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Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases.

Activation of large-conductance Ca2+-activated K+ (BKCa) channels evokes cell survival programs that mitigate intestinal ischemia and reperfusion (I/R) inflammation and injury 24 h later. The goal of the present study was to determine the roles of reactive oxygen species (ROS) and heme oxygenase (HO)-1 in delayed acquisition of tolerance to I/R induced by pretreatment with the BKCa channel opener NS-1619. Superior mesentery arteries were occluded for 45 min followed by reperfusion for 70 min in wild-type (WT) or HO-1-null (HO-1−/−) mice that were pretreated with NS-1619 or saline vehicle 24 h earlier. Intravital microscopy was used to quantify the numbers of rolling and adherent leukocytes. Mucosal permeability, tumor necrosis factor-α (TNF-α) levels, and HO-1 activity and expression in jejunum were also determined. I/R induced leukocyte rolling and adhesion, increased intestinal TNF-α levels, and enhanced mucosal permeability in WT mice, effects that were largely abolished by pretreatment with NS-1619. The anti-inflammatory and mucosal permeability-sparing effects of NS-1619 were prevented by coincident treatment with the HO-1 inhibitor tin protoporphyrin-IX or a cell-permeant SOD mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), in WT mice. NS-1619 also increased jejunal HO-1 activity in WT animals, an effect that was attenuated by treatment with the BKCa channel antagonist paxilline or MnTBAP. I/R also increased postischemic leukocyte rolling and adhesion and intestinal TNF-α levels in HO-1−/− mice to levels comparable to those noted in WT animals. However, NS-1619 was ineffective in preventing these effects in HO-1-deficient mice. In summary, our data indicate that NS-1619 induces the development of an anti-inflammatory phenotype and mitigates postischemic mucosal barrier disruption in the small intestine by a mechanism that may involve ROS-dependent HO-1 activity. NEW & NOTEWORTHY Antecedent treatment with the large-conductance Ca2+-activated K+ channel opener NS-1619 24 h before ischemia-reperfusion limits postischemic tissue injury by an oxidant-dependent mechanism. The present study shows that NS-1619-induced oxidant production prevents ischemia-reperfusion-induced inflammation and mucosal barrier disruption in the small intestine by provoking increases in heme oxygenase-1 activity.

Purinergic signaling is a major pathway in regulating bladder function, and mechanical force stimulates urothelial ATP release, which plays an important role in bladder mechanotransduction. Although urothelial ATP release was first reported almost 20 years ago, the way in which release is regulated by mechanical force, and the presence of ATP-converting enzymes in regulating the availability of released ATP is still not well understood. Using a set of custom-designed Ussing chambers with the ability to manipulate mechanical forces applied on the urothelial tissue, we have demonstrated that it is stretch and not hydrostatic pressure that induces urothelial ATP release. The experiments reveal that urothelial ATP release is tightly controlled by stretch speed, magnitude, and direction. We have further shown that stretch-induced urothelial ATP release is insensitive to temperature (4°C). Interestingly, stretch-induced ATP release shows polarized distribution, with the ATP concentration in mucosal chamber (nanomolar level) about 10 times higher than the ATP concentration in serosal chamber (subnanomolar level). Furthermore, we have consistently observed differential ATP lifetime kinetics in the mucosal and serosal chambers, which is consistent with our immunofluorescent localization data, showing that ATP-converting enzymes ENTPD3 and alkaline phosphatase are expressed on urothelial basal surface, but not on the apical membrane. In summary, our data indicate that urothelial ATP release is finely regulated by stretch speed, magnitude, and direction, and extracellular ATP signaling is likely to be differentially regulated by ectonucleotidase, which results in temporally and spatially distinct ATP kinetics in response to mechanical stretch.

Patients with nonerosive reflux disease exhibit impaired esophageal mucosal integrity, which may underlie enhanced reflux perception. In vitro topical application of an alginate solution can protect mucosal biopsies against acid-induced changes in transepithelial electrical resistance (TER). We aimed to confirm this finding in a second model using 3D cell cultures and to assess prolonged protection in a biopsy model. We assessed the protective effect of a topically applied alginate solution 1 h after application. 3D cell cultures were grown by using an air-liquid interface and were studied in Ussing chambers. The apical surface was “protected” with 200 μl of either alginate or viscous control or was unprotected. The tissue was exposed to pH 3 + bile acid solution for 30 min and TER change was calculated. Distal esophageal mucosal biopsies were taken from 12 patients and studied in Ussing chambers. The biopsies were coated with either alginate or viscous control solution. The biopsies were then bathed in pH 7.4 solution for 1 h. The luminal chamber solution was replaced with pH 2 solution for 30 min. Percentage changes in TER were recorded. In five biopsies fluorescein-labeled alginate solution was used to allow immunohistological localization of the alginate after 1 h. In the cell culture model, alginate solution protected tissue against acid-induced change in TER. In biopsies, 60 min after protection with alginate solution, the acidic exposure caused a −8.3 ± 2.2% change in TER compared with −25.1 ± 4.5% change after protection with the viscous control ( P < 0.05). Labeled alginate could be seen coating the luminal surface in all cases. In vitro, alginate solutions can adhere to the esophageal mucosa for up to 1 h and exert a topical protectant effect. Durable topical protectants can be further explored as first-line/add-on therapies for gastroesophageal reflux disease.

Little is known about the mucosal phenotype of the proximal human esophagus. There is evidence to suggest that the proximal esophagus is more sensitive to chemical and mechanical stimulation compared with the distal. This may have physiological relevance (e.g., in prevention of aspiration of gastroesophageal refluxate), but also pathological relevance (e.g., in reflux perception or dysphagia). Reasons for this increased sensitivity are unclear but may include impairment in mucosal barrier integrity or changes in sensory innervation. We assessed mucosal barrier integrity and afferent nerve distribution in the proximal and distal esophagus of healthy human volunteers. In 10 healthy volunteers baseline proximal and distal esophageal impedance was measured in vivo. Esophageal mucosal biopsies from the distal and proximal esophagus were taken, and baseline transepithelial electrical resistance (TER) was measured in Ussing chambers. Biopsies were examined immunohistochemically for presence and location of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers. In a further four healthy volunteers we investigated for colocalization of CGRP and protein gene product (PGP) 9.5 immunoreactivity in nerve fibers. Baseline impedance was higher in the proximal than in the distal esophagus [2,936 Ω (SD578) vs. 2,229 Ω (SD821); P = 0.03], however, baseline TER was not significantly different between them. Mucosal CGRP-immunoreactive nerves were found in the epithelium of both proximal and distal esophagus, but were located more superficially in the proximal mucosa compared with the distal [11.5 (SD7) vs. 21.7 (SD5) cell layers from lumen, P = 0.002] 19% of proximal, and 10% of distal mucosal PGP-immunoreactive fibers colocalized with CGRP. PGP-immunoreactive fibers were also significantly closer to the luminal surface in the proximal compared with the distal esophagus ( P < 0.001). We conclude that mucosal barrier integrity is similar in proximal and distal esophagus, but proximal mucosal afferent nerves are in a more superficial location. The enhanced sensitivity to reflux-evoked symptoms of the proximal esophagus most likely has an anatomical basis.

Increased esophageal sensitivity and impaired mucosal integrity have both been described in patients with gastroesophageal reflux disease, but the relationship between hypersensitivity and mucosal integrity is unclear. The aim of the present study was to investigate acid sensitivity in patients with erosive and nonerosive reflux disease and control subjects to determine the relation with functional esophageal mucosal integrity changes as well as to investigate cellular mechanisms of impaired mucosal integrity in these patients. In this prospective experimental study, 12 patients with nonerosive reflux disease, 12 patients with esophagitis grade A or B, and 11 healthy control subjects underwent an acid perfusion test and upper endoscopy. Mucosal integrity was measured during endoscopy by electrical tissue impedance spectroscopy and biopsy specimens were analyzed in Ussing chambers for transepithelial electrical resistance, transepithelial permeability and gene expression of tight junction proteins and filaggrin. Patients with nonerosive reflux disease and esophagitis were more sensitive to acid perfusion compared with control subjects, having a shorter time to perception of heartburn and higher perceived intensity of heartburn. In reflux patients, enhanced acid sensitivity was associated with impairment of in vivo and vitro esophageal mucosal integrity. Mucosal integrity was significantly impaired in patients with esophagitis, displaying higher transepithelial permeability and lower extracellular impedance. Although no significant differences in the expression of tight junction proteins were found in biopsies among patient groups, mucosal integrity parameters in reflux patients correlated negatively with the expression of filaggrin. In conclusion, sensitivity to acid is enhanced in patients with gastroesophageal reflux disease, irrespective of the presence of erosions, and is associated with impaired esophageal mucosal integrity. Mucosal integrity of the esophagus is associated with the expression of filaggrin.

Squamous esophageal epithelium adapts to acid reflux-mediated injury by proliferation and differentiation via signal transduction pathways. Induction of the Wnt antagonist Dickkopf-1 (Dkk1) is involved in tissue repair during inflammation and cellular injury. In this study, we aimed to identify the biological role of Dkk1 in human reflux esophagitis with respect to cell growth and regulation of Wnt signaling. Esophageal biopsies from reflux-esophagitis patients ( n = 15) and healthy individuals ( n = 10) were characterized in terms of Dkk1 expression. The role of Dkk1 in response to acid-mediated epithelial injury was analyzed by cellular assays in vitro utilizing squamous esophageal epithelial cell lines (EPC1-hTERT, EPC2-hTERT, and HEEC). Dkk1 was significantly overexpressed in human reflux-esophagitis tissue compared with healthy esophageal mucosa at transcriptional and translational levels. After acute and chronic acid (pH 4) exposure, esophageal squamous epithelial cell lines expressed and secreted high levels of Dkk1 in response to stress-associated DNA injury. High extracellular levels of human recombinant Dkk1 inhibited epithelial cell growth and induced cellular senescence in vitro, as demonstrated by reduced cell proliferation, G0/G1cell cycle arrest, elevated senescence-associated β-galactosidase activity, and upregulation of p16. Acid pulsing induced Dkk1-mediated senescence, which was directly linked to the ability of Dkk1 to antagonize the canonical Wnt/β-catenin signaling. In healthy esophageal mucosa, Dkk1 expression was associated with low expression of transcriptionally active β-catenin, while in reflux-esophagitis tissue, Dkk1 overexpression correlated with increased senescence-associated β-galactosidase activity and p16 upregulation. The data indicate that, in human reflux esophagitis, Dkk1 functions as a secreted growth inhibitor by suppressing Wnt/β-catenin signaling and promoting cellular senescence. These findings suggest a significant role for Dkk1 and cellular senescence in esophageal tissue homeostasis during reflux esophagitis.

Necrotizing enterocolitis (NEC) is typified by mucosal destruction, which subsequently can lead to intestinal necrosis. Prematurity, enteral feeding, and bacterial colonization are the main risk factors and, combined with other stressors, can cause increased intestinal permeability, injury, and an exaggerated inflammatory response. Heme oxygenase-1 (HO-1) mediates intestinal protection due to anti-inflammatory, antioxidative, and antiapoptotic effects of its products carbon monoxide, biliverdin, and bilirubin. This study investigates a possible role of HO-1 in the pathogenesis of NEC using a newborn mouse model. We induced NEC-like intestinal injury in 7-day-old HO-1 heterozygous (HO-1 Het, Hmox1+/-) and wild-type (Wt, Hmox1+/+) mice by gavage feeding and hypoxic exposures. Control (Con) pups of both genotypes were dam-fed. Intestines of HO-1 Het Con pups appeared predisposed to injury, with higher histological damage scores, more TUNEL-positive cells, and a significant reduction in muscularis externa thickness compared with Wt Con pups. The increase in HO activity after HO-1 induction by the substrate heme or by hypoxic stress was significantly impaired in HO-1 Het pups. After induction of intestinal injury, HO-1 Het pups displayed significantly higher NEC incidence (78 vs. 43%), mortality (83 vs. 54%), and median scores (2.5 vs. 1.5) than Wt NEC pups. PCR array analyses revealed increased expressions of IL-1β, P-selectin, matrix metallopeptidase 2, collagen type XVIII-α1, serpine 1, and others in NEC-induced HO-1 Het ileal and jejunal tissues. We conclude that a partial HO-1 deficiency promotes experimental NEC-like intestinal injury, possibly mediated by exaggerated inflammation and disruption in tissue repair.

Spinal cord transection (SCT) leads to an increase in spontaneous contractile activity in the isolated bladder that is reminiscent of an overactive bladder syndrome in patients with similar damage to the central nervous system. An increase in interstitial cell number in the suburothelial space between the urothelium and detrusor smooth muscle layer occurs in SCT bladders, and these cells elicit excitatory responses to purines and pyrimidines such as ATP, ADP, and UTP. We have investigated the hypothesis that these agents underlie the increase in spontaneous activity. Rats underwent lower thoracic spinal cord transection, and their bladder sheets or strips, with intact mucosa except where specified, were used for experiments. Isometric tension was recorded and propagating Ca2+ and membrane potential ( Em) waves were recorded by fluorescence imaging using photodiode arrays. SCT bladders were associated with regular spontaneous contractions (2.9 ± 0.4/min); ADP, UTP, and UDP augmented the amplitude but not their frequency. With strips from such bladders, a P2Y6-selective agonist (PSB0474) exerted similar effects. Fluorescence imaging of bladder sheets showed that ADP or UTP increased the conduction velocity of Ca2+/ Em waves that were confined to regions of the bladder wall with an intact mucosa. When transverse bladder sections were used, Ca2+/ Em waves originated in the suburothelial space and propagated to the detrusor and urothelium. Analysis of wave propagation showed that the suburothelial space exhibited properties of an electrical syncitium. These experiments are consistent with the hypothesis that P2Y-receptor agonists increase spontaneous contractile activity by augmenting functional activity of the cellular syncitium in the suburothelial space.

Human esophageal epithelium is continuously exposed to physical stimuli or to gastric acid that sometimes causes inflammation of the mucosa. Transient receptor potential vanilloid 1 (TRPV1) is a nociceptive, Ca2+-selective ion channel activated by capsaicin, heat, and protons. It has been reported that activation of TRPV1 expressed in esophageal mucosa is involved in gastroesophageal reflux disease (GERD) or in nonerosive GERD symptoms. In this study, we examined the expression and function of TRPV1 in the human esophageal epithelial cell line Het1A, focusing in particular on the role of oxidative stress. Interleukin-8 (IL-8) secreted by Het1A cells upon stimulation by capsaicin or acid with/without 4-hydroxy-2-nonenal (HNE) was measured by ELISA. Following capsaicin stimulation, the intracellular production of reactive oxygen species (ROS) was determined using a redox-sensitive fluorogenic probe, and ROS- and HNE-modified proteins were determined by Western blotting using biotinylated cysteine and anti-HNE antibody, respectively. HNE modification of TRPV1 proteins was further investigated by immunoprecipitation after treatment with synthetic HNE. Capsaicin and acid induced IL-8 production in Het1A cells, and this production was diminished by antagonists of TRPV1. Capsaicin also significantly increased the production of intracellular ROS and ROS- or HNE-modified proteins in Het1A cells. Moreover, IL-8 production in capsaicin-stimulated Het1A cells was enhanced by synthetic HNE treatment. Immunoprecipitation studies revealed that TRPV1 was modified by HNE in synthetic HNE-stimulated Het1A cells. We concluded that TRPV1 functions in chemokine production in esophageal epithelial cells, and this function may be regulated by ROS via posttranslational modification of TRPV1.

Exposure of esophageal mucosa to hydrochloric acid (HCl) is a crucial factor in the pathogenesis of reflux disease. We examined supernatant of HCl-exposed rabbit mucosa for inflammatory mediators enhancing migration of leukocytes and production of H2O2as an indicator of leukocyte activation. A tubular segment of rabbit esophageal mucosa was tied at both ends to form a sac, which was filled with HCl-acidified Krebs buffer at pH 5 (or plain Krebs buffer as control) and kept oxygenated at 37°C. The medium around the sac (supernatant) was collected after 3 h. Rabbit peripheral blood leukocytes (PBL) were isolated, and sac supernatant was used to investigate PBL migration and H2O2production. HCl-exposed esophageal mucosa released substance P (SP), CGRP, platelet-activating factor (PAF), and IL-8 into the supernatant. PBL migration increased in response to IL-8 or to supernatant of the HCl-filled mucosal sac. Supernatant-induced PBL migration was inhibited by IL-8 antibodies and by antagonists for PAF (CV3988) or neurokinin 1 (i.e., SP), but not by a CGRP antagonist. Supernatant of the HCl-filled mucosal sac increased H2O2release by PBL that was significantly reduced by CV3988 and by a SP antagonist but was not affected by IL-8 antibodies or by a CGRP antagonist. We conclude that IL-8, PAF, and SP are important inflammatory mediators released by esophageal mucosa in response to acid that promote PBL migration. In addition, PAF and SP induce production of H2O2by PBL. These findings provide a direct link between acid exposure and recruitment and activation of immune cells in esophageal mucosa.

Despite its position at the front line against ingested pathogens, very little is presently known about the role of the esophageal epithelium in host innate immune defense. As a key player in the innate immune response, Toll-like receptor (TLR) signaling has not been well characterized in human esophageal epithelial cells. In the present study, we investigated the inflammatory response and signaling pathways activated by TLR stimulation of human esophageal cells in vitro. Using quantitative RT-PCR, we profiled the expression pattern of human TLRs 1-10 in primary esophageal keratinocytes (EPC2), immortalized nontransformed esophageal keratinocytes (EPC2-hTERT), and normal human esophageal mucosal biopsies and found that TLRs 1, 2, 3, and 5 were expressed both in vivo and in vitro. Using the cytokine IL-8 as a physiological read out of the inflammatory response, we found that TLR3 is the most functional of the expressed TLRs in both primary and immortalized esophageal epithelial cell lines in response to its synthetic ligand polyinosinic polycytidylic acid [poly(I:C)]. Through reporter gene studies, we show that poly(I:C)-induced NF-kappaB activation is critical for the transactivation of the IL-8 promoter in vitro and that nuclear translocation of NF-kappaB occurs at an early time point following poly(I:C) stimulation of esophageal epithelial cells. Importantly, we also show that poly(I:C) stimulation induces the NF-kappaB-dependent esophageal epithelial expression of TLR2, leading to enhanced epithelial responsiveness of EPC2-hTERT cells to TLR2 ligand stimulation, suggesting an important regulatory role for TLR3-mediated NF-kappaB signaling in the innate immune response of esophageal epithelial cells. Our findings demonstrate for the first time that TLR3 is highly functional in the human esophageal epithelium and that TLR3-mediated NF-kappaB signaling may play an important regulatory role in esophageal epithelial homeostasis.

Systemic viral infections produce a highly regulated set of responses in sickness behavior, such as fever, anorexia, and adipsia. Toll-like receptor (TLR)7, activated by viral RNA during infection, potently stimulates the innate and adaptive immune responses that aid in viral clearance. However, the physiological consequences of TLR7 activation have not been thoroughly studied. In these experiments, we used a potent synthetic TLR7 ligand, 9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine ( SM360320 ; 1V136), to investigate the consequences of TLR7 activation in genetically defined strains of mice. Administration of the drug by the nasal, intragastric, or intraperitoneal routes caused transient hypophagia, hypodypsia, and hypothermia. Analyses of mutant mouse strains indicated that these effects were dependent on the expression of TLR7, its adaptor protein MyD88, and TNF-α, and independent of IL-1β, IL-6 and cyclo-oxygenase-1 (COX1). Partial roles were also implied for mast cells and COX2. Although plasma TNF-α levels were significantly higher after systemic drug delivery, the behavioral effects were maximal when the agent was administered to the mucosa. Tissue and mucosal mast cells are known to express high levels of TLR7 and to rapidly release TNF-α upon TLR7 ligation. Mice deficient in tissue mast cells, W/W(v), had significantly less anorexia after TLR7 activation, and this response was restored with mast cell reconstitution. Our results thus suggest that tissue mast cells may play a role in the anorexia induced by mucosal activation of TLR7.

Background and aims: the neural mechanisms of distension-induced esophagoupper esophageal sphincter (UES) reflexes have not been explored in humans. We investigated the modulation of these reflexes by mucosal anesthesia, acid exposure, and GABAB receptor activation. In 55 healthy human subjects, UES responses to rapid esophageal air insufflation and slow balloon distension were examined before and after pretreatment with 15 ml of topical esophageal lidocaine, esophageal HCl infusion, and baclofen 40 mg given orally. In response to rapid esophageal distension, UES can variably relax or contract. Following a mucosal blockade by topical lidocaine, the likelihood of a UES relaxation response was reduced by 11% ( P < 0.01) and the likelihood of a UES contractile response was increased by 14% ( P < 0.001) without alteration in the overall UES response rate. The UES contractile response to rapid esophageal air insufflation was also increased by 8% ( P < 0.05) following sensitization by prior mucosal acid exposure. The UES contractile response, elicited by balloon distension, was regionally dependent ( P < 0.05) (more frequent and of higher amplitude with proximal esophageal distension), and the response was attenuated by topical lidocaine ( P < 0.05). Baclofen (40 mg po) had no effect on these UES reflexes. Abrupt gaseous esophageal distension activates simultaneously both excitatory and inhibitory pathways to the UES. Partial blockade of the mucosal mechanosensitive receptors permits an enhanced UES contractile response mediated by deeper esophageal mechanoreceptors. Activation of acid-sensitive esophageal mucosal chemoreceptors upregulates the UES contractile response, suggestive of a protective mechanism.

Mucosal repair is a complex event that immediately follows acute injury induced by ischemia and noxious luminal contents such as bile. In the small intestine, villous contraction is the initial phase of repair and is initiated by myofibroblasts that reside immediately beneath the epithelial basement membrane. Subsequent events include crawling of healthy epithelium adjacent to the wound, referred to as restitution. This is a highly regulated event involving signaling via basement membrane integrins by molecules such as focal adhesion kinase and growth factors. Interestingly, however, ex vivo studies of mammalian small intestine have revealed the importance of closure of the interepithelial tight junctions and the paracellular space. The critical role of tight junction closure is underscored by the prominent contribution of the paracellular space to measures of barrier function such as transepithelial electrical resistance. Additional roles are played by subepithelial cell populations, including neutrophils, related to their role in innate immunity. The net result of reparative mechanisms is remarkably rapid closure of mucosal wounds in mammalian tissues to prevent the onset of sepsis.

Control of the regenerative properties of urothelial tissue would greatly aid the clinician in the management of urinary tract disease and disorders. Fibroblast growth factor 10 (FGF-10) is a mitogen which is particularly promising as a protein therapy for urothelial injury. The spatial synthesis, transport, targeting, and mechanistic pathway of FGF-10 and its receptor were studied in a human urothelial cell culture model and in fixed sections of lower urinary tract tissue. Synthesis of FGF-10 was restricted to mesenchymal fibroblasts, and secreted FGF-10 exhibited paracrine transport to two proximal sites, transitional epithelium and smooth muscle cell bundles, both of which were also the exclusive sites of FGF-10 receptor synthesis. The addition of recombinant FGF-10 to quiescent urothelial cells in vitro was sufficient to stimulate DNA synthesis. This stimulation was through a pathway independent of the epidermal growth factor receptor pathway. Deconvolution, light and transmission electron microscopic studies captured FGF-10 and its receptor in association with the urothelial cell surface, in cytoplasm, and within nuclei, observations that describe the mechanism that transduces the mitogenic signal in these tissues. Localization of the FGF-10 receptor to the superficial urothelial layer is clinically significant because intravesical administration of FGF-10 may provide the clinician a means to control the turnover of transitional epithelium in bladder disorders such as interstitial cystitis.

Recent reports have proposed that the urothelium can sense mechanical stretch and communicate this information to sensory afferent neurons by the release of ATP into the vicinity of P2X-containing neurons. This report investigates the bidirectional release of ATP by in vitro rabbit urothelium. ATP was measured using the luciferin-luciferase assay. Immediately after washing of both sides of the epithelium, there was a linear increase in ATP content in the mucosal compartment with a rate of 23 ± 6.5 fmol·min−1·cm−2 ( n = 18). Serosal ATP content increased as a saturating exponential function, suggesting a constant rate of release and degradation of ATP by ectonucleotidases/exonucleotidases. The presence of a serosal ectonucleotidase/exonucleotidases was demonstrated by the time-dependent decrease in exogenously added ATP. The maximum rate of hydrolysis was 11 pmol·min−1·cm−2 with a Km of 0.49 μM. The time course of serosal ATP release was modeled as a constant rate of release ( d: mol·min−1·cm−2) and rate constant of hydrolysis ( kh: min−). In control conditions d was 18 fmol·min−1·cm−2 and kh of 0.056 ± 0.01 min− ( n = 18). Steady-state serosal chamber content is 370 ± 90 fmol/cm2, and concentration is 50 ± 1.2 × 10−12 M. Stretching the tissue resulted in a transient fivefold increase in the rate of mucosal ATP release and a transient sixfold increase in serosal ATP release. Half-osmotic strength solutions increased mucosal release by 10-fold and serosal release by 5-fold. Tissue damage resulted in a step-increase in mucosal chamber ATP content by 6.6 ± 1 pmol/cm2 and serosal chamber ATP by 0.1 ± 0.06 pmol/cm2 ( n = 5).

Naturally arising CD4+CD25+ regulatory T (TR) cells have been shown to prevent and cure murine T cell-mediated colitis. However, their exact mechanism of controlling colitogenic memory CD4+ T cells in in vivo systems excluding the initial process of naive T cell activation and differentiation has not been examined to date. Using the colitogenic effector memory (TEM) CD4+ cell-mediated colitis model induced by adoptive transfer of colitogenic CD4+CD44highCD62L− lamina propria (LP) T cells obtained from colitic CD4+CD45RBhigh T cell-transferred mice, we have shown in the present study that CD4+CD25+ TR cells are able not only to suppress the development of colitis, Th1 cytokine production, and the expansion of colitogenic LP CD4+ TEM cells but also to expand these cells by themselves extensively in vivo. An in vitro coculture assay revealed that CD4+CD25+ TR cells proliferated in the presence of IL-2-producing colitogenic LP CD4+ TEM cells at the early time point (48 h after culture), followed by the acquisition of suppressive activity at the late time point (96 h after culture). Collectively, these data suggest the distinct timing of the IL-2-dependent expansion of CD4+CD25+ TR cells and the their suppressive activity on colitogenic LP CD4+ TEM cells.

The IGF-II gene normally exhibits genomic imprinting, a DNA modification that allows the expression of only one of the two inherited alleles. With loss of imprinting, there is a gain of allelic gene expression (GOAGE) due to IGF-II being expressed by both alleles. GOAGE for IGF-II has been demonstrated in a number of malignancies and in normal epithelia surrounding malignancies, but not in epithelia without associated neoplasia. We hypothesized that nonneoplastic Barrett's epithelium might have GOAGE for IGF-II that could facilitate its progression to neoplasia. Endoscopic biopsies were obtained from metaplastic esophageal, normal gastric, and normal duodenal epithelia from 43 patients with Barrett's esophagus. Genomic DNA were analyzed using PCR followed by ApaI restriction enzyme digestion or allele-specific PCR to identify an ApaI polymorphism of IGF-II. cDNA from patients with the ApaI polymorphism were analyzed for IGF-II GOAGE using exon connection PCR, followed by a secondary nested PCR and ApaI restriction enzyme digestion. We found that 13 (30%) of 43 samples of Barrett's metaplasia contained the ApaI polymorphism and were thus informative for IGF-II, and sufficient material was available for GOAGE analysis in 9 of those 13 cases. GOAGE for IGF-II was demonstrated in five (56%) of those nine cases. All patients with GOAGE in Barrett's metaplasia also demonstrated GOAGE in the gastric and duodenal epithelia. In contrast, patients without GOAGE in Barrett's metaplasia also had no GOAGE in their gastric and duodenal epithelia. We conclude that in patients with Barrett's esophagus, GOAGE for IGF-II is found frequently in the metaplastic esophageal epithelium as well as in normal gastric and duodenal epithelia.

Mammalian intestinal surfaces are in constant and intimate contact with a vast consortium of indigenous commensal bacteria. As a result, gut epithelia have evolved an array of strategies for limiting bacterial invasion into deeper tissues, helping to preserve the mutually beneficial nature of intestinal host-microbial relationships. In this review, we discuss a growing body of evidence indicating that commensal bacteria are actively involved in shaping the very barriers that confine them to the gut lumen. By modulating epithelial inflammatory responses, antimicrobial protein expression, and tissue repair functions, indigenous microbial populations are essential for the maintenance of healthy mucosal surfaces.

The mammalian esophagus has the capacity to secrete a HCO3−and mucin-rich fluid in the esophageal lumen. These secretions originate from the submucosal glands (SMG) and can contribute to esophageal protection against refluxed gastric acid. The cellular mechanisms by which glandular cells achieve these secretions are largely unknown. To study this phenomenon, we used the pH-stat technique to measure luminal alkali secretion in an isolated, perfused pig esophagus preparation. Immunohistochemistry was used to localize receptors and transporters involved in HCO3−transport. The SMG-bearing esophagus was found to have significant basal alkali secretion, predominantly HCO3−, which averaged 0.21 ± 0.04 μeq·h−1·cm−2. This basal secretion was doubled when stimulated by carbachol but abolished by HCO3−or Cl−removal. Basal- and carbachol-stimulated secretions were also blocked by serosal application of atropine, pirenzipine, DIDS, methazolamide, and ethoxzolamide. The membrane-impermeable carbonic anhydrase inhibitor benzolamide, applied to the serosal bath, partially inhibited basal HCO3−secretion and blocked the stimulation by carbachol. Immunohistochemistry using antibodies to M1cholinergic receptor or carbonic anhydrase-II enzyme showed intense labeling of duct cells and serous demilunes but no labeling of mucous cells. Labeling with an antibody to Na+-(HCO3−)n(rat kidney NBC) was positive in ducts and serous cells, whereas labeling for Cl−/HCO3−exchanger (AE2) was positive in duct cells but less pronounced in serous cells. These data indicate that duct cells and serous demilunes of SMG play a role in HCO3−secretion, a process that involves M1cholinergic receptor stimulation. HCO3−transport in these cells is dependent on cytosolic and serosal membrane-bound carbonic anhydrase. HCO3−secretion is also dependent on serosal Cl−and is mediated by DIDS-sensitive transporters, possibly NBC and AE2.

The purpose of the present study was to compare the effect of 24 h of exposure to 7% O2(normal middle ear physiological conditions) vs. 21% O2(found in the middle ear after ventilation tube placement) on transepithelial Na+absorption and Cl–secretion in cultured gerbil middle ear epithelial cell monolayers. Although no difference in apical Na+absorption was identified, the UTP-induced stimulation of apical Cl–secretion in the presence of apical Na+channel blockade with amiloride was significantly enhanced after exposure to 21% O2compared with 7% O2exposure. In the presence of a calcium-activated Cl–channel inhibitor, DIDS, UTP-induced stimulation of Cl–secretion after 21% O2exposure was decreased, suggesting a role for calcium-activated Cl–channels in middle ear Cl–secretion in response to relative hyperoxia.

NFS/N- sld mice harbor a spontaneous autosomal recessive mutation, sld (sublingual gland differentiation arrest) and histologically display attenuated mucous cell expression in sublingual glands (Hayashi et al. Am J Pathol 132: 187–191, 1988). Because altered serous demilune cell expression is unknown, we determined the phenotypic expression of this cell type in mutants. Moreover, we evaluated whether absence of glycoconjugate staining in 3-day-old mutant glands is related to disruption in apomucin gene expression and/or to posttranslational glycosylation events. Serous cell differentiation is unaffected, determined morphologically and by serous cell marker expression (PSP, parotid secretory protein; and Dcpp, demilune cell and parotid protein). Conversely, apical granules in “atypical” exocrine cells of mutant glands are PSP and mucin negative, but contain abundant SMGD (mucous granule marker). Age-related appearance of mucous cells is associated with expression of apomucin gene products, whereas SMGD expression is unaltered. “Atypical” cells thus appear specified to a mucous cell fate but do not synthesize mucin glycoproteins unless selectively induced postnatally, indicating the sld mutation disrupts apomucin transcriptional regulation and/or decreases apomucin mRNA stability.

Development of otitis media has been related to abnormal Eustachian tube (ET) mechanics. ET is a collapsible tube that is periodically opened to regulate middle ear pressure and to clear middle ear fluid into the nasopharynx. The ability to perform these physiological functions depends on several mechanical properties, including the ET's opening pressure (Popen), compliance (ETC), and hysteresis (η). In this study, a previously developed modified force-response protocol was used to determine ET mechanical properties after experimental manipulation of the mucosal surface condition. Specifically, these properties were measured in the right ear of six cynomologous monkeys under baseline conditions after “washing out” the normal ET mucous layer and after instillation of a pulmonary surfactant, Infasurf. Removal of the normal mucosa did not significantly alter Popen but did result in a decrease in ETC and η ( P < 0.05). Treatment of the mucosa with Infasurf was effective in reducing Popen and increasing both ETC and η to baseline values ( P < 0.05). These results indicate that the mucosa-air surface tension can affect the overall ETC and η properties of the ET. In addition, this study indicates that surfactant therapy may only be beneficial in patients with rigid or inelastic ETs (large Popen and low ETC and η).

Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.

We investigated the mechanisms of esophageal distension-induced reflexes in decerebrate cats. Slow air esophageal distension activated esophago-upper esophageal sphincter (UES) contractile reflex (EUCR) and secondary peristalsis (2P). Rapid air distension activated esophago-UES relaxation reflex (EURR), esophago-glottal closure reflex (EGCR), esophago-hyoid distraction reflex (EHDR), and esophago-esophagus contraction reflex (EECR). Longitudinal esophageal stretch did not activate these reflexes. Magnitude and timing of EUCR were related to 2P but not injected air volume. Cervical esophagus transection did not affect the threshold of any reflex. Bolus diversion prevented swallow-related esophageal peristalsis. Lidocaine or capsaicin esophageal perfusion, esophageal mucosal layer removal, or intravenous baclofen blocked or inhibited EURR, EGCR, EHDR, and EECR but not EUCR or 2P. Thoracic vagotomy blocked all reflexes. These six reflexes can be activated by esophageal distension, and they occur in two sets depending on inflation rate rather than volume. EUCR was independent of 2P, but 2P activated EUCR; therefore, EUCR may help prevent reflux during peristalsis. All esophageal peristalsis may be secondary to esophageal stimulation in the cat. EURR, EHDR, EGCR, and EECR may contribute to belching and are probably mediated by capsaicin-sensitive, rapidly adapting mucosal mechanoreceptors. GABA-B receptors also inhibit these reflexes. EUCR and 2P are probably mediated by slowly adapting muscular mechanoreceptors. All six reflexes are mediated by vagal afferent fibers.

We describe an optical method to quantify single- gland secretion. Isolated tracheal mucosa were mounted at the air-Krebs interface and coated with oil. Gland secretions formed spherical bubbles that were digitally imaged at intervals, allowing rates of secretion to be calculated. We monitored 340 glands in 54 experiments with 12 sheep. Glands secreted basally at low rates (0.57 ± 0.04 nl · min−1 · gland−1, 123 glands) in tissues up to 9 h postharvest and at lower rates for up to 3 days. Carbachol (10 μM) stimulated secretion with an early transient and a sustained or oscillating phase. Peak secretion was 15.7 ± 1.2 nl · min−1 · gland−1 (60 glands); sustained secretion was 4.5 ± 0.5 nl · min−1 · gland−1 (10 glands). Isoproterenol and phenylephrine (10 μM each) stimulated only small, transient responses. We confirmed that cats have a large secretory response to phenylephrine (11.6 ± 3.7 nl · min−1 · gland−1, 12 glands), but pigs, sheep, and humans all have small responses (−1 · gland−1). Carbachol-stimulated peak secretion was inhibited 56% by bumetanide, 67% by HCO[Formula: see text] replacement with HEPES, and 92% by both. The distribution of secretion rates was nonnormal, suggesting the existence of subpopulations of glands.

A number of studies show a close temporal relationship between the rate of change in muscle thickness as detected by high-frequency intraluminal ultrasonography (HFIUS) and intraluminal pressure measured by manometry. There is a marked variability in esophageal contraction amplitude from one swallow to another at a given level in the esophagus and along the length of the esophagus. Furthermore, peristaltic pressures are higher in the distal compared with the proximal esophagus. The goal of this study was to evaluate the relationship between the baseline and peak muscle thickness and the contraction amplitude during swallow-induced contractions along the length of the esophagus. Fifteen normal subjects were studied using simultaneous esophageal pressures and HFIUS or HFIUS alone. Recordings were made during baseline and standardized swallows in the lower esophageal sphincter (LES) and at 2, 4, 6, 8, and 10 cm above the LES. HFIUS images were digitized, and esophageal muscle thickness and peak contraction amplitudes were measured. In the resting state, muscle thickness is higher in the LES compared with the rest of the esophagus. Baseline muscle thickness is also significantly higher at 2 cm vs. 10 cm above the LES. In a given subject and among different subjects, there is a good relationship between peak muscle thickness and peak peristaltic pressures (r = 0.55) at all sites along the length of the esophagus. The positive correlation between pressure and muscle thickness implies that the mean circumferential wall stress is fairly uniform from one swallow to another, irrespective of the contraction amplitude.

In an earlier neonatal porcine model of smoke inhalation injury (SII), immediate postinjury application of partial liquid ventilation (PLV) had dramatic beneficial effects on lung compliance, oxygenation, and survival over a 24-h period. To explore the efficacy of PLV following SII, we treated animals at 2 and 6 h after SII and followed them for 72 h. Pigs weighing 8–12 kg were sedated and pharmacologically paralyzed, given a SII, and placed on volume-cycled, pressure-limited ventilation. Animals were randomized to three groups: group I (+SII, no PLV, n = 8), group II(+SII, PLV at 2 h, n = 6), and group III (+SII, PLV at 6 h, n = 7). Ventilatory parameters and arterial blood gasses were obtained at scheduled intervals. The PLV animals ( groups II and III) followed a worse course than group I (no PLV); PLV groups had higher peak and mean airway pressures, oxygenation index, and rate-pressure product (a barotrauma index) and lower lung compliance and arterial partial pressure of oxygen-to-inspired oxygen fraction ratio (all P < 0.05). PLV conferred no survival advantage. The reported beneficial effects of PLV with other models of acute lung injury do not appear to extend to the treatment of SII when PLV is instituted in a delayed manner. This study was not able to validate the previously reported beneficial effects of PLV in SII and actually found deleterious effects, perhaps reflecting the predominance of airway over alveolar disease in SII.

Environmental toxins, infection, and allergens lead to a transient mucous cell hyperplasia (MCH) in airway epithelia; however, the mechanisms for reducing mucous cell numbers during recovery are largely unknown. This study investigated Bcl-2 expression in mucous cells induced by a neutrophilic or eosinophilic inflammatory response. Brown Norway rats intratracheally instilled with lipopolysaccharide (LPS) showed an inflammatory response characterized primarily by neutrophils. Secreted mucin was increased fourfold at 1 day, and the number of mucous cells was increased fivefold 2, 3, and 4 days post-LPS instillation compared with those in noninstilled rats. None of the mucous cells in non- or saline-instilled control animals expressed Bcl-2, whereas 20–30% of mucous cells were Bcl-2 positive 1 and 2 days post-LPS instillation. Brown Norway rats immunized and challenged with ovalbumin (OVA) for 2, 4, and 6 days showed an inflammatory response characterized primarily by eosinophils. Secreted mucin increased fivefold, and mucous cell number increased fivefold after 4 and 6 days of OVA exposure compared with water-immunized control rats challenged with OVA aerosols. Approximately 10–25% of mucous cells were Bcl-2 positive in OVA-immunized and -challenged rats. These data demonstrate Bcl-2 expression in hyperplastic mucous cells of Brown Norway rats regardless of the type of inflammatory response and indicate that apoptotic mechanisms may be involved in the resolution of MCHs.

The opossum esophagus, like that of humans, contains a network of submucosal glands with the capacity to secrete bicarbonate ions into the esophageal lumen. To evaluate the role of these glands in protecting the epithelial surface from acid insult, we measured the lumen-to-surface pH gradient in opossum esophagus at different luminal pH and compared it to that of rabbit esophagus, an organ devoid of submucosal glands. Sections of opossum and rabbit esophageal epithelium were mounted luminal side up in a modified Ussing chamber. pH-sensitive microelectrodes, positioned within 5 μm of the epithelial cell surface, were used to monitor surface pH during perfusion with solutions of different pH. At luminal pH 7.5, the pHsof both opossum and rabbit were similar (pHs= 7.5). Lowering luminal pH from 7.5 to 3.5 in opossum decreased pHsto 4.2 ± 0.16, a value significantly higher than pH of perfusate, whereas in rabbit this maneuver decreased pHsto 3.69 ± 0.08, a value not significantly different from pH of perfusate. In opossum but not in rabbit, addition of carbachol to the serosal solution increased basal pHsto 7.8 ± 0.1 and significantly blunted the decline in pHson perfusion with acidic Ringer solution (pH 3.5), with pHsfalling to 5.6 ± 0.45. The effect of carbachol on surface buffering was inhibited by prior treatment with atropine. Luminal acidification to pH 2.0 in opossum (as in rabbit) abolished the lumen-to-surface pH gradient even after addition of serosal carbachol. We conclude that the presence of submucosal glands in esophagus contributes through bicarbonate secretion to creation of a lumen-to-surface pH gradient. Although this gradient can be modulated by carbachol, its capacity to buffer (and therefore to protect) the epithelial surface against back-diffusing H+is limited and dissipated at pH 2.0.

Previous studies demonstrated that ACh-induced liquid secretion by porcine bronchi is driven by active Cl− and H[Formula: see text] secretion. The present study was undertaken to determine whether this process was localized to submucosal glands and mediated by the cystic fibrosis transmembrane conductance regulator (CFTR). When excised, cannulated, and treated with ACh, porcine bronchi secreted 15.6 ± 0.6 μl ⋅ cm−2 ⋅ h−1. Removal of the surface epithelium did not significantly affect the rate of secretion, indicating that the source of the liquid was the submucosal glands. Pretreatment with diphenylamine-2-carboxylate, a relatively nonselective Cl−-channel blocker, significantly reduced liquid secretion by 86%, whereas pretreatment with DIDS, which inhibits a variety of Cl− channels but not CFTR, had no effect. When bronchi were pretreated with glibenclamide or 5-nitro-2-(3-phenylpropylamino)benzoic acid (both inhibitors of CFTR), the rate of ACh-induced liquid secretion was significantly reduced by 39 and 91%, respectively, compared with controls. Agents that blocked liquid secretion also caused disproportionate reductions in H[Formula: see text] secretion. Polyclonal antibodies to the CFTR bound preferentially to submucosal gland ducts and the surface epithelium, suggesting that this channel was localized to these sites. These data suggest that ACh-induced gland liquid secretion by porcine bronchi is driven by active secretion of both Cl− and H[Formula: see text] and is mediated by the CFTR.

The mucosal defensive mechanisms of the esophagus against acid and pepsin remain to be elucidated. In the present study, we investigated the contribution of the salivary mucin in maintaining the integrity of the esophageal mucosa. When an everted esophageal sac, isolated from normal rat, was treated with N-acetyl-L-cysteine, a mucolytic agent, the amount of glycoprotein in the gel layer adherent to the epithelium was completely depleted and the susceptibility of the mucosa against acidified pepsin-induced digestion increased. In sialoadenectomized rats, 7 days after extirpation, the amount of glycoprotein adherent to the esophageal epithelium was definitely reduced, and the esophageal mucosa was significantly vulnerable to acidified pepsin-induced digestion compared with the sham-operated rats. Induction of regurgitation of the gastric juices into the esophagus resulted in the development of severe hemorrhagic esophageal lesions only in the sialoadenectomized rats but not in the sham-operated rats. In conclusion, the glycoprotein in the adherent gel layer in rat esophagus, which mainly derives from salivary glands, plays an important role in the preepithelial defense to maintain the integrity of the esophageal mucosa against acid and pepsin.

To further understand the early biochemical events that occur in infected surface epithelium, we developed for the first time a model in which a respiratory submucosal gland cell population can be infected with rhinovirus (RV). Viral infection was confirmed by demonstrating with PCR that viral titers in supernatants and lysates from infected cells increased with time. Infection by RV14 upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, the major RV receptor, on submucosal gland cells, and it increased production of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor in supernatants. Antibodies to ICAM-1 inhibited RV infection of submucosal gland cells and decreased the production of cytokines after RV infection. Both IL-1alpha and IL-1beta upregulated ICAM-1 mRNA expression and increased susceptibility to RV infection, whereas other cytokines failed to alter ICAM-1 mRNA expression. Furthermore, neutralizing antibodies to IL-1alpha and IL-1beta significantly decreased the viral titers in supernatants and ICAM-1 mRNA expression after RV infection, but a neutralizing antibody to tumor necrosis factor-alpha was without effect. These findings suggest that respiratory submucosal gland cells play an important role in the initial stages of inflammation and provide useful insights into the pathogenesis of RV infection.

The humoral mucosal immune response of the kidney involves the transport of secretory IgA (S-IgA) through renal epithelial cells by the polymeric immunoglobulin receptor (pIgR). The pIgR is cleaved and released as free secretory component (FSC) or attached to IgA (S-IgA). We examined the effects of an ischemic model of acute renal failure (ARF) on the expression of pIgR and the secretion of FSC and S-IgA in the urine. Kidney pIgR mRNA levels decreased in ischemic animals by 55% at 4 h and by 85% at 72 h compared with controls. pIgR protein expression in the medullary thick ascending limb (TAL) decreased within 24 h and was nearly undetectable by 72 h. Urinary S-IgA and FSC concentrations decreased by 60% between days 3 and 6. pIgR mRNA and pIgR protein in the kidney returned to approximately 90% of control levels and urinary FSC and S-IgA concentrations returned to approximately 55% of control levels by day 7. We demonstrate that ischemic ARF decreases renal mucosal S-IgA transport in vivo and may contribute to the increased incidence of urinary tract infections.

Asthma is characterized by both local infiltration of eosinophils in the bronchial mucosa and bronchial hyperreactivity (BHR). A detailed characterization of BHR implies analysis of a histamine or methacholine dose-response curve yielding not only the dose at 20% fall of baseline forced expiratory volume in 1 s (FEV1), but also a plateau (P) representing the maximal narrowing response in terms of percent change in FEV1 and reactivity as the steepest slope at 50% of P (%FEV1/doubling dose). In the baseline condition, the specific airway conductance (sGaw) may be considered closely related to airway lumen diameter. In 20 nonsmoking asthmatic patients, methacholine dose-response curves were obtained, and a sigmoid model fit yielded the BHR indexes. Immunohistochemistry with the monoclonal antibodies (EG1 and EG2) was used to recognize the total number of eosinophils and activated eosinophils, respectively. The number of activated eosinophils was significantly correlated to both P ( r = 0.62; P < 0.05) and sGaw ( r = −0.52; P < 0.05), whereas weaker and nonsignificant correlations were found for dose at 20% fall of baseline FEV1 and the total number of eosinophils. We conclude that the number of activated eosinophils can be considered a marker of the inflammation-induced decrease of airway lumen diameter as represented by the plateau index and sGaw.

Continuous measurements were made of bladder blood flow by laser Doppler flowmetry in anesthetized dogs during bladder filling and emptying. In both mucosa and muscle, perfusion was inversely proportional to intravesical pressure. There was significantly greater perfusion in the bladder mucosa of males than females at baseline and up to 10 cm water filling pressure but not in the muscle. Intra-arterial infusion of the nitric oxide synthase inhibitor N G-nitro-l-arginine produced a significant decrease in resting bladder perfusion in the mucosa only, with no differences seen in the response to intravesical pressure. Intra-arterial infusion ofl-arginine produced a significant increase in the level of perfusion in the mucosa seen immediately after the bladder was drained. No changes were observed in muscle perfusion afterl-arginine. These results suggest that the perfusion of the bladder mucosa differs by gender and is regulated differently than the bladder muscle, possibly related to the different function of the two layers.

Functional gastrointestinal disorders are the most common problem in gastroenterological practice. They are defined by chronic abdominal symptom complexes that occur in the absence of underlying structural abnormalities. The pathogenesis of these disorders is heterogeneous and involves behavioral, infective, and inflammatory components. Common symptoms are abdominal pain, diarrhea, constipation, and bloating. Mechanisms underlying these symptoms include alterations in gastrointestinal motility, visceral perception, altered epithelial function, and disturbances in fermentation activity by gut commensal bacteria.

The objectives of this study were to identify and to characterize the pharyngoesophageal inhibitory reflex (PEIR) in an animal model. Thirty-one cats (2.4–5.0 kg) were anesthetized using α-chloralose (45 mg/kg ip), and esophageal peristalsis was recorded manometrically. Secondary peristalsis was activated by rapid air injection (8–20 ml) at midesophagus or slow infusion of water through the manometric catheters. Neither stimulus activated primary peristalsis. The PEIR was activated by rapid water injection or focal mechanical stimulation of the pharynx. Rapid air injection activated secondary peristalsis in 92% of the trials, and slow water infusion activated 1 secondary peristalsis every 3.2 min. Pharyngeal stimulation by 0.3, 0.5, 0.8, or 1.0 ml of water inhibited or blocked ongoing secondary peristalsis in 67, 82, 97, or 93% of trials, respectively. Mechanical stimulation of the posterior wall of the pharynx with 11–20 g pressure attenuated secondary peristalsis in 96% of the trials or blocked secondary peristalsis in 41% of the trials. Centripetal electrical stimulation at 30 Hz, 0.2 ms, 2 V for 4 s of the superior laryngeal (SLN) or glossopharyngeal (GPN) nerves blocked or inhibited secondary peristalsis in 100% of the trials. Bilateral transection of the GPN ( n = 8), but not the SLN ( n = 6), blocked the PEIR. Anesthetization of the pharyngeal mucosa using lidocaine (2%) blocked the PEIR ( n = 3). We concluded that 1) the PEIR exists in the cat, 2) mechanical stimulation of the pharynx more strongly activates the PEIR than water, 3) activation of either SLN or GPN afferents attenuates ongoing secondary peristalsis, 4) the receptors mediating the PEIR are located in the pharyngeal mucosa, and 5) both SLN and GPN contribute to the PEIR, but the GPN is the major afferent limb of this reflex.

The intent of this work was to evaluate the role of cAMP in regulation of ciliary activity in frog mucociliary epithelium and to examine the possibility of cross talk between the cAMP- and Ca2+-dependent pathways in that regulation. Forskolin and dibutyryl cAMP induced strong transient intracellular Ca2+concentration ([Ca2+]i) elevation and strong ciliary beat frequency enhancement with prolonged stabilization at an elevated plateau. The response was not affected by reduction of extracellular Ca2+concentration. The elevation in [Ca2+]iwas canceled by pretreatment with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Under those experimental conditions, forskolin raised the beat frequency to a moderately elevated plateau, whereas the initial strong rise in frequency was completely abolished. All effects were canceled by H-89, a selective protein kinase A (PKA) inhibitor. The results suggest a dual role for PKA in ciliary regulation. PKA releases Ca2+from intracellular stores, strongly activating ciliary beating, and, concurrently, produces moderate prolonged enhancement of the beat frequency by a Ca2+-independent mechanism.

We postulated that prolonged exposure to intermittent positive-pressure ventilation (IPPV) with high pressure (HIPPV) alone without hyperoxia promotes the development of airway hyperresponsiveness and remodeling. To test this hypothesis, young rats were ventilated under halothane anesthesia with HIPPV (maximum inspiratory pressure at 32–35 cmH2O in 70% nitrous oxide and 30% O2) for 3.5–4 h daily for 6 days. Control rats were ventilated with low IPPV (maximum inspiratory pressure < 13 cmH2O) during the same time period with the same gas mixture. With the use of tracheal rings isolated from these rats and a setup in tissue baths, contractile responses to carbachol (10−6 to 10−2 mM), 5-hydroxytryptamine (5-HT; 10−9 to 10−5 mM) and KCl (1–100 mM) were examined isometrically. In tracheal rings from HIPPV rats compared with low-pressure IPPV rats, the concentration tension curves showed a significantly enhanced response to all agonists ( P < 0.005). Sensitivity to carbachol, 5-HT, and KCl was also significantly increased ( P < 0.05) compared with control rats as evidenced by decreases in EC50. Maximum tension (reactivity) to 5-HT and KCl in the HIPPV group increased significantly ( P < 0.05), and there was a trend ( P = 0.07) toward increased reactivity to carbachol in this group as well. Histological examinations of tracheal rings demonstrated epithelial squamous metaplasia in the HIPPV group. Morphometric studies demonstrated tracheal smooth muscle thickening ( P < 0.05) without changes in the thickness of the mucosa or the lamina propria. When contractile responses were normalized for the smooth muscle cross-sectional area (i.e., stress), reactivity to all contractile agents was reduced, whereas reactivity to 5-HT still demonstrated significant increase ( P < 0.005). Sensitivity of tracheal segments to all three agents was not affected by this normalization. These findings suggest that prolonged exposure to HIPPV without hyperoxia and the resultant overdistension of lung tissues (volutrauma) induced airway remodeling and airway hyperreactivity.

Acid produces a dynamic effect on the cell phenotype of Barrett’s esophagus (BE) ex vivo. An acid pulse induces hyperproliferation, whereas continuous acid exposure promotes differentiation. To examine the mechanism for acid pulse-induced hyperproliferation, we studied the Na+/H+exchanger (NHE), which plays a role in the control of intracellular pH and cell proliferation. NHE was inhibited pharmacologically in endoscopic BE biopsies using amiloride analogs. Cell proliferation was assessed after pulsed or continuous acid exposure using tritiated thymidine incorporation assays and immunohistochemical analysis of proliferating cell nuclear antigen expression. The NHE-dependent intracellular pH response to an acid pulse was examined by pH-sensitive microfluorimetry using a Barrett’s adenocarcinoma cell line TE7. NHE inhibition significantly reduced the hyperproliferative acid-pulse effect. Furthermore, the acid-pulse activation of NHE occurred via increased transporter activity (22Na uptake) without any change in NHE-1 protein levels. Inhibition of protein kinase C (PKC), an NHE activator, also reduced the hyperproliferative response. The response of TE7 cells to an acid pulse was similar to that of BE biopsies in terms of cell proliferation and NHE and PKC dependence. Acid-pulse exposure of TE7 cells resulted in intracellular acidification followed by reneutralization to an intracellular pH greater than preacidosis values. We conclude that NHE may mediate the hyperproliferative response of BE to an acid pulse via changes in intracellular pH.

We have investigated the role in vivo of mineralocorticoid and glucocorticoid hormones in regulating the rate of electrogenic amiloride-sensitive Na+ absorption across murine airway tissue studied in vivo (nasal potential difference) and in vitro (Ussing chambers). We found that elevating the plasma aldosterone concentration 10-fold (low-Na+diet) had no significant effect on amiloride-sensitive Na+ absorption across tracheal or nasal epithelia. High doses of dexamethasone for 1 wk likewise did not change the rate of amiloride-sensitive Na+ absorption across airway epithelia. In contrast, both hormonal manipulations elevated the rate of colonic Na+ absorption. Furthermore, adrenalectomy (both normal and cystic fibrosis mice) also failed to alter Na+ absorption across airway epithelia. We conclude that, in vivo, neither the mineralocorticoid nor the glucocorticoid hormones significantly regulate the rates of amiloride-sensitive electrogenic Na+ absorption across airway epithelia in the adult mouse.

The eosinophil granule protein major basic protein (MBP) is toxic to a wide variety of cell types, by a poorly understood mechanism. To determine whether the action of MBP involves an alteration in membrane permeability, we tested purified MBP on rabbit urinary bladder epithelium using transepithelial voltage-clamp techniques. Addition of nanomolar concentrations of MBP to the mucosal solution caused an increase in apical membrane conductance only when the voltage across the apical membrane was cell interior negative. The magnitude of the MBP-induced conductance was a function of MBP concentration, and the rate of the initial increase in conductance was a function of the transepithelial voltage. The MBP-induced conductance was nonselective for K+and Cl−. Mucosal Ca2+reversed the induced conductance, whereas mucosal Mg2+partially blocked the induced conductance and slowed the rate of the increase in conductance. The induced conductance was partially reversed by changing the voltage gradient across the apical membrane to cell interior positive. Prolonged exposure resulted in an irreversible loss of the barrier function of the urinary bladder epithelium. These results suggest that an increase in cell membrane ion permeability is an initial step in MBP-induced loss of barrier function.

In porcine bronchi, inhibition of both Cl− and[Formula: see text] transport is required to block the anion secretion response to ACh and to cause mucus accumulation within ACh-treated submucosal gland ducts [S. K. Inglis, M. R. Corboz, A. E. Taylor, and S. T. Ballard. Am. J. Physiol. 272 ( Lung Cell. Mol. Physiol. 16): L372–L377, 1997]. In this previous study, a combination of three potential [Formula: see text] transport inhibitors [1 mM acetazolamide, 1 mM DIDS, and 0.1 mM dimethylamiloride (DMA)] was used to block carbonic anhydrase, Cl−/[Formula: see text]exchange, and Na+/H+exchange, respectively. The aim of the present study was to obtain a better understanding of the mechanism of ACh-induced[Formula: see text] secretion in airway glands by determining which of the three inhibitors, in combination with bumetanide, is required to block anion secretion and so cause ductal mucin accumulation. Gland duct mucin content was measured in distal bronchi isolated from domestic pigs. Addition of either bumetanide alone, bumetanide plus acetazolamide, or bumetanide plus DIDS had no significant effect on ACh-induced mean gland duct mucin content. In contrast, glands treated with bumetanide plus DMA as well as glands treated with all four anion transport blockers were almost completely occluded with mucin after the addition of ACh. These data suggest that mucin is cleared from the ducts of bronchial submucosal glands by liquid generated from Cl−- and DMA-sensitive [Formula: see text] transport.

To determine whether dopamine D1receptor antagonist impairs the swallowing reflex and reduces substance P (SP) in the peripheral organs, the swallowing reflex in terms of the number of swallows elicited by injections of three different volumes (0.2, 0.4, and 0.6 ml) of distilled water into the pharynx through a catheter was examined in anesthetized guinea pigs pretreated with Sch-23390. Animals were pretreated with either subcutaneous Sch-23390 (200 μg/kg) or a vehicle of Sch-23390 every 12 h for 7 days. The number of swallows was counted by submental electromyographic activity and visual observation of characteristic laryngeal movement. Injections of distilled water caused a volume-dependent increase in the number of swallows in animals without Sch-23390 treatment. Sch-23390 significantly decreased and exogenously administered SP increased the number of swallows elicited by all volumes of distilled water. FK-888 (10−5M, 1 ml), a specific inhibitor of the NK1receptor, reduced the number of swallows to a greater degree than Sch-23390. Sch-23390 significantly reduced SP content in the laryngeal and pharyngeal mucosa compared with control. These results suggest that inhibition of the dopamine D1receptor may impair the swallowing reflex and reduce SP content in the peripheral organs.

Neuropeptides stimulate airway epithelial cell proliferation and migration in vitro, but the role of neuropeptides in the repair of the epithelium after injury in vivo is not clear. We studied epithelial proliferation and repair in 83 male Hartley guinea pigs. Animals received capsaicin weekly for 3 wk to deplete airway neuropeptides. One week later, the dorsal aspect of the trachea was injured with a metal stylette. Animals were killed 1 h to 1 wk later, after which epithelial cell proliferation was assessed for the presence of proliferating cell nuclear antigen (PCNA). PCNA labeling was

Skin from larval bullfrogs was mounted in an Ussing-type chamber in which the apical surface was bathed with a Ringer solution containing 115 mM K+and the basolateral surface was bathed with a Ringer solution containing 115 mM Na+. Ion transport was measured as the short-circuit current ( Isc) with a low-noise voltage clamp, and skin resistance ( Rm) was measured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine waves to the command stage of the voltage clamp. From the ratio of the Fourier-transformed voltage and current signals, it was possible to calculate the resistance and capacitance of the apical and basolateral membranes of the epithelium ( Raand Rb, Caand Cb, respectively). With [Formula: see text] as the anion, Rmdecreased rapidly within 5 min following the addition of 150 U/ml nystatin to the apical solution, whereas Iscincreased from 0.66 to 52.03 μA/cm2over a 60-min period. These results indicate that nystatin becomes rapidly incorporated into the apical membrane and that the increase in basolateral K+permeability requires a more prolonged time course. Intermediate levels of Iscwere obtained by adding 50, 100, and 150 U/ml nystatin to the apical solution. This produced a progressive decrease in Raand Rbwhile Caand Cbremained constant. With Cl−as the anion, Iscvalues increased from 2.03 to 89.57 μA/cm2following treatment with 150 U/ml nystatin, whereas with gluconate as the anion Iscwas only increased from 0.63 to 11.64 μA/cm2. This suggests that the increase in basolateral K+permeability produced by nystatin treatment, in the presence of more permeable anions, is due to swelling of the epithelial cells of the tissue rather than the gradient for apical K+entry. Finally, Cbwas not different among skins exposed to Cl−,[Formula: see text], or gluconate, despite the large differences in Isc, nor did inhibition of Iscby treatment with hyperosmotic dextrose cause significant changes in Cb. These results support the hypothesis that increases in cell volume activate K+channels that are already present in the basolateral membrane of epithelial cells.

The human esophagus is exposed daily to luminal contents that are hypertonic with respect to blood. Because the effects of such exposure are unknown, we investigated the impact of luminal hypertonicity on esophageal epithelial structure and (barrier and transport) function. Rabbit esophageal epithelium was mounted in Ussing chambers and exposed luminally to hypertonic (1,200 mosmol/kgH 2 O) solutions of mannitol, urea, or NaCl or to normal Ringer solution (300 mosmol/kgH 2 O). The potential difference (PD), short-circuit current (I sc ), and resistance (R) were monitored and, after exposure, tissues were assessed morphologically by light and electron microscopy. Hypertonic mannitol had no significant effect on the electrical or morphological properties of the tissue. However, hypertonic urea and NaCl increased I sc by 91 ± 31% and 305 ± 51%, respectively, and decreased R by 20 ± 6% and 57 ± 3%, respectively (both P < 0.05 vs. Ringer or mannitol). Furthermore, serosal ouabain blocked the urea-induced increase in I sc completely and the NaCl-induced increase partially (65%) but had no effect on the decline in R. The decline in R for both urea and NaCl was associated with increased [ 14 C]mannitol fluxes and, morphologically, with dilated intercellular spaces but not cell necrosis. These data suggest that hypertonic urea and NaCl, but not hypertonic mannitol, stimulate net Na + transport in esophageal epithelium and increase permeability via the paracellular route. Notably, the effects of hypertonic urea and NaCl occur rapidly at clinically relevant osmolalities and, though reversible, resolve more slowly. We conclude that hypertonic luminal environments can significantly alter esophageal epithelial structure and function, but that the impact of such exposure depends as much on the nature of the solute as on its osmolality and duration of contact.