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The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCTll6-derived Hlda2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1- and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines. caveolin-1; NOS2; SLC3A2; gastrointestinal tissue doi: 10.1152/ajpgi.00169.2010.

Nitric oxide (NO) and ATP mediate smooth muscle relaxation in the gastrointestinal tract. However, the involvement of these neurotransmitters in spontaneous neuronal activity is unknown. The aim of the present work was to study spontaneous neuromuscular transmission in the rat midcolon. Microelectrode experiments were performed under constant stretch both in circular and longitudinal directions. Spontaneous inhibitory junction potentials (sIJP) were recorded. Tetrodotoxin (1 [micro]M) and apamin (1 [micro]M) depolarized smooth muscle cells and inhibited sIJP. [N.sup.[omega]]-nitro-L-arginine (L-NNA, 1 mM) depolarized smooth muscle cells but did not modify sIJP. In contrast, the [P2Y.sub.1] antagonist MRS-2500 (1 [micro]M) did not modify the resting membrane potential (RMP) but reduced sIJP ([IC.sub.50] = 3.1 nM). Hexamethonium (200 [micro]M), NF-023 (10 [micro]M), and ondansetron (1 [micro]M) did not modify RMP and sIJP. These results correlate with in vitro (muscle bath) and in vivo (strain gauges) data where L-NNA but not MRS-2500 induced a sustained increase of spontaneous motility. We concluded that, in the rat colon, inhibitory neurons regulate smooth muscle RMP and cause sIJP. In vitro, the release of inhibitory neurotransmitters is independent of nicotinic, P2X, and 5-hydroxytryptamine type 3 receptors. Neuronal NO causes a sustained smooth muscle hyperpolarization that is responsible for a constant inhibition of spontaneous motility. In contrast, ATP acting on [P2Y.sub.1] receptors is responsible for sIJP but does not mediate inhibitory neural tone. ATP and NO have complementary physiological functions in the regulation of gastrointestinal motility. gastrointestinal; [P2Y.sub.1] receptors; nitric oxide; spontaneous inhibitory junction potential doi: 10.1152/ajpgi.00448.2009.

In smooth muscle, the gating of dihydropyridine-sensitive [Ca.sup.2+] channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for [Ca.sup.2+] influx and determining the bulk average cytoplasmic Ca concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to [Ca.sup.2+] signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (-70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent [Ca.sup.2+] current ([I.sub.Ca]) and evoked a rise in [[Ca.sup.2+]] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [[Ca.sup.2+]] increase ([[[Ca.sup.2+]].sub.c]) was approximately uniform, whereas that of the subplasma membrane space ([[[Ca.sup.2+]].sub.PM]) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active [Ca.sup.2+] channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive [Ca.sup.2+] channels were not normally constitutively active. The repetitive localized [[[Ca.sup.2+]].sub.PM] rises ('persistent [Ca.sup.2+] sparklets') that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active [Ca.sup.2+] channels regulate the bulk average [[[Ca.sup.2+]].sub.c], A dihydropyridine blocker of [Ca.sup.2+] channels, nimodipine, which blocked [I.sub.Ca] and accompanying [[[Ca.sup.2+]].sub.c] rise, reduced neither the resting bulk average [[[Ca.sup.2+]].sub.c] (at -70 mV) nor the rise in [[[Ca.sup.2+]].sub.c], which accompanied an increased electrochemical driving force on the ion by hyperpolarization (-130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent [Ca.sup.2+] channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [[[Ca.sup.2+]].sub.c] regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [[Ca.sup.2+]].

The actions of cholecystokinin (CCK) on gastrointestinal functions occur mainly via paracrine effects on peripheral sensory vagal fibers, which engage vago-vagal brain stem circuits to convey effector responses back to the gastrointestinal tract. Recent evidence suggests, however, that CCK also affects brain stem structures directly. Many electrophysiological studies, including our own, have shown that brain stem vagal circuits are excited by sulfated CCK (CCK-8s) directly, and we have further demonstrated that CCK-8s induces a remarkable degree of plasticity in GABAergic brain stem synapses. In the present study, we used fasted, anesthetized Sprague-Dawley rats to investigate the effects of brain stem administration of CCK-8s on gastric tone before and after activation of the esophageal-gastric reflex. CCK-8s microinjected in the dorsal vagal complex (DVC) or applied on the floor of the fourth ventricle induced an immediate and transient decrease in gastric tone. Upon recovery of gastric tone to baseline values, the gastric relaxation induced by esophageal distension was attenuated or even reversed. The effects of CCK-8s were antagonized by vagotomy or fourth ventricular, but not intravenous, administration of the CCK-A antagonist lorglumide, suggesting a central, not peripheral, site of action. The gastric relaxation induced by DVC microinjection of CCK-8s was unaffected by pretreatment with systemic bethanecol but was completely blocked by [N.sup.G]-nitro-L-arginine methyl ester, suggesting a nitrergic mechanism of action. These data suggest that 1) brain stem application of CCK-8s induces a vagally mediated gastric relaxation; 2) the CCK-8s-induced gastric relaxation is mediated via activation of nonadrenergic, noncholinergic pathways; and 3) CCK-8s reverses the esophageal-gastric reflex transiently. vago-vagal reflexes; gastric reflexes

In Kruppel-like factor (KLF)-4-deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. The aim of this study was to examine whether Notch represses KLF4 expression to regulate goblet cell differentiation. We first detected that KLF4 gene expression was upregulated in a human progastrin-overexpressing mouse model where goblet cell hyperplasia has been observed. We then found that mice treated with a [gamma]-secretase inhibitor (compound E, 10 [micro]mol/kg) for 24 h, which inhibits the Notch signaling pathway, had significantly increased KLF4 mRNA levels in small intestine and colon, accompanied by an increased number of KLF4-expressing cells at the bottom of crypts in small intestine and colon. In a colon cancer cell line (HCT116 cells), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1) by transient cotransfection assays. This inhibition was significantly compromised by a dominant-negative RBPjk, a repressive mediator of the Notch signaling pathway. An ICN1-responsive element was then mapped in the human KLF4 promoter between -151 and -122 nucleotides upstream of the transcriptional start site. It was also found that an intact ICN1-responsive element is required for ICN1 to inhibit KLF4 promoter activity by transient cotransfection assays. Our findings thus reveal a possible mechanism by which KLF4 is inhibited by Notch, which controls goblet cell differentiation in mouse gastrointestinal tract. Kruppel-like factor-4; notch signaling; gene expression; goblet cells

ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland. gastric gland structure; tight junction; development; ezrin; radixin

The gastrointestinal tract of premature infants is a highly fragile organ due to numerous developmental immaturities. Exposure to luminal microbes in the first several weeks of these infants' lives may play a significant role in the development of short-term disease and may have profound effects on long-term health. New non-culture-based techniques are providing exciting new insights into how the intestinal microbiota of premature infants develops and relates to health. A brief summary of recent research in this area is presented, which may be adapted to nutritional strategies for disease prevention.

The current study aimed to evaluate the variation in fermentation activity along the distal canine gastrointestinal tract (GIT, Exp. 1). It also aimed to assess fermentation kinetics and end product profiles of 16 dietary fibers for dog foods using canine fecal inoculum (Exp. 2). For Exp. 1, digesta were collected from the distal ileum, proximal colon, transverse colon, and rectum of 3 adult dogs. Digesta per part of the GIT were pooled for 3 dogs, diluted (1:25, wt/vol), mixed, and filtered for the preparation of inoculum. A fructan, ground soy hulls, and native potato starch were used as substrates and incubated for cumulative gas production measurement as an indicator of the kinetics of fermentation. In addition, fermentation bottles with similar contents were incubated but were allowed to release their gas throughout incubation. Fermentation fluid was sampled at 4, 8, 12, 24, 48, and 72 h after initiation of incubation, and short-chain fatty acids and ammonia were measured. Results showed comparable maximal fermentation rates for rectal and proximal colonic inocula (P > 0.05). Production of short-chain fatty acids was least for the ileal and greatest for the rectal inoculum (P < 0.05). Therefore, for in vitro studies, fecal microbiota can be used as an inoculum source but may slightly overestimate in vivo fermentation. Experiment 2 evaluated the gas production, fermentation kinetics, and end product profiles at 8 and 72 h of incubation for citrus pectin, 3 fructans, gum arabic, 3 guar gums, pea fiber, peanut hulls, soy fiber, sugar beet fiber, sugar beet pectin, sugar beet pulp, wheat fiber, and wheat middlings. Feces of 4 adult dogs were used as an inoculum source. Similar techniques were used as in Exp. 1 except for the dilution factor used (1:10, wt/vol). Among substrates, large variations in fermentation kinetics and end product profiles were noted. Sugar beet pectin, the fructans, and the gums were rapidly fermentable, indicated by a greater maximal rate of gas production ([R.sub.max]) compared with all other substrates (P < 0.05), whereas peanut hulls and wheat fiber were poorly fermentable, indicated by the least amount of gas produced (P < 0.05). Sugar beet fiber, sugar beet pulp, soy fiber, and wheat middlings were moderately fermentable with a low [R.sub.max]. Citrus pectin and pea fiber showed a similar low [R.sub.max], but time at which this occurred was later compared with sugar beet fiber, sugar beet pulp, soy fiber, and wheat middlings (P < 0.05). Results of this study can be used to formulate canine diets that stimulate dietary fiber fermentation along the distal GIT that may optimize GIT health and stimulate the level of satiety in dogs. Key words: dog, fiber, in vitro fermentation kinetics, short-chain fatty acid

The mechanism responsible for fluorescein isothiocyanate (FITC)-albumin internalization by columnar cells in culture obtained from the midgut of Bombyx mori larvae was examined by confocal laser scanning microscopy. Protein uptake changed over time, and it appeared to be energy dependent, since it was strongly reduced by both low temperatures and metabolic inhibitors. Labeled albumin uptake as a function of increasing protein concentration showed a saturation kinetics with a Michaelis constant value of 2.0 [+ or -] 0.6 [micro]M. These data are compatible with the occurrence of receptor-mediated endocytosis. RT-PCR analysis and colocalization experiments with an anti-megalin primary antibody indicated that the receptor involved was a putative homolog of megalin, the multi-ligand endocytic receptor belonging to the low-density lipoprotein receptor family, responsible for the uptake of various molecules, albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required [Ca.sup.2+] for albumin internalization and was inhibited by gentamicin. FITC-albumin internalization was clathrin mediated, since two inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalized in the intermicrovillar areas of the apical plasma membrane. The integrity of actin and microtubule organization was essential for the correct functioning of the endocytic machinery. lepidopteran larval midgut; columnar ceils in culture; albumin endocytosis; clathrin-mediated endocytosis; megalin

Researching the functions of serotonin, or 5-hydroxytryptamine (5-HT), in the gut has helped define new 5-HT receptors, increased the understanding of the side effects of numerous drugs and, via development of drugs, brought relief to millions of people suffering from disorders such as gastroparesis, dyspepsia, constipation, diarrhoea, irritable bowel syndrome and cancer. However, safety issues associated with alosetron and tegaserod (key drugs that modulate 5-HT function) have brought 5-HT and gastrointestinal research to a crossroad--is it now too hard to develop drugs in this area or is there a way forward? In this review, I describe the background to 5-HT in gastrointestinal physiology and disease, and the actions of drugs that interact with [5-HT.sub.3] and [5-HT.sub.4] receptors. Future research directions include modulating 5-HT availability by inhibition of tryptophan hydroxylase, understanding the functions of receptors such as [5-HT.sub.2B], [5-HT.sub.7] and the recently described [5-HT.sub.3]-receptor subunits, and investigating how receptors activated by other products of tryptophan catabolism interact with gastrointestinal functions of 5-HT.

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in [H.sup.+]/[K.sup.+]ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparinbinding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer. gastric cancer; primary gastric epithelial cells; cagE; HB-EGF

This investigation demonstrates the presence and binding of the protein LC8 (described as 'protein inhibitor of nNOS' or PIN in some reports) to different components of neuronal nitric oxide synthase (nNOS) in nitrergic varicosities of mice gut. Whole varicosity extracts showed three (320-, 250-, and 155-kDa) nNOS bands with anti-[nNOS.sub.1422-1433] antibody and a 10-kDa band with anti-LC8 antibody. The LC8 immunoprecipitate (IP) showed three nNOS bands, suggesting that LC8 was bound with all three forms of nNOS but dissociated from them during SDS-PAGE. Studies using LC8 IP and supernatant and probed with anti-CaM showed that LC8 was not associated with CaM-bound 320-kDa nNOS but was present in the CaM-lacking fraction. Probing these fractions with anti-serine847-P-nNOS showed that 320-kDa serine847-phosphorylated-nNOS consisted of LC8-bound and LC8-1acking components. Subsequent studies with varicosity membrane and cytosolic fractions separately showed that membrane contained CaM-bound and CaM-lacking, serine847-phosphorylated 320-kDa nNOS; both these fractions lacked LC8. On the other hand, the cytosolic fraction contained CaM-lacking, serine847-phosphorylated 320-kDa, 250-kDa, and 155-kDa nNOS bands that were all associated with LC8. These studies, along with in vitro nitric oxide assays, show that in gut nitrergic nerve varicosities 1) all cytosolic serine847-phosphorylated nNOS was catalytically inactive and bound with LC8, and 2) membrane-associated nNOS consisted of catalytically active, CaM-bound and catalytically inactive, CaM-lacking, serine847-phosphorylated nNOS[alpha] dimers, both of which lacked LC8. These results suggest that LC8 may dissociate from the 320-kDa nNOS[alpha] dimer upon binding to membrane, thus supporting the view that LC8 may transport nNOS[alpha] dimer to the varicosity membrane for participation in nitrergic neurotransmission. protein inhibitor of nNOS (PIN), dynein light chain (DLC8 or LC8), nNOS; membrane-bound nNOS; cytosolic nNOS; nitrergic neurotransmission

Chronic administration of anorexigenic substances to experimental animals by injections or continuous infusion typically produces either no effect or a transient reduction in food intake and body weight. Our aim here was to identify an intermittent dosing strategy for intraperitoneal infusion of peptide YY(3-36) [PYY(3-36)] that produces a sustained reduction in daily food intake and adiposity in diet-induced obese rats. Rats (665 [+ or -] 10 g body wt, 166 [+ or -] 7 g body fat) with intraperitoneal catheters tethered to infusion swivels had free access to a high-fat diet. Vehicle-treated rats (n = 23) had relatively stable food intake, body weight, and adiposity during the 9-wk test period. None of 15 PYY(3-36) dosing regimens administered in succession to a second group of rats (n = 22) produced a sustained 15-25% reduction in daily food intake for >5 days, although body weight and adiposity were reduced across the 9-wk period by 12% (594 [+ or -] 15 vs. 672 [+ or -] 15 g) and 43% (96 [+ or -] 7 vs. 169 [+ or -] 9 g), respectively. The declining inhibitory effect of PYY(3-36) on daily food intake when the interinfusion interval was [greater than or equal to] 3 h appeared to be due in part to an increase in food intake between infusions. The declining inhibitory effect of PYY(3-36) on daily food intake when the interinfusion interval was < 3 h suggested possible receptor downregulation and tolerance to frequent PYY(3-36) administration; however, food intake significantly increased when PYY(3-36) treatments were discontinued for 1 day following apparent loss in treatment efficacies. Together, these results demonstrate the development of a potent homeostatic response to increase food intake when PYY(3-36) reduces food intake and energy reserves in diet-induced obese rats. gastrointestinal; peptide; intraperitoneal administration; anorexia; body composition

Endogenous ghrelin causes interdigestive contractions of the stomach in rats. In contrast, previous studies showed that 5-HT3 and 5-H[T.sub.4] receptors were involved in regulating intestinal interdigestive contractions. We studied the possible role of endogenous ghrelin and 5-HT regulating interdigestive gastrointestinal (GI) contractions in rats. Four strain gauge transducers were implanted on the antrum, duodenum, and proximal and distal jejunum. After an overnight fast, GI contractions were recorded in freely moving conscious rats and ghrelin receptor antagonists [(D-lys3)GHRP6; 1 [micro]mol/kg], 5-H[T.sub.3] antagonists (Ondansetron; 0.5 mg/kg) and 5-HT4 antagonists (GR 125,487; 1 mg/kg) were administered (bolus iv). To evaluate the relationship between the luminal concentrations of 5-HT and phase III-like contractions of the duodenum, duodenal juice was collected via the intraduodenal catheter. 5-HT content of the duodenal juice was measured by HPLC. (D-Iys3)GHRP6 significantly attenuated the occurrence and amplitude of phase III-like contractions of the antrum, but not the duodenum and jejunum. 5-HT4 antagonists significantly reduced spontaneous phase III-like contractions of the jejunum, without affecting those of the antrum and duodenum. In contrast, 5-H[T.sub.3] antagonists did not affect phase III-like contractions in GI tract. Luminal concentration of 5-HT at the phase III-like contraction (36.0 [+ or -] 13.3 ng/ml, n = 9) was significantly higher than that at the phase I-like contractions of the duodenum (4.9 [+ or -] 1.6 ng/ml, n = 9, P < 0.05). It is suggested that released ghrelin from the gastric mucosa mediates gastric phase III-like contractions, whereas 5-HT released from enterochromaffin cells of the duodenal mucosa mediates intestinal phase III-like contractions via 5-H[T.sub.4] receptors. 5-H[T.sub.3] receptors; 5-H[T.sub.4] receptors; enterochromaffin cells

Mechanisms by which gut luminal content regulates secretion and motility are ill understood. We evaluated whether neuroendocrine enterochromaffin (EC) cells act as luminal sensors for a wide variety of nutrients and defined the secretory mechanisms of this process. Pure (98-99%) FACS-sorted human EC cells and neoplastic EC cells (KRJ-I) were studied. RT-PCR identified transcripts for T2R1 (bitter), OR1G1 (class II olfactory) and trace amine (TAR1) G protein-coupled receptors (GPCRs) and transporters for glutamine (SNAT1/ 2), glucose (GLUT1/3/SGLT1), and bile salts (ABST). Glutamine and sodium deoxycholate stimulated 5-HT release (E[C.sub.50] = 0.002-0.2 [micro]M; 2-fold release) but were 10-100 times more potent in neoplastic EC cells, which also secreted 6-13 times more 5-HT. Tastants (caffeine, tyramine, octopamine) and olfactants (thymol and eugenol) also stimulated normal and neoplastic EC cell 5-HT secretion (E[C.sub.50] = 1.2 nM to 2.1 [micro]M and 0.05 [micro]M to 0.1 [micro]M release, respectively); 2-deoxyglucose and the artificial sweetener sucralose also stimulated (E[C.sub.50] = 9.2 and 0.38 nM). 5-HT release was associated with ERK pbosphorylation (1.5-fold, P < 0.02) and could be inhibited by a somatostatin analog (I[C.sub.50] = 1 pM). Eleven secretory associated genes including the vesicle docking inhibitor STXBP3 were upregulated in response to glutamine and bile salt stimulation in neoplastic EC cells. Targeting STXBP3 expression by use of antisense knockdown significantly (P < 0.05) reduced 5-HT secretion. In conclusion, EC cells express GPCRs and transporters for luminal tastants, olfactants, glutamine, glucose, and bile salts. Activation includes a panel of secretory genes, ERK phosphorylation, and 5-HT secretion. Luminal EC cell regulation is likely to be as important as G cell regulation in gastric acid secretion; development of agents to target EC cell function is therefore a critical therapeutic goal. gastrointestinal; neuroendocrine; olfactant; secretion; tastant; enterochromaffin cells

Food and parasites can independently play a role in destabilizing population fluctuations of animals, and yet, more than 50 years ago, David Lack proposed that these two factors should act in concert. We examined the role of these factors on the vital rates of freeliving white-footed mice (Peromyscus leucopus) over the summer and autumn months. We used a replicated factorial experiment in which deer exclosures doubled acorn availability and anthelmintic application reduced gastrointestinal helminths. Specifically, we wanted to know if either factor or an interaction between the two accounted for the midsummer breeding hiatus observed in this species. We found no influence of habitat quality on mouse breeding, vital rates, or demography; however, anthelmintic treatment resulted in mice continuing to reproduce during the hiatus at the same rate as previously, and they also exhibited increased body condition, growth rate, and survival. These results provide evidence that gastrointestinal helminths reduce P. leucopus reproductive output in central Pennsylvania, and these effects on reproduction could play a role in the unstable dynamics of small mammals. Key words: acorn; anthelmintic treatment; body condition; gastrointestinal helminths; habitat; mouse; nematode; parasite; Peromyscus leucopus; reproduction; small mammal.

Aquaporin (AQP)-mediated intestinal water absorption may play a major osmoregulatory role in euryhaline teleosts, although the molecular identity and anatomical distribution of AQPs in the fish gastrointestinal tract is poorly known. Here, we have investigated the functional properties and cellular localization in the intestine of two gilthead seabream (Sparus aurata) homologs of mammalian aquaporin-1 (AQPI), named SaAqp1a and SaAqp1b. Heterologous expression in Xenopus laevis oocytes showed that SaAqp1a and SaAqp1b were water-selective channels. Real-time quantitative RT-PCR and Western blot using specific antisera indicated that abundance of SaAqp1a mRNA and protein was higher in duodenum and hindgut than in the rectum, whereas abundance of SaAqp1b was higher in rectum. In duodenum and hindgut, SaAqp1a localized at the apical brush border and lateral membrane of columnar enterocytes, whereas SaAqp1b was detected occasionally and at very low levels at the apical membrane. In the rectum, however, SaAqp1a was mainly accumulated in the cytoplasm of a subpopulation of enterocytes spread in groups over the surface of the epithelia, including the intervillus pockets, whereas SaAqp1b was detected exclusively at the apical brush border of all rectal enterocytes. Freshwater acclimation reduced the synthesis of SaAqp1a protein in all intestinal segments, but it only reduced SaAqp1b abundance in the rectum. These results show for the first time in teleosts a differential distribution and regulation of two functional AQP1 homologs in the intestinal epithelium, which suggest that they may play specialized functions during water movement across the intestine. gilthead seabream; SaAqp1a; SaAqp1b; functional expression; salinity; gastrointestinal tract

We studied the effect of dietary free peptides vs. peptides released naturally during digestion on gut endogenous nitrogen (N) flow (ENFL) and amino acid (AA) flow (EAAFL). Semisynthetic diets containing 110 g/kg diet of the same casein, intact (C) or hydrolyzed (HC), were formulated with Ti[O.sub.2] as a dietary marker. Sprague-Dawley rats were fed the diets hourly (0800-1500 h) for 10 min each hour for 7 d. Rats received unlabeled diets for 6 d and [sup.15]N-labeled diets on d 7, whereby they were killed and digesta sampled (6 observations per group) along the intestinal tract. EAAFL and ENFL were determined by [sup.15]N-isotope dilution (ID) for C or by ID or after centrifugation and ultrafiltration (UF) for HC. Ileal EAAFL and ENFL (ID) were not enhanced with diet HC compared with diet C. The AA compositions (g/16 g N) of ileal ENFL did not differ between rats fed HC and C except for Asp, Phe, Tyr, and Ser, for which contributions were relatively lower (P < 0.05) for rats fed C. Ileal EAAFL and ENFL (HC) were considerably lower (P< 0.05) with ID than with UF, but flows of Gly, Phe, and His were similar. There was no stimulatory effect of dietary peptides from HC on endogenous ileal protein flow compared with C, but the result is tentative given the high degree of dietary N recycled within endogenous protein and which could have occurred at a differential rate between rats fed diets Cand HC.

We have previously shown that plasma protein supplementation prevents the activation of lymphocyte populations of Peyer's patches and mesenteric lymph nodes, which is known as organized gut-associated lymphoid tissue (GALT). Here, we examined the effects of spray-dried plasma proteins (SDAP) and Ig concentrate (IgC) supplements on lamina propria and intraepithelial lymphocytes (diffuse GALT) in a model of mild intestinal inflammation induced by the intraperitoneal administration of Staphylococcus aureus enterotoxin B (SEB). Wistar-Lewis rats were fed diets supplemented with SDAP (8% wt:wt), IgC (1.5% wt:wt), or bovine milk proteins (control diet) from weaning (d 21) to d 34 after birth. On d 30 and 33, rats were given SEB (0.5 mg/kg body weight) or PBS (control). Experimental groups were designated control, SEB, SEB-SDAP, and SEB-IgC. Lymphocyte populations were analyzed by immunohistochemistry. In lamina propria, SEB increased the cytotoxic lymphocyte populations of T-[gamma][delta] cells (38%; P < 0.001 ) and natural killer cells (59%; P < 0.05) and the number of activated T lymphocytes (148%; P < 0.001 ). Both SDAP and IgC decreased the effects of SEB on these lymphocyte subsets (P < 0.05). In the epithelium, SEB induced a 117% increase in intraepithelial-activated lymphocytes that was reduced by SDAP supplementation (P < 0.01). The effects of plasma supplements on intestinal lymphocyte populations suggest that oral plasma proteins can modulate the degree of activation of diffuse GALT.

Using a comparative approach, we examined the gut morphology of 6 ecologically similar rodent species coexisting in an African savanna. Although all species are primarily granivores, we aimed to establish whether modifications of the gut morphology exist that would promote the potential for exploiting different food types, particularly foliage. The species examined exhibited a gradation in gut structure, with some showing modifications more toward granivory and others toward folivory. These findings are supported by studies of food preferences in captivity. Modifications of the gut, enabling the potential for exploitation of a greater folivorous diet in some species compared to others, may be 1 way in which dietary partitioning as a method of coexistence may be promoted. Nonetheless, other mechanisms, such as spatial and temporal segregation, social tolerance, and behavioral differences, also may determine coexistence in this community. Key words: coexistence, dietary partitioning, food preferences, folivory, granivory, gut morphology

Purinergic receptors comprise a family of transmembrane receptors that are activated by extracellular nucleosides and nucleotides. The two major classes of purinergic receptors, P1 and P2, are expressed widely in the gastrointestinal tract as well as immune cells. The purinergic receptors serve a variety of functions from acting as neurotransmitters, to autocoid and paracrine signaling, to cell activation and immune response. Nucleosides and nucleotide agonist of purinergic receptors are released by many cell types in response to specific physiological signals, and their levels are increased during inflammation. In the past decade, the advent of genetic knockout mice and the development of highly potent and selective agonists and antagonists for the purinergic receptors have significantly advanced the understanding of purinergic receptor signaling in health and inflammation. In fact, agonist/antagonists of purinergic receptors are emerging as therapeutic modalities to treat intestinal inflammation. In this article, the distribution of the purinergic receptors in the gastrointestinal tract and their physiological and pathophysiological role in intestinal inflammation will be reviewed. adenosine receptor; P2 receptor; inflammation

Hedgehog (Hh) proteins are members of a family of secreted signaling factors that orchestrate the development of many organs and tissues including those of the gastrointestinal (GI) tract. The requirement for Hh activity is not limited to early development but underlies the homeostasis of a number of tissues, and abnormal activity of the Hh pathway is associated with several GI malignancies. Understanding the roles and mechanisms of action of Hh signaling both in development and postnatally should thus give novel insights into potential treatments for these diseases. Here we focus on the Hh signaling pathway and its role in GI tract development and maintenance and consider the diseases resulting from aberrant Hh activity. gastrointestinal tract; VACTERL; pancreas; cyclopamine; anorectal malformation

A fundamental way in which animal-dispersed plants can influence the viability and distribution of dispersed seeds is through control of retention time in the guts of dispersers. Using two species of wild chilies and their dispersers, we examined how chemical and physical properties of fruits and seeds mediate this interaction. Capsicum chacoense is polymorphic for pungency, occurs in Bolivia, and is dispersed mostly by elaenias. Capsicum annuum is not polymorphic, occurs in Arizona (USA), and is dispersed mostly by thrashers. We first tested whether capsaicin, the substance responsible for the pungency of chilies, affects gut retention time of seeds in primary dispersers. Capsaicin slowed gut passage of seeds but did so in a manner that differed greatly between bird species because the constipative effects of capsaicin occurred only after an 80-minute time lag. Elaenias in Bolivia held only 6% of C. chacoense seeds for >80 minutes, whereas thrashers in Arizona held 78% of C. annuum seeds for >80 minutes. Next we examined the effects of retention time on seed viability and germination. Increased retention resulted in a greater proportion of seeds germinating in C. annuum, had no effects on non-pungent C. chacoense, and had negative effects on pungent C, chacoense. These divergent effects are explained by differences in seed coat morphology: seed coats of pungent C. chacoense are 10-12% thinner than those of the other two types of seeds. Thus, longer retention times damaged seeds with the thinnest seed coats. In C. annuum, seed viability remained high regardless of retention time, but germination increased with retention, suggesting a role for scarification. Thus, in C. annuum, fruit chemistry appears well matched with seed morphology and disperser physiology: capsaicin extends gut retention for most seeds, resulting in greater seed scarification and higher germination rates. Increased retention of pungent C. chacoense seeds is detrimental, but because the primary consumers have short retention times, capsaicin slows only a small proportion of seeds, minimizing negative effects. These results illustrate the importance of context in studies of fruit secondary metabolites. The same chemical can have different impacts on plant fitness depending on its morphological, physiological, and ecological context. Key words: capsaicin capsaicinoids; Capsicum annuum; Capsicum chacoense, chilies; Elaenia parvirostris; frugivory; fruit-frugivore interactions; fruit secondary metabolites; gut retention; scarification; seed germination; Toxostoma curvirostre.

Using a recently developed in vitro preparation of vagal afferent pathways, we examined the role of TRPV1 and ASIC3 on the mechano- and chemosensitive properties of gastroesophageal sensory neurons. Esophagus, stomach, and the intact vagus nerves up to the central terminations were carefully dissected from TRPV1 and ASIC3 knockout mice and wild-type controls. The organ preparation was placed in a superfusion chamber to obtain intracellular recordings from the soma of nodose neurons during luminal stimulation of esophagus and stomach. The proximal esophagus and distal stomach were separately intubated to allow perfusion and graded luminal distension. In wild-type mice, mechanosensitive neurons were activated by low distension pressures and encoded stimulus intensity over the entire range tested. Luminal acidification significantly transiently increased the resting frequency but did not alter responses to subsequent mechanical stimulation. ASIC3 and TRPV1 knockout significantly blunted responses to distension compared with wild-type controls, with deletion of TRPV1 having a more significant effect than ASIC3 deletion. Luminal acidification did not activate mechanosensory neurons in ASIC3 and TRPV1 knockout mice. Our data demonstrate a role of TRPV1 in chemo- and mechanosensation of gastroesophageal afferents. ASIC3 may contribute to acid sensation but plays a more subtle role in responses to distending stimuli. Considering the importance of acid in dyspeptic symptoms and gastroesophageal reflux, TRPV1 or ASIC3 may be an attractive target for treatment strategies in patients who do not respond to acid suppressive therapy. visceral sensation; electrophysiology; mechanosensation; chemosensation

Supplementation of prebiotic compounds, including short-chain fructooligosaccharides (scFOS) has been shown to confer benefits on nutrient utilization, growth, and disease resistance of various animal species through improved gastrointestinal (GI) microbiota. However, potential uses of prebiotics for shrimp have not been defined. A 6-wk feeding trial was conducted in a recirculating system to determine the effects of scFOS supplementation on growth performance, immune functions, and GI microbiota composition of Pacific white shrimp (Litopenaeus vannamei), scFOS was supplemented in a nutritionally complete diet (35% crude protein) at 0.025, 0.0500, 0.075, 0.100, 0.200, 0.400, and 0.800% by weight. After 6 wk of feeding, shrimp fed 0, 0.1, and 0.8% scFOS were sampled for assays of immune function and GI microbiota. Dietary supplementation of scFOS did not improve weight gain, feed conversion ratio, or survival of shrimp. Denaturing gradient gel electrophoresis analysis suggested the intestinal tract microbial community from shrimp fed the basal diet was different from that of shrimp fed the scFOS diets [similarity coefficient (SC) = 74.9%)], although the intestinal tract microbial community from shrimp fed the scFOS-supplemented diets was very similar (SC = 92.3%). All the bacterial species contributing to the GI microbial differences were identified, although most of them are uncultured species. Both total hemocyte count and hemocyte respiratory burst increased (P < 0.05) by incremental dietary supplementation of scFOS (0-0.8%). This study is the first to our knowledge to show that dietary scFOS can selectively support growth of certain bacterial species in the G I tract of shrimp and enhance immunity, which may facilitate development of alternative strategies, including novel probiotics and synbiotics, for shrimp growth and health management. J. Nutr. 137: 2763-2768, 2007.

Several findings suggest the existence of a 'fatty' taste, and the CD36 fatty acid translocase is a candidate taste receptor. The present study compared fat preference and acceptance in CD36 knockout (KO) and wild-type (WT) mice using nutritive (triglyceride and fatty acid) and nonnutritive (Sefa Soyate oil) emulsions. In two-bottle tests (24 h/day) naive KO mice, unlike WT mice, displayed little or no preference for dilute soybean oil, linoleic acid, or Sefa Soyate emulsions. At high concentrations (2.5-20%), KO mice developed significant soybean oil preferences, although they consumed less oil than WT mice. The postoral actions of fat likely conditioned these preferences. KO mice, like WT mice, learned to prefer a flavored solution paired with intragastric soybean oil infusions. These findings support CD36 mediation of a gustatory component to fat preference but demonstrate that it is not essential for fat-conditioned flavor preferences. The finding that oil-naive KO mice failed to prefer a nonnutritive oil, assumed to provide texture rather than taste cues, requires explanation. Finally, CD36 deletion decreased fat consumption and enhanced the ability of the mice to compensate for the calories provided by their optional fat intake. linoleic acid; soybean oil; Sefa Soyate oil; flavor conditioning; gastric infusions

The effects of ground flaxseed (FS) and a multicarbohydrase enzyme (C) supplement on piglet performance, gastrointestinal microbial activity, and nutrient digestibility were investigated in a 28-d trial. The enzyme supplement provided 500 units of pectinase, 50 units of cellulase, 400 units of mannanase, 1,200 units of xylanase, 450 units of glucanase, and 45 units of galactanase per kilogram of diet. Ninety-six pigs were weaned at 17 d of age (BW, 6.1 [+ or -] 0.4 kg, mean [+ or -] SD) and assigned to treatments based on a 2 x 2 factorial arrangement in a completely randomized design, with 6 pens per diet (4 pigs per pen). The diets contained wheat, barley, peas, soybean meal, and canola meal with 0 or 12% FS, and were fed without or with C. Flaxseed was included by changing the levels of the other ingredients to balance the diets for DE and nutrients. Diets had similar nutrient contents and met the NRC (1998) nutrient specifications, with the exception of DE, CP, and AA, which were 95, 94, and 97% of the NRC requirements, respectively. Diets were fed in a 2-phase feeding program (2 wk/phase). Feed intake and BW were measured weekly, and 1 pig per pen with a BW nearest the pen average was bled weekly to evaluate plasma urea nitrogen. On d 28, fresh fecal samples were collected from each pen and 1 pig per pen with a BW nearest the pen average was killed to evaluate intestinal microbial activity and nutrient digestibility. A dietary effect on piglet performance was observed only in wk 3, when the FS diets decreased (P = 0.005) ADG and G:F, tended to decrease (P = 0.070) ADFI, and increased (P = 0.027) plasma urea nitrogen. An interaction between FS and C was observed for ileal digesta viscosity (P = 0.045), such that C increased viscosity in the FS diet but had no effect in the non-FS diet. Flaxseed and C interacted to affect ileal ammonia content (P = 0.049), such that in the absence of FS, pigs fed the diet with C had lower ammonia than those on the diet without C. Flaxseed and C affected other ileal parameters independently. Pigs fed the FS diets had decreased (P = 0.003 to 0.033) anaerobic spore counts, organic acid, DM, CP, and nonstarch polysaccharide (NSP) digestibility compared with pigs fed the non-FS diets, whereas pigs fed the C-supplemented diets had greater (P = 0.009 to 0.061) lactobacilli counts, lactate, DM, and NSP digestibility than pigs fed the unsupplemented diets. In conclusion, FS reduced ileal microbial activity, nutrient digestibilities, and piglet performance in wk 3. The multicarbohydrase supplement increased ileal DM and NSP digestibilities as well as lactobacilli counts and lactate. Key words: carbohydrase, early-weaned pig, flaxseed, growth performance, intestinal microbial activity, nutrient digestibility

To investigate the microbial and nutritional characteristics of dry feed, liquid feed containing fermented liquid cereal grains, and fermented liquid feed, and their effect on gastrointestinal ecology and growth performance, 120 piglets from 40 litters were used and housed in pens with 5 animals in each. The 3 dietary treatments (all nonheated and nonpelleted diets) were: a dry meal diet (DRY); a fermented, liquid cereal grain feed (FLG); and a fermented liquid feed (FLF). The FLG diet was prepared by storing the dietary cereals (barley and wheat) and water (1:2.5, wt/ wt) in a closed tank at 20[degrees]C and adding the remaining dietary ingredients immediately before feeding. The FLF diet was prepared by storing compound feed and water (1:2.5, wt/wt) in a closed tank at 20[degrees]C. Three times daily, 50% of the fermented cereals or compound feed and water stored in the tanks was removed and replaced with an equal amount of fresh cereals or feed and water. On d 14, 1 piglet from each pen was killed and samples from the gastrointestinal tract were obtained. The pH of the fermented cereals was 3.85 (SD = 0.10), that of the FLG diet was 5.00 (SD = 0.18), and the pH of the FLF diet was 4.45 (SD = 0.11). The dietary concentration of lysine (g/16 g of N) pointed to a decreased concentration in the FLF (5.46, SD = 0.08) com pared with the DRY (6.01) and FLG (6.21, SD = 0.27) diets, and the concentration of cadaverine was greater in the FLF diet (890 mg/kg, SD = 151.3) than in the DRY (32 mg/kg) or FLG (153 mg/kg, SD = 18.7) diets. Fermenting only the cereal component of the diet (FLG) promoted the growth of yeasts to a greater extent than fermenting the whole diet (FLF). Terminal RFLP profiles of diets and digesta from the stomach and midcolon showed differences among dietary groups. The number of yeasts able to grow at 37[degrees]C in the stomach and caudal small intestine was greatest in the FLG group compared with the other 2 dietary groups (P < 0.01). In the cecum and colon, the differences were only significant between piglets fed the FLG and the FLF diets (P < 0.05). The greatest number of yeasts able to grow at 20[degrees]C was detected in the animals fed the FLG diet. However, the values were different from the FLF-fed piglets only in the stomach (P < 0.05) and midcolon (P < 0.05). There was a tendency (P < 0.10) for greater ADG of the piglets fed the FLG compared with the FLF diet. Feeding liquid feed containing fermented, liquid cereal grains as a means of avoiding microbial decarboxylation of free amino acids in the feed and increasing feed intake by improving palatability seems promising but requires further investigation. Key words: digestive tract, fermented cereal, growth, liquid diet, microbiota, piglet