Actions of vitamin D are mediated by the TLR4 pathway in inflammation-induced colon cancer.

Many chronic inflammatory diseases are associated with increased risk of developing cancer. In the colon, strong support for a link between chronic inflammation and cancer extends, in part, from population-based studies of persons with inflammatory bowel disease (IBD). Patients with IBD are at increased risk of developing colorectal cancer (CRC). The general consensus is that IBD results from the combined effects of genetics and environment factors known to affect the immune system. Vitamin D, an important regulator of the immune system, has been linked to IBD. Despite the strong potential reported for 1,25-dihydroxyvitamin D (1,25-OH)2D), its effects on calcium metabolism limits its application. Recently, less active vitamin D metabolites, cholecalciferol and 25-hydroxyvitamin D (25(OH)D), have gained considerable attention as promising agents against IBD-related colon cancer. Yet, their anti-proliferative properties and mechanism of action remain to be better defined. We present several signaling pathways commonly regulated by vitamin D compounds and highlight their regulation on TLR4. The efficacy of 25(OH)D and 1alpha-hydroxyviatmin D5 are evaluated using the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced IBD-related colon carcinogenesis model. In summary, vitamin D supplementation may provide a cost-effective approach to reduce IBD related colon cancer.

Extent of prevalence and size of flat neoplasms in a heterogeneous population undergoing routine colorectal cancer screening.

BACKGROUND: The importance of identifying flat colorectal neoplasms is increasingly appreciated, although the extent of prevalence of these lesions in a general population is not known. OBJECTIVE: To determine the extent of prevalence of flat neoplasms in a diverse population undergoing routine endoscopic screening for colorectal cancer. DESIGN: Patients referred to the Colorectal Cancer Screening Clinic over a 12-month period (n = 642). RESULTS: The patient population was 56% African American and 21% Caucasian; with a mean age of 59 + or - 9 years. Flat neoplasms were detected in 5.5% of all patients, similar to that reported elsewhere, with extent of prevalence being similar regardless of gender or race. Average size of flat neoplasms was of 2.8 + or - 2.3 mm (range 1-20 mm). However, there was no evidence of advanced pathology in any of the flat neoplasms identified. CONCLUSIONS: Flat neoplasms are common but may not be associated with advanced pathology in a population undergoing routine screening.

Age-dependent differences in galanin-dependent colonic fluid secretion after infection with Salmonella typhimurium.

Epithelial cells lining the colon do not normally express galanin type 1 receptors (Gal1Rs). However, subsequent to infection with enteric pathogens such as Salmonella typhimurium, the Gal1R is rapidly upregulated in colonocytes where it contributes to the excess fluid production associated with diarrhoea. Humans infected with non-typhoid Salmonella respond differently according to age: infants develop diarrhoea but not bacteraemia and survive, while the elderly become bacteraemic and die. Thus the aim of this study was to determine if age-related differences exist in response to S typhimurium infection in mice, and whether these differences are due to altered Gal1R expression. Wild-type C57BL/6J mice that were 2 and 15 months old, as well as 2-month-old Gal1R knockout mice, were infected by gavage. Young wild-type mice expressed Gal1R in response to infection, had increased colonic fluid secretion, low rates of bacteraemia and survived. In contrast, 15-month-old wild-type mice expressed fewer Gal1Rs in response to infection, had attenuated increases in colonic fluid secretion, high rates of bacteraemia and died. A similar profile was noted in 2-month-old Gal1R knockout mice. Addition of polyethylene glycol to the drinking water of 15-month-old wild-type mice increased colonic fluid secretion and reduced rates of bacteraemia to those observed in 2-month-old wild-type mice and eliminated fatalities. The difference in response to S typhimurium infection with age may be due, at least in part, to decreased Gal1R expression and decreased amounts of colonic fluid secretion.

International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states.

The mammalian bombesin receptor family comprises three G protein-coupled heptahelical receptors: the neuromedin B (NMB) receptor (BB(1)), the gastrin-releasing peptide (GRP) receptor (BB(2)), and the orphan receptor bombesin receptor subtype 3 (BRS-3) (BB(3)). Each receptor is widely distributed, especially in the gastrointestinal (GI) tract and central nervous system (CNS), and the receptors have a large range of effects in both normal physiology and pathophysiological conditions. The mammalian bombesin peptides, GRP and NMB, demonstrate a broad spectrum of pharmacological/biological responses. GRP stimulates smooth muscle contraction and GI motility, release of numerous GI hormones/neurotransmitters, and secretion and/or hormone release from the pancreas, stomach, colon, and numerous endocrine organs and has potent effects on immune cells, potent growth effects on both normal tissues and tumors, potent CNS effects, including regulation of circadian rhythm, thermoregulation; anxiety/fear responses, food intake, and numerous CNS effects on the GI tract as well as the spinal transmission of chronic pruritus. NMB causes contraction of smooth muscle, has growth effects in various tissues, has CNS effects, including effects on feeding and thermoregulation, regulates thyroid-stimulating hormone release, stimulates various CNS neurons, has behavioral effects, and has effects on spinal sensory transmission. GRP, and to a lesser extent NMB, affects growth and/or differentiation of various human tumors, including colon, prostate, lung, and some gynecologic cancers. Knockout studies show that BB(3) has important effects in energy balance, glucose homeostasis, control of body weight, lung development and response to injury, tumor growth, and perhaps GI motility. This review summarizes advances in our understanding of the biology/pharmacology of these receptors, including their classification, structure, pharmacology, physiology, and role in pathophysiological conditions.

The case for gastrin-releasing peptide acting as a morphogen when it and its receptor are aberrantly expressed in cancer.

Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are frequently expressed by cancers of the gastrointestinal tract, breast, lung, and prostate. Most studies have found that GRP and its amphibian homologue bombesin act to increase tumor cell proliferation, leading to the hypothesis that this peptide hormone is a mitogen important for the growth of various cancers. Yet GRP/GRP-R co-expression in cancer promotes the development of a well-differentiated phenotype; while multiple studies suggest that the presence of these 2 proteins confer a survival advantage. Along with recent reports showing that GRP and its receptor critically regulate aspects of colon and lung organogenesis, we argue that these proteins do not function primarily as mitogens when aberrantly expressed in cancer. Rather, we postulate that GRP/GRP-R are onco-fetal antigens that function as morphogens, with their effect on tumor cell proliferation being a component property of their ability to regulate differentiation. Thus aberrant GRP/GRP-R expression in cancer recapitulates, albeit in a dysfunctional manner, their normal role in development.

Glycosylation of the gastrin-releasing peptide receptor and its effect on expression, G protein coupling, and receptor modulatory processes.

Many gastrointestinal G protein-coupled receptors are glycosylated; however, which potential glycosylation sites are actually glycosylated and their role in receptor transduction or receptor modulation (internalization, down-regulation, desensitization) is largely unknown. We used site-directed mutagenesis to address these issues with the gastrin-releasing peptide receptor (GRP-R). Each of the four potential glycosylation sites was mutated by converting the Asn (N) to Gln (Q). Transient expression in CHOP cells demonstrated that changing Asn(24) or Asn(191) inhibited GRP-R cell surface expression, whereas elimination of Asn(5) and Asn(20) had no effect. Using ligand cross-linking studies in stable mutants expressed in Balb 3T3 cells, all four potential extracellular sites were glycosylated with carbohydrate residues of approximately 13 kDa on Asn(5), 10 kDa on Asn(20), 5 kDa on Asn(24), and 9 kDa on Asn(191). Removal of three glycosylation sites (N5,20,24,Q mutant) did not alter receptor affinity or G protein coupling; therefore, it could be speculated that deglycosylation at Asn(191) might be responsible for the altered G protein coupling seen with complete enzymatic deglycosylation of the native receptor previously reported. Removal of any single glycosylation site did not interfere with GRP-R induced chronic desensitization or down-regulation. However, elimination of all three NH(2)-terminal sites (N5,20,24) markedly attenuated both processes, with no effect on acute homologous desensitization and with only a minimal alteration of GRP-R internalization, supporting the findings of other studies that suggest that chronic desensitization and down-regulation are functionally coupled, distinct from acute desensitization and distinct from internalization. These data show that separate and specific glycosylation sites are important for GRP-R trafficking to the cell surface, ligand binding, G protein coupling, chronic desensitization, and down-regulation.

Galanin-1 receptor up-regulation mediates the excess colonic fluid production caused by infection with enteric pathogens.

Galanin is widely distributed in enteric nerve terminals lining the gastrointestinal tract. We previously showed that pathogenic Escherichia coli, but not normal commensal organisms, increase galanin-1 receptor expression by epithelial cells lining the colon (i.e., colonocytes). When present, galanin-1 receptor activation by ligand causes colonocyte Cl- secretion. We herein demonstrate that disparate pathogens including Salmonella typhimurium and Shigella flexerii also increase colonocyte galanin-1 receptor expression, whose activation is responsible for a principal component of the increased colonic fluid secretion observed. Although eliminating the GAL1R gene by homologous recombination does not alter basal colonic fluid secretion, removal of one or both alleles completely attenuates the increase in fluid secretion due to infection with enteric pathogens. Galanin-1 receptor up-regulation therefore represents a novel mechanism accounting for the increased colonic fluid secretion observed in infectious diarrhea due to several different pathogens.

Characterization of gastrin-releasing peptide and its receptor aberrantly expressed by human colon cancer cell lines.

Gastrin-releasing peptide (GRP) is a mitogen and morphogen important in the development of human colon cancers. Although epithelial cells lining the colon do not normally express GRP or its receptor (GRP-R), most human tumors express GRP-R mRNA. Yet functional protein has only been detected in 24 to 40% of colon cancers. To elucidate the reason for the difference between the expression of GRP/GRP-R mRNA and protein, we studied nine human colon cancer cell lines. Quantitative polymerase chain reaction revealed that all colon cancer cell lines expressed similar amounts of mRNA for both GRP as well as GRP-R. Yet binding studies using (125)I-Tyr(4)-bombesin detected functional receptors on only five of the nine cell lines studied. Conformational fragment-length polymorphism analysis indicated that although mRNA for the ligand GRP was never mutated, mRNA for the GRP-R was always mutated. Sequencing revealed that the message for GRP-R contained between two and seven separate mutations at the nucleotide level. This resulted in 14 separate coding mutations, 2 of which were observed in more than one cell line. Each mutation was individually recreated by site-directed mutagenesis and studied in transiently transfected Chinese hamster ovary-K1 cells. Alteration of Pro(145) into a tyrosine, of Val(317) into a glutamic acid, and insertion of a 32-nucleotide segment resulting in a frameshift distal to Asp(137) all resulted in GRP receptors incapable of binding ligand. Thus, these data indicate that human colon cancers commonly express GRP and GRP-R mRNA but that receptor mutations account for the failure of functional protein to be generated.

Gastrin-releasing peptide is a mitogen and a morphogen in murine colon cancer.

Little is known about the factors involved in regulating the appearance, or differentiation, of solid tumors including those arising from the colon. We herein demonstrate that the mitogen gastrin-releasing peptide (GRP) is a morphogen, critically important in regulating the differentiation of murine colon cancer. Although epithelial cells lining the mouse colon do not normally express GRP and its receptor (GRP-R), both are aberrantly expressed by all better differentiated cancers in wild-type C57BL/6J mice treated with the carcinogen azoxymethane. Whereas small tumors in both wild-type and GRP-R-deficient (i.e., GRP-R-/-) mice are histologically similar, larger tumors become better differentiated in the former but degenerate into more poorly differentiated mucinous adenocarcinomas in the latter. This alteration in phenotype is attributable to GRP increasing focal adhesion kinase expression in GRP-R-expressing tumors. Consistent with GRP acting as a mitogen, GRP/GRP-R coexpressing tumors in wild-type animals also contain more proliferating cells than those occurring in GRP-R-/- mice. Yet tumors are similarly sized in animals of either genotype receiving azoxymethane for identical times, a finding attributable to the significantly higher number of apoptotic cells detected in GRP/GRP-R coexpressing cancers. Thus, these findings indicate that although GRP is a mitogen, aberrant expression does not result in increased tumor growth. Rather, the mitogenic properties of GRP are subordinate to it acting as a morphogen, where it and its receptor are critically involved in regulating colon cancer histological progression by promoting a well-differentiated phenotype.

Dextran sulfate sodium-induced murine colitis activates NF-kappaB and increases galanin-1 receptor expression.

Galanin is widely distributed in enteric nerve terminals and acts to modulate intestinal motility by altering smooth muscle contraction. This ligand causes Cl(-) secretion when colonic epithelial cells express the galanin-1 receptor (Gal1-R) subtype. Because Gal1-R expression by colonic epithelia is upregulated by the transcription factor nuclear factor-kappaB (NF-kappaB), increasingly appreciated as critical in the pathophysiology of inflammatory bowel disease, we wondered whether the diarrhea associated with this condition could be due to NF-kappaB-mediated increases in Gal1-R expression. To test this hypothesis, we provided oral dextran sulfate sodium (DSS) to C57BL/6J mice. Although Gal1-R are not normally expressed by epithelial cells lining the mouse colon, DSS treatment resulted in increased NF-kappaB activation and Gal1-R expression. Whereas galanin had no effect on murine colonic tissues studied ex vivo, it progressively increased short-circuit current and colonic fluid secretion in DSS-treated mice. Concomitant parenteral administration of the NF-kappaB inhibitor dexamethasone attenuated the activation of this transcription factor by DSS, inhibiting the increase in Gal1-R expression. Although Gal1-R-specific antagonists do not exist, intracolonic administration of commercially available galanin antibody diminished the DSS-induced increase in colonic fluid accumulation. Overall, these data demonstrate that a significant component of the excessive fluid secretion observed in DSS-treated mice is due to increased Gal1-R expression.

Why are internal medicine residents at university medical centers not pursuing fellowship training in gastroenterology? A survey analysis.

OBJECTIVE: The decrease in available GI fellowship positions appears to be associated with a disproportionate decrease in the quality of applicants. Thus, the aims of this study were: 1) to determine the current interest in advanced training of nonprimary care internal medicine residents at university medical centers, and 2) to identify the reasons why fellowship-bound residents are not pursuing GI. METHODS: Postage prepaid survey cards were distributed directly to the campus mailboxes of 1862 internal medicine residents at 61 university medical centers at the beginning of their second postgraduate year of training. E-mail questionnaires were then sent to 144 residents planning fellowship training and careers in academia other than in GI/hepatology. RESULTS: A total of 592 residents (32% response) returned completed survey cards. Overall, 392 (66%) indicated that they will pursue fellowship training and approximately 60% wanted to remain in academia. However, <10% indicated an interest in GI/hepatology. E-mail replies were then obtained from 122 residents (87% response) planning academic careers but not in GI. The major reasons for disinterest in GI/hepatology include the perception that jobs are not widely available, that the field is intellectually unchallenging, as well as that is too procedure-oriented. CONCLUSIONS: The majority of residents at university training programs plan advanced training and want to pursue careers in academia, but not in GI/hepatology. Efforts to attract highly qualified residents to GI must emphasize the improved job market, especially as it exists in academia; must advertise research opportunities; and must de-emphasize the procedural nature of this subspecialty.

Quantitative immunohistochemistry by measuring cumulative signal strength using commercially available software photoshop and matlab.

Currently available techniques for performing quantitative immunohistochemistry (Q-IHC) rely upon pixel-counting algorithms and therefore cannot provide information as to the absolute amount of chromogen present. We describe a novel algorithm for true Q-IHC based on calculating the cumulative signal strength, or energy, of the digital file representing any portion of an image. This algorithm involves subtracting the energy of the digital file encoding the control image (i.e., not exposed to antibody) from that of the experimental image (i.e., antibody-treated). In this manner, the absolute amount of antibody-specific chromogen per pixel can be determined for any cellular region or structure. (J Histochem Cytochem 48:303-311, 2000)

Azoxymethane-induced fulminant hepatic failure in C57BL/6J mice: characterization of a new animal model.

Without transplantation, approximately 50-90% of all patients with fulminant hepatic failure (FHF) die. This poor outcome is due in part to the absence of an appropriate animal model, which would allow for a greater understanding of the pathophysiology of this syndrome. Given the reports of liver injury in humans and livestock fed cycad palm nuts on the island of Guam, we hypothesized that the active ingredient azoxymethane (AOM) could cause FHF. We therefore evaluated AOM in C57BL/6J mice. Histologically, we observed microvesicular steatosis 2 h, sinusoidal dilatation 4 h, and centrilobular necrosis 20 h after AOM administration, and transmission electron microscopy showed that this agent causes mitochondrial injury. FHF was associated with all four stages of encephalopathy, as well as by a prodromal period of decreased eating and drinking lasting approximately 15 h before the development of stage I encephalopathy (i.e., loss of scatter reflex). Late encephalopathy was associated with increased arterial ammonia, decreased serum glucose, and evidence of brain edema (astrocyte swelling). We show that AOM-induced FHF is highly reproducible, without evidence of lot-to-lot variability, and is dose dependent. These findings therefore suggest that AOM is an excellent agent for the study of FHF, as well as indicate that Guamanian FHF may be due to AOM found in unwashed cycad palm nuts.

Pathogenic Escherichia coli increase Cl- secretion from intestinal epithelia by upregulating galanin-1 receptor expression.

Galanin is widely distributed in enteric nerve terminals lining the human gastrointestinal (GI) tract. We have shown previously that galanin-1 receptors (Gal1-R) are expressed by epithelial cells lining the human GI tract, and upon activation cause Cl- secretion. Because expression of this receptor is transcriptionally regulated by nuclear factor-kappa B (NF-kappa B), which is activated by enteric pathogens as a part of the host epithelial response to infection, we investigated whether such bacterial pathogens could directly increase Gal1-R expression in the T84-cell model system. Pathogenic Escherichia coli, but not nonpathogenic E. coli, activate a p50/p65 NF-kappa B complex that binds to oligonucleotides corresponding to a recognition site located within the 5' flanking region of the human GAL1R gene. Pathogenic E. coli, but not normal commensal organisms, increase Gal1-R mRNA synthesis and [(125)I]galanin binding sites. Whereas galanin increases short-circuit current (Isc) approximately 5-fold in uninfected T84 cells, exposure to pathogenic, but not nonpathogenic, E. coli results in galanin increasing Isc approximately 20-fold. To confirm the validity of these in vitro observations, we also studied C57BL/6J mice infected with enterohemorrhagic E. coli (EHEC) by gavage. Infection caused a progressive increase in both NF-kappa B activation and Gal1-R expression, with maximal levels of both observed 3 days after gavage. Ussing chamber studies revealed that colons infected with EHEC, but not those exposed to normal colonic flora, markedly increased Isc in response to galanin. These data indicate that pathogen-induced increases in Gal1-R expression by epithelial cells lining the colon may represent a novel unifying pathway responsible for at least a portion of the excessive fluid secretion observed during infectious diarrhea.

Characterization of gastrin-releasing peptide receptors aberrantly expressed by non-antral gastric adenocarcinomas.

Epithelial cells lining the GI tract except in the gastric antrum do not normally express gastrin-releasing peptide receptors (GRP-R). Because GRP-R activation causes the proliferation of many GI cancer cell lines, aberrant expression has been presumed to negatively influence patient survival. We therefore determined the incidence and quality of GRP-R aberrantly expressed by non-antral gastric adenocarcinomas, and evaluated the impact of receptor expression on patient survival. We studied RNA isolated from 20 consecutive non-antral gastric adenocarcinomas, and determined that 8 (40%) aberrantly expressed GRP-R. Of these, 6 (75%) were found to be mutated. Pharmacologically, the effect of these mutations ranged from rendering the GRP-R non-functional to constitutively active. Contrary to expectations, however, survival of patients whose tumor expressed functional GRP-R (18.5 +/- 9.8 months) was not statistically different from those that did not (8.3 +/- 1.8 months; p = 0.24). Thus our data indicate that mutated isoforms of GRP-R are commonly expressed by non-antral gastric adenocarcinomas. However, expression of functional GRP-R does not alter patient survival, suggesting that this receptor may not be clinically important to the growth of gastric cancers.

Aberrant expression of gastrin-releasing peptide and its receptor by well-differentiated colon cancers in humans.

Epithelial cells lining the adult human colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, approximately one-third of human colon cancers and cancer cell lines have been shown to express GRP-binding sites. Because GRPR activation causes the proliferation of many cancer cell lines, GRP has been presumed to act as a clinically significant growth factor. Yet GRP has not been shown to be expressed by colon cancers in humans nor has the effect of GRP and/or GRPR coexpression on tumor behavior been investigated. We therefore determined GRP and GRPR expression by immunohistochemistry in 50 randomly selected colon cancers resected between 1980 and 1997, all 37 associated lymph node and liver metastases, and 20 polyps. Tumor sections studied were those that contained the margin and adjacent nonmalignant epithelium. Overall, 84% of cancers aberrantly expressed GRP or GRPR, with 62% expressing both ligand and receptor, whereas expression was not observed in adjacent normal epithelium. Consistent with the previously established mitogenic capabilities of GRP, tissues coexpressing GRP and GRPR were more likely to express proliferating cell nuclear antigen than tissues not expressing both ligand and receptor. Yet GRP/GRPR coexpression was seen with equal frequency in stage A as in stage D cancers and was only detected in 1 in 37 metastases. Furthermore, Kaplan-Meier analysis did not reveal any difference in patient survival between those whose tumors did or did not express GRP/GRPR. In contrast, GRP/GRPR coexpression was found in all well-differentiated tumor regions, whereas poorly differentiated tissues never coexpressed GRP/GRPR. Overall, these data indicate that, although GRP is a mitogen, it is not a clinically significant growth factor in human colon cancers. Rather, the strong association of GRP/GRPR coexpression with tumor differentiation raises the possibility that these proteins primarily act in vivo as morphogens.

Human colonic epithelial cells express galanin-1 receptors, which when activated cause Cl- secretion.

Galanin is a peptide hormone widely expressed in the central nervous system and gastrointestinal (GI) tract. Within the GI tract galanin is present in enteric nerve terminals where it is known to modulate intestinal motility by altering smooth muscle contraction. Recent studies also show that galanin can alter intestinal short-circuit current (Isc) but with differing results observed in rats, rabbits, guinea pigs, and pigs. In contrast, nothing is known about the ability of galanin to alter ion transport in human intestinal epithelial tissues. By RT-PCR, we determined that these tissues express only the galanin-1 receptor (Gal1-R) subtype. To evaluate Gal1-R pharmacology and physiology, we studied T84 cells. Gal1-R expressed by these cells bound galanin rapidly (half time 1-2 min) and with high affinity (inhibitor constant 0.7 +/- 0.2 nM). T84 cells were then studied in a modified Ussing chamber and alterations in Isc, a measure of all ion movement across the tissue, were determined. Maximal increases in Isc were observed in a concentration-dependent manner around 2 min after stimulation with peptide, with 1 microM galanin causing Isc to rise more than eightfold and return to baseline occurring within 10 min. The increase in galanin-induced Isc was shown by 125I efflux studies to be due to Cl- secretion, which occurred independently of alterations in cAMP and phospholipase C. Rather, Cl- secretion is mediated via a Ca2+-dependent, pertussis toxin-sensitive mechanism. These data suggest that galanin released by enteric nerves may act as a secretagogue in the human colon by activating Gal1-R.

Electrophysiological characterization of human distal colon epithelium isolated using a novel technique.

Electrophysiological studies of human colonic epithelia traditionally have been hampered by the lack of tissue availability and by poor tissue quality. Human colonic epithelium is usually obtained surgically from individuals with underlying disease, while surgery itself can injure or alter the resected tissue. As a result, a wide range in electrophysiological parameters is reported in previous studies of human colonic epithelium. Such factors may also account for differences in measurements between humans and the few other species studied. We therefore devised a novel and rapid endoscopic technique, endoscopic mucosal resection (EMR), that allows for the removal and study of intestinal mucosal epithelium from normal volunteers. Using EMR we rapidly (7.2+/-2.4 min) isolated surgical-sized epithelial sheets from the distal colon (1.4+/-0.4 by 1.3+/-0.4 cm) that were readily mounted in a 0.64-cm2 Ussing chamber. We observed stable resistance (289+/-30 omega cm2), potential difference (1.6+/-0.6 mV), and I(SC)(24+/-9 microA/cm2) for at least 90 min, after which all experiments were terminated. Exposure to carbachol increased I(SC)2.2+/-0.5-fold, while forskolin increased I(SC) 4.4+/-0.5-fold. These data show that the electrophysiological characteristics of the human distal colon removed by EMR more closely approximate values reported for other mammals than when removed using other techniques. Thus EMR represents a significant advance over traditional techniques for isolating human tissues and will increase the availability of this tissue for future studies.

Galanin causes Cl- secretion in the human colon. Potential significance of inflammation-associated NF-kappa B activation on galanin-1 receptor expression and function.

Galanin is widely distributed in enteric nerves and nerve terminals throughout the gastrointestinal (GI) tract. Within the GI tract galanin is best known for its ability to alter smooth muscle contractility and regulate intestinal motility. However, recent studies also indicate that galanin can modulate epithelial ion transport. We previously showed that epithelial cells lining the human GI tract, including those of colonic origin, express Gal1 galanin receptors (Gal1-R). We herein demonstrate that epithelial cells lining the human colon only express Gal1-R receptors and do not express other galanin receptor subtypes. We previously showed that Gal1-R expression was transcriptionally regulated by the transcription factor NF-kappa B. Consistent with this transcription factor being activated in a number of inflammatory conditions, we show increased colonic Gal1-R expression in patients with colitis due to a variety of causes. To further evaluate the physiology of Gal1-R activation, we studied this receptor expressed by the human colon epithelial cell line T84. Gal1-R activation resulted in a dose-dependent increase in Cl- secretion; whereas infection of T84 cells with pathogens known to activate NF-kappa B augmented Gal1-R expression and Cl- secretion. Thus, galanin acts as a secretagogue in epithelial cells lining the human colon, with alterations in Gal1-R expression possibly playing an important role in the diarrhea associated with various inflammatory processes affecting the GI tract.