI endoscopy centers in the United States follow ASGE or other guidelines for granting and renewing endoscopic privileges., Results: Completed surveys were received from 479 respondents in 46 states, Puerto Rico, and the District of Columbia. Most respondents were either the physician director (24%) or nurse manager (57%) of the endoscopy center. Most centers have more than 5 endoscopists (62%), and gastroenterologists performed procedures in the majority of the centers (89%). For initial endoscopic credentialing, few centers require a minimum number of procedures and only 10% meet ASGE criteria. To maintain credentials, less than one third require a minimum number and only 2% require more than 25 procedures/year. Although three fourths report periodic review of procedures, less than 5% review them more frequently than every 6 months. Only 20% of centers had ever denied endoscopic privileges (poor skills [80%], no references [27%], poor communication [7%], and excess complications [6%]) for which half faced litigation., Conclusions: Most GI endoscopy centers responding to this survey have no minimum standards for determining endoscopic competence and may credential GI endoscopists with suboptimal training. Only 10% adhere to ASGE guidelines. Moreover, there is lack of uniformity to application of these guidelines, and few centers use resulting data to deny or renew credentials. To guarantee high-quality endoscopic practice, more stringent, universal credentialing standards are required.

Acetylcholine interacts with endothelial muscarinic receptors to enhance nitric oxide (NO) release and thereby cause vasodilation. The present study was designed to determine if this effect of acetylcholine is mediated by muscarinic M3 receptors. Thoracic aortae were isolated from wild-type (WT) and M3 receptor knock out (M3R-/-) male mice, and endothelium-intact (I) and -denuded (D) aortic rings were bathed in physiological buffer. Preparations were utilized to examine the contractile response to phenylephrine (1 x 10(-8) - 3 x 10(-4) M added cumulatively) and the vasodilatory actions of acetylcholine (10(-8) - 10(-4) M), carbachol (10(-9) - 10(-4) M), ATP (3 x 10(-5) M) and the NO donor SIN-1 (10(-4) M), each added in the presence of phenylephrine. Endothelium-dependent vasodilatory effects of acetylcholine and carbachol were obvious in aortae isolated from WT mice (56.3 +/- 9.8% and 49.1 +/- 4.1% reductions, respectively, in phenylephrine-induced contraction; p < 0.05), while acetylcholine and carbachol-associated relaxations observed in endothelium-intact M3R-/- preparations (17.9 +/- 2.6% and 13.5 +/- 4.2% reductions, respectively) did not differ significantly from time-control values. ATP-induced, endothelium-dependent vasodilation was similar in preparations from M3R-/- and WT mice, and SIN-1 elicited similar dilatory effects in intact and denuded WT and M3R-/- segments. Phenylephrine concentration-response curves were shifted leftwards by removal of the endothelium in both groups (EC50 values: WT-I/D--25.59 +/- 6.86/3.13 +/- 1.01 x 10(-7) M; M3R-/-I/D--13.92 +/- 4.21/1.52 +/- 0.46 x 10(-7) M; both p < 0.05); however, the phenylephrine response did not differ significantly when compared between the WT and M3R-/- groups. These results indicate that the attenuated vasodilatory effect of acetylcholine in endothelium-intact aortae from M3R-/- mice is due to the absence of muscarinic M3 receptors, and thus suggest that in mouse aorta, muscarinic M3 receptors play a major role in the endothelium-dependent acetylcholine-induced vasodilation., (Copyright 2004 Elsevier B.V.)

Some human colon cancer cell lines (e.g., H508 cells) express M3 subtype muscarinic receptors that are activated by cholinergic agonists. The objective of the present study was to determine the cellular mechanisms underlying M3 muscarinic receptor-mediated proliferation of H508 human colon cancer cells. In H508 cells, but not in SNU-C4 cells that do not express muscarinic receptors, acetylcholine stimulated calcium-dependent phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and p90 ribosomal S6 kinase and consequent cell proliferation. Atropine or inhibitors of MAPK phosphorylation blocked these effects. Conversely, the actions of epidermal growth factor (EGF) on H508 cells were neither calcium dependent nor mediated by cholinergic mechanisms. Both acetylcholine- and EGF-induced phosphorylation of p44/42 MAPK was abolished in the presence of EGF receptor (EGFR) inhibitors (AG1478 and PD168393). In Chinese hamster ovary cells transfected with the rat M3 muscarinic receptor, which lack EGFR, acetylcholine-induced MAPK phosphorylation was not altered in the presence of EGFR inhibitors. In H508 cells, protein kinase C inhibitors did not alter acetylcholine- or EGF-induced MAPK phosphorylation. Finally, inhibition of EGFR activation abolished acetylcholine-induced H508 cell proliferation. These data indicate that, in H508 human colon cancer cells, cholinergic ligand interaction with M3 muscarinic receptors results in transactivation of EGFR, thereby stimulating cellular proliferation.

Bile acids alter regulatory pathways in several cell types. The molecular basis for these actions is not fully elucidated, but lithocholyltaurine interacts functionally with muscarinic receptors on gastric chief cells. In the present report, we demonstrate selective interaction of bile acids with Chinese hamster ovary (CHO) cells expressing each of the five muscarinic receptors. Lithocholyltaurine decreases binding of a radioligand to muscarinic M3 receptors, but not to other muscarinic receptors. Sulfated lithocholyltaurine, the major human metabolite, inhibits radioligand binding to muscarinic M1, but not to M2 or M3 receptors. Post-receptor actions of lithocholyltaurine include modulation of acetylcholine-induced increases in inositol phosphate formation and mitogen-activated protein (MAP) kinase phosphorylation. Molecular modeling suggests that the specific and functional interaction of lithocholyltaurine with muscarinic receptors is most likely due to similar shape and surface charge distribution of portions of acetylcholine and the bile acid. We propose that bile acids are signaling molecules whose effects may be mediated by interaction with muscarinic receptors.

Previous work from our laboratory indicates that bile acids, specifically lithocholic acid conjugates, interact with muscarinic receptors on gastric chief cells. Structural similarities between acetylcholine and lithocholyltaurine suggest a potential molecular basis for their interaction with the same receptor. We synthesized a hybrid molecule consisting of the steroid nucleus of lithocholyltaurine and the choline moiety of acetylcholine. The new molecule, lithocholylcholine, is hydrolyzed by acetyl-cholinesterase. Lithocholylcholine inhibited binding of a cholinergic radioligand to Chinese hamster ovary cells expressing each of the five muscarinic receptor subtypes. The binding affinities (K(i); micromolar) of lithocholylcholine for these receptors were: M3 (1.0) > M1 (2.7) > M2 (4.1) = M4 (4.9) > M5 (6.2). Lithocholylcholine inhibited intracellular signaling pathways mediated by interaction with M1, M2, and M3 muscarinic receptors. Regarding M3 receptors, lithocholylcholine was 10-fold more potent than lithocholyltaurine in terms of binding affinity and inhibition of acetylcholine-induced increases in inositol phosphate formation and mitogen-activated protein kinase phosphorylation. In a functional assay, lithocholylcholine inhibited acetylcholine-induced relaxation of rat aortic rings. These observations indicate that lithocholylcholine is a muscarinic receptor antagonist and provide further evidence that bile acids may have gastrointestinal signaling functions that extend beyond their effects on sterol metabolism, lipid absorption, and cholesterol elimination. Hybrid molecules created from bile acids and acetylcholine may be used to develop selective muscarinic receptor ligands.

In the course of examining the actions of major human bile acids on cholinergic receptors, we discovered that conjugates of lithocholic acid are partial muscarinic agonists. In the present communication, we report that conjugates of deoxycholic acid (DC) act as cholinergic muscarinic receptor antagonists. Chinese hamster ovary (CHO) cells expressing rat M3-muscarinic receptors were used to test bile acids for inhibition of radioligand [N- (3)H-methylscopolamine ((3)H-NMS)] binding; alteration of inositol phosphate (IP) formation; mitogen-activated protein (MAP) kinase phosphorylation and cell toxicity. We observed approximately 18.8, 30.3 and 37.1% inhibition of (3)H-NMS binding with DC and its glycine (DCG) and taurine (DCT) conjugates, respectively (all 100 micromol/l, p < 0.01). DCT and DCG inhibited acetylcholine-induced increases in IP formation and MAP kinase phosphorylation (p44 and p42 ERK). DCG and DCT did not alter trypan blue exclusion or lactate dehydrogenase release from CHO-M3 cells. We observed the following rank order of potency (IC(50) micromol/l) for inhibition of (3)H-NMS by muscarinic antagonists and bile acids: NMS (0.0004) > 4-DAMP (0.009) > atropine (0.012) > DCT (170) > DCG (250). None of the bile acids tested were hydrolyzed by recombinant cholinesterase. At concentrations achieved in human bile, DC derivatives are natural muscarinic antagonists., (Copyright 2002 S. Karger AG, Basel)