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Glucagon-like peptide-2 (GLP-2) reduces mouse gastric tone and small intestine transit, but its action on large intestine motility is still unknown. The purposes of the present study were 1) to examine the influence of GLP-2 on spontaneous mechanical activity and on neurally evoked responses, by recording intraluminal pressure from mouse isolated colonic segments; 2) to characterize GLP-2 mechanism of action; and 3) to determine the distribution of GLP-2 receptor (GLP-2R) in the mouse colonic muscle coat by immunohistochemistry. Exogenous GLP-2 (0.1-300 nM) induced a concentration-dependent reduction of the spontaneous mechanical activity, which was abolished by the desensitization of GLP-2 receptor or by tetrodotoxin, a voltage-dependent [Na.sup.+]-channel blocker. GLP-2 inhibitory effect was not affected by [N.sup.[omega]]-nitro-L-arginine methyl ester (a nitric oxide synthase inhibitor), apamin (a blocker of small conductance [Ca.sup.2+]-dependent [K.sup.+] channels), or [Lys1,Pro2,5,Arg3,4,Tyr6][VIP.sup.7-28] (a VIP receptor antagonist), but it was prevented by atropine or pertussis toxin (PTX), a [G.sub.i/o] protein inhibitor. Proximal colon responses to electrical field stimulation were characterized by nitrergic relaxation, which was followed by cholinergic contraction. GLP-2 reduced only the cholinergic evoked contractions. This effect was almost abolished by GLP-2 receptor desensitization or PTX. GLP-2 failed to affect the contractile responses to exogenous carbachol. GLP-2R immunoreactivity (IR) was detected only in the neuronal cells of both plexuses of the colonic muscle coat. More than 50% of myenteric GLP-2R-IR neurons shared the choline acetyltransferase IR. In conclusion, the activation of GLP-2R located on cholinergic neurons may modulate negatively the colonic spontaneous and electrically evoked contractions through inhibition of acetylcholine release. The effect is mediated by [G.sub.i] protein. enteric nervous system; gastrointestinal hormones; colonic motility; acetylcholine doi: 10.1152/ajpgi.00282.2010.

The colon is believed to absorb NaCl via the coupled operation of apical Na/H exchanger-3 (NHE3) and Cl/HC[O.sub.3] exchanger SLC26A3 (DRA). Efficient coupling requires that NHE3 and DRA operate in close proximity within common luminal and cytosolic microenvironments. Thus we examined whether these proteins coexist along the apical margin of surface enterocytes by quantitative immunofluorescence microscopy in consecutive colon segments from nonfasted mice and rats. The cecocolonic profiles of NHE3 and DRA expression were roughly inverse; NHE3 was highest in proximal colon (PC) and negligible in distal colon, whereas DRA was absent in early PC and highest in the late midcolon, and DRA was prominent in the cecum whereas NHE3 was not. NHE3 and DRA coexisted only in the middle third of the colon. The consequences of unpaired NHE3/DRA expression on mucosal surface (subscript MS) pH and [Na.sup.+] concentration ([[Na.sup.+]]) were assessed in nonfasted rats in situ using miniature electrodes. In the cecum, where only DRA is expressed, prims was ~7.5, markedly higher than underlaying stool (6.3), consistent with net HC[O.sup.-.sub.3] secretion. In the early PC, where NHE3 is not expressed with DRA, prims was acidic (6.2), consistent with unopposed [H.sup.+] secretion. [[Na.sup.+]]Ms was ~60 mM in the cecum, decreased along the PC to ~20 mM, and declined further to ~10 mM distally. [C1.sup.-] was secreted into the PC, then reabsorbed distally. Our results suggest a model in which 1) unpaired DRA activity in the cecum maintains an alkaline mucosal surface that could neutralize fermentative [H.sup.+]; 2) unpaired NHE3 activity in the early PC preserves an acidic mucosal surface that could energize short-chain fatty acid absorption; and 3) coupled NHE3/ DRA activities in the midcolon allow for vigorous NaC1 absorption at a neutral prims. mouse; rat; intestine; electroneutral NaCl absorption doi: 10.1152/ajpgi.00151.2010.

Information about colorectal distension (i.e., colorectal dilation by increased intraluminal pressure) is primarily encoded by stretch-sensitive colorectal afferents in the pelvic nerve (PN). Despite anatomic differences between rectum and distal colon, little is known about the functional roles of colonic vs. rectal afferents in the PN pathway or the quantitative nature of mechanosensory encoding. We utilized an in vitro mouse colorectum-PN preparation to investigate pressure-encoding characteristics of colorectal afferents. The colorectum with PN attached was dissected, opened longitudinally, and pinned flat in a Sylgard-lined chamber. Action potentials of afferent fibers evoked by circumferential stretch (servo-controlled force actuator) were recorded from the PN. Stretch-sensitive fibers were categorized into the following four groups: colonic muscular, colonic muscular/mucosal, rectal muscular, and rectal muscular/mucosal. Seventy-nine stretch-sensitive PN afferents evenly distributed into the above four groups were studied. Rectal muscular afferents had significantly greater stretch-responses than the other three groups. Virtually all rectal afferents (98%) had low thresholds for response and encoded stimulus intensity into the noxious range without obvious saturation. Most colonic afferents (72%) also had low thresholds (< 14 mmHg), but a significant proportion (28%) had high thresholds (>18 mmHg) for response. These high-threshold colonic afferents were sensitized to stretch by inflammatory soup; response threshold was significantly reduced (from 23 to 12 mmHg), and response magnitude significantly increased. These results suggest that the encoding of mechanosensory information differs between colonic and rectal stretch-sensitive PN afferents. Rectal afferents have a wide response range to stretch, whereas high-threshold colonic afferents likely contribute to visceral nociception. muscular afferents; muscular/mucosal afferents; single-fiber recording; irritable bowel syndrome doi:10.1152/ajpgi.00487.2009.

Facilitative UT-B urea transporters enable the passage of urea across cell membranes. Gastrointestinal urea transporters are thought to play a significant role in the urea nitrogen salvaging process that occurs between mammalian hosts and their gut bacteria. This study investigated the expression of UT-B urea transporters in different segments of human colon. Immunoblot analysis showed that human colon expressed a 35-kDa glycosylated UT-B protein in the colonic mucosa. The 35-kDa UT-B transporter was predominantly located in plasma membrane-enriched samples (P < 0.001; n = 6), and its expression was greater in the ascending colon compared with the descending colon (P < 0.01 ; n = 3). At the cellular level, UT-B transporters were located throughout colonocytes situated in the upper portion of the colonic crypts. Bidirectional trans-epithelial urea transport was significantly greater in the ascending colon than the descending colon (P < 0.05; n = 6). In addition, the facilitative urea transporter inhibitor 1,3,dimethylurea significantly reduced urea transport in the ascending colon (P < 0.05; n = 6) but had no effect in the descending colon (NS; n = 6). These results illustrate differential protein abundance of functional UT-B protein in different sections of the human colon, strongly correlating to regions that contain the largest populations of intestinal bacteria. This study suggests an important role for UT-B urea transporters in maintaining the symbiotic relationship between humans and their gut bacteria. gastrointestinal tract; ascending colon; descending colon doi:10.1152/ajpgi.00405.2009.

Lin L, Xu L-M, Zhang W, Ge Y-B, Tang Y-R, Zhang H-J, Li X-L, Chen JDZ. Roles of stem cell factor on the depletion of interstitial cells of Cajal in the colon of diabetic mice. Am J Physiol Gastrointest Liver Physiol 298: G241-G247, 2010. First published October 29, 2009; doi:10.1152/ajpgi.90706.2008.--The aim of this study was to investigate the effects of stem cell factor (SCF) on interstitial cell of Cajal (ICC) depletion in the colon of diabetic mice. Male C57/BL6 mice were treated by a single intraperitoneally injected dose of streptozotocin, and those displaying sustained high blood glucose were selected as diabetes mellitus models. Six groups of mice were used: three groups of normal nondiabetic mice (untreated and treated with IgG or SCF antibody), and three groups of diabetic mice (untreated and treated with vehicle or SCF). Changes of the ICC quantities were analyzed by immunohistochemistry. ICC morphologies were observed with transmission electron microscopy. The SCF levels in sera and colon tissues were detected by ELISA and Western blot, respectively. The nondiabetic mice treated with SCF antibody and the untreated diabetic mice showed decreased SCF levels in the sera and colonic tissues, reduced numbers of ICC, and pathological changes of the ICC ultrastructures, whereas the nondiabetic mice treated with mouse IgG showed no significant changes compared with the nondiabetic mice. The diabetic mice treated with exogenous SCF showed restored SCF levels in both sera and colon tissues and improvement in the numbers of ICC and the damages of ICC ultrastructures, whereas the vehicle control of diabetic mice showed no significant changes compared with the diabetic mice. The blood glucose remained high and unchanged with the treatment of SCF or vehicle in the diabetic mice. These results indicate that diabetic mice show a decline in the number of ICC and impairment in the ultrastructures of ICC, and these abnormalities are attributed to a deficiency in the endogenous SCF but are not related to hyperglycemia. Exogenous SCF partially reverses the pathological changes of ICC in diabetic mice. diabetes mellitus; colon motility; ultrastructures; gastrointestinal motility doi: 10.1152/ajpgi.90706.2008.

Am J Physiol Gastrointest Liver Physiol 298: G45-G56, 2010. First published November 5, 2009; doi: 10.1152/ajpgi.00349.2009.--Peptide YY (PYY) antisecretory effect on intestinal epithelia is well established, whereas less is known about its actions to influence colonic motility in conscious animals. We characterized changes in basal function and stimulated colonic motor function induced by PYY-related peptides in conscious mice. [PYY.sub.3-36,] PYY, and neuropeptide Y (NPY) (8 nmol/ kg) injected intraperitoneally inhibited fecal pellet output (FPO) per hour during novel environment stress by 90%, 63%, and 57%, respectively, whereas the [Y.sub.1]-preferring agonists, [[Pro.sup.34]]pyY and [[Leu.sup.31,[Pro.sup.34]]]NpY, had no effect. Corticotrophin-releasing factor 2 receptor antagonist did not alter [PYY.sub.3-36] inhibitory action. PYY and PYY3-36 significantly reduced restraint-stimulated defecation, and PYY3-36 inhibited high-amplitude distal colonic contractions in restrained conscious mice for 1 h, by intraluminal pressure with the use of a microtransducer. PYY suppression of intraperitoneal 5-hydroxytryptophan induced FPO and diarrhea was blocked by the [Y.sub.2] antagonist, BIIE0246, injected intraperitoneally and mimicked by [PYY.sub.3-36,] but not [Leu31 ,Pro34]NpY. [PYY.sub.3-36] also inhibited bethanechol-stimulated FPO and diarrhea. [PYY.sub.3-36] inhibited basal FPO during nocturnal feeding period and light phase in fasted/refed mice for 2-3 h, whereas the reduction of food intake lasted for only 1 h. [PYY.sub.3-36] delayed gastric emptying after fasting-refeeding by 48% and distal colonic transit time by 104%, whereas [[Leu.sup.31,][Pro.sup.34]]NpY had no effect. In the proximal and distal colon, higher [Y.sub.2] mRNA expression was detected in the mucosa than in muscle layers, and [Y.sub.2] immunoreactivity was located in nerve terminals around myenteric neurons. These data established that PYY/[PYY.sub.3-36] potently inhibits basal and stress/serotonin/cholinergic-stimulated propulsive colonic motor function in conscious mice, likely via [Y.sub.2] receptors. [PYY.sub.3-36;] stress; serotonin; bethanechol

Yu H, Riederer B, Stieger N, Boron WF, Shull GE, Manns MP, Seidler UE, Bachmann O. Secretagogue stimulation enhances NBCe1 (electrogenic [Na.sup.+]/HC[O.sup.-.sub.3] cotransporter) surface expression in murine colonic crypts. Am J Physiol Gastrointest Liver Physiol 297: G1223-G1231, 2009. First published September 24, 2009; doi: 10.1152/ajpgi.00157.2009.--A [Na.sup.+]/HC[O.sup.-.sub.3] co- transporter (NBC) is located in the basolateral membrane of the gastrointestinal epithelium, where it imports HC[O.sup.-.sub.3] during stimulated anion secretion. Having previously demonstrated secretagogue activation of NBC in murine colonic crypts, we now asked whether vesicle traffic and exocytosis are involved in this process. Electrogenie NBCel-B was expressed at significantly higher levels than electroneutral NBCnl in colonic crypts as determined by QRT-PCR. In cell surface biotinylation experiments, a time-dependent increase in biotinylated NBCel was observed, which occurred with a peak of +54.8% after 20 min with forskolin (P < 0.05) and more rapidly with a peak of +59.8% after 10 min with carbachol (P < 0.05) and which corresponded well with the time course of secretagogue-stimulated colonic bicarbonate secretion in Ussing chamber experiments. Accordingly, in isolated colonic crypts pretreated with forskolin and carbachol for 10 min, respectively, and subjected to immunohistochemistry, the NBCel signal showed a markedly stronger colocalization with the E-cadherin signal, which was used as a membrane marker, compared with the untreated control. Cytochalasin D did not change the observed increase in membrane abundance, whereas colchicine alone enhanced NBCel membrane expression without an additional increase after carbachol or forskolin, and LY294002 had a marked inhibitory effect. Taken together, our results demonstrate a secretagogue-induced increase of NBCel membrane expression. Vesicle traffic and exocytosis might thus represent a novel mechanism of intestinal NBC activation by secretagogues. colon; membrane expression doi: 10.1152/ajpgi.00157.2009

Brumovsky PR, Feng B, Xu L, McCarthy C J, Gebhart GF. Cystitis increases colorectal afferent sensitivity in the mouse. Am J Physiol Gastrointest Liver Physiol 297: G1250-G1258, 2009. First published October 1, 2009; doi: 10.1152/ajpgi.00329.2009.--Studies in humans and rodents suggest that colon inflammation promotes urinary bladder hypersensitivity and, conversely, that cystitis contributes to colon hypersensitivity, events referred to as cross-organ sensitization. To investigate a potential peripheral mechanism, we examined whether cystitis alters the sensitivity of pelvic nerve colorectal afferents. Male C57BL/6 mice were treated with cyclophosphamide (CYP) or saline, and the mechanosensitive properties of single afferent fibers innervating the colorectum were studied with an in vitro preparation. In addition, mechanosensitive receptive endings were exposed to an inflammatory soup (IS) to study sensitization. Urinary bladder mechanosensitive afferents were also tested. We found that baseline responses of stretch-sensitive colorectal afferents did not differ between treatment groups. Whereas IS excited a proportion of colorectal afferents CYP treatment did not alter the magnitude of this response. However, the number of stretch-sensitive fibers excited by IS was increased relative to saline-treated mice. Responses to IS were not altered by CYP treatment, but the proportion of IS-responsive fibers was increased relative to saline-treated mice. In bladder, IS application increased responses of muscular afferents to stretch, although no differences were detected between saline- and CYP-treated mice. In contrast, their chemosensitivity to IS was decreased in the CYP-treated group. Histological examination revealed no changes in colorectum and modest edema and infiltration in the urinary bladder of CYP-treated mice. In conclusion, CYP treatment increased mechanical sensitivity of colorectal muscular afferents and increased the proportion of chemosensitive colorectal afferents. These data support a peripheral contribution to cross-organ sensitization of pelvic organs. sensitization; pelvic nerve; bladder; colon; inflammatory soup doi: 10.1152/ajpgi.00329.2009

Hasler WL, Saad RJ, Rao SS, Wilding GE, Parkman HP, Koch KL, McCallum RW, Kuo B, Sarosiek I, Sitrin MD, Semler JR, Chey WD. Heightened colon motor activity measured by a wireless capsule in patients with constipation: relation to colon transit and IBS. Am J Physiol Gastrointest Liver Physiol 297: G1107-G1114, 2009. First published October 1, 2009; doi: 10.1152/ajpgi.00136.2009.--Relationships of regional colonic motility to transit in health, constipation, and constipation-predominant irritable bowel syndrome (CIBS) are poorly characterized. This study aimed to 1) characterize regional differences in colon pressure, 2) relate motor differences in constipation to colon transit, and 3) quantify the role of IBS in altered contractility with constipation. Colon pH and pressure were measured by wireless capsules in 53 healthy and 36 constipated subjects. Numbers of contractions >25 mmHg and areas under curves (AUC) were calculated for colon transit quartiles by time. Constipation was classified as normal transit ( 100 h). Twelve out of 36 constipated subjects had C-IBS; 24 had functional constipation. Numbers of contractions and AUCs increased from the first to the fourth quartile in health (P < 0.0001). Mean numbers of contractions in constipated subjects were similar to controls. Mean AUCs with normal transit (P = 0.01) and moderate STC (P = 0.004) but not severe STC (P = NS) were higher than healthy subjects. IBS was associated with greater mean numbers of contractions (P = 0.05) and AUCs (P = 0.0006) vs. controls independent of transit. Numbers of contractions increased from the first to fourth quartiles in moderate STC, C-IBS, and functional constipation; AUCs increased from the first to fourth quartiles in all groups (all P < 0.05). In conclusion, colon pressure activity is greater distally than proximally in health. Constipated patients with normal or moderately delayed transit show increased motor activity that is partly explained by IBS. These findings emphasize differential effects on transit and motility in different constipation subtypes. gastrointestinal motility; irritable bowel syndrome; functional outlet obstruction doi: 10.1152/ajpgi.00136.2009

Chronic stress precipitates or exacerbates the symptoms of functional bowel disorders, including motility dysfunction. The cellular mechanisms of these effects are not understood. We tested the hypothesis that heterotypic chronic stress (HeCS) elevates the release of norepinephrine from the adrenal medulla, which enhances transcription of the gene-regulating expression of [Ca.sub.v]l.2 (L-type) channels in colonic circular smooth muscle cells, resulting in enhanced colonic motor function. The experiments were performed in rats using a 9-day heterotypic chronic stress (HeCS) protocol. We found that HeCS, but not acute stress, time dependently enhances the contractile response to ACh in colonic circular smooth muscle strips and in single dissociated smooth muscle cells, the plasma levels of norepinephrine and the mRNA and protein expressions of the [[alpha].sub.1c] subunit of [Ca.sub.v] 1.2 channels. These effects result in faster colonic transit and increase in defecation rate. The effects of HeCS are blocked by adrenalectomy but not by depletion of norepinephrine in sympathetic neurons. The inhibition of receptors for glucocortocoids, corticotropin-releasing hormone or nicotine also does not block the effects of heterotypic chronic stress. Norepinephrine acts on [alpha]- and [[beta].sub.3]-adrenergic receptors to induce the transcription of [[alpha].sub.1c] subunit. We conclude that HeCS alters colonic motor function by elevating the plasma levels of norepinephrine. Colonic motor dysfunction is associated with enhanced gene transcription of [Ca.sub.v]1.2 channels in circular smooth muscle cells. These findings suggest the potential cellular mechanisms by which heterotypic chronic stress may exacerbate motility dysfunction in patients with irritable bowel syndrome. corticotropin-releasing hormone; corticosterone; enteric neurotransmitters; smooth muscle

There is a glaring lack of knowledge on mouse colonic motility in vivo, primarily due to unavailability of adequate recording methods. Using a noninvasive miniature catheter pressure transducer inserted into the distal colon, we assessed changes in colonic motility in conscious mice induced by various acute or chronic stressors and determined the neurotransmitters mediating these changes. Mice exposed to restraint stress (RS) for 60 min displayed distal colonic phasic contractions including high-amplitude giant migrating contractions (GMCs), which had peak amplitudes >25 mmHg and occurred at a rate of 15-25 [h.sup.-1] of which over 50% were aborally propagative. Responses during the first 20-min of RS were characterized by high-frequency and high-amplitude contractions that were correlated with defecation. RS-induced GMCs and fecal pellet output were blocked by atropine (0.5 mg/kg ip) or the corticotrophin releasing factor (CRF) receptor antagonist astressin-B (100 [micro]g/kg ip). RS activated colonic myenteric neurons as shown by Fos immunoreactivity. In mice previously exposed to repeated RS (60 min/day, 14 days), or in transgenic mice that overexpress CRF, the duration of stimulation of phasic colonic contractions was significantly shorter (10 vs. 20 min). In contrast to RS, abdominal surgery abolished colonic contractions including GMCs. These findings provide the first evidence for the presence of frequent cholinergic-dependent GMCs in the distal colon of conscious mice and their modulation by acute and chronic stressors. Noninvasive colonic manometry opens new venues to investigate colonic motor function in genetically modified mice relevant to diseases that involve colonic motility alterations. colon; mice; giant migrating contractions; manometry; stress; corticotropin releasing factor-overexpressing

The study was designed to investigate the effect of estradiol on the excitatory effect of oxytocin (OT) on colon motility. Female Wistar rats were used, and some of them were ovariectomized (OVX) and treated with vehicle or estradiol (Ez). A plastic balloon made of condom was inserted into colon to monitor the change of colonic pressure in vivo. Longitudinal muscle strips of distal colon were prepared to monitor the spontaneous contraction of colon in vitro. Expression of OT receptor (OTR) was investigated by Western blot analysis. Expression of OTR mRNA was detected by RT-PCR. Immunohistochemistry was used to locate OTR. In OVX rats, pretreatment of [E.sub.2] (4-100 [micro]g/kg sc) dose-dependently increased the excitatory effect of OT on colon motility both in vivo and in vitro and increased the expression of OTR and OTR mRNA in colon. Systemic administration of OT excited the colon motility in vivo in rats at perioda of proestrus and estrus but did not influence it at diestrus period, when the concentration of plasma [E.sub.2] was lowest in the estrous cycle. Pretreatment of atosiban, the specific OTR antagonist, and TTX, the blocker of voltage-dependent sodium channel on nerve fiber, attenuated the excitatory effect of OT on colon motility. OTR was located in myenteric plexus of colon. These results suggested that [E.sub.2] increased the excitatory effect of OT on colon motility by upregulating the expression of OTR in myenteric plexus. colon; motility

Cannabinoid-1 ([CB.sub.1]) receptors on myenteric neurons are involved in the regulation of intestinal motility. Our aim was to investigate [CB.sub.1] receptor involvement in ascending neurotransmission in mouse colon and to characterize the involved structures by functional and morphological means. Presence of the [CB.sub.1] receptor was investigated by RT-PCR, and immunohistochemistry was used for colabeling studies. Myenteric reflex responses were initiated by electrical stimulation (ES) at different distances, and junction potentials (JP) were recorded from circular smooth muscle cells by intracellular recording in an unpartitioned and a partitioned recording chamber. In vivo colonic propulsion was tested in wild-type and [CB.sub.1.sup.-/-] mice. Immunostaining with the cytoskeletal marker peripherin showed [CB.sub.1] immunoreactivity both on Dogiel type I and type II neurons. Further neurochemical characterization revealed [CB.sub.1] on choline acetyltransferase-, calretinin-, and 5-HT-immunopositive myenteric neurons, but nitrergic neurons appeared immunonegative for [CB.sub.1] immunostaining. Solitary spindle-shaped [CB.sub.1]-immunoreactive cells in between smooth muscle cells lacked specific markers for interstitial cells of Cajal or glial cells. ES elicited neuronally mediated excitatory JP (EJP) and inhibitory JP. Gradual increases in distance resulted in a wave-like EJP with EJP amplitudes being maximal at the location of stimulating electrode 6 and a maximal EJP projection distance of ~18 mm. The [CB.sub.1] receptor agonist WIN 55,212-2 reduced the amplitude of EJP and was responsible for shortening the oral spreading of the excitatory impulse. In a partitioned chamber, WIN 55,212-2 reduced EJP at the separated oral sites, proving that [CB.sub.1] activation inhibits interneuron-mediated neurotransmission. These effects were absent in the presence of the [CB.sub.1] antagonist SR141716A, which, when given alone, had no effect. WIN 55,212-2 inhibited colonic propulsion in wild-type mice but not in SR141716A-pretreated wild-type or [CB.sub.1.sup.-/-] mice. Activation of the CBI receptor modulates excitatory cholinergic neurotransmission in mouse colon by reducing amplitude and spatial spreading of the ascending electrophysiological impulses. This effect on electrophysiological spreading involves [CB.sub.1]-mediated effects on motor neurons and ascending interneurons and is likely to underlie the here reported in vivo reduction in colonic propulsion. peristaltic reflex; enteric nervous system; electrophysiology

The role of specific PKC isoforms in the regulation of epithelial [Cl.sup.-] secretion by [Ca.sup.2+]-dependent secretagogues remains controversial. In the developing rabbit distal colon, the bile acid taurodeoxycholate (TDC) acts via intracellular calcium to stimulate [Cl.sup.-] transport in adult, but not in young, animals, whereas the PKC activator phorbol dibutyrate (PDB) stimulates [Cl.sup.-] transport at all ages. We tested the hypothesis that specific PKC isoforms account for the age-specific effects of TDC. The effects of conventional (cPKC) and novel (nPKC) PKC-specific inhibitors on TDC- and PDB-stimulated [Cl.sup.-] transport in adult and weanling colonocytes were assessed by using 6-methoxy-quinolyl acetoethyl ester. In adult colonocytes, the cPKC inhibitor G6-6976 inhibited PDB action but not TDC action, whereas the cPKC and nPKC inhibitor Go-6850 blocked both TDC and PDB actions. Additionally, rottlerin and the PKC-[delta]-specific inhibitor peptide ([delta]V1-1) inhibited TDC- and PDB-stimulated [Cl.sup.-] transport in adult colonocytes. Rottlerin also decreased TDC-stimulated short-circuit current in intact colonic epithelia. Only G0-6976, but neither rottlerin nor [delta]V1-1, inhibited PDB-stimulated transport in weanling colonocytes. Colonic lysates express PKC-[alpha], -[lambda], and -[??] protein equally at all ages, but they do not express PKC-[gamma] or -[theta] at any age. Expression of PKC-[beta] and PKC-[epsilon] protein was newborn>adult>weanling, whereas PKC-[delta] was expressed in adult but not in weanling or newborn colonocytes. TDC (1.6-fold) and PDB (2.0-fold) stimulated PKC-[delta] enzymatic activity in adult colonocytes but failed to do so in weanling colonocytes, PKC-[delta] mRNA expression showed age dependence, Thus PKC-[delta] appears critical for the action of TDC in the adult colon, and its low expression in young animals may account for their inability to secrete in response to bile acids. epithelial chloride transport; taurodeoxycholate; signal transduction

Older age and inadequate folate intake are strongly implicated as important risk factors for colon cancer and each is associated with altered DNA methylation. This study was designed to determine the effects of aging and dietary folate on select features of DNA methylation in the colon that are relevant to carcinogenesis. Old (18 mo; n = 34) and young (4 mo; n = 32) male C57BL/6 mice were randomly divided into 3 groups and fed diets containing 0, 4.5, or 18 [micro]mol folate/kg (deplete, replete, and supplemented groups, respectively) for 20 wk. Genomic DNA methylation and p16 promoter methylation in the colonic mucosa were analyzed by liquid chromatography/electrospray ionization/MS and methylation-specific PCR, respectively. p16 gene expression was determined by real-time RT-PCR. Old mice had significantly lower genomic DNA methylation compared with young mice at each level of dietary folate (4.5 [+ or -] 0.2, 4.8 [+ or -] 0.1, and 4.9 [+ or -] 0.1 vs. 6.0 [+ or -] 0.1, 5.3 -+ 0.2, and 5.9 [+ or -] 0.2%, in folate-deplete, -replete, and -supplemented groups, respectively, P< 0.05) and markedly higher p16 promoter methylation (61.0 [+ or -] 2.7, 69.7 [+ or -] 6.9, and 87.1 [+ or -] 13.4 vs. 10.8 [+ or -] 3.6, 8.4 [+ or -] 1.8, and 4.9 [+ or -] 1.7 %, respectively, P < 0.05). In old mice, genomic and p16 promoter DNA methylation each increased in a manner that was directly related to dietary folate ([P.sub.trend] = 0.009). Age-related enhancement of p16 expression occurred in folate-replete (P = 0.001 ) and folate supplemented groups (P = 0.041), but not in the folate-deplete group. In conclusion, aging decreases genomic DNA methylation and increases promoter methylation and expression of p16 in mouse colons. This effect is dependent on the level of dietary folate.

Reduced colonic motility has been observed in aged rats with a parallel reduction in acetylcholine (ACh)-induced myosin light chain (ML[C.sub.20]) phosphorylation. ML[C.sub.20] phosphorylation during smooth muscle contraction is maintained by a coordinated signal transduction cascade requiring both PKC-[alpha] and RhoA. Caveolae are membrane microdomains that permit rapid and efficient coordination of different signal transduction cascades leading to sustained smooth muscle contraction of the colon. Here, we show that normal physiological contraction can be reinstated in aged colonic smooth muscle cells (CSMCs) upon transfection with wild-type caveolin-1 through the activation of both the RhoA/Rho kinase and PKC pathways. Our data demonstrate that impaired contraction in aging is an outcome of altered membrane translocation of PKC-[alpha] and RhoA with a concomitant reduction in the association of these molecules with the caveolae-specific protein caveolin-1, resulting in a parallel decrease in the myosin phosphatase-targeting subunit (MYPT) and CPI-17 phosphorylation. Decreased MYPT and CPI-17 phosphorylation activates MLC phosphatase activity, resulting in ML[C.sub.20] dephosphorylation, which may be responsible for decreased colonic motility in aged rats. Importantly, transfection of CSMCs from aged rats with wild-type yellow fluorescent protein-caveolin-1 cDNA restored translocation of RhoA and PKC-[alpha] and phosphorylation of MYPT, CPI-17, and ML[C.sub.20], thereby restoring the contractile response to levels comparable with young adult rats. Thus, we propose that caveolin-1 gene transfer may represent a promising therapeutic treatment to correct the age-related decline in colonic smooth muscle motility. aging; myosin phosphorylation; RhoA; PKC doi:10.1152/ajpgi.00064.2007

Neonatal maternal separation (MS) predisposes adult rats to develop stress-induced mucosal barrier dysfunction/visceral hypersensitivity and rat pups to develop colonic epithelial dysfunction. Our aim was to examine if enhanced epithelial permeability in such pups resulted from abnormal regulation by enteric nerves. Pups were separated from the dam for 3 h/day (dab, s 4-20); nonseparated (NS) pups served as controls. On day 20, colonic tissues were removed and mounted in Using chambers. Horseradish peroxidase (HRP) flux was used to measure macromolecular permeability. HRP flux was increased in MS versus NS pups. The enhanced flux was inhibited by the cholinergic muscarinic antagonist atropine and the nicotinic antagonist hexamethonium. The cholinergic component was greater in tissues from MS versus NS pups, suggesting that increased cholinergic activity was responsible for the MS elevated permeability. Western blots and immunohistochemistry of colonic tissues demonstrated increased expression of choline acetyltransferase (CHAT) in MS pups, indicating greater synthesis of acetylcholine. Since a previous study indicated that corticotrophin-releasing factor (CRF) mediates barrier dysfunction in MS pups, we examined if the two pathways were linked. In MS tissues, nonselective CRF receptor antagonism inhibited the enhanced flux, and the addition of atropine did not produce further inhibition. Using selective receptor antagonists, we identified that CRF receptor 2 was involved in mediating this effect. These findings suggest that CRF, via CRF receptor 2, acts on cholinergic nerves to induce epithelial barrier dysfunction. Our study provides evidence that MS stimulates synthesis of acetylcholine, which, together with released CRF, creates a condition conducive to the development of epithelial barrier defects. stress; colonic; barrier doi:10.1152/ajpgi.00392.2006

Cannabinoid receptors (CBR) are located on cholinergic neurons in the brain stem, stomach, and colon. CBR stimulation inhibits motility in rodents. Effects in humans are unclear. Dronabinol (DRO), a nonselective CBR agonist, inhibits colonic motility and sensation. The aim of this study was to compare effects of DRO and placebo (PLA) on colonic motility and sensation in healthy volunteers. Fifty-two volunteers were randomly assigned (double-blind) to a single dose of 7.5 mg DRO or PLA postoperative with concealed allocation. A balloon-manometric assembly placed into the descending colon allowed assessment of colonic compliance, motility, tone, and sensation before and 1 h after oral ingestion of medication, and during fasting, and for 1 h after 1,000-kcal meal. There was an overall significant increase in colonic compliance (P = 0.045), a borderline effect of relaxation in fasting colonic tone (P = 0.096), inhibition of postprandial colonic tone (P = 0.048), and inhibition of fasting and postprandial phasic pressure (P = 0.008 and 0.030, respectively). While DRO did not significantly alter thresholds for first gas or pain sensation, there was an increase in sensory rating for pain during random phasic distensions at all pressures tested and in both genders (P = 0.024). In conclusion, in humans the nonselective CBR agonist, DRO, relaxes the colon and reduces postprandial colonic motility and tone. Increase in sensation ratings to distension in the presence of relaxation of the colon suggests central modulation of perception. The potential for CBR to modulate colonic motor function in diarrheal disease such as irritable bowel syndrome deserves further study. dronabinol; compliance; tone; pain doi:10.1152/ajpgi.00565.2006

Calcitonin gene-related peptide (CGRP) mediates neurogenic inflammation and modulates intestinal motility. The CGRP receptor is a heterodimer of calcitonin receptor-like receptor (CLR) and receptor-associated modifying protein 1. We used RNA interference to elucidate the specific role of CLR in colonic motility and inflammation. Intramural injection of double-stranded RNA (dsRNA) against CLR (dsCLR) into the colonic wall at two sites caused the spatial and temporal downregulation of CLR in the colon within 1 day of dsRNA injection. Knockdown of CLR persisted for 7-9 days, and the effect of knockdown spread to ~2 cm proximal and distal to the injection sites, whereas control dsRNA injection did not affect CLR expression. Measurement of isometric contractions of isolated colonic muscle segments revealed that in control dsRNA-injected rats, CGRP abrogated contractions entirely and decreased resting muscular tone, whereas in dsCLR-injected rats, CGRP decreased muscle tone but slow-wave contractions of varying amplitude persisted. In trinitrobenzene sulfonic acid-induced colitis, rats with knockdown of CLR displayed a significantly greater degree of edema and necrosis than saline- or control dsRNA-injected rats. Levels of the proinflammatory cytokines TNF-[alpha] and IL-6 were markedly upregulated by trinitrobenzene sulfonic acid treatment. TNF-[alpha] mRNA levels were further increased in CLR knockdown rats, whereas levels of IL-6 were unaltered. Thus this study demonstrates that CLR is a functional receptor for CGRP. calcitonin gene-related peptide; RNA interference; trinitrobenzene sulfonic acid; cytokines; colon-specific; tumor necrosis factor-[alpha] doi:10.1152/ajpgi.00464.2006

Kruppel-like factor 9 (Klf9), a zinc-finger transcription factor, is implicated in the control of cell proliferation, cell differentiation, and cell fate. Using Klf9-null mutant mice, we have investigated the involvement of Klf9 in intestine crypt-villus cell renewal and lineage determination. We report the predominant expression of Klf9 gene in small and large intestine smooth muscle (muscularis externa). Jejunums null for Klf9 have shorter villi, reduced crypt stem/transit cell proliferation, and altered lineage determination as indicated by decreased and increased numbers of goblet and Paneth cells, respectively. A stimulatory role for Klf9 in villus cell migration was demonstrated by bromodeoxyuridine labeling. Results suggest that Klf9 controls the elaboration, from intestine smooth muscle, of molecular mediator(s) of crypt cell proliferation and lineage determination and of villus cell migration. Krtippel-like; Igfbp4; Ptk6; stem cells; colon; smooth muscle doi:10.1152/ajpgi.00013.2007

Our previous studies have shown that stereotaxic implantation of corticosterone (Cort) onto the central amygdaloid nucleus increases both anxiety and colonic sensitivity. The goal of this study was to examine the relative importance of amygdaloid glucocorticoid (GR) and mineralocorticoid receptor (MR)-mediated mechanisms in the induction of anxiety and colonic hypersensitivity. In male Fischer 344 rats, Cort or cholesterol micropellets were stereotaxically implanted bilaterally at the dorsal boundary of the central amygdaloid nucleus either alone or in combination with a GR antagonist, mifepristone, or a MR antagonist, spironolactone. Anxiety was assessed on the elevated plus maze and quantified as the percentage of time spent in open arm exploration. Colonic sensitivity was measured by recording a visceromotor response, the number of abdominal muscle contractions in response to colorectal distension. In Cort-implanted rats there was a significant reduction in the percentage of time spent in the open arms of the elevated plus maze compared with cholesterol controls, indicating increased anxiety. Furthermore, colonic hypersensitivity was observed in response to colorectal distension compared with rats with cholesterol implants. In rats with Cort implants combined with either a GR or MR antagonist, there was a significant inhibition of anxiety and colonic hypersensitivity. Our data suggest that both GR and MR play a critical role in Cort-induced anxiety and colonic hypersensitivity. corticosterone; hypothalamic-pituitary-adrenal axis; irritable bowel syndrome doi:10.1152/ajpgi.00080.2007

The aim of this study was to characterize the pacemaker activity and inhibitory neurotransmission in the colon of Ws/Ws mutant rats, which harbor a mutation in the c-kit gene that affects development of interstitial cells of Cajal (ICC). In Ws/Ws rats, the density of KIT-positive cells was markedly reduced. Wild-type, but not Ws/Ws, rats showed low- and high-frequency cyclic depolarization that were associated with highly regular myogenic motor patterns at the same frequencies. In Ws/Ws rats, irregular patterns of action potentials triggered irregular muscle contractions occurring within a bandwidth of 10-20 cycles/min. Spontaneous activity of nitrergic nerves caused sustained inhibition of muscle activity in both wild-type (+/+) and Ws/Ws rats. Electrical field stimulation of enteric nerves, after blockade of cholinergic and adrenergic activity, elicited inhibition of mechanical activity and biphasic inhibitory junction potentials both in wild-type and Ws/Ws rats. Apamin-sensitive, likely purinergic, inhibitory innervation was not affected by loss of ICC. Variable presence of nitrergic innervation likely reflects the presence of direct nitrergic innervation to smooth muscle cells as well as indirect innervation via ICC. In summary, loss of ICC markedly affects pacemaker and motor activities of the rat colon. Inhibitory innervation is largely maintained but nitrergic innervation is reduced possibly related to the loss of ICC-mediated relaxation. c-kit; slow waves; interstitial cells of Cajal; inhibitory junction potential; stereology; fast Fourier transform; colonic motility doi:10.1152/ajpgi.00136.2006

In mammals that develop rectal aganglionosis, the aganglionic segment still exhibits spontaneous phasic contractions that contribute to dysmotility and pseudoobstruction in this region. However, almost nothing is known about the mechanisms that generate these myogenic contractions or the effects of aganglionosis on the generation of [Ca.sup.2+] waves that underlie contractions of the longitudinal muscle (LM) and circular muscle (CM). In a mouse model of Hirschsprung's disease [endothelin type B receptor-deficient ([Ednrb.sup.1-l]/[Ednrb.sup.s-l]) mice], the [Ca.sup.2+] indicator fluo-4 was used to simultaneously monitor the temporal activation and spread of intercellular [Ca.sup.2+] waves in the LM and CM during spontaneous colonic motor activities. During the intervals between colonic migrating motor complexes (CMMCs) in control mice, [Ca.sup.2+] waves discharged asynchronously between the LM and CM. However, in these same mice, during CMMCs, a burst of discreet [Ca.sup.2+] waves fired simultaneously in both muscle layers, where the propagation velocity of [Ca.sup.2+] waves significantly increased, as did the rate of initiation and number of collisions between [Ca.sup.2+] waves. Hexamethonium (300 [micro]M) or atropine (1 [micro]M) prevented synchronized firing of [Ca.sup.2+] waves. In the aganglionic distal colon of [Ednrb.sup.s-l]/[Ednrb.sup.s-l] mice, not only were CMMCs absent, but [Ca.sup.2+] waves between the two muscle layers fired asynchronously, despite increased propagation velocity. The generation of CMMCs in control mice involves synchronized firing of enteric motor nerves to both the LM and CM, explaining the synchronized firing of discreet [Ca.sup.2+] waves between the two muscle layers. Aganglionosis results in a sporadic and sustained asynchrony in [Ca.sup.2+] wave firing between the LM and CM and an absence of CMMCs. colon; myenteric; peristalsis; colonic migrating motor complexes

Using the membrane-bending elasticity theory and a simple effective model of adhesion, we study the morphology of lipid vesicle doublets. In the weak adhesion regime, we find flat-contact axisymmetric doublets, whereas at large adhesion strengths, the vesicle aggregates are nonaxisymmetric and characterized by a sigmoidally curved, S-shaped contact zone with a single invagination and a complementary evagination on each vesicle. The sigmoid-contact doublets agree very well with the experimentally observed shapes of erythrocyte aggregates. Our results show that in identical vesicles with large to moderate surface-to-volume ratio, the sigmoid-contact shape is the only bound morphology. We also discuss the role of sigmoid contacts in the formation of multicellular aggregates such as erythrocyte rouleaux. lipid vesicle | vesicle doublet | sigmoid-contact doublet | rouleau

Central nervous system neurons process information converging from the uterus, colon, and bladder, partly via the hypogastric nerve. This processing is influenced by the estrous cycle, suggesting the existence of an estrous-modifiable central nervous system substrate by which input from one pelvic organ can influence functioning of other pelvic organs. Here, we tested predictions from this hypothesis that acute inflammation of colon, uterine horn, or bladder would produce signs of inflammation in the other uninflamed organs (increase vascular permeability) and that cross-organ effects would vary with estrous and be eliminated by hypogastric neurectomy (HYPX). Under urethane anesthesia, the colon, uterine horn, or bladder of rats in proestrus or metestrus, with or without prior HYPX, was treated with mustard oil or saline. Two hours later, Evans Blue dye extravasation was measured to assess vascular permeability. Extravasation was increased in all inflamed organs, regardless of estrous stage. For rats in proestrus, but not metestrus, either colon or uterine horn inflammation significantly increased extravasation in the uninflamed bladder. Much smaller cross-organ effects were seen in colon and uterine horn. HYPX reduced extravasation in the inflamed colon and inflamed uterine horn, but not the inflamed bladder. HYPX eliminated the colon-to-bladder and uterine horn-to-bladder effects. These results demonstrate that inflaming one pelvic organ can produce estrous-modifiable signs of inflammation in other pelvic organs, particularly bladder, and suggest that the cross-organ effects involve the hypogastric nerve and are at least partly centrally mediated. Such effects could contribute to cooccurrence and cyclicity of distressing pelvic disorders in women. pelvic pain; inflammation; interstitial cystitis; uterus; colon

The effects of fibroblast growth factor 10 on colonic epithelium development, using hypomorphic mice studies, are presented.

The logistic sigmoid function curve provides an accurate description of the baroreflex input-output relationship and is the most commonly used equation for this purpose. The threshold (Thr) and saturation (Sat) values for the baroreflex are commonly defined as the values of mean arterial pressure (MAP) at which the reflexly controlles variable (e.g., heart rate or sympathetic nerve activity) is within 5% of the upper or lower plateau, respectively, of the sigmoid function, These values are referred to here as [Thr.sub.5%] and [Sat.sub.5%]. In many studies, Thr and Sat are calculated with the equations Thr = [A.sub.3] - 2.0/[a.sub.2] and Sat = [A.sub.3] - 2.0/[a.sub.2], where [A.sub.3] is the value of MAP at the point where the reflexly controlled variable is at the midpoint of its range and [A.sub.2] is the gain coefficient. Although it is commonly stated that the values of Thr and Sat calculated with these equations represent [Thr.sub.5%] and [Sat.sub.5%], we show here that instead they are significantly greater and less than [Thr.sub.5%] and [Sat.sub.5%], respectively. Furthermore, the operating range (difference between Thr and Sat) calculated with these equations is] 32% less than the difference between [Thr.sub.5%] and [Sat.sub.5%]. We further show that the equations that provide correct valued of [Thr.sub.5%] and [Sat.sub.5%] are [Thr.sub.5%] = [A.sub.3] - 2.944/[A.sub.2] and [Sat.sub.5%]= [A.sub.3] + 2.944/[A.sub.2]. We propose that these be used as the standard equations for calculating threshold and saturation values when a logistic sigmoid function is used to model the open-loop baroreflex function curve. mathematical modeling; input-output relationship; baroreflex operating range; regression analysis

The mechanism of apical [Na.sup.+]-dependent [H.sup.+] extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH ([pH.sub.i]) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal [Na.sup.+] stimulated similar rates of [pH.sub.i] recovery in cells at the base of distal colonic crypts of wild-type or [Na.sup.+]/[H.sup.+] exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 [micro]M HOE694 (NHE2 inhibitor) blocked 68-75% of the [pH.sub.i] recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 [micro]M), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 [micro]M), inhibited [pH.sub.i] recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial [Na.sup.+] leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of [Na.sup.+]-dependent [pH.sub.i] recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive [pH.sub.i] recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a [Na.sup.+]-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters. S1c9a2; S1c9a3; BCECF; SNARF-5F; intracellular pH; laser scanning confocal microscopy; colon; [Na.sup.+]/[H.sup.+] exchanger

Indirect evidence suggests that ATP is a neurotransmitter involved in inhibitory pathways in the neuromuscular junction in the gastrointestinal tract. The aim of this study was to characterize purinergic inhibitory neuromuscular transmission in the human colon. Tissue was obtained from colon resections for neoplasm. Muscle bath, microelectrode experiments, and immunohistochemical techniques were performed. 2'-deoxy-[N.sup.6]-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) was used as a selective inhibitor of P2YI receptors. We found that 1) ATP (1 mM) and adenosine 5'-[beta]-2-thiodiphosphate (ADP[beta]S) (10 [micro]M), a preferential P2Y agonist, inhibited spontaneous motility and caused smooth muscle hyperpolarization (about -12 mV); 2) MRS 2179 (10 [micro]M) and apamin (1 [micro]M) significantly reduced these effects; 3) both the fast component of the inhibitory junction potential (IJP) and the nonnitrergic relaxation induced by electrical field stimulation were dose dependently inhibited ([IC.sub.50] -1 [micro]M) by MRS 2179; 4) ADP[beta]S reduced the IJP probably by a desensitization mechanism; 5) apamin (1 [micro]M) reduced the fast component of the IJP (by 30-40%) and the inhibitory effect induced by electrical field stimulation; and 6) [P2Y.sub.1] receptors were localized in smooth muscle cells as well as in enteric neurons. These results show that ATP or a related purine is released by enteric inhibitory motoneurons, causing a fast hyperpolarization and smooth muscle relaxation. The high sensitivity of MRS 2179 has revealed, for the first time in the human gastrointestinal tract, that a [P2Y.sub.1] receptor present in smooth muscle probably mediates this mechanism through a pathway that partially involves apamin-sensitive calcium-activated potassium channels. [P2Y.sub.1] receptors can be an important pharmacological target to modulate smooth muscle excitability. smooth muscle relaxation; purinergic receptors; inhibitory junction potential

Nitrone-related therapeutics (NRTs) represent a new class of small molecules potentially effective in the treatment of inflammatory bowel disease (IBD) by protecting cells from damage caused by excess inflammation and/or oxidative stress. The goal of this study was to determine the efficacy and potency of CPI-1189, a novel therapeutic agent, in dextran sulfate sodium (DSS)-induced colitis in mice. Mice received oral doses of either CPI-1189 (3, 10, or 30 mg [kg.sup.-1]) or the methyl cellulose vehicle along with 3% dextran sulfate in their drinking water. Assessment of colitis was by calculation of a disease activity index (DAI) and by histological observations. Signs of colitis in vehicle-treated mice were evident by day 3 using the DAI and with histological confirmation on day 7. In mice given CPI-1189, there was a significant and dose-dependent improvement in all signs of colitis with an overall protection of approximately 50%. These observations suggest that CPI-1189 is a novel, orally active, therapeutic agent that could be developed for the treatment of crohn's disease and ulcerative colitis in humans. Key words: Colon, inflammation, nitrone-related therapeutics, INTRODUCTION Inflammatory bowel disease (IBD) is a blanket term for the chronic, inflammatory condition experienced by individuals with either Crohn's disease or ulcerative colitis. The etiology and pathophysiology of IBD [...]

Our colons harbor trillions of microbes including a prominent archaeon, Methanobrevibacter smithii. To examine the contributions of Archaea to digestive health, we colonized germ-free mice with Bacteroides thetaiotaomicron, an adaptive bacterial forager of the polysaccharides that we consume, with or without M. smithii or the sulfate-reducing bacterium Desulfovibrio piger. Whole-genome transcriptional profiling of B. thetaiotaomicron, combined with mass spectrometry, revealed that, unlike D. piger, M. smithii directs B. thetaiotaomicron to focus on fermentation of dietary fructans to acetate, whereas B. thetaiotaomicron-derived formate is used by M. smithii for methanogenesis. B. thetaiotaomicron-M. smithii cocolonization produces a significant increase in host adiposity compared with monoassociated, or B. thetaiotaomicron-D. piger biassociated, animals. These findings demonstrate a link between this archaeon, prioritized bacterial utilization of polysaccharides commonly encountered in our modern diets, and host energy balance. adiposity | energy homeostasis | gut microbial ecology | polysaccharide metabolism | Methanobrevibactersmithii

In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N- and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel. colon | parasite

Several papers report that the colon is one of the tissues regulated by estrogen receptor (ER)[beta]. To better understand the physiological role of ER[beta] in colonic tissue, we have compared morphology, proliferation, and differentiation of colonic epithelium in ER[[beta].sup.-/-] mice and WT littermates. BrdUrd labeling revealed that the number of proliferating cells was higher in ER[[beta].sup.-/-] mice and that the migration of labeled cells toward the luminal surface was faster in ER[[beta].sup.-/-] mice than in WT littermates. Additionally, in the absence of ER[beta], there was a decrease in apoptosis, which was measured by immunohistochemical staining of cleaved caspase-3. The state of differentiation of the colonic epithelial cells was studied by using epithelial markers. In ER[[beta].sup.-/-] mice, there was a significant decrease in the expression of the differentiation marker cytokeratin (CK)20 and in the cellular adhesion molecules [alpha]-catenin (an adherens junction protein) and plectin (a hemidesmosomal protein). These changes were also evident by electron microscopy as abnormalities in tight junctions and in the number and shape of desmosomes in ER[[beta].sup.-/-] mice. These findings suggest a role for ER[beta] in the organization and architectural maintenance of the colon. Furthermore, our results indicate that the rapidly proliferating cells of the colonic epithelium in ER[[beta].sup.-/-] mice are lost by increased shedding and not by increased apoptosis. In this way, hyperproliferative cells that lack ER[beta] do not form hyperplastic lesions and do not accumulate in the superficial epithelium. adhesion | cancer | colon | differentiation | proliferation

During acute exacerbations of inflammatory bowel diseases, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the colon mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (N[H.sub.2]Cl), to serve as agonists of [Ca.sup.2+] and [Zn.sup.2+] accumulation within the colonocyte. Individual colon crypts prepared from Sprague-Dawley rats were mounted in perfusion chambers after loading with fluorescent reporters fura 2-AM and fluozin 3-AM. These reporters were characterized, in situ, for responsiveness to [Ca.sup.2+] and [Zn.sup.2+] in the cytoplasm. Responses to different concentrations of N[H.sub.2]Cl (50, 100, and 200 [micro]M) were monitored. Subsequent studies were designed to identify the sources and mechanisms of N[H.sub.2]Cl-induced increases in [Ca.sup.2+] and [Zn.sup.2+] in the cytoplasm. Exposure to N[H.sub.2]Cl led to dose-dependent increases in intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]]i) in the range of 200-400 nM above baseline levels. Further studies indicated that N[H.sub.2]Cl-induced accumulation of [Ca.sup.2+] in the cytoplasm is the result of release from intracellular stores and basolateral entry of extracellular [Ca.sup.2+] through store-operated channels. In addition, exposure to N[H.sub.2]Cl resulted in dose-dependent and sustained increases in intracellular [Zn.sup.2+] concentration ([[Zn.sup.2+]]i) in the nanomolar range. These alterations were neutralized by dithiothreitol, which shields intracellular thiol groups from oxidation. We conclude that [Ca.sup.2+]- and [Zn.sup.2+]-handling proteins are susceptible to oxidation by chloramines, leading to sustained, but not necessarily toxic, increases in [[Ca.sup.2+]]i and [[Zn.sup.2+]]i. Under certain conditions, N[H.sub.2]Cl may act not as a toxin but as an agent that activates intracellular signaling pathways. key words

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain ([MLC.sub.20]) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-[alpha]. Presently, we examined the cross talk between RhoA and PKC-[alpha] in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-[alpha], CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21 [+ or -] 3.52 and 42.19 [+ or -] 3.85%nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12 [+ or -] 46.60% and 290.59 [+ or -] 22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-[alpha] by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-[alpha] pathways, suggesting cross talk between RhoA and PKC-[alpha] resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of [MLC.sub.20]. myosin light chain; contraction; CPI-17; acetylcholine; Rho kinase; colon; phosphatase; protein kinase C; heat shock protein

We have previously shown that sigmoid circular muscle cells from patients with ulcerative colitis (UC) exhibit reduced contraction and [Ca.sup.2+] signaling in response to the neurotransmitter neurokinin A (NKA) and that IL-[beta] and [H.sub.2][O.sub.2] may contribute to these reduced responses in UC. In addition, we have found that nitric oxide (NO) levels were significantly increased in UC circular muscle. To establish the site of origin for IL-1[beta], [H.sub.2][O.sub.2], and NO, we assembled an in vitro system in which normal or UC mucosa were sealed between two chambers filled with oxygenated Krebs solution. Because the mucosa consists of fullthickness mucosa and submucosa, it is expected that whatever is released into the undernatant from the submucosal side may diffuse to the circular muscle layer in the intact colon. Treatment of normal sigmoid circular muscle cells for 2 h with undernatants collected from the UC submucosal side (UCS) significantly decreased contraction induced by NKA and thapsigargin and the NKA- and caffeine-induced [Ca.sup.2+] signal in [Ca.sup.2+]-free medium. In addition, UC mucosa released into the undernatant on its submucosal side significantly more [H.sub.2][O.sub.2], IL-[beta], and NO than normal mucosa. The reduction in contraction and [Ca.sup.2+] signal induced by UCS was partially reversed by pretreatment with an IL-1[beta] antibody or with catalase. The NO scavenger hemoglobin partially prevented UCS-induced reduction in contraction and [Ca.sup.2+] signaling in response to NKA but not the reduced response to thapsigargin or caffeine. Sodium nitroprusside inhibited NKA but not the caffeine-induced [Ca.sup.2+] signal. We conclude that in UC the mucosa releases IL-1[beta], [H.sub.2][O.sub.2], and NO, which may contribute to the impaired [Ca.sup.2+] release and altered sigmoid muscle contractility. neurokinin A; calcium; human; colon

Neural crest cells leave the hindbrain, enter the gut mesenchyme at the pharynx, and migrate as strands of cells to the terminal bowel to form the enteric nervous system. We generated embryos containing fluorescent enteric neural crest-derived cells (ENCCs) by mating Wntl-Cre mice with Rosa-floxed-YFP mice and investigated ENCC behavior in the intact gut of mouse embryos using time-lapse fluorescent microscopy. With respect to the entire gut, we have found that ENCCs in the cecum and proximal colon behave uniquely. ENCCs migrating caudally through either the ileum, or caudal colon, are gradually advancing populations of strands displaying largely unpredictable local trajectories. However, in the cecum, advancing ENCCs pause for approximately 12 h, and then display an invariable pattern of migration to distinct regions of the cecum and proximal colon. In addition, while most ENCCs migrating through other regions of the gut remain interconnected as strands; ENCCs initially migrating through the cecum and proximal colon fragment from the main population and advance as isolated single cells. These cells aggregate into groups isolated from the main network, and eventually extend strands themselves to reestablish a network in the mid-colon. As the advancing network of ENCCs reaches the terminal bowel, strands of sacral crest cells extend, and intersect with vagal crest to bridge the small space between. We found a relationship between ENCC number, interaction, and migratory behavior by utilizing endogenously isolated strands and by making cuts along the ENCC wavefront. Depending on the number of cells, the ENCCs aggregated, proliferated, and extended strands to advance the wavefront. Our results show that interactions between ENCCs are important for regulating behaviors necessary for their advancement. Keywords: Enteric nervous system; Neural development; YFP; Wntl; Cre recombinase; Neural crest; Strand migration; Cecum; Population pressure; GDNF; Endothelin

Water is an essential nutrient because it must be introduced from exogenous sources to satisfy metabolic demand. Under physiologic conditions, the colon can absorb and secrete considerable amounts of water even against osmotic gradients, thus helping to maintain the body fluid balance. Here we describe studies on both aquaporin (AQP) expression and function using cells isolated from the superficial and lower crypt regions of the rat proximal colon. The expression of AQP-3, -4, and -8 in isolated colonocytes was determined by semiquantitative RT-PCR and by immunoblotting. The localization of AQP-8 in the colon was evaluated by immunohistochemistry. A stopped-flow light scattering method was used to examine osmotic water movement in isolated colonocytes. Moreover, the contribution of AQP-8 to overall water movement through isolated colonocytes was studied using RNA interference technology. Colonocytes from the proximal colon express AQP-3, -4, and -8 with increasing concentrations from the lower crypt cells toward those on the surface. Osmotic water permeability was higher in surface than in crypt colonocytes (P < 0.05); it was significantly inhibited by the water channel blocker dimethyl sulfoxide, and reversed by [beta]-mercaptoethanol (P < 0.05). Immunohistochemistry revealed a strong AQP-8 labeling in the apical membrane of the superficial colonocytes. Inhibition of aquaporin-8 expression by small interfering RNA significantly decreased osmotic water permeability (~38%; P < 0.05). Current results indicate that aquaporin-8 may play a major role in water movement through the colon by acting on the apical side of the superficial cells. KEY WORDS: * aquaporins * water channel * small interfering RNA * colon

The cellular mechanism for [Cl.sup.-] and [K.sup.+] secretion in the colonic epithelium requires [K.sup.+] channels in the basolateral and apical membranes. Colonic mucosa from guinea pig and rat were fixed, sectioned, and then probed with antibodies to the [K.sup.+] channel proteins [K.sub.v]LQT1 (Kcnq1) and minK-related peptide 2 (MiRP2, Kcne3). Immunofluorescence labeling for Kcnq1 was most prominent in the lateral membrane of crypt cells in rat colon. The guinea pig distal colon had distinct lateral membrane immunoreactivity for Kcnq1 in crypt and surface cells. In addition, Kcne3, an auxiliary subunit for Kcnq1, was detected in the lateral membrane of crypt and surface cells in guinea pig distal colon. Transepithelial short-circuit current ([I.sub.sc]) and transepithelial conductance ([G.sub.t]) were measured for colonic mucosa during secretory activation by epinephrine (EPI), prostaglandin [E.sub.2] (PG[E.sub.2]), and carbachol (CCh). HMR1556 (10 [micro]M), an inhibitor of Kcnq1 channels (Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Briiggemann A, Busch A, Suessbrich H, Bleich M, and Greger R. J Med Chem 44: 3831-3837, 2001), partially (~50%) inhibited [Cl.sup.-] secretory [I.sub.sc] and Gt activated by PGE2 and CCh in rat colon with an I[C.sub.50] of 55 nM, but in guinea pig distal colon [Cl.sup.-] secretory [I.sub.sc] and [G.sub.t] were unaltered. EPI-activated [K.sup.+]-secretory [I.sub.sc] and [G.sub.t] also were essentially unaltered by HMR1556 in both rat and guinea pig colon. Although immunofluorescence labeling with a Kcnq1 antibody supported the basolateral membrane presence in colonic epithelium of the guinea pig as well as the rat, the Kcnq1 [K.sup.+] channel is not an essential component for producing [Cl.sup.-] secretion. Other [K.sup.+] channels present in the basolateral membrane presumably must also contribute directly to the [K.sup.+] conductance necessary for [K.sup.+] exit during activation of Cl secretion in the colonic mucosa. HMR1556; [K.sup.+] secretion; epinephrine; prostaglandin [E.sub.2]; cholinergic

In vitro experiments have shown that fermentation of carbohydrates prevents accumulation of nitrogen in the colon. Variable results have been obtained on modulation of dietary intakes in vivo. Lactose[[sup.15]N, [sup.15]N]-labeled ureide has been proposed as a tool to study colonic nitrogen metabolism. However, on oral administration of the marker, different urinary excretion patterns of the [sup.15]N label have been found. In this study, 50 mg lactose[[sup.15]N, [sup.15]N]ureide was directly instilled in the colon through an orocecal tube to investigate the colonic handling of this molecule in a direct way. In basal conditions, 42% (range, 37-48%) of labeled nitrogen administered as lactose[[sup.15]N, [sup.15]N]ureide was retrieved in urine after 72 h. A substantial variability in total urinary excretion of the label was found, but the urinary excretion pattern of the label was similar in all volunteers. When inulin, a fermentable carbohydrate, was administered together with the labeled marker, a significant decrease in urinary excretion of [sup.15]N after 72 h was found, to 29% (range, 23-34%). The effect of a smaller dose of inulin (250 mg) on colonic handling of lactose[[sup.15]N, [sup.15]N]ureide (50 mg), was investigated in another group of volunteers, and this time, fecal excretion of the marker was also evaluated. The results seem to indicate that fermentation of inulin causes an increased fecal excretion of the marker, thereby reducing urinary excretion but not retention in the human nitrogen pool. This instillation study shows that lactose[[sup.15]N, [sup.15]N]ureide is a tool with good properties to investigate the effect of different types of carbohydrates on nitrogen metabolism in the proximal colon in vivo. carbohydrate fermentation; stable isotopes

This study was setup to investigate the design and performance of a shape memory alloy clip for colonic anastomosis. The thermomechanical properties of the shape memory alloy material were studied and the data were used to derive a nonlinear material model. This enabled the development of computer computer aided design models and finite element analysis of the clip and tissue compression. The maximum strain of the anastomosis clip was within the recoverable range, and it exerted parallel compression of the colonic walls with a uniform pressure distribution. The design of the anastomosis clip was optimized for safe, simple, and effective use in colon surgery. [DOI: 10.1115/1.1871195] Keywords: Shape Memory Alloy, Finite Element Analysis, Anastomosis, Colon Surgery

This study was initiated to evaluate the efficacy of negative contrast-enhanced microcomputed tomography (microCT) colonography for the noninvasive detection of colonic tumors in living mice. After colonic preparation, 20 anesthetized congenic mice were scanned with high-resolution microCT. Images were displayed by using commercial visualization software and interpreted by two gastrointestinal radiologists, who were unaware of tumor prevalence and findings at gross pathology. Two-dimensional multiplanar images were assessed by using a five-point scale to distinguish colonic tumors (polyps) from fecal pellets (5 = definitely a tumor, 4 = probably a tumor, 3 = indeterminate, 2 = probably not a tumor, 1 = definitely not a tumor). Gross pathologic evaluation of excised mouse colons served as the reference standard. Data analysis included dichotomizing results, with 1-2 indicating no tumor and 3-5 indicating tumor and also receiver operator characteristic curve analysis with area under the curve for threshold-independent assessment. A total of 41 colonic polyps in 18 of the 20 mice were identified at gross examination on necropsy, of which 30 measured 2-5 mm and 11 measured < 2 mm in size. The pooled per-polyp sensitivity for lesions > 2 mm was 93.3% (56/60). The pooled per-mouse sensitivity for polyps > 2 mm was 97.1% (33/34). Pooled specificity for distinguishing fecal pellets from tumor was 98.5% (65/66). The combined area under the curve from receiver operator characteristic curve analysis was 0.810 [+ or -] 0.038 (95% confidence interval, 0.730-0.890). These findings indicate that accurate noninvasive longitudinal monitoring of colon tumor progression or response to various therapies is now technically feasible in live mice by using this microCT colonography method. colonic neoplasm

Mice carrying heterozygous mutations in the Sox10 gene display aganglionosis of the colon and represent a model for human Hirschsprung disease. Here, we show that the closely related Sox8 functions as a modifier gene for Sox10-dependent enteric nervous system defects as it increases both penetrance and severity of the defect in Sox10 heterozygous mice despite having no detectable influence on enteric nervous system development on its own. Sox8 exhibits an expression pattern very similar to Sox10 with occurrence in vagal and enteric neural crest cells and later confinement to enteric glia. Loss of Sox8 alleles in Sox10 heterozygous mice impaired colonization of the gut by enteric neural crest cells already at early times. Whereas proliferation, apoptosis, and neuronal differentiation were normal for enteric neural crest cells in the gut of mutant mice, apoptosis was dramatically increased in vagal neural crest cells outside the gut. The defects in enteric nervous system development of mice with Sox10 and Sox8 mutations are therefore likely caused by a reduction of the pool of undifferentiated vagal neural crest cells. Our study suggests that Sox8 and Sox10 are jointly required for the maintenance of these vagal neural crest stem cells. Keywords: Sry; High-mobility-group; Sox10; Enteric nervous system; Hirschsprung disease; Megacolon