Nelia A. Tobey

Dilated Intercellular Spaces and Shunt Permeability in Nonerosive Acid-Damaged Esophageal Epithelium

OBJECTIVES:It has recently been established that patients with nonerosive reflux disease have on biopsy within esophageal epithelium a lesion known as dilated intercellular spaces (DIS).METHODS:To further explore the nature and implications of this lesion, in vitro models of nonerosive acid and acid-pepsin damage were created in Ussing chamber-mounted rabbit esophageal epithelium. Using these models circuit analysis and permeability studies were carried out, the latter using dextran of varying size and human epidermal growth factor (EGF).RESULTS:Luminal HCl, pH 1.1, or HCl, pH 2.0 + pepsin, 1 mg/ml, for 30 min significantly reduced transepithelial electrical resistance (RT) but produced no gross erosions or histologic evidence of cell necrosis. Transmission electron microscopy, however, documented the presence of DIS. Circuit analysis on healthy esophageal epithelium showed that shunt resistance (RS) was much lower than apical membrane, basolateral membrane and transcellular resistances (Ra, Rb, and Rcell, respectively) and approached that of RT. Further, circuit analysis on acid and acid-pepsin damaged tissues showed that the declines in RT resulted from declines in RS. Moreover, the declines in RT (and so RS) were associated with a linear increase in permeability to 4 kD dextrans as well as an increase in permeability to 6 kD EGF and dextrans as large as 20 kD.CONCLUSIONS:In nonerosive acid-damaged esophageal epithelium DIS develop in association with and as a marker of reduced transepithelial resistance and increased shunt permeability. This change in shunt permeability upon acid or acid-pepsin exposure is substantial, permitting dextran molecules as large as 20 kD (33 Å) to diffuse across the epithelium. Also, this shunt leak enables luminal EGF at 6 kD to diffuse across the acid-damaged epithelium and by so doing enables it to access its receptors on epithelial basal cells. We hypothesize that the shunt leak of EGF may in part account for the development of a reparative phenomenon on esophageal biopsy in patients with nonerosive reflux disease known as basal cell hyperplasia.

Barriers to paracellular permeability in rabbit esophageal epithelium

Morphological and electrophysiological techniques were used to define the location and nature of the barriers to diffusion across the intercellular space (paracellular pathway) of rabbit esophageal epithelium. Transmission electron microscopy and light microscopy coupled with histochemistry identified a series of tight junctions and an intercellular material staining positively for neutral and acidic glycoconjugates as likely barrier candidates. Additional studies with lanthanum and horseradish peroxidase showed that the barrier to diffusion of tracers was present throughout the stratum corneum and extended to the upper three to seven layers of stratum spinosum and that these findings were most compatible with the presence of the intercellular glycoconjugate material but not the tight junctions. Further positive staining for carbohydrate moieties at the electron microscopic level with periodic acid-thiocarbohydrazide-silver proteinate suggested that the glycoconjugate material was synthesized in the cells of the barrier layers and packaged in intracellular membrane-bound vesicles before secretion into the intercellular space. Although tight junctions were present in series within stratum corneum and, less commonly, extended to two to three cell layers of upper stratum spinosum, analysis of tracer studies, freeze-fracture replicas, electrophysiological data, and mannitol fluxes, while not conclusive, provided little to support a major role for these junctions in barrier function in this tissue.

Role of e-cadherin in the pathogenesis of gastroesophageal reflux disease

OBJECTIVES:An early event in the pathogenesis of gastroesophageal reflux disease (GERD) is an acid-induced increase in junctional (paracellular) permeability in esophageal epithelium (EE). The molecular events that account for this change are unknown. E-cadherin is a junctional protein important in barrier function in EE. Therefore, defects in barrier function in EE were sought in GERD as well as whether their presence correlated with abnormalities in e-cadherin.METHODS:Endoscopic biopsies of EE from GERD (n=20; male 10; female 10; mean age 50±10 years) and subjects with a healthy esophagus (controls; n=23; male 11; female 12; mean age 51±11 years) were evaluated in mini-Ussing chambers and by western blot and immunochemistry; and serum analyzed by enzyme-linked immunosorbent assay (ELISA). A role for e-cadherin was also assessed using a unique conditional knockout of e-cadherin in adult mouse esophagus.RESULTS:EE from GERD patients had lower electrical resistance and higher fluorescein flux than EE from controls; and the findings in GERD associated with cleavage of e-cadherin. Cleavage of e-cadherin in GERD was documented in EE by the presence of a 35-kDa, C-terminal fragment of the molecule on western blot and by an increase in soluble N-terminal fragments of the molecule in serum. Activation of the membrane metalloproteinase, A Disintegrin And Metalloproteinase (ADAM-10), was identified as a likely cause for cleavage of e-cadherin by western blot and immunostaining and a role for e-cadherin in the increased junctional permeability in EE from GERD supported by showing increased permeability after deletion of e-cadherin in mouse EE.CONCLUSIONS:The EE in GERD has increased junctional permeability and this is in association with proteolytic cleavage of e-cadherin. As loss of e-cadherin can, alone, account for the increase in junctional permeability, cleavage of e-cadherin likely represents a critical molecular event in the pathogenesis of GERD, and identification of cleaved fragments may, if confirmed in larger studies, be valuable as a biomarker of GERD.

Esophageal exposure to ethanol increases risk of acid damage in rabbit esophagus

Heavy alcohol consumption is associated with thedevelopment of reflux esophagitis. Among the reasons forthis are impairment of the antireflux barrier,stimulation of acid secretion, and altered tissue resistance. To explore the contribution ofaltered tissue resistance to the development ofesophagitis, sections of rabbit esophageal epitheliumwere mounted in Ussing chambers and exposed luminally to 10% ethanol, acid (HCl, pH 2), or combinationsof both. Tissue injury was assessed by measurements ofpotential difference (PD), short circuit current(Isc) and electrical resistance (R) and byhistology. Tissues exposed luminally to HCl for 1 hrexhibited little or no change electrically ormorphologically compared to Ringer controls, whileluminal exposure to 10% ethanol for 1 hr lowered PD (53± 4%), Isc (30 ± 1%), and R (31± 5%) and produced cellular edema in the upperlayers. Simultaneous exposure to ethanol and acidresulted in significantly greater declines in PD (81± 1%) and Isc (70 ± 2%), but not R (40± 4%), and greater morphologic damage. Moreover,this vulnerability of ethanol-exposed tissues to acidwas demonstrable at generally innocuous levels ofacidity (pH 2-4), after only short periods of ethanol exposure (10 min)and with delays for acid exposures of up to 1 hrfollowing ethanol removal from the bathing solution. Inconclusion, ethanol has a direct noxious effect on esophageal epithelium, which predisposes thetissue to acid injury. Tissue vulnerability developswith even short exposures to clinically relevantconcentrations of ethanol, lasts for at least 1 hr after ethanol clearance, and transforms relativelyinnocuous concentrations of acid into damaging agents.These results support the likelihood that ethanolsability to alter tissue resistance plays an important role in the development of reflux esophagitisin humans.

Human Intestinal Brush Border Peptidases

Hydrolysis of small peptides, like disaccharide hydrolysis, is an important function of the intestinal brush border, but little is known of the individual human peptidases. The purposes of this study were to detect all human brush border enzymes hydrolyzing dipeptides and tripeptides, identify the most discriminating substrate for each enzyme in order to permit assays in crude mixtures, and begin biochemical characterization of each enzyme. Four brush border peptidases were identified. Enzymes I (aspartate aminopeptidase, E.C. 3.4.11.7) and III (amino-oligopeptidase, E.C. 3.4.11.2) are known brush border enzymes. Enzymes II (membrane Gly-Leu peptidase) and IV (zinc stable Asp-Lys peptidase) have not been identified in human brush border previously. They are distinct from dipeptidyl aminopeptidase IV, carboxypeptidase, and gamma-glutamyl transferase. The substrate most discriminating for each enzyme is alpha-Glu-beta-naphthylamide for I (100% of the brush border activity for this substrate is due to enzyme I), glycylleucine for II (80%), leucyl-beta-naphthylamide for III (91%), and aspartyl-lysine in 5 mM Zn2+ for IV (63%). The enzymes are immunologically distinct and antibodies to each one localize to the brush border on immunohistochemical staining. Purification of 142-, 79-, 158-, and 46-fold was achieved for enzymes I through IV, respectively. Biochemical characteristics include slightly alkaline pH optima, molecular weights of 91,000-190,000, and evidence of metal ion involvement in activity. These studies provide necessary information for determining the role of brush border peptidase deficiencies in human disease.

Human esophageal epithelial cells possess an Na + /H + exchanger for H + extrusion

Objective:The human esophagus is regularly exposed to refluxed gastric acid. Therefore, its epithelial cells require for survival a means of extruding excess H+ from the cytoplasm. Because Na+/H+ exchange activity has been observed in many mammalian cell types, including that of rabbit esophagus, we sought its presence in human esophageal epithelium.Methods:Human esophageal epithelial cells derived from endoscopic biopsy specimens or surgical esophagectomy specimens were grown in primary culture and loaded with the fluorescent dye, 2′7′-bis(carboxyethyl)-5(6)-carboxyfluorescein, to monitor intracellular pH (pHi).Results:Resting pHi in bicarbonate-free N′-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid was 7.5 ± 0.03 (n = 50). Acidification using the NH4Cl prepulse technique lowered pHi by 0.6 ± 0.02 pH units, with recovery ensuing at an initial rate of 0.09 ± 0.04 pH units/min. Notably, the rate of recovery was faster the more acidic the pHi, and recovery was abolished by amiloride or replacement with an Na+-free buffer. Acidification by lowering pHo with HCl resulted in a similarly rapid rate of return as with the NH4Cl technique, and resting cells acidified by 0.17 ± 0.02 pH units/5 min upon exposure to amiloride.Conclusions:Human esophageal cells possess an H+-extruding mechanism consistent with an Na+/H+ exchanger. This mechanism is active in resting cells, adapts to the degree of pHi lowering, and extrudes H+ efficiently whether loaded by intracellular or extracellular means, making it well suited for epithelial defense against acid injury.

HCl-induced cell edema in rabbit esophageal epithelium: A bumetanide-sensitive process

BACKGROUND & AIMS The morphology of acid damage to esophageal epithelium is characterized by marked cell (swelling) edema. This observation suggests that, in the process of acid damage, an increase in osmotic forces develops within the cell that accounts for the increase in cell water. The aim of this study was to document that esophageal cells swell at acidic pH and to explore the nature of the osmolytes and mechanisms responsible for it. METHODS Cell edema was assessed in sections of rabbit esophageal epithelium by correlating morphological change with change in tissue wet weight after immersion in acidic solutions for up to 4 hours. RESULTS At pH < or = 2 for 2 hours, tissues gained weight and showed cell edema on both light and electron microscopy. In addition to being time- and pH-dependent, cell edema was dependent on bathing solution osmolytes, specifically Na+, K+, and Cl-, and could be inhibited by tissue pretreatment with bumetanide (or ethacrynic acid). CONCLUSIONS HCl exposure can spontaneously produce cell edema in esophageal epithelium. The phenomenon is pH- and time-dependent and requires acid stimulation of osmolyte absorption through a bumetanide-sensitive process compatible with an NaK2Cl cotransporter in the epithelial cell membrane.

Effect of heat stress on rabbit esophageal epithelium

Hot beverages expose the esophageal epithelium to temperatures as high as 58 degrees C. To study the impact of such temperatures, rabbit esophageal epithelium was exposed to luminal heat or both luminal and serosal heat while mounted in Ussing chambers. Luminal heat, mimicking exposure to hot beverages, reduced potential difference (PD) and resistance (R) when applied at >/=49 degrees C and reduced short-circuit current (Isc) at >/=60 degrees C. At >/=60 degrees C, subepithelial blisters developed. Higher temperatures reduced R only moderately and reversibly. In contrast, the Isc declined sharply and irreversibly once threshold was reached. Luminal and serosal heat also reduced PD, Isc, and R, although the threshold for reduction in Isc was now similar to that for R. Additionally, luminal and serosal heat reduced Isc more than R for any given temperature and resulted in blisters at lower temperatures (50 degrees C) than luminal heat alone. The heat-induced decline in Isc was attributed in part to inactivation of Na-K-ATPase activity, although other transport systems could have been equally affected, and the decline in R to an increase in paracellular permeability. The latter effect on R also contributed to an increase in tissue sensitivity to luminal acid damage. Consumption of hot beverages exposes the esophagus to temperatures that can negatively impact epithelial structure and function. Impaired barrier function by heat increases the risk of esophageal damage by subsequent contact with (refluxed) gastric acid. These findings help explain in part the association between esophageal disease and consumption of hot beverages.Hot beverages expose the esophageal epithelium to temperatures as high as 58°C. To study the impact of such temperatures, rabbit esophageal epithelium was exposed to luminal heat or both luminal and serosal heat while mounted in Ussing chambers. Luminal heat, mimicking exposure to hot beverages, reduced potential difference (PD) and resistance ( R) when applied at ≥49°C and reduced short-circuit current ( I sc) at ≥60°C. At ≥60°C, subepithelial blisters developed. Higher temperatures reduced R only moderately and reversibly. In contrast, the I sc declined sharply and irreversibly once threshold was reached. Luminal and serosal heat also reduced PD, I sc, and R, although the threshold for reduction in I scwas now similar to that for R. Additionally, luminal and serosal heat reduced I sc more than R for any given temperature and resulted in blisters at lower temperatures (50°C) than luminal heat alone. The heat-induced decline in I sc was attributed in part to inactivation of Na-K-ATPase activity, although other transport systems could have been equally affected, and the decline in R to an increase in paracellular permeability. The latter effect on R also contributed to an increase in tissue sensitivity to luminal acid damage. Consumption of hot beverages exposes the esophagus to temperatures that can negatively impact epithelial structure and function. Impaired barrier function by heat increases the risk of esophageal damage by subsequent contact with (refluxed) gastric acid. These findings help explain in part the association between esophageal disease and consumption of hot beverages.

Effect of ethanol on the structure and function of rabbit esophageal epithelium

Epidemiological studies indicate a relationship between alcohol consumption and esophageal epithelial disease. We therefore sought the contribution of the direct effects of ethanol on esophageal epithelial structure and (transport and barrier) function. Epithelium from the rabbit was mounted in Ussing chambers and exposed luminally for 1 h to 1-40% ethanol. At concentrations of 1-5% potential difference (PD) increased, and at 10-40% PD decreased. The increase in PD with 1-5% ethanol was accompanied by an increase in short-circuit current (Isc), and this increase in Isc could be blocked by ouabain pretreatment. The decrease in PD with 10-40% ethanol was associated with a decrease in electrical resistance (R), and this decrease in R was paralleled by an increase in transepithelial [14C]mannitol flux. Reversibility of these changes was limited at ethanol concentrations > or = 10%, and these were associated morphologically by patchy or diffuse tissue edema. Moreover, as with ethanol exposure in vitro, exposure in vivo produced dose-dependent changes in PD, Isc, R, and morphology. These observations indicate that exposure to ethanol in concentrations and under conditions reflecting alcohol consumption in humans can alter and impair esophageal epithelial transport and barrier functions. Such impairments are likely to contribute to the observed increase in risk of esophageal disease with regular consumption of alcoholic beverages.Epidemiological studies indicate a relationship between alcohol consumption and esophageal epithelial disease. We therefore sought the contribution of the direct effects of ethanol on esophageal epithelial structure and (transport and barrier) function. Epithelium from the rabbit was mounted in Ussing chambers and exposed luminally for 1 h to 1-40% ethanol. At concentrations of 1-5% potential difference (PD) increased, and at 10-40% PD decreased. The increase in PD with 1-5% ethanol was accompanied by an increase in short-circuit current ( I sc), and this increase in I sccould be blocked by ouabain pretreatment. The decrease in PD with 10-40% ethanol was associated with a decrease in electrical resistance ( R), and this decrease in R was paralleled by an increase in transepithelial [14C]mannitol flux. Reversibility of these changes was limited at ethanol concentrations ≥10%, and these were associated morphologically by patchy or diffuse tissue edema. Moreover, as with ethanol exposure in vitro, exposure in vivo produced dose-dependent changes in PD, I sc, R, and morphology. These observations indicate that exposure to ethanol in concentrations and under conditions reflecting alcohol consumption in humans can alter and impair esophageal epithelial transport and barrier functions. Such impairments are likely to contribute to the observed increase in risk of esophageal disease with regular consumption of alcoholic beverages.

The Acute Effects of Smokeless Tobacco on Transport and Barrier Function of Buccal Mucosa

The acute effects of smokeless tobacco (ST) on buccal mucosal transport and barrier function were studied by means of in vivo and in vitro techniques. In humans, in vivo exposure to 0.5 g ST transiently increased the transmural electrical potential difference (PD). However, despite continued exposure, PD returned to baseline within 20 min. The mechanisms for these changes were explored by use of dog buccal mucosa mounted in Ussing chambers. Luminal exposure to a Ringer-extract of ST (EOST) increased PD and short-circuit current (Isc) and decreased electrical resistance (R), with changes reversible upon removal of EOST from the bath. Further, radioisotopic fluxes showed that the increase in Isc in EOST-exposed tissues was accompanied by increased absorption of electrolytes (Na, Cl, and other ions), and the decrease in R was accompanied by increased permeability to mannitol. Light microscopy of tissues exposed to EOST showed no morphological changes after exposure to 0.5 g of ST, but after exposure to 1.5-2.5 g of ST, dilated intercellular spaces were identified. Contact of aqueous media with ST led to the release of electrolytes and other soluble compounds into solution. To determine the effect of electrolyte release on buccal function, we exposed mucosae luminally to a solution with ion composition and/or osmolality similar to EOST or to one with an EOST previously dialyzed against Ringer. Solutions with similar ion composition and/or osmolarity changed PD, Isc, and R in a manner similar to EOST, while dialyzed-EOST had no effect. In addition, luminal nicotine produced effects different from EOST, decreasing PD and Isc and increasing R. These results indicate that ST can acutely alter buccal transport and barrier function by creating, through electrolyte release, electrochemical and osmolar gradients across the tissue.