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Bacterial or ingested food-derived short-chain fatty acids (SCFAs) are present in the duodenal lumen. Acetate, the most abundant SCFA in the foregut lumen, is absorbed immediately after ingestion, although the mechanism by which this absorption occurs is not fully understood. We investigated the distribution and function of candidate SCFA transporters in rat duodenum. The Na+-coupled monocarboxylate transporter-1 (SMCT1) was localized to the brush border, whereas the pH-dependent monocarboxylate transporter (MCT) 1 and MCT4 were localized to the duodenocyte basolateral membrane. In Ussing chambered duodenal mucosa, luminal acetate dose-dependently increased short-circuit current ( Isc) in the presence of serosal bumetanide and indomethacin by a luminal Na+-dependent, ouabain-sensitive mechanism. The Isc response was inhibited dose-dependently by the SMCT1 nonsubstrate inhibitor ibuprofen, consistent with net electrogenic absorption of acetate via SMCT1. Other SCFAs and lactate also increased Isc. Furthermore, duodenal loop perfusion of acetate increased portal venous acetate concentration, inhibited by coperfusion of ibuprofen or a MCT inhibitor. Luminal acetate perfusion increased duodenal HCO3− secretion via capsaicin-sensitive afferent nerve activation and cyclooxygenase activity, consistent with absorption-mediated HCO3− secretion. These results suggest that absorption of luminal SCFA via SMCT1 and MCTs increases duodenal HCO3− secretion. In addition to SCFA sensing via free fatty acid receptors, the presence of rapid duodenal SCFA absorption may be important for the suppression of luminal bacterial colonization and implicated in the generation of functional dyspepsia due to bacterial overgrowth.

Intraduodenal fatty acids (FA) and bacterial overgrowth, which generate short-chain FAs (SCFAs), have been implicated in the generation of functional dyspepsia symptoms. We studied the mechanisms by which luminal SCFA perfusion affects duodenal HCO3− secretion (DBS), a measure of mucosal neurohumoral activation. Free fatty acid receptor (FFAR) 1 (FFA1), which binds long-chain FA (LCFA), and SCFA receptors FFA2 and FFA3 were immunolocalised to duodenal enteroendocrine cells. FFA3 colocalised with glucagon-like peptide (GLP)-1, whereas FFA2 colocalised with 5-HT. Luminal perfusion of the SCFA acetate or propionate increased DBS, enhanced by dipeptidyl peptidase-IV (DPPIV) inhibition, at the same time as increasing GLP-2 portal blood concentrations. Acetate-induced DBS was partially inhibited by monocarboxylate/HCO3− exchanger inhibition without affecting GLP-2 release, implicating acetate absorption in the partial mediation of DBS. A selective FFA2 agonist dose-dependently increased DBS, unaffected by DPPIV inhibition or by cholecystokinin or 5-HT3 receptor antagonists, but was inhibited by atropine and a 5-HT4 antagonist. By contrast, a selective FFA1 agonist increased DBS accompanied by GLP-2 release, enhanced by DPPIV inhibition and inhibited by a GLP-2 receptor antagonist. Activation of FFA1 by LCFA and presumably FFA3 by SCFA increased DBS via GLP-2 release, whereas FFA2 activation stimulated DBS via muscarinic and 5-HT4 receptor activation. SCFA/HCO3− exchange also appears to be present in the duodenum. The presence of duodenal fatty acid sensing receptors that signal hormone release and possibly signal neural activation may be implicated in the pathogenesis of functional dyspepsia.

Luminal ATP increases duodenal bicarbonate secretion (DBS) via brush border P2Y receptors. Because ATP is sequentially dephosphorylated to adenosine (ADO) and the brush border highly expresses adenosine deaminase (ADA), we hypothesized that luminal [ADO] regulators and sensors, including P1 receptors, ADA, and nucleoside transporters (NTs) regulate DBS. We measured DBS with pH and CO(2) electrodes, perfusing ADO ± adenosine receptor agonists or antagonists or the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR(inh)-172 on DBS. Furthermore, we examined the effect of inhibitors of ADA or NT on DBS. Perfusion of AMP or ADO (0.1 mM) uniformly increased DBS, whereas inosine had no effect. The A(1/2) receptor agonist 5'-(N-ethylcarboxamido)-adenosine (0.1 mM) increased DBS, whereas ADO-augmented DBS was inhibited by the potent A(2B) receptor antagonist N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-acetamide (MRS1754) (10 μM). Other selective adenosine receptor agonists or antagonists had no effect. The A(2B) receptor was immunolocalized to the brush border membrane of duodenal villi, whereas the A(2A) receptor was immunolocalized primarily to the vascular endothelium. Furthermore, ADO-induced DBS was enhanced by 2'-deoxycoformycin (1 μM) and formycin B (0.1 mM), but not by S-(4-nitrobenzyl)-6-thioinosine (0.1 mM), and it was abolished by CFTR(inh)-172 pretreatment (1 mg/kg i.p). Moreover, ATP (0.1 mM)-induced DBS was partially reduced by (1R,2S,4S,5S)-4-2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS2500) or 8-[4-[4-(4-chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine (PSB603) and abolished by both, suggesting that ATP is sequentially degraded to ADO. Luminal ADO stimulates DBS via A(2B) receptors and CFTR. ATP release, ecto-phosphohydrolases, ADA, and concentrative NT may coordinately regulate luminal surface ADO concentration to modulate ADO-P1 receptor signaling in rat duodenum.

Regulation of localized extracellular pH (pHo) maintains normal organ function. An alkaline microclimate overlying the duodenal enterocyte brush border protects the mucosa from luminal acid. We hypothesized that intestinal alkaline phosphatase (IAP) regulates pHo due to pH-sensitive ATP hydrolysis as part of an ecto-purinergic pH regulatory system, comprised of cell-surface P2Y receptors and ATP-stimulated duodenal bicarbonate secretion (DBS). To test this hypothesis, we measured DBS in a perfused rat duodenal loop, examining the effect of the competitive alkaline phosphatase inhibitor glycerol phosphate (GP), the ecto-nucleoside triphosphate diphosphohydrolase inhibitor ARL67156, and exogenous nucleotides or P2 receptor agonists on DBS. Furthermore, we measured perfusate ATP concentration with a luciferin–luciferase bioassay. IAP inhibition increased DBS and luminal ATP output. Increased luminal ATP output was partially CFTR dependent, but was not due to cellular injury. Immunofluorescence localized the P2Y1 receptor to the brush border membrane of duodenal villi. The P2Y1 agonist 2-methylthio-ADP increased DBS, whereas the P2Y1 antagonist MRS2179 reduced ATP- or GP-induced DBS. Acid perfusion augmented DBS and ATP release, further enhanced by the IAP inhibitor l-cysteine, and reduced by the exogenous ATPase apyrase. Furthermore, MRS2179 or the highly selective P2Y1 antagonist MRS2500 co-perfused with acid induced epithelial injury, suggesting that IAP/ATP/P2Y signalling protects the mucosa from acid injury. Increased DBS augments IAP activity presumably by raising pHo, increasing the rate of ATP degradation, decreasing ATP-mediated DBS, forming a negative feedback loop. The duodenal epithelial brush border IAP–P2Y–HCO3− surface microclimate pH regulatory system effectively protects the mucosa from acid injury.

Background Intermittent warm-water infusion in lieu of air insufflation permitted 52% of patients who accepted sedation on demand to complete colonoscopy without sedation. Objective To test the hypothesis that the water method enhances cecal intubation and increases the proportion of patients who report willingness to repeat a scheduled unsedated colonoscopy. Design Observational study. Patients Two consecutive groups of veterans. Interventions From June 2005 to May 2006, the usual air insufflation method was used to aid colonoscope insertion. From June 2006 to October 2007, the water method was used. Main Outcome Measurements Cecal intubation; report of willingness to repeat unsedated colonoscopy. Results Sixty-two and 63 veterans were examined by the air method and the water method, respectively. Intention-to-treat analysis revealed that the cecal intubation rate with the water method (97% [61/63]) was significantly higher than that with the air method (76% [47/62]). The proportion of patients who reported willingness to repeat unsedated colonoscopy was significantly higher with the water method (90% [57/63]) compared with the air method (69% [43/62]). Limitations Single site, nonrandomized, unblinded, small number of elderly male veterans. Conclusion The effects of the water method in the group for scheduled unsedated colonoscopy were sufficiently provocative to warrant calling for their confirmation by a randomized controlled trial.

The Center for Ulcer Research and Education (CURE) from its onset was primarily the work of one man: Professor Morton Grossman, or “Mort” as he was known and called by all. Mort's legacy includes a large body of scientific publications, the first National Institutes of Health Digestive Diseases Center (CURE), and, most importantly, a group of scientists who have become academic leaders and who have made important contributions in the fields of upper gastrointestinal (GI) tract secretion, hormones and receptors, mucosal defense mechanisms, the design and conduct of randomized clinical trials, and ulcer epidemiology. Indeed, Mort is considered to be a founding father of modern academic GI research. I was fortunate to have known and worked with Mort and would like to memorialize his contributions so that his memory can inspire the next generation of academicians.

In 1951, I began a 2-year postdoctoral research fellowship under Dr Simon Komarov at Temple University. Later, as a staff gastroenterologist at the Orange County General Hospital in California, I was interested in the possible role of blood flow alterations in peptic ulcer disease. We adapted Silvio Baez's just described in vivo microscopy technique to study blood flow in the gastric microcirculation in the anesthetized cat. In 1969, I joined the Gastroenterology Service at the West Los Angeles Veterans Administration (VA), primarily because of the presence there of Dr Morton Grossman, one of the leading researchers in gastrointestinal physiology. The most important lesson I learned from Dr Grossman was the importance of openness--investigators sharing their ideas, thoughts, and findings. Dr Harold Wayland, Professor, California Institute of Technology, helped me in setting up my laboratory at the VA and arranged with his friend Dr Masaharu Tsuchiya, Chair of Gastroenterology at Keio University, Tokyo, Japan, for a collaborative effort with fellows from Keio coming for 2-year fellowships with us (under my grant). Harold introduced us to the fluorescein-labeled albumen technique for the study of microvascular permeability. Later, Dr Jonathon Kaunitz, a colleague at the VA, and Kotaro Kaneko, my Keio University fellow, succeeded in modifying this technique to measure pHi in surface gastric cells in vivo. When I retired in 1993, Dr Kaunitz took charge of my laboratory. He and his associate Dr Akiba have completed extensive studies comparing and contrasting how stomach and duodenal surface cells defend themselves against acid loads. In the course of this work, they have become deeply involved in cell signaling and intracellular pathways when nutrients enter the duodenum. They have developed a technique for using confocal microscopy in vivo in this work. Dr Grossman was right: when investigators share their ideas, everyone benefits and science advances.

The duodenum must absorb ∼450 mmol of H+ per 24 h in order to decrease [H+] of the luminal content by 6 log orders over its 15 cm length (Feldman & Colturi, 1984). HCl in the duodenal lumen is neutralized by HCO3− secreted by the pancreas and duodenal epithelium, generating extremely high luminal CO2 pressures (PCO2 > 30 kPa) which dissipate by the proximal jejunum (Rune & Henriksen, 1969; Winship & Robinson, 1974). Gastric mucosal CO2 and H+ permeability is low, since pyloric obstruction leads to severe metabolic alkalosis due to the inability of the stomach to absorb substantial quantities of H+ or CO2 (Gamble & Ross, 1925; Javaheri & Nardell 1981). Thus, the duodenum is the major site for intestinal H+ and CO2 absorption. Transmucosal bulk absorption of CO2 and H+ is likely to follow the sequence of transport across the apical cell membrane into the cytosol, transport across the basolateral membrane of the epithelium into the subepithelial interstitium, followed by transport into the portal vein. The mechanism for CO2 and H+ transport across cell membranes remains incompletely understood. In terms of transapical CO2 and H+ transport, one accepted model involves conversion of luminal H+ and HCO3− to CO2 and H2O, diffusion of CO2 across the apical plasma membrane, and hydration of CO2 to HCO3− and H+ in the cytoplasm. This process, sometimes termed the Jacobs-Stewart cycle after its original description in red blood cells (Jacobs & Stewart, 1942), requires the presence of intracellular and extracellular carbonic anhydrases (CAs) and a plasma membrane anion exchanger. Since the duodenal epithelium has abundant cytoplasmic and membrane-associated CA activity (Sugai et al. 1994; Lonnerholm et al. 1989; Parkkila et al. 1994; Saarnio et al. 1998; Purkerson & Schwartz, 2005; Leppilampi et al. 2005) and apical anion exchangers (Wang et al. 2002; Spiegel et al. 2003), we hypothesized that most of the excess duodenal luminal H+ was absorbed through neutralization of secreted HCO3−, yielding luminal CO2. CO2 is the molecular species that then traverses the apical membrane, which enters the cytoplasm, and is hydrated to H2CO3, which then dissociates in the cytoplasm to H+ and HCO3−. HCO3− is transported into the lumen whereas H+ is transported into the submucosal space by membrane transport proteins. In essence, luminal H+ is transported through the apical membrane as CO2, but HCO3− is simultaneously secreted in its anionic form. Several observations support this hypothesis. We, and others have found that luminal acidification or elevation of luminal PCO2 provokes several epithelial responses, such as acidification of the cytoplasm and subepithelial interstitial fluid, increased mucosal blood flow, increased HCO3− secretion, and increased mucus secretion (Flemstrom & Kivilaakso, 1983; Flemstrom, 1994; Seno et al. 1998; Paimela et al. 1990, 1992; Akiba & Kaunitz, 1999; Akiba et al. 2000, 2001a,b, 2006). Since these responses appear to be dependent on acidification of the subepithelial space, and since elevated luminal CO2 or H+ provoke similar responses, it appears that luminal CO2 must be converted to subepithelial H+, which then signals these protective mechanisms (Allen & Flemstrom, 2005). The further characterization of transmucosal H+ and CO2 movement thus has larger implications for the understanding of mucosal protective mechanisms and the signalling pathways coordinating mucosal responses to acid perfusion. In order to further test our hypothesis, we devised a system in which the movement of CO2 and H+ between lumen, mucosa and the portal vein was measured. In order to determine the contribution of cytoplasmic and extracellular CAs towards H+ and CO2 movement, we used cell-permeant and -impermeant CA inhibitors. Finally, transmucosal tracer carbon movement was measured using 13C. Our results support our hypothesis that luminal H+, neutralized by secreted HCO3−, is converted to CO2 prior to entry into the cytoplasm of the epithelial cells, and that cellular CO2 is reconverted to H+, which then is transported into the portal vein.

RX 77368 (RX) increases gastric mucosal blood flow by a vagal cholinergic mechanism. The relative roles of mucosal and connective tissue mast cells (MMC and CTMC) were investigated in RX-injected rats. Blood flow and mast cell degranulation were measured after intracisternal RX. RX significantly increased gastric mucosal blood flow, and sequentially degranulated CTMC and MMC. Ketotifen or doxantrazole inhibited the hyperemic response. Ondansetron, RS-039604-90, or famotidine, but not ketanserin or pyrilamine, reduced hyperemia. Mast cells mediate RX-induced gastric hyperemia via 5-HT3, 5-HT4, and H2 receptors; initial increase depends upon CTMC whereas MMC contributes to the later response.

The proximal duodenum is exposed to extreme elevations of Pco2because of the continuous mixture of secreted HCO3−with gastric acid. These elevations (up to 80 kPa) are likely to place the mucosal cells under severe acid stress. Furthermore, we hypothesized that, unlike most other cells, the principal source of CO2for duodenal epithelial cells is from the lumen. We hence examined the effect of elevated luminal Pco2on duodenal HCO3−secretion (DBS) in the rat. DBS was measured by the pH-stat method. For CO2challenge, the duodenum was superfused with a high Pco2solution. Intracellular pH (pHi) of duodenal epithelial cells was measured by ratio microfluorometry. CO2challenge, but not isohydric solutions, strongly increased DBS to approximately two times basal for up to 1 h. Preperfusion of the membrane-permeant carbonic anhydrase inhibitor methazolamide, or continuous exposure with indomethacin, fully inhibited CO2-augmented DBS. Dimethyl amiloride (0.1 mM), an inhibitor of the basolateral sodium-hydrogen exchanger 1, also inhibited CO2-augumented DBS, although S-3226, a specific inhibitor of apical sodium-hydrogen exchanger 3, did not. DIDS, an inhibitor of basolateral sodium-HCO3−cotransporter, also inhibited CO2-augemented DBS, as did the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid. CO2decreased epithelial cell pHi, followed by an overshoot after removal of the CO2solution. We conclude that luminal CO2diffused in the duodenal epithelial cells and was converted to H+and HCO3−by carbonic anhydrase. H+initially exited the cell, followed by secretion of HCO3−. Secretion was dependent on a functioning basolateral sodium/proton exchanger, a functioning basolateral HCO3−uptake mechanism, and submucosal prostaglandin generation and facilitated hydration of CO2into HCO3−and H+.

Luminal lipid in the duodenum modulates gastroduodenal functions via the release of gut hormones and mediators such as cholecystokinin and 5-HT. The effects of luminal short-chain fatty acids (SCFAs) in the foregut are unknown. Free fatty acid receptors (FFARs) for long-chain fatty acids (LCFAs) and SCFAs are expressed in enteroendocrine cells. SCFA receptors, termed FFA2 and FFA3, are expressed in duodenal enterochromaffin cells and L cells, respectively. Activation of LCFA receptor (FFA1) and presumed FFA3 stimulates duodenal HCO3(-) secretion via a glucagon-like peptide (GLP)-2 pathway, whereas FFA2 activation induces HCO3(-) secretion via muscarinic and 5-HT4 receptor activation. The presence of SCFA sensing in the duodenum with GLP-2 and 5-HT signals further supports the hypothesis that luminal SCFA in the foregut may contribute towards the generation of functional symptoms.Intraduodenal fatty acids (FA) and bacterial overgrowth, which generate short-chain FAs (SCFAs), have been implicated in the generation of functional dyspepsia symptoms. We studied the mechanisms by which luminal SCFA perfusion affects duodenal HCO3(-) secretion (DBS), a measure of mucosal neurohumoral activation. Free fatty acid receptor (FFAR) 1 (FFA1), which binds long-chain FA (LCFA), and SCFA receptors FFA2 and FFA3 were immunolocalised to duodenal enteroendocrine cells. FFA3 colocalised with glucagon-like peptide (GLP)-1, whereas FFA2 colocalised with 5-HT. Luminal perfusion of the SCFA acetate or propionate increased DBS, enhanced by dipeptidyl peptidase-IV (DPPIV) inhibition, at the same time as increasing GLP-2 portal blood concentrations. Acetate-induced DBS was partially inhibited by monocarboxylate/HCO3(-) exchanger inhibition without affecting GLP-2 release, implicating acetate absorption in the partial mediation of DBS. A selective FFA2 agonist dose-dependently increased DBS, unaffected by DPPIV inhibition or by cholecystokinin or 5-HT3 receptor antagonists, but was inhibited by atropine and a 5-HT4 antagonist. By contrast, a selective FFA1 agonist increased DBS accompanied by GLP-2 release, enhanced by DPPIV inhibition and inhibited by a GLP-2 receptor antagonist. Activation of FFA1 by LCFA and presumably FFA3 by SCFA increased DBS via GLP-2 release, whereas FFA2 activation stimulated DBS via muscarinic and 5-HT4 receptor activation. SCFA/HCO3(-) exchange also appears to be present in the duodenum. The presence of duodenal fatty acid sensing receptors that signal hormone release and possibly signal neural activation may be implicated in the pathogenesis of functional dyspepsia.

We studied the role of duodenal cellular ion transport in epithelial defense mechanisms in response to rapid shifts of luminal pH. We used in vivo microscopy to measure duodenal epithelial cell intracellular pH (pHi), mucus gel thickness, blood flow, and HCO[Formula: see text] secretion in anesthetized rats with or without the Na+/H+exchange inhibitor 5-( N, N-dimethyl)-amiloride (DMA) or the anion transport inhibitor DIDS. During acid perfusion pHidecreased, whereas mucus gel thickness and blood flow increased, with pHiincreasing to over baseline (overshoot) and blood flow and gel thickness returning to basal levels during subsequent neutral solution perfusion. During a second brief acid challenge, pHidecrease was lessened (adaptation). These are best explained by augmented cellular HCO[Formula: see text] uptake in response to perfused acid. DIDS, but not DMA, abolished the overshoot and pHiadaptation and decreased acid-enhanced HCO[Formula: see text] secretion. In perfused duodenum, effluent total CO2output was not increased by acid perfusion, despite a massive increase of titratable alkalinity, consistent with substantial acid back diffusion and modest CO2back diffusion during acid perfusions. Rapid shifts of luminal pH increased duodenal epithelial buffering power, which protected the cells from perfused acid, presumably by activation of Na+-HCO[Formula: see text] cotransport. This adaptation may be a novel, important, and early duodenal protective mechanism against rapid physiological shifts of luminal acidity.

We previously showed that the duodenal hyperemic response to acid occurs through activation of capsaicin-sensitive afferent nerves with subsequent release of vasodilatory substances such as calcitonin gene-related peptide (CGRP) and nitric oxide. We then tested the hypothesis that similar factors regulate duodenal mucus gel thickness. Gel thickness was optically measured using in vivo microscopy in anesthetized rats. Duodenal mucosae were superfused with pH 7.0 buffer with vanilloid receptor agonist capsaicin, bradykinin, or PGE2 injection or were challenged with pH 2.2 solution, with or without the vanilloid antagonist capsazepine, human CGRP-(8–37), N G-nitro-l-arginine methyl ester, and indomethacin. Other rats underwent sensory ablation with high-dose capsaicin pretreatment. Acid, bradykinin, capsaicin, and PGE2 all quickly thickened the gel. Antagonism of vanilloid and CGRP receptors, inhibition of nitric oxide synthase, and sensory deafferentation delayed gel thickening, suggesting that the capsaicin pathway mediated the initial burst of mucus secretion that thickened the gel. Indomethacin abolished gel thickening due to acid, bradykinin, and capsaicin. Inhibition of gel thickening by indomethacin in response to multiple agonists suggests that cyclooxygenase activity is essential for duodenal gel thickness regulation. Duodenal afferent neural pathways play an important role in the modulation of cyclooxygenase-mediated physiological control of gel thickness.

Mechanisms responsible for the rapid tissue destruction in gas gangrene are not well understood. To examine the early effects of Clostridium perfringens exotoxins on tissue perfusion, a rat model of muscle blood flow was developed. Intramuscular injection of a clostridial toxin preparation containing both phospholipase C (PLC) and theta-toxin caused a rapid (1-2 min) and irreversible decrease in blood flow that paralleled formation of activated platelet aggregates in venules and arterioles. Later (20-40 min), aggregates contained fibrin and leukocytes, and neutrophils accumulated along vascular walls. Flow cytometry confirmed that these clostridial toxins or recombinant PLC induced formation of P-selectin-positive platelet aggregates. Neutralization of PLC activity in the clostridial toxin preparation completely abrogated human platelet responses and reduced perfusion deficits. It is concluded that tissue destruction in gas gangrene is related to profound attenuation of blood flow initiated by activation of platelet responses by PLC.

We tested the hypothesis that the duodenal hyperemic response to acid occurs through activation of capsaicin-sensitive afferent nerves with subsequent release of vasodilatory substances such as calcitonin gene-related peptide (CGRP) and nitric oxide (NO). Laser-Doppler flowmetry was used to measure duodenal blood flow in urethan-anesthetized rats. Duodenal mucosa was superfused with pH 7.0 buffer with capsaicin or bradykinin or was acid challenged with pH 2.2 solution, with or without vanilloid receptor antagonists, a CGRP receptor antagonist, an NO synthase (NOS) inhibitor, or a cyclooxygenase inhibitor. The selective vanilloid receptor antagonist capsazepine (CPZ) dose dependently inhibited the hyperemic response to acid and capsaicin but did not affect bradykinin-induced hyperemia. Ruthenium red was less inhibitory than capsazepine. Selective ablation of capsaicin-sensitive nerves, CGRP-(8—37), and N G-nitro-l-arginine methyl ester inhibited acid-induced hyperemia, but indomethacin did not. We conclude that luminal acid, but not bradykinin, stimulates CPZ-sensitive receptors on capsaicin-sensitive afferent nerves of rat duodenum. Activation of these receptors produces vasodilation via the CGRP-NO pathway but not via the cyclooxygenase pathway. Acid appears to be the endogenous ligand for duodenal vanilloid receptors.

The purpose of this study was to evaluate the role of nitric oxide (NO) in the maintenance of basal endometrial blood flow of ovariectomized rats and in the increase of endometrial blood flow after administration of estradiol 17β (E2β). Endometrial blood flow was repeatedly measured with the H2 gas clearance technique in ovariectomized rats. N ω-nitro-l-arginine methyl ester (l-NAME) dose dependently reduced basal endometrial blood flow and increased mean arterial blood pressure and endometrial vascular resistance. E2β (1 μg/kg iv) increased endometrial blood flow and reduced endometrial vascular resistance, which peaked by 2 h after the injection. The vasoconstrictive activity of l-NAME (an inhibitor for NO synthesis) was compared with that of phenylephrine (PE, an α-receptor agonist acting through an NO-independent mechanism). Doses ofl-NAME (1 and 3 mg/kg iv) were matched with those of PE (3.2 and 6.4 mg ⋅ kg−1 ⋅ h−1iv), as they induced an approximately equivalent percent increase in basal endometrial vascular resistance. The percent increases of endometrial vascular resistance in E2β-treated animals by the two agents in matched doses were also of a similar magnitude. When animals were first treated with l-NAME or PE, E2β lost the ability to reduce endometrial vascular resistance. Enzyme activity and gene expression of NO synthase in the rat uterine tissue were also examined after E2β treatment, and no significant changes were observed. These data raise doubts about the role of NO in the regulation of endometrial blood flow after acute administration of E2β and suggest that other mechanisms may be involved.

Gastric hyperemic and acid responses to the stable thyrotropin-releasing hormone (TRH) analog RX-77368 injected intracisternally at a cytoprotective dose were investigated, as well as the underlying mechanisms of the responses. Gastric acid secretion (GAS), mucosal blood flow (GMBF; measured by the hydrogen gas clearance technique), and mucosal vascular resistance (GMVR) and mean arterial pressure (MAP) were assessed simultaneously for 30 min before and after RX-77368 (1.5 ng) administration in urethan-anesthetized rats. RX-77368 increased GMBF from 46.8 +/- 5.3 to 100.6 +/- 20.9 ml.min-1.100 g-1 and MAP from 70.3 +/- 2.1 to 84.3 +/- 5.9 mmHg and decreased GMVR from 1.50 +/- 0.33 to 0.84 +/- 0.08 mmHg.ml-1.min.100 g, whereas GAS was not significantly altered (1.8 +/- 0.4 vs. 4.7 +/- 1.7 mumol/30 min) in vehicle-pretreated rats. The GMBF, MAP, and GMVR responses to RX-77368 were not modified by indomethacin (5 mg/kg ip), whereas GAS was increased. In rats pretreated with capsaicin (125 mg/kg sc) or calcitonin gene-related peptide (CGRP) antagonist hCGRP-(8-37), intracisternal RX-77368 did not increase GMBF or decrease GMVR but did stimulate GAS. These data show that vagal stimulation by the TRH analog RX-77368 injected intracisternally at a nonacid secretory dose increases GMBF. Gastric hyperemia is mediated by CGRP contained in capsaicin-sensitive afferent fibers, whereas acid secretion is under the inhibitory influence of prostaglandins and CGRP.

KirALy, A., G. SUTO, P. Guth and Y. TachE. Influence of [4Cl-D-Phe 6 ,Leu 17 ]VIP on VIP- and central TRH-induced gastric hyperemia. Peptides 18(9) 1321–1325, 1997.—The specific VIP receptor antagonist, [4Cl-D-Phe 6 ,Leu 17 ]VIP, infused iv blocked close-intra-arterial infusion of VIP-induced increase in gastric mucosal blood flow (GMBF, measured by the hydrogen gas clearance), and decrease in mean arterial blood pressure while not influencing basal levels in urethane-anesthetized rats. The thyrotropin-releasing hormone (TRH) stable analog, RX 77368, injected intracisternally (IC, 30 ng) increased GMBF and blood pressure. The VIP antagonist did not significantly reduce the GMBF response to IC RX 77368 while enhancing the rise in blood pressure. These findings indicate that [4Cl-D-Phe 6 ,Leu 17 ]VIP is an antagonist for exogenous VIP-induced gastric hyperemia and hypotension and that VIP modulates the systemic blood pressure response to IC RX 77368 at 30 ng while not playing a primary role in the increase of GMBF.

We studied the intestinotrophic hormone glucagon-like peptide-2 (GLP-2) as a possible therapy for non-steroidal anti-inflammatory drug (NSAID)-induced intestinal ulcers. Luminal nutrients release endogenous GLP-2 from enteroendocrine L cells. Since GLP-2 is degraded by dipeptidyl peptidase IV (DPPIV), we hypothesized that DPPIV inhibition combined with luminal administration of nutrients potentiates the effects of endogenous GLP-2 on intestinal injury. Intestinal injury was induced by indomethacin (10 mg/kg, sc) in fed rats. The long-acting DPPIV inhibitor K579 was given intragastrically (ig) or intraperitoneally (ip) before or after indomethacin treatment. l-Alanine (l-Ala) and inosine 5′-monophosphate (IMP) were co-administered ig after the treatment. Indomethacin treatment induced intestinal ulcers that gradually healed after treatment. Pretreatment with ig or ip K579 given at 1 mg/kg reduced total ulcer length, whereas K579 at 3 mg/kg had no effect. Exogenous GLP-2 also reduced intestinal ulcers. The preventive effect of K579 was dose-dependently inhibited by a GLP-2 receptor antagonist. Daily treatment with K579 (1 mg/kg), GLP-2, or l-Ala + IMP after indomethacin treatment reduced total ulcer length. Co-administration (ig) of K579 and l-Ala + IMP further accelerated intestinal ulcer healing. DPPIV inhibition and exogenous GLP-2 prevented the formation and promoted the healing of indomethacin-induced intestinal ulcers, although high-dose DPPIV inhibition reversed the preventive effect. Umami receptor agonists also enhanced the healing effects of the DPPIV inhibitor. The combination of DPPIV inhibition and luminal nutrient-induced GLP-2 release may be a useful therapeutic tool for the treatment of NSAIDs-induced intestinal ulcers.

BACKGROUND: Antisecretory and bismuth compounds protect the gastric mucosa from injury resulting from non-steroidal anti-inflammatory drugs. AIM: To study the mechanism underlying the gastroprotective effects of ranitidine bismuth citrate (GG311) in rats. METHODS: Indomethacin rat injury model and in vivo microscopy in which acid output, surface cell intracellular pH (pHi), gastric mucus gel thickness, and mucosal blood flow were measured simultaneously. RESULTS: In injury studies, GG311 dose dependently protected against severe injury induced by indomethacin (60 mg/kg subcutaneously). In in vivo microscopic studies, indomethacin significantly decreased mucus gel thickness and increased the initial rate of acidification of gastric surface cells when the superfusate pH was lowered from 7.4 to 1.0, and impaired pHi during acid exposure. Indomethacin had no effect on mucosal blood flow or acid output. GG311 alone had no effect on gel thickness, blood flow, or pHi homeostasis during acid exposure, but improved the initial acidification rate and pHi during superfusion with pH 1.0 solutions in the presence of indomethacin. In separate experiments, indomethacin pretreatment considerably increased gastric mucus bismuth concentrations in rats given GG311. CONCLUSIONS: The gastroprotective effect of GG311 against indomethacin induced gastric injury is associated with high and prolonged gastric mucus bismuth concentrations, which may impair proton permeation across the mucus gel.

The gastric epithelium is covered by a continuous layer of secreted mucus and bicarbonate. The function of this mucobicarbonate layer in terms of protecting the epithelial cells from luminal acid is controversial. Several studies conducted in vitro have shown that gastric mucus can slow proton diffusion and can enable the formation of a pH gradient across the mucobicarbonate layer. In our laboratory, simultaneous measurements of intracellular pH and the thickness of the mucus gel overlying gastric surface cells in vivo indicated that surface cell acidification rates and mucus gel thickness were inversely related. This suggests that the gastric mucobicarbonate layer delays proton permeation into gastric surface cells, enabling secreted bicarbonate to neutralize luminal acid. Several theoretical models, including the effects of mucus and bicarbonate secretion, convection, stirring, and lipids are offered as a possible explanation for the experimental observations. Lipid content and additional unstirred layers outside of the mucus gel are offered as possible explanations for the experimental observations. On the basis of the available data and theoretical considerations, we can conclude that all of these factors probably interact in an integrated manner to protect the gastric epithelial cells from damage due to luminal acid.

Stimulation of capsaicin-sensitive sensory nerves induces gastric mucosal hyperemia, which is mediated in part by both calcitonin gene-related peptide (CGRP) and nitric oxide (NO). In the present study, we used in vivo microscopy in anesthetized rats to determine 1) whether these agents were released locally at the submucosal level and, if so, 2) whether CGRP dilates arterioles via release of endothelium-derived NO. Intragastric capsaicin (160 microM) dilated submucosal arterioles from 25 +/- 3 to 67 +/- 8 microns. The intragastric capsaicin-induced vasodilation was markedly reversed not only by intravenous administration of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) but also by submucosal suffusion of either L-NAME or the CGRP receptor antagonist human CGRP-(8-37). The latter findings indicate that both NO and CGRP are released locally at the submucosal level. Submucosal application of CGRP induced dose-dependent dilation of gastric submucosal arterioles, which was significantly attenuated by L-NAME. However, at the same degree of vasodilation (42 microns), the dilation induced with submucosal CGRP was much less attenuated by NO synthesis inhibition (-28%) compared with that induced with intragastric capsaicin (-79%). This indicates that endothelium-derived NO released by CGRP was not the only source of submucosal NO in the latter response. There must be another as yet undetermined source of submucosal NO, e.g., possibly nitroxidergic nerves.

The H2 gas clearance technique was employed to measure uterine blood flow (UBF) in ovariectomized rats. A needle-type platinum electrode (125 microns diam) was inserted into the rat uterine wall to measure the tissue blood flow surrounding the electrode. The electrode can be placed in individual layers of the uterus to measure the endometrial blood flow (EBF) or the myometrial blood flow (MBF). By use of this technique, baseline EBF and MBF were 37.8 +/- 3.53 (n = 21) and 47.2 +/- 4.56 (n = 5) ml.min-1.100 g-1, respectively, with an EBF/MBF ratio of 0.8. Intravenous bolus injection of 17 beta-estradiol (1 microgram/kg) induced a significant increase in UBF. Phenylephrine, an alpha-adrenergic receptor agonist, reduced UBF. In some animals, a second platinum electrode was used to measure gastric mucosal blood flow simultaneously with UBF. While 17 beta-estradiol selectively increased UBF, pentagastrin selectively increased gastric mucosal blood flow. To further validate the baseline UBF distribution between endometrial and myometrial layers, iodo[14C]antipyrine autoradiography was employed. With the iodo[14C]antipyrine technique, the EBF/MBF ratio was 0.91 +/- 0.07 (n = 5), which is similar to that obtained with the H2 gas clearance technique.

Intestinal endocrine cells release gut hormones, including glucagon-like peptides (GLPs), in response to luminal nutrients. Luminal l-glutamate (l-Glu) and 5′-inosine monophosphate (IMP) synergistically increases duodenal HCO3− secretion via GLP-2 release. Since L cells express the bile acid receptor TGR5 and dipeptidyl peptidase (DPP) IV rapidly degrades GLPs, we hypothesized that luminal amino acids or bile acids stimulate duodenal HCO3− secretion via GLP-2 release, which is enhanced by DPPIV inhibition. We measured HCO3− secretion with pH and CO2 electrodes using a perfused rat duodenal loop under isoflurane anesthesia. l-Glu (10 mM) and IMP (0.1 mM) were luminally coperfused with or without luminal perfusion (0.1 mM) or intravenous (iv) injection (3 μmol/kg) of the DPPIV inhibitor NVP728. The loop was also perfused with a selective TGR5 agonist betulinic acid (BTA, 10 μM) or the non-bile acid type TGR5 agonist 3-(2-chlorophenyl)- N-(4-chlorophenyl)- N,5-dimethylisoxazole-4-carboxamide (CCDC; 10 μM). DPPIV activity visualized by use of the fluorogenic substrate was present on the duodenal brush border and submucosal layer, both abolished by the incubation with NVP728 (0.1 mM). An iv injection of NVP728 enhanced l-Glu/IMP-induced HCO3− secretion, whereas luminal perfusion of NVP728 had no effect. BTA or CCDC had little effect on HCO3− secretion, whereas NVP728 iv markedly enhanced BTA- or CCDC-induced HCO3− secretion, the effects inhibited by a GLP-2 receptor antagonist. Coperfusion of the TGR5 agonist enhanced l-Glu/IMP-induced HCO3− secretion with the enhanced GLP-2 release, suggesting that TGR5 activation amplifies nutrient sensing signals. DPPIV inhibition potentiated luminal l-Glu/IMP-induced and TGR5 agonist-induced HCO3− secretion via a GLP-2 pathway, suggesting that the modulation of the local concentration of the endogenous secretagogue GLP-2 by luminal compounds and DPPIV inhibition helps regulate protective duodenal HCO3− secretion.

Background/Aims: Uremia increases gastric mucosal H + permeability and acid back-diffusion-related injury in rats. The aim of this study was to examine the effect of the synthetic gastroprotective compound 16,16-di-methyl prostaglandin E 2 (16,16-dm PGE 2 ) on the gastric barrier to acid injury in uremic rats. Methods: Chronic renal failure was induced by subtotal nephrectomy. Acid back-diffusion injury was induced by superfusion with 15% ethanol in 0.15N HCl and was assessed by image analysis. Intracellular pH, initial surface cell acidification rate, and thickness of mucous gel layer were measured with in vivo microscopy. Gastric mucosal blood flow was measured in separate experiments by laser-Doppler flowmetry. Results: Pretreatment with 16,16-dm PGE 2 attenuated H + back-diffusion and prevented the production of gross lesions. 16,16-dm PGE 2 increased gastric mucous gel thickness, decreased initial acidification rate, and maintained intracellular pH homeostasis during exposure to luminal acid. Gastric mucosal blood flow was not changed during superfusion with a neutral buffer but increased during acid exposure in rats treated with 16,16-dm PGE 2 . Conclusions: 16,16-dm PGE 2 attenuated H + back-diffusion injury in uremic rats. This effect was associated with blunting of the initial decrease of intracellular pH and enhanced surface cell intracellular pH homeostasis during acid exposure. These effects were associated with an increased mucous gel layer thickness and an acid-related increase in blood flow.

Patients with cirrhosis have an increased incidence of gastric ulcers and erosions. We evaluated the effect of carbon tetrachloride—induced cirrhosis on rat gastric mucosal defense mechanisms using our recently developed in vivo fluorescence microscopy technique. Cirrhotic rats had increased portal vein pressure, increased serum aminotransferase concentrations and decreased serum albumin concentrations. We noted significantly more spontaneous gross gastric lesions in the cirrhotic rats. In vivo microscopic measurements revealed that cirrhotic rats had (a) a significantly thinner gastric mucous gel layer, (b) a much greater decrease in surface mucosal cell intracellular pH in response to an acid load, (c) decreased gastric mucosal blood flow and (d) decreased surface cell viability. We conclude that spontaneous gastric mucosal lesions in cirrhotic rats may be related to more rapid penetration of acid through a thinner gastric mucous gel layer and a lower mucosal blood flow. These changes are associated with a decreased ability of the surface cells to maintain intracellular pH homeostasis, increased initial gastric surface cell acidification, decreased surface cell viability and a lower blood flow that probably is inadequate to remove the increased acid. (Hepatology 1994;20:445-452.)

The aim of this study was to determine if hypertrophy of different tissues seen in uremic rats included gastrointestinal hypertrophy and an increase in parietal cell mass that might explain the increased acid secretion we previously reported. Chronic renal failure was induced by subtotal nephrectomy. Despite a lower total body weight, uremic rats had a significantly greater stomach weight (33%), corpus area (13%), corpus mucosal height (19%), and parietal (32%) and enterochromaffin-like (ECL, 54%) cell density, but a 16% decrease in mucous neck cell region height. These findings suggest that uremia leads to gastric stem cell stimulation with differentiation favoring parietal and ECL cells over mucous cells. In addition, in uremic rats there was an increase in height of the duodenal mucosa, but not of the ileal or transverse colon mucosa. In conclusion, the present study shows that uremia in the rat promotes hypertrophy of the stomach with cell differentiation favoring parietal cells over mucus cells. The increase in parietal cell mass may explain the increased acid secretion in these rats.

Luminal nutrient chemosensing during meal ingestion is mediated by intestinal endocrine cells, which regulate secretion and motility via the release of gut hormones. We have reported that luminal coperfusion of L-Glu and IMP, common condiments providing the umami or proteinaceous taste, synergistically increases duodenal bicarbonate secretion (DBS) possibly via taste receptor heterodimers, taste receptor type 1, member 1 (T1R1)/R3. We hypothesized that glucose-dependent insulinotropic peptide (GIP) or glucagon-like peptide (GLP) is released by duodenal perfusion with L-Glu/IMP. We measured DBS with pH and CO(2) electrodes through a perfused rat duodenal loop in vivo. GIP, exendin (Ex)-4 (GLP-1 receptor agonist), or GLP-2 was intravenously infused (0.01-1 nmol/kg/h). l-Glu (10 mM) and IMP (0.1 mM) were luminally perfused with or without bolus intravenous injection (3 or 30 nmol/kg) of the receptor antagonists Pro(3)GIP, Ex-3(9-39), or GLP-2(3-33). GIP or GLP-2 infusion dose-dependently increased DBS, whereas Ex-4 infusion gradually decreased DBS. Luminal perfusion of l-Glu/IMP increased DBS, with no effect of Pro(3)GIP or Ex-3(9-39), whereas GLP-2(3-33) inhibited L-Glu/IMP-induced DBS. Vasoactive intestinal peptide (VIP)(6-28) intravenously or N(G)-nitro-L-arginine methyl ester coperfusion inhibited the effect of L-Glu/IMP. Perfusion of L-Glu/IMP increased portal venous concentrations of GLP-2, followed by a delayed increase of GLP-1, with no effect on GIP release. GLP-1/2 and T1R1/R3 were expressed in duodenal endocrine-like cells. These results suggest that luminal L-Glu/IMP-induced DBS is mediated via GLP-2 release and receptor activation followed by VIP and nitric oxide release. Because GLP-1 is insulinotropic and GLP-2 is intestinotrophic, umami receptor activation may have additional benefits in glucose metabolism and duodenal mucosal protection and regeneration.

In rat colon damaged by 10% acetic acid and by dinitrochlorobenzene, we test the following hypotheses: (1) mucosal hemodynamic changes are significantly different at the ulcer base, the ulcer margin, and the inflamed non-ulcer-bearing mucosa; and (2) these mucosal hemodynamic changes also vary with time after induction of the colonic injury. Mucosal hemodynamic changes were documented by reflectance spectrophotometry, and variations in gross mucosal morphology were confirmed by hematoxylin and eosin histologic sections. Results revealed that in the acute stage, the ulcer base, which was covered by necrotic debris, showed ischemia without congestion. The ulcer margin at the edge of the ulcer base showed ischemia with congestion. The nonulcerated mucosa, which appeared erythematous, showed increased perfusion. In the convalescent stage, all the altered perfusion patterns returned to normal. These observations offer plausible explanations for the variability in colonic perfusion observed in experimentally damaged colons.

Cold water immersion restraint (CWIR) is associated with gastric hypercontractility and gastric corpus erosions in the rat. Because the gastric blood flow response to CWIR has not been well defined, we performed the following study. Rats were implanted with force transducers, subjected to CWIR for 2 hr, and then blood flow was determined by the iodo[14C]antipyrine autoradiographic (IAP) technique. When compared to control animals, the CWIR-treated animals displayed foci of gastric corpus hyperemia with a marked and significant increase in blood flow in all layers of the gastric corpus. There was approximately a 100% increase in the mucosa and a 50% increase in the muscularis externa. The hyperemia was not uniform, but rather alternated every 2.1 +/- 0.2 mm with regions of low blood flow. Blood flow in the antrum and duodenum was unaffected by CWIR. We conclude that CWIR is associated with alternating regions of high and low blood flow only in the gastric corpus. Reduction of corpus mucosal blood flow might be due to the powerful gastric contractions associated with CWIR.

Background An observational study in veterans showed that a novel water method (water infusion in lieu of air insufflation) enhanced cecal intubation and willingness to undergo a repeat scheduled unsedated colonoscopy. Objective To confirm these beneficial effects and significant attenuation of discomfort in a randomized, controlled trial (RCT). Design Prospective RCT, intent-to-treat analysis. Setting Veterans Affairs ambulatory care facility. Patients Veterans undergoing scheduled unsedated colonoscopy. Interventions During insertion, the water and traditional air methods were compared. Main Outcome Measurements Discomfort and procedure-related outcomes. Results Eighty-two veterans were randomized to the air (n = 40) or water (n = 42) method. Cecal intubation (78% vs 98%) and willingness to repeat (78% vs 93%) were significantly better with the water method ( P P = .002 (Student t test), and the median overall discomfort after colonoscopy was 3 versus 2, P = .052 (Mann-Whitney U test), respectively. The method, but not patient characteristics, was a predictor of discomfort ( t = −1.998, P = .049, R 2 = 0.074). The odds ratio for failed cecal intubation was 2.09 (95% CI, 1.49-2.93) for the air group. Fair/poor previous experience increased the risk of failed cecal intubation in the air group only. The water method numerically increased adenoma yield. Limitations Single site, small number of elderly men, unblinded examiner, possibility of unblinded subjects, restricted generalizability. Conclusions The RCT data confirmed that the water method significantly enhanced cecal intubation and willingness to undergo a repeat colonoscopy. The decrease in maximum discomfort was significant; the decrease in overall discomfort approached significance. The method, but not patient characteristics, was a predictor of discomfort. (Clinical trial registration number NCT00747084).

Neutrophils (PMN) have been implicated as mediators of the “no-reflow” phenomenon seen in skeletal muscle during reperfusion after ischemia. In order to evaluate the PMN contribution to the changes seen in the microcirculation of skeletal muscle after ischemia, we evaluated PMN velocity using in vivo microscopy in a rat model. After the induction of anesthesia, the right iliac and femoral arteries were isolated. The right anterior tibialis muscle was exposed in situ, covered with a plexiglass disc, and perfused with Kreb's solution. Fluorescein-labeled bovine albumin was given intravenously, which identified the capillaries under microscopic magnification as viewed on the video screen. Acridine orange was then administered intravenously, which selectively fluoresced the PMN. The right iliac and femoral arteries were clamped for ischemia intervals of 5, 10, 15, 20, 25, and 30 min. Acridine orange was given immediately after the arteries were unclamped, after 30 min of reperfusion and after 60 min of reperfusion. PMN velocity was determined by the distance traveled by the PMN over time using the videotape and frame-by-frame review. Results demonstrated no change in PMN velocity (mm/sec) after 5 min of ischemia. After 10, 15, and 20 min of ischemia, PMN velocity initially slowed and then recovered, which was not statistically significant. After 25 min of ischemia, PMN velocity decreased significantly, which persisted (P < 0.05 compared to 5-min ischemia by AN-OVA). No flow was seen after 30 min of ischemia. In conclusion: (1) Skeletal muscle capillaries can be visualized using this technique of in vivo microscopy and (2) changes in PMN velocity during reperfusion may contribute to areas of “no-reflow” as seen after only 30 min of ischemia in this rat model.

We investigated whether the recently described endothelium-derived nitric oxide-mediated gastric hyperemia in the uremic rat protects the gastric mucosa against ethanol injury. Uremia was induced by subtotal nephrectomy. Basal gastric mucosal blood flow, measured by a hydrogen gas clearance technique, was significantly higher in uremic than control rats. Continuous intragastric perfusion with 40% ethanol produced significantly less gross and histological lesions in uremic than in control rats. The administration of 3 mg/kg of NW-nitro-L-arginine methyl ester, a specific inhibitor of nitric oxide biosynthesis, decreased resting gastric mucosal blood flow to control levels in uremic rats, but had no effect on basal gastric blood flow in control rats. This pretreatment with the inhibitor of nitric oxide biosynthesis increased 40% ethanol-induced gastric mucosal lesions in uremic rats to the same level as that observed in control rats, but had no effect on lesions in control rats. In conclusion, this study suggests that in the uremic rat, gastric hyperemia, mediated by increased endothelium-derived nitric oxide, attenuates ethanol-induced gastric mucosal injury.

Intracellular pH (pHi) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na+/H+ exchange in maintaining pHi homeostasis were studied in vivo using a fluorescent microscopic technique. pHi was measured during superfusion with buffers of pH 1.2-7.4. When the pH of the superfusate was 7.4, baseline pHi was unchanged. Superfusion with pH 3 buffer rapidly decreased pHi to 6.7, with subsequent recovery to baseline pHi within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pHi continuously to below 6.2 with no recovery observed. Despite the relentless decline in pHi during superfusion with pH-1.2 and -1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na+/H+ exchange in the regulation of pHi. Superfusion with amiloride did not affect recovery of pHi from intracellular acidification induced by a NH4Cl prepulse. Exposure to the potent, lipophilic Na+/H+ exchange inhibitor 5-(N,N-hexamethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pHi from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pHi to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na+/H+ exchanger, most probably localized to the basolateral membrane.

Disruption of the gastric mucosal barrier with resultant increased acid back-diffusion leads to a marked increase in gastric mucosal blood flow (GMBF). This increase in GMBF is blocked by ablation of capsaicin-sensitive sensory neurons. The gastric arterioles are densely innervated by afferent neurons containing vasodilator peptides, calcitonin gene-related peptide (CGRP) being the most potent of these. We investigated (a) whether CGRP is the vasodilator mediator released by acid stimulation of capsaicin-sensitive sensory neurons and (b) whether the resultant hyperemia protects against the acid-induced mucosal injury. When the stomach was perfused with 0.15N HCl plus 15% ethanol, GMBF significantly increased by 70%. This hyperemic response was completely blocked by intra-arterial infusion of human CGRP8-37 (500 pmol/min), a CGRP-receptor antagonist, close to the stomach. With the blockade of the hyperemic response to acid back-diffusion, gross and histological mucosal damage were significantly aggravated. It is concluded that CGRP mediates the gastric hyperemic response to acid back-diffusion and that this gastric hyperemic response is an important protective factor against acid-induced injury. (Gastroenterology 1992 Apr;102(4 Pt 1):1124-8)

Regulation of localized extracellular pH (pH(o)) maintains normal organ function. An alkaline microclimate overlying the duodenal enterocyte brush border protects the mucosa from luminal acid. We hypothesized that intestinal alkaline phosphatase (IAP) regulates pH(o) due to pH-sensitive ATP hydrolysis as part of an ecto-purinergic pH regulatory system, comprised of cell-surface P2Y receptors and ATP-stimulated duodenal bicarbonate secretion (DBS). To test this hypothesis, we measured DBS in a perfused rat duodenal loop, examining the effect of the competitive alkaline phosphatase inhibitor glycerol phosphate (GP), the ecto-nucleoside triphosphate diphosphohydrolase inhibitor ARL67156, and exogenous nucleotides or P2 receptor agonists on DBS. Furthermore, we measured perfusate ATP concentration with a luciferin-luciferase bioassay. IAP inhibition increased DBS and luminal ATP output. Increased luminal ATP output was partially CFTR dependent, but was not due to cellular injury. Immunofluorescence localized the P2Y(1) receptor to the brush border membrane of duodenal villi. The P2Y(1) agonist 2-methylthio-ADP increased DBS, whereas the P2Y(1) antagonist MRS2179 reduced ATP- or GP-induced DBS. Acid perfusion augmented DBS and ATP release, further enhanced by the IAP inhibitor l-cysteine, and reduced by the exogenous ATPase apyrase. Furthermore, MRS2179 or the highly selective P2Y(1) antagonist MRS2500 co-perfused with acid induced epithelial injury, suggesting that IAP/ATP/P2Y signalling protects the mucosa from acid injury. Increased DBS augments IAP activity presumably by raising pH(o), increasing the rate of ATP degradation, decreasing ATP-mediated DBS, forming a negative feedback loop. The duodenal epithelial brush border IAP-P2Y-HCO(3-) surface microclimate pH regulatory system effectively protects the mucosa from acid injury.

Head injury is frequently accompanied by an increase in intracranial pressure and gastric lesion formation. We used a model of controlled intracranial pressure to investigate the effect of elevated intracranial pressure on gastric acid secretion and mucosal blood flow and on the susceptibility of the gastric mucosa to lesion formation. With increasing intracranial pressure, there was a corresponding increase in gastric acid output but no significant change in gastric mucosal blood flow. This imbalance between acid secretion and blood flow could be a factor in the pathogenesis of the gastric lesions seen with head injury. Susceptibility to gastric mucosal injury then was studied in a model that is independent of the acid secretory state--exogenous intragastric HCl plus ethanol. Elevated intracranial pressure did render the gastric mucosa more susceptible to injury in this model, but there was no impairment of the increased gastric mucosal blood flow response to the increased acid back-diffusion. In this situation, factors other than altered overall blood flow appear to be responsible for the increased lesion formation.

Submucosal blood vessels of the mammalian stomach are densely innervated by neurons containing calcitonin gene-related peptide (CGRP), substance P, neurokinin A, and vasoactive intestinal polypeptide (VIP). Because all these peptides are vasodilators in certain vascular beds, we tested the hypothesis that rat alpha-CGRP, rat VIP, substance P, and neurokinin A are candidate mediators of noncholinergic vasodilator neurons in the gastric mucosa and submucosa. The experiments were performed on urethane-anesthetized Sprague-Dawley rats. Gastric mucosal blood flow (GMBF) was measured by the hydrogen gas clearance technique, and the peptides were infused close arterially to the stomach via a catheter inserted retrogradely in the splenic artery. Basal GMBF was in the range of 35-50 ml/min/100 g. Infusion of rat alpha-CGRP (15 and 75 pmol/min) significantly increased GMBF in a dose-dependent manner, whereas mean arterial blood pressure was significantly lowered only by the higher dose of CGRP. Substance P (125 and 625 pmol/min) and neurokinin A (50 and 250 pmol/min) failed to alter GMBF, although the higher dose of each peptide led to a significant decrease in mean arterial blood pressure. Infusion of rat VIP (25 pmol/min) failed to affect GMBF and mean arterial blood pressure, whereas a fivefold higher dose of VIP (125 pmol/min) led to a significant rise of GMBF and to significant hypotension. These findings indicate that substance P and neurokinin A are unlikely to be of physiological significance for the regulation of GMBF. CGRP and VIP, however, can be considered as candidate mediators of submucosal nerve endings involved in the neural control of GMBF.(ABSTRACT TRUNCATED AT 250 WORDS)

Access to potentially life-saving screening colonoscopy is limited by the high cost of sedation. We explored the practicability of having supervised trainees perform unsedated colonoscopies.A nursing shortage at our Veterans Administration gastroenterology training program necessitated discontinuing sedated colonoscopy. We offered the procedure without sedation to restore local access to screening colonoscopy.From September 2002 to June 2005, 145 of 483 patients accepted the unsedated option. The procedure was done by second-year gastroenterology (GI) fellows who had performed about 100 sedated colonoscopies in their first year of training. Cecal intubation was achieved in 81% of 138 well purged patients without obstructive lesions. Implementation obviated the need for 2 registered nurses, the escort requirement, and postprocedure activity restriction. It also eliminated sedation-related complications.This report confirms the feasibility of unsedated colonoscopy performed by supervised trainees. The unsedated option minimizes direct and indirect costs of colonoscopy. Describing unsedated screening colonoscopy to patients as a "sedation risk-free" procedure encouraged them to consider the benefits. We recommend that future studies test primary care providers' willingness to inform patients of the feasibility of this nonstandard option, and perhaps reshape the practice of colonoscopy for colorectal cancer screening.

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H+absorption from the lumen. We thus examined the effect of inhibition of Na+/H+exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO2diffusion and HCO3−secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO2solution or pH 2.2 low CO2solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTRinh-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO2]). Luminal high CO2solution increased CO2absorption and HCO3−secretion, accompanied by PV acidification and PV Pco2increase. During CO2challenge, CFTRinh-172 induced HCO3−absorption, while inhibiting PV acidification. S3226 reversed CFTRinh-associated HCO3−absorption. Luminal pH 2.2 challenge increased H+and CO2absorption and acidified the PV, inhibited by CFTRinh-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO3−secretion to absorption and inhibited PV acidification during CO2challenge, suggesting that HCO3−secretion helps facilitate CO2/H+absorption. Furthermore, CFTR inhibition prevented CO2-induced cellular acidification reversed by S3226. Reversal of increased HCO3−loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H+available to neutralize luminal HCO3−with consequent CO2absorption. NHE3, by secreting H+into the luminal microclimate, facilitates net transmucosal HCO3−absorption with a mechanism similar to proximal tubular HCO3−absorption.

Laparotomy significantly attenuates ethanol-induced gastric mucosal lesions in the rat. The effects of sensory denervation by capsaicin, indomethacin, atropine, guanethidine, and hexamethonium on laparotomy-induced protection were studied in the rat. Gastric mucosal injury was induced by the intragastric instillation of 1 mL of 75% ethanol. The laparotomy-induced protection against ethanol injury was abolished by sensory denervation by capsaicin (total dose, 125 mg/kg, SC) and also by pretreatment with indomethacin (5 mg/kg, SC). In contrast, pretreatment with atropine (0.5 mg/kg, IP), guanethidine (total dose, 20 mg/kg, SC), or hexamethonium (20 mg/kg, IP) had no significant effect on laparotomy-induced protection. These data indicate that capsaicin-sensitive sensory afferent neurons, but not cholinergic or adrenergic autonomic neurons, mediate laparotomy-induced protection against ethanol injury. The hypothesis is put forward that the protective response to laparotomy arises from a somatovisceral and/or viscerovisceral axon reflex of capsaicin-sensitive afferent neurons. Prostaglandins might play a mediator role in the activation by laparotomy of somatic and/or visceral branches of the afferent neurons.

We hypothesized that duodenal HCO3−secretion alkalinizes the microclimate surrounding intestinal alkaline phosphatase (IAP), increasing its activity. We measured AP activity in rat duodenum in situ in frozen sections with the fluorogenic substrate ELF-97 phosphate and measured duodenal HCO3−secretion with a pH-stat in perfused duodenal loops. We examined the effects of the IAP inhibitors l-cysteine or l-phenylalanine (0.1–10 mM) or the tissue nonspecific AP inhibitor levamisole (0.1–10 mM) on AP activity in vitro and on acid-induced duodenal HCO3−secretion in vivo. AP activity was the highest in the duodenal brush border, decreasing longitudinally to the large intestine with no activity in stomach. Villous surface AP activity measured in vivo was enhanced by PGE2intravenously and inhibited by luminal l-cysteine. Furthermore, incubation with a pH 2.2 solution reduced AP activity in vivo, whereas pretreatment with the cystic fibrosis transmembrane regulator (CFTR) inhibitor CFTRinh-172 abolished AP activity at pH 2.2. l-Cysteine and l-phenylalanine enhanced acid-augmented duodenal HCO3−secretion. The nonselective P2 receptor antagonist suramin (1 mM) reduced acid-induced HCO3−secretion. Moreover, l-cysteine or the competitive AP inhibitor glycerol phosphate (10 mM) increased HCO3−secretion, inhibited by suramin. In conclusion, enhancement of the duodenal HCO3−secretory rate increased AP activity, whereas inhibition of AP activity increased the HCO3−secretory rate. These data support our hypothesis that HCO3−secretion increases AP activity by increasing local pH at its catalytic site and that AP hydrolyzes endogenous luminal phosphates, presumably ATP, which increases HCO3−secretion via activation of P2 receptors.

The duodenum is the site of mixing of massive amounts of gastric H+ with secreted HCO3-, generating CO2 and H2O accompanied by the neutralization of H+. We examined the role of membrane-bound and soluble carbonic anhydrases (CA) by which H+ is neutralized, CO2 is absorbed, and HCO3- is secreted. Rat duodena were perfused with solutions of different pH and PCO2 with or without a cell-permeant CA inhibitor methazolamide (MTZ) or impermeant CA inhibitors. Flow-through pH and PCO2 electrodes simultaneously measured perfusate and effluent pH and PCO2. High CO2 (34.7 kPa) perfusion increased net CO2 loss from the perfusate compared with controls (pH 6.4 saline, PCO2 approximately 0) accompanied by portal venous (PV) acidification and PCO2 increase. Impermeant CA inhibitors abolished net perfusate CO2 loss and increased net HCO3- gain, whereas all CA inhibitors inhibited PV acidification and PCO2 increase. The changes in luminal and PV pH and [CO2] were also inhibited by the Na+-H+ exchanger-1 (NHE1) inhibitor dimethylamiloride, but not by the NHE3 inhibitor S3226. Luminal acid decreased total CO2 output and increased H+ loss with PV acidification and PCO2 increase, all inhibited by all CA inhibitors. During perfusion of a 30% CO2 buffer, loss of CO2 from the lumen was CA dependent as was transepithelial transport of perfused 13CO2. H+ and CO2 loss from the perfusate were accompanied by increases of PV H+ and tracer CO2, but unchanged PV total CO2, consistent with CA-dependent transmucosal H+ and CO2 movement. Inhibition of membrane-bound CAs augments the apparent rate of net basal HCO3- secretion. Luminal H+ traverses the apical membrane as CO2, is converted back to cytosolic H+, which is extruded via NHE1. Membrane-bound and cytosolic CAs cooperatively facilitate secretion of HCO3- into the lumen and CO2 diffusion into duodenal mucosa, serving as important acid-base regulators.

Background & Aims: The duodenal mucosa is exposed to Pco2 >200 mm Hg due to the luminal mixture of gastric acid with secreted bicarbonate, which augments mucosal protective mechanisms. We examined the hyperemic response to elevated luminal Pco2 in the duodenum of anesthetized rats luminally exposed to high co2 saline to help elucidate luminal acid-sensing mechanisms. Methods: Blood flow was measured by laser Doppler, and intracellular pH of epithelial cells by measured by ratio microimaging. The permeant carbonic anhydrase (CA) inhibitor methazolamide, relatively impermeant CA inhibitor benzolamide, vanilloid receptor antagonist capsazepine, or sodium-hydrogen exchanger 1 (NHE-1) inhibitor dimethyl amiloride were perfused with or without the high co2 solution. Results: The high co2 solution increased duodenal blood flow, which was abolished by pretreatment with methazolamide or capsazepine or by dimethyl amiloride coperfusion. Sensory denervation with capsaicin also abolished the co2 effects. Benzolamide dose-dependently inhibited co2-induced hyperemia and at 100 nmol/L inhibited co2-induced intracellular acidification. The membrane-bound CA isoforms IV, IX, XII, and XIV and cytosolic CA II and the vanilloid receptor 1 (TRPV1) were expressed in duodenum and stomach. Dorsal root ganglion and nodose ganglion expressed all isoforms except for CA IX. Conclusions: The duodenal hyperemic response to luminal co2 is dependent on cytosolic and membrane-bound CA isoforms, NHE-1, and TRPV1. co2-induced intracellular acidification was inhibited by selective extracellular CA inhibition, suggesting that co2 diffusion across the epithelial apical membrane is mediated by extracellular CA. NHE-1 activation preceding TRPV1 stimulation suggests that luminal co2 is sensed as H+ in the subepithelium.