Solange Abdulnour-Nakhoul

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.

Alterations in junctional proteins, inflammatory mediators and extracellular matrix molecules in eosinophilic esophagitis

Eosinophilic esophagitis (EoE), an inflammatory atopic disease of the esophagus, causes massive eosinophil infiltration, basal cell hyperplasia, and sub-epithelial fibrosis. To elucidate cellular and molecular factors involved in esophageal tissue damage and remodeling, we examined pinch biopsies from EoE and normal pediatric patients. An inflammation gene array confirmed that eotaxin-3, its receptor CCR3 and interleukins IL-13 and IL-5 were upregulated. An extracellular matrix (ECM) gene array revealed upregulation of CD44 & CD54, and of ECM proteases (ADAMTS1 & MMP14). A cytokine antibody array showed a marked decrease in IL-1α and IL-1 receptor antagonist and an increase in eotaxin-2 and epidermal growth factor. Western analysis indicated reduced expression of intercellular junction proteins, E-cadherin and claudin-1 and increased expression of occludin and vimentin. We have identified a number of novel genes and proteins whose expression is altered in EoE. These findings provide new insights into the molecular mechanisms of the disease.

Substrate specificity of Rhbg: ammonium and methyl ammonium transport

Rhbg is a nonerythroid membrane glycoprotein belonging to the Rh antigen family. In the kidney, Rhbg is expressed at the basolateral membrane of intercalated cells of the distal nephron and is involved in NH4+ transport. We investigated the substrate specificity of Rhbg by comparing transport of NH3/NH4+ with that of methyl amine (hydrochloride) (MA/MA+), often used to replace NH3/NH4+, in oocytes expressing Rhbg. Methyl amine (HCl) in solution exists as neutral methyl amine (MA) in equilibrium with the protonated methyl ammonium (MA+). To assess transport, we used ion-selective microelectrodes and voltage-clamp experiments to measure NH3/NH4+- and MA/MA+-induced intracellular pH (pH(i)) changes and whole cell currents. Our data showed that in Rhbg oocytes, NH3/NH4+ caused an inward current and decrease in pH(i) consistent with electrogenic NH4+ transport. These changes were significantly larger than in H2O-injected oocytes. The NH3/NH4+-induced current was not inhibited in the presence of barium or in the absence of Na+. In Rhbg oocytes, MA/MA+ caused an inward current but an increase (rather than a decrease) in pH(i). MA/MA+ did not cause any changes in H2O-injected oocytes. The MA/MA+-induced current and pH(i) increase were saturated at higher concentrations of MA/MA+. Amiloride inhibited MA/MA+-induced current and the increase in pH(i) in oocytes expressing Rhbg but had no effect on control oocytes. These results indicate that MA/MA+ is transported by Rhbg but differently than NH3/NH4+. The protonated MA+ is likely a direct substrate whose transport resembles that of NH4+. Transport of electroneutral MA is also enhanced by expression of Rhbg.

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.

Characterization of esophageal submucosal glands in pig tissue and cultures.

The submucosal glands (SMGs) of the pig esophagus, like the human, secrete mucin and bicarbonate, which help in luminal acid clearance and epithelial protection. The aim of this study was to characterize histochemically the esophageal SMGs and a primary culture obtained from these glands. Tissues and cultures were stained with hematoxylin and eosin, periodic acid Schiff, Alcian blue, lectins, or cytokeratins. In the perfused esophagus, addition of carbachol increased mucin secretion by approximately 2-fold. The results indicate that [1] a method for culturing SMG cells was developed; [2] conventional staining indicates the presence of sulfated, acidic, and neutral mucopolysaccharides in glands and cultures; [3] lectin binding indicates the presence of N-acetyl glucosamine, N-acetyl neuraminic acid, N-acetyl galactosamine, and α-l-fucose in mucous cells and cultures; [4] cytokeratin and lectin staining indicated similarities with Barrett epithelium (columnar metaplasia of the esophagus); and [5] cholinergic agonists enhance mucin secretion and this could play a significant role in esophageal protection.

Mechanisms of ammonia and ammonium transport by rhesus-associated glycoproteins.

In this study we characterized ammonia and ammonium (NH3/NH4(+)) transport by the rhesus-associated (Rh) glycoproteins RhAG, Rhbg, and Rhcg expressed in Xenopus oocytes. We used ion-selective microelectrodes and two-electrode voltage clamp to measure changes in intracellular pH, surface pH, and whole cell currents induced by NH3/NH4(+) and methyl amine/ammonium (MA/MA(+)). These measurements allowed us to define signal-specific signatures to distinguish NH3 from NH4(+) transport and to determine how transport of NH3 and NH4(+) differs among RhAG, Rhbg, and Rhcg. Our data indicate that expression of Rh glycoproteins in oocytes generally enhanced NH3/NH4(+) transport and that cellular changes induced by transport of MA/MA(+) by Rh proteins were different from those induced by transport of NH3/NH4(+). Our results support the following conclusions: 1) RhAG and Rhbg transport both the ionic NH4(+) and neutral NH3 species; 2) transport of NH4(+) is electrogenic; 3) like Rhbg, RhAG transport of NH4(+) masks NH3 transport; and 4) Rhcg is likely to be a predominantly NH3 transporter, with no evidence of enhanced NH4(+) transport by this transporter. The dual role of Rh proteins as NH3 and NH4(+) transporters is a unique property and may be critical in understanding how transepithelial secretion of NH3/NH4(+) occurs in the renal collecting duct.

pH sensitivity of ammonium transport by Rhbg

Rhbg is a membrane glycoprotein that is involved in NH(3)/NH(4)(+) transport. Several models have been proposed to describe Rhbg, including an electroneutral NH(4)(+)/H(+) exchanger, a uniporter, an NH(4)(+) channel, or even a gas channel. In this study, we characterized the pH sensitivity of Rhbg expressed in Xenopus oocytes. We used two-electrode voltage clamp and ion-selective microelectrodes to measure NH(4)(+)-induced [and methyl ammonium (MA(+))] currents and changes in intracellular pH (pH(i)), respectively. In oocytes expressing Rhbg, 5 mM NH(4)Cl (NH(3)/NH(4)(+)) at extracellular pH (pH(o)) of 7.5 induced an inward current, decreased pH(i), and depolarized the cell. Raising pH(o) to 8.2 significantly enhanced the NH(4)(+)-induced current and pH(i) changes, whereas decreasing bath pH to 6.5 inhibited these changes. Lowering pH(i) (decreased by butyrate) also inhibited the NH(4)(+)-induced current and pH(i) decrease. In oocytes expressing Rhbg, 5 mM methyl amine hydrochloride (MA/MA(+)), often used as an NH(4)Cl substitute, induced an inward current, a pH(i) increase (not a decrease), and depolarization of the cell. Exposing the oocyte to MA/MA(+) at alkaline bath pH (8.2) enhanced the MA(+)-induced current, whereas lowering bath pH to 6.5 inhibited the MA(+) current completely. Exposing the oocyte to MA/MA(+) at low pH(i) abolished the MA(+)-induced current and depolarization; however, pH(i) still increased. These data indicate that 1) transport of NH(4)(+) and MA/MA(+) by Rhbg is pH sensitive; 2) electrogenic NH(4)(+) and MA(+) transport are stimulated by alkaline pH(o) but inhibited by acidic pH(i) or pH(o); and 3) electroneutral transport of MA by Rhbg is likely but is less sensitive to pH changes.

Delayed administration of pituitary adenylate cyclase-activating polypeptide 38 ameliorates renal ischemia/reperfusion injury in mice by modulating Toll-like receptors.

We investigated whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) ameliorates kidney injury after ischemia/reperfusion (IR) by modulating Toll-like receptor (TLR)-associated signaling pathways. Male C57BL/6 mice were subjected to bilateral renal ischemia for 45 min. PACAP38, 20 μg in 100 μl of saline, was administered i.p. at 24 and 48 h after IR, and mice were euthanized at 72h. In IR mice, PACAP38 maintained serum creatinine near control levels (0.81 ± 0.08 vs. 0.69 ± 0.17 mg/dl in controls, p=NS, vs. 1.8 ± 0.03 in saline-treated IR mice, p<0.01) and significantly reduced the expression of kidney injury biomarkers. PACAP38 significantly reduced the levels of apoptosis and neutrophil infiltration, and protected against tubular damage. With PCR arrays, 59 of 83 TLR-related genes significantly changed their expression after IR. TLR2 increased 162 fold, followed by Fas-associated death domain (37 fold) and TLR6 (24 fold), while ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) decreased 55 fold. PACAP38 given 24 and 48 h after IR injury significantly reversed these changes in 56 genes, including TLR2, TLR3, TLR4, TLR6, and genes in the NF-κB pathways. The alterations in TLR2, TLR3, TLR6, and UBE2V1 were confirmed by RT-PCR. After IR, PACAP38 also suppressed protein levels of TLR-associated cytokines. PACAP38 reversed the changes in IR-activated TLR-associated NF-κB signaling pathways even when treatment was delayed 24h. Therefore, PACAP38 could be an effective therapeutic for unexpected IR-mediated renal injury. The prominently IR-induced TLR-related genes identified in this study could be novel drug targets.

The Effect of Tegaserod on Esophageal Submucosal Glands Bicarbonate and Mucin Secretion

Tegaserod, a 5-HT4 partial agonist, was shown to reduce esophageal acid exposure in patients with gastroesophageal reflux disease; however, its mechanism of action is poorly understood. Therefore, we have examined the effect of tegaserod on luminal bicarbonate and mucin secretion in the isolated perfused pig esophagus. We also studied its role in esophageal protection using SMG-bearing pig esophagus in comparison to the rabbit esophagus, which is devoid of them. The tissues were mounted in Ussing chambers, and acid injury was replicated by exposing the lumen to acid (pH 1.6) or acid/pepsin (pH 2.5). In pig esophagus, tegaserod increased bicarbonate secretion, but had no effect on basal mucin secretion. In Ussing chambers, tegaserod reduced injury to pig, but not rabbit esophagus exposed to acid (pH 2.5) plus pepsin. These results indicate that tegaserod stimulates SMG bicarbonate secretion, an effect that likely accounts for the observed protection against acid–pepsin injury to pig, but not rabbit, esophagus.

Effect of norepinephrine on intracellular pH in kidney proximal tubule: role of Na+-(HCO-3)n cotransport.

We examined the effect of norepinephrine (NE) on intracellular pH (pHi) and activity of Na+([Formula: see text]) in the isolated perfused kidney proximal tubule of Ambystoma, using single-barreled voltage and ion-selective microelectrodes. In control[Formula: see text] Ringer, addition of 10-6 M NE to the bath reversibly depolarized the basolateral membrane potential ( V 1), the luminal membrane potential ( V 2), and the transepithelial potential difference ( V 3) and increased pHi by 0.14 ± 0.02. These effects were mimicked by isoproterenol but were abolished after pretreatment with SITS or in the absence of CO2/[Formula: see text]. Removal of bath Na+ depolarized V 1 and V 2, hyperpolarized V 3, and decreased pHi. These effects are largely mediated by the electrogenic Na+-([Formula: see text]) n cotransporter. In the presence of NE, the effects of Na+ removal on membrane potential differences and the rate of change of pHi were significantly smaller. Reducing bath [Formula: see text] concentration from 10 to 2 mM at constant CO2 (pH 6.8) depolarized V 1 and V 2, decreased pHi, and lowered[Formula: see text]. These changes are also due to Na+-([Formula: see text]) n . In the presence of NE, reducing bath [[Formula: see text]] caused a smaller depolarizations of V 1 and V 2, and the rate of pHi decrease was significantly reduced. Our results indicate: 1) NE causes an increase in pHi; 2) the NE-induced alkalinization is mediated by a SITS-sensitive and[Formula: see text]-dependent transporter on the basolateral membrane; and 3) in the presence of NE, the reduced effects caused by basolateral[Formula: see text] changes or Na+ removal are indicative of an inhibitory effect of NE on Na+-([Formula: see text]) n cotransport.