Yasutada Akiba

Increased expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in colonic mucosa of patients with active ulcerative colitis

Background—Increased production of reactive metabolites of oxygen and nitrogen has been implicated in chronic inflammation of the gut. The object of this study was to examine the magnitude and location of nitric oxide synthase (NOS) activity and peroxynitrite formation in the colonic mucosa of patients with ulcerative colitis in relation to the degree of inflammation. Subjects—Thirty three patients with active ulcerative colitis (17 with mild or moderate inflammation, 16 with severe inflammation). Methods—Inducible NOS activity was determined in the colonic mucosa by measuring the conversion ofl-arginine to citrulline in the absence of calcium. The localisation of NOS and nitrotyrosine immunoreactivity was assessed immunohistochemically using the labelled streptavidin biotin method. Results—Inducible NOS activity increased in parallell with the degree of inflammation of the mucosa. Expression of inducible NOS was found not only in the lamina propria, but also in the surface of the epithelium. Peroxynitrite formation as assessed by nitrotyrosine staining was frequently observed in the lamina propria of actively inflamed mucosa. Conclusions—Nitric oxide and peroxynitrite formation may play an important role in causing irreversible cellular injury to the colonic mucosa in patients with active ulcerative colitis.

Increased Nitric Oxide Production and Inducible Nitric Oxide Synthase Activity in Colonic Mucosa of Patients with Active Ulcerative Colitis and Crohn's Disease

It is postulated that an enhanced production ofnitric oxide by inflamed intestine plays a role in thepathophysiology of active inflammatory bowel disease. Inthis study, systemic NOx concentrations and colonic nitric oxide synthase activity weredetermined in patients with ulcerative colitis orCrohns disease. The relationship between these twoparameters and disease activity, as well as differences in nitric oxide synthase activity betweenulcerative colitis and Crohns disease, were areas ofspecific focus. Patients with active ulcerative colitisand Crohns disease had significantly elevated plasma NOx concentrations; a positivecorrelation was found between NOx values andinducible nitric oxide synthase activities in the activemucosa of these patients. In active ulcerative colitis,levels of inducible nitric oxide synthase were significantlyelevated in both normal and inflamed mucosa, althoughinducible nitric oxide synthase activity was higher inthe latter. These colonic inducible nitric oxidesynthase activities correlated well with the results ofendoscopic and histologic grading of inflammation. Therewas no increase in constitutive nitric oxide synthaseactivity in patients with active ulcerative colitis. However, constitutive nitric oxidesynthase activity was significantly increased in theinflamed mucosa in patients with Crohns disease. InCrohns disease, elevated inducible nitric oxidesynthase activity was found in both normal and inflamedmucosa, with no significant difference between thetissues. Such differences in nitric oxide production inthe colonic mucosa possibly reflect the significant differences in the pathophysiology andcharacteristic clinical features between ulcerativecolitis and Crohns disease.

Intestinal alkaline phosphatase regulates protective surface microclimate pH 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.

Luminal chemosensing and upper gastrointestinal mucosal defenses

The upper gastrointestinal mucosa is exposed to endogenous and exogenous substances, including gastric acid, carbon dioxide, and foodstuffs. Physiologic processes such as secretion, digestion, absorption, and motility occur in the gastrointestinal tract in response to ingested substances, which implies the presence of mucosal sensors. We hypothesize that mucosal acid sensors and tastelike receptors are important components of the mucosal chemosensing system. We have shown that luminal acid/carbon dioxide is sensed via ecto- and cytosolic carbonic anhydrases and ion transporters in the epithelial cells and via acid sensors on the afferent nerves in the duodenum and esophagus. Furthermore, a luminal l-glutamate signal is mediated via mucosal l-glutamate receptors with activation of afferent nerves and cyclooxygenase in the duodenum, which suggests the presence of luminal l-glutamate sensing. These luminal chemosensors help to activate mucosal defense mechanisms to maintain the mucosal integrity and physiologic responses of the upper gastrointestinal tract. Because neural pathways are components of the luminal chemosensory system, investigation of these pathways may help to identify novel molecular targets in the treatment and prevention of mucosal injury and visceral sensation.

Acid-sensing pathways of rat duodenum

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.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.

Epithelial carbonic anhydrases facilitate PCO2 and pH regulation in rat duodenal mucosa

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 P  CO 2 with or without a cell‐permeant CA inhibitor methazolamide (MTZ) or impermeant CA inhibitors. Flow‐through pH and P  CO 2 electrodes simultaneously measured perfusate and effluent pH and P  CO 2. High CO2 (34.7 kPa) perfusion increased net CO2 loss from the perfusate compared with controls (pH 6.4 saline, P  CO 2 ≈ 0) accompanied by portal venous (PV) acidification and P  CO 2 increase. Impermeant CA inhibitors abolished net perfusate CO2 loss and increased net HCO3− gain, whereas all CA inhibitors inhibited PV acidification and P  CO 2 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 P  CO 2 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.

Umami receptor activation increases duodenal bicarbonate secretion via glucagon-like peptide-2 release in 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 CO2 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 Pro3GIP, 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 Pro3GIP or Ex-3(9-39), whereas GLP-2(3-33) inhibited l-Glu/IMP-induced DBS. Vasoactive intestinal peptide (VIP)(6–28) intravenously or NG-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.

Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury

Secretion of bicarbonate from epithelial cells is considered to be the primary mechanism by which the duodenal mucosa is protected from acid-related injury. Against this view is the finding that patients with cystic fibrosis, who have impaired duodenal bicarbonate secretion, are paradoxically protected from developing duodenal ulcers. Therefore, we hypothesized that epithelial cell intracellular pH regulation, rather than secreted extracellular bicarbonate, was the principal means by which duodenal epithelial cells are protected from acidification and injury. Using a novel in vivo microscopic method, we have measured bicarbonate secretion and epithelial cell intracellular pH (pH(i)), and we have followed cell injury in the presence of the anion transport inhibitor DIDS and the Cl(-) channel inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). DIDS and NPPB abolished the increase of duodenal bicarbonate secretion following luminal acid perfusion. DIDS decreased basal pH(i), whereas NPPB increased pH(i); DIDS further decreased pH(i) during acid challenge and abolished the pH(i) overshoot over baseline observed after acid challenge, whereas NPPB attenuated the fall of pH(i) and exaggerated the overshoot. Finally, acid-induced epithelial injury was enhanced by DIDS and decreased by NPPB. The results support the role of intracellular bicarbonate in the protection of duodenal epithelial cells from luminal gastric acid.

Acid-sensing pathways in rat gastrointestinal mucosa

The gastrointestinal mucosa serves as the interface between the luminal contents, including nutrients and injurious substances, and submucosal structures. Secreted gastric acid is one of the principal injurious components of the luminal contents. To be protected against harm from this acid, the epithelium has an “early warning” system that can activate potent defense mechanisms. We studied the mechanisms that defend the epithelium against luminal acid-induced injury, including the regulation of epithelial intracellular pH (pHi), blood flow, and mucus gel secretion in the perfused rat duodenum, and the pathways involved in the activation and regulation of these mechanisms. Physiological concentrations of luminal acid acidified the epithelial cells and increased blood flow (hyperemic response) and mucus gel thickness. The hyperemic response to acid was abolished by inhibitors of the Na+/H+ exchange, vanilloid receptors (VR), calcitonin gene-related peptide (CGRP) receptors, and nitric oxide (NO) synthase, and also by sensory afferent denervation, but not by pretreatment with a nonselective cyclooxygenase (COX) inhibitor. Mucus secretion in response to luminal acid was delayed by an interruption to the capsaicin pathway, which includes VR, capsaicin-sėnsitive afferent nerves, CGRP, and NO, and was abolished by COX inhibition. These observations support the hypothesis that the capsaicin pathway is an acid-sensing pathway that promotes hyperemia and mucus secretion in response to luminal acid. The COX pathway is a secondary regulatory system for mucus secretion. A similar acid-sensing capsaicin pathway is also present in the colon, suggesting that the gastrointestinal mucosa “tastes” luminal acidity through epithelial-VR communication.

CO2 chemosensing in rat oesophagus

Background: Acid in the oesophageal lumen is often sensed as heartburn. It was hypothesised that luminal CO2, a permeant gas, rather than H+, permeates through the epithelium, and is converted to H+, producing an afferent neural signal by activating chemosensors. Methods: The rat lower oesophageal mucosa was superfused with pH 7.0 buffer, and pH 1.0 or pH 6.4 high CO2 (PCO2 = 260 Torr) solutions with or without the cell-permeant carbonic anhydrase (CA) inhibitor methazolamide (MTZ, 1 mM), the cell-impermeant CA inhibitor benzolamide (BNZ, 0.1 mM), the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (CPZ, 0.5 mM) or the acid-sensing ion channel (ASIC) inhibitor amiloride (0.1 mM). Interstitial pH (pHint) was measured with 5′,6′-carboxyfluorescein (5 mg/kg intravenously) loaded into the interstitial space, and blood flow was measured with laser-Doppler. Results: Perfusion of a high CO2 solution induced hyperaemia without changing pHint, mimicking the effect of pH 1.0 perfusion. Perfused MTZ, BNZ, CPZ and amiloride all inhibited CO2-induced hyperaemia. CA XIV was expressed in the prickle cells, with CA XII in the basal cells. TRPV1 was expressed in the stratum granulosum and in the muscularis mucosa, whereas all ASICs were expressed in the prickle cells, with ASIC3 additionally in the muscularis mucosa. Conclusions: The response to CO2 perfusion suggests that CO2 diffuses through the stratum epithelium, interacting with TRPV1 and ASICs in the epithelium or in the submucosa. Inhibition of the hyperaemic response to luminal CO2 by CA, TRPV1 and ASIC inhibitors implicates CA and these chemosensors in transduction of the luminal acid signal. Transepithelial CO2 permeation may explain how luminal H+ equivalents can rapidly be transduced into hyperaemia, and the sensation of heartburn.