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JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2008, 59, 3, 525–542 www.jpp.krakow.pl

B.R. LIN1, 2, 3, H.T. HSIEH1, J.M. LEE4, I.R. LAI4, C.F. CHEN1, L.C.H. YU1*

LUMINAL HYDROCHLORIC ACID STIMULATES RAPID TRANSEPITHELIAL ION FLUXES IN RODENT ESOPHAGEAL STRATIFIED SQUAMOUS EPITHELIUM Graduate Institute of Physiology, National Taiwan University College of Medicine; 2Department of Integrated Diagnostics and Therapeutics, National Taiwan University Hospital; 3Department of Internal Medicine, 4Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan

1

It remains unclear whether enhanced ion fluxes occur in the esophageal stratified squamous epithelium upon acid exposure. Rat esophageal tissues devoid of submucosal glands displayed basal short-circuit current (Isc) of 5.03 ± 1.93 µA/cm2 and lumen-negative potential difference (PD) in association with net absorption of Na+ and Cl-, and secretion of HCO3-. Luminal hydrochloric acid (HCl) challenge (pH = 1.6) triggered an acute rise of the Isc and increment of negative PD to seven-fold of baseline, which was diminished in HCO3--free, but not Na+- free buffer. The rise of Isc was inhibited by pretreatment with di-isothiocyanatostilbene-2, 2'-disulphonic acid (DIDS) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). Topical carbachol, capsaicin, forskolin or CFTRinh-172 had no effect on basal Isc. CFTRinh-172 did not reduce the acid-increased Isc. Functional ablation of capsaicin-sensitive nerves had no effect on the acid-induced Isc. The phenomenon of enhanced ion fluxes upon acid stimulation was confirmed in human esophageal specimens. Our results demonstrated that the mechanism of acid-induced rapid transepithelial ion fluxes is dependent on the presence of bicarbonate ions as well as functional anion transporters and Na+/H+ exchanger, but independent of cystic fibrosis transmembrane conductance regulator (CFTR). The capsaicin-sensitive and muscarinic-dependent nerve pathways did not play roles in the mechanism. K e y w o r d s : hydrochloric acid, short-circuit current, ion transporter, esophageal epithelium, bicarbonate ions

526 INTRODUCTION

The esophageal epithelium normally acts as a protective barrier against tiding hydrochloric acid (HCl) challenges during daily life. Only a proportion of the individuals develop esophageal epithelial injuries and gastroesophageal reflux disease (GERD) (1). The occurrence, severity, and progression of GERD are determined by the balance between the aggressive factors, e.g. HCl, and the protective factors, e.g. epithelial barrier (2). Substantial literatures underlined the physical resistance conferred by esophageal epithelium, focusing on the presence of tight paracellular junctions in the stratified squamous epithelium in rabbit models and human subjects (3 - 5). Others have identified bicarbonate ion secretion from the esophageal submucosal glands and the duodenal columnar epithelium that are crucial for the neutralization of gastric acid (6 - 10). Earlier studies have documented bicarbonate ion secretion upon acid challenge in esophageal tissues in human, pig and opossum, and attributed this phenomenon to the presence of submucosal glands in the tissues (7, 10, 11). It is noteworthy that much discrepancy and controversy exists in the number and distribution of esophageal submucosal glands in human. Uneven spacing of submucosal glands was documented along the longitudinal axis of the esophagus with extreme variability among individuals (12). A topographic study using human necropsy specimens confirmed the large spatial variation of glands ranging from complete absence to one third of the total surface in different areas of the esophagus. In the upper, middle and lower third segments of the esophagus, the median percent surface of submucosal glands varies from 1.68 % (0 35.53%), 0.03 % (0 - 13.84%), and 1.00 % (0 - 9.24%), respectively (13). It has long been recognized that none of the rodent species, including marmot, squirrel, gopher, porcupine, rat and mouse, possess structures of esophageal glands (12). In the case of rabbits, some laboratories denies while others reported the presence of glands in the esophagus (10, 12). In comparison to the gastric and duodenal mucosa, the mucous layer covering the esophageal epithelium is much thinner and without an efficient pH buffering capacity (5, 6, 9). Therefore, defense mechanism contributed by the esophageal squamous epithelium may play crucial roles in areas deficient of glands. A recent study documented basal short-circuit current (Isc) and transmural lumen-negative potential difference (PD) in endoscopic biopies of stratified squmous epithelium from normal human subjects using modified mini-Ussing Chambers (14). In patients with Barrett's specialized columnar epithelium, increased Isc associated with anion secretion was found mediated by an apical anion channel in the esophageal epithelium (14), which was suggested to be a protective mechanism against gastric acid exposure. In addition, a previous study in rabbit esophageal tissues has shown that luminal acid at pH 1.6 triggers an increase of the Isc (15). Up to date, there are limited reports characterizing the

527 acid-induced electrogenic ion fluxes in esophageal stratified squamous epithelium. A number of ion transporters have been identified so far on the esophageal epithelium. Basolateral Na+/H+ exchanger (NHE) and anion exchanger, e.g. Cl/HCO3- antiport, are responsible for acid extrusion for recovery from low intracellular pH in rabbit and human primary esophageal epithelial cell culture (16 - 20). In the intestinal epithelium, cystic fibrosis transmembrane conductance regulator (CFTR) channels were known to mediate electrogenic Cl- as well as HCO3- secretion (21 - 24). It is unknown whether CFTR channel mediates ion fluxes in the esophageal epithelium. The sensory afferent innervation in the esophagus includes both divisions of the autonomic nervous system (25, 26). Both vagal and spinal afferents in the esophageal mucosa respond to pH and chemicals, and their hypersensitivity was incriminated in augmented transmission of pain perception in GERD (26). Capsaicin-sensitive nerve endings were identified within the stratified epithelial layer in mammalian esophagus as well as in human esophageal mucosal biopsies (27 - 30). Their expression was up-regulated in inflamed and acid-exposed tissues, which may trigger symptoms of burning pain (29, 30). In the gastroduodenum, luminal acidification augments bicarbonate ion secretion via both capsaicin TRPV1-dependent and -independent afferent pathway (8, 31 - 35). Whether capsaicin-dependent nerve pathway is involved in the mechanism of acid-induced ion fluxes in esophageal epithelia remains unclear. The aim of this study was to investigate the mechanism of enhanced ion fluxes in response to luminal hydrochloric acid challenge in esophageal epithelium using an ex vivo rat model. The type of ions and potential transporters involved were examined by ion replacement experiments and pharmacological blockage studies. The role of capsaicin-sensitive afferent and muscarinic nerve pathways in esophageal acid sensing and ion fluxes was also elucidated. MATERIALS AND METHODS

Animals Male Wistar rats (280 - 350 g) obtained from the Animal Center of the National Taiwan University were used for the study. Animals were raised in a temperature-controlled room (20 ± 2°C) with 12-hr light-dark cycles, and fed with regular rat show and water. Animal experiments were approved and monitored by Institutional Animal Care and Use Committee (IACUC), National Taiwan University College of Medicine and College of Public Health, Taiwan. The animals were fasted overnight with free access to water the day before the experiments. Rats were anesthetized with intraperitoneal administration of pentobarbital (50 mg/kg, i.p.), and thoracotomy and laparotomy were performed to expose the full length of the esophagus and the intestine. The lower (intra-abdominal) and middle (intra-thoracic, beneath the level of aortic arch and above diaphragm) portions of the esophagus, and in some experiments, the colonic segments were dissected. Tissues were immediately placed in prewarmed and oxygenated Krebs buffer (pH

528 7.35 ± 0.02) for Ussing Chamber studies (see below). The whole procedure from dissection of tissues to mounting on Ussing Chambers was completed within 30 min.

Human surgical specimens Esophageal surgical specimens were obtained from six adult subjects, ages 18 - 75 years old, three undergoing gastrectomy for gastric cardiac cancer and three undergoing esophagectomy for esophageal cancer. Tissues were placed in ice-cold, oxygenated Krebs buffer (pH 7.35 ± 0.02) for transportation and then mounted on Ussing Chambers (see below). The whole course from dissection to mounting was completed within 30 min. The esophageal specimens from non-tumor parts consists the mucosa and submucosal regions with intact lining of squamaous epithelium, which were histologically verified. This protocol was approved by the Internal Review Board for Human Subjects at the National Taiwan University College of Medicine and College of Public Health, Taipei, Taiwan, and all subjects provided written informed consent.

Ussing Chamber studies The rat esophageal segments were excised longitudinally to expose the luminal and serosal sides, and tissues of full thickness were mounted on Ussing Chambers (World Precision Instruments, Sarasota, FL, USA). In some experiments, the external muscle layers of the rat esophageal and colonic segments were stripped off, leaving the mucosa intact, and mounted on Ussing Chambers. An area of 0.75 cm2 of esophagus was exposed to 5 mL of prewarmed Krebs buffer bubbling with 95% O2 and 5% CO2 at 37°C. Krebs buffer contained 115 mM NaCl, 8 mM KCl, 1.25 mM CaCl2, 1.2 mM MgCl2, 2 mM KH2PO4, and 25 mM NaHCO3, pH 7.35 ± 0.02. The serosal buffer contains 10 mM of glucose as an energy source and osmotically balanced with 10 mM of mannitol in the luminal buffer (final solution 290 mOsm/Kg H2O). All of the chemicals in the study were purchased from Sigma Chemicals, St Louis, Missouri, USA, unless otherwise noted. The tissue was clamped at 0 V using a voltage clamp instrument (WPI). The short-circuit current (Isc, in microamperes) was recorded continuously, and PD measurements (in mV) were taken in 5 min intervals during the experiments. Tissues were allowed to equilibrate and the basal Isc and PD values were determined. Following equilibration, esophageal tissues were luminally exposed to acid at pH 1.6 by titration with 10 N HCl (final concentration of 60 mM) and the changes of the Isc were measured for 30 min post challenge. In some experiments, to examine the role of chloride gradient in the stimulation, tissues were luminally exposed to HCl at pH 1.6 and balanced with the same amount of chloride ions by adding choline chloride to the serosal buffer. In other settings, esophageal tissues were luminally exposed to acid at pH 1.6 by titration with H2SO4 to verify the role of hydrogen ions in the stimulation. To control for the osmolarity changes induced by exposure to luminal HCl, equimolar concentration of choline chloride was added in place of HCl in the luminal buffer and the Isc was measured for 30 min post challenge. The osmolarity of the luminal Krebs buffer containing either 60 mM of HCl or choline chloride was measured by an osmometer and the values are 378 and 390 mOsm/KgH2O, respectively. Moreover, to evaluate the involvement of ion transporters and nerve pathway in acid-induced modification of Isc, tissues were pretreated with various agonists and inhibitors prior to HCl challenge for 30 min (see below).

Measurement of the ion concentrations in luminal and serosal buffers The transepithelial transport of chloride, bicarbonate or sodium ions in normal esophageal tissues was assessed. The luminal and serosal Krebs buffer on the Ussing Chambers were collected after incubating with the esophageal tissues for one hour, and their ion concentrations were measured by using blood gas analyzers. The concentrations of Na+ and K+ ions were measured by

529 TBA-120FR autoanalyzer, Toshiba, Tokyo, Japan; HCO3- ion concentration was measured by Radiometer ABL520, Copenhagen, Denmark. The direction of transepithelial ion fluxes in esophageal tissues was determined by subtracting the ion concentrations in the luminal buffer to those in the serosal buffer after one hour of incubation.

Ion substitution experiments The dependency of different ions in the phenomenon of acid-induced changes of Isc was examined by bathing esophageal tissues with Krebs buffer deficient in chloride, bicarbonate or sodium ions. The Cl--free, HCO3--free, and Na+-free solutions were prepared following the formula described in previous references (7, 36, 37). These specific ion-free buffers were used instead of Krebs buffer in Ussing Chamber studies for the measurement of Isc. The serosal buffer contains 10 mM glucose, and the luminal buffer contains 10 mM mannitol. Chloride-free solution with equimolar substitution of acetate ions were adjusted to pH 7.35 ± 0.02 with sulfuric acid. The ionic composition (in mM) of Cl--free buffer was 115 Na(C2H3O2), 8 K(C2H3O2), 1.25 Ca(C2H3O2)2, 1.2 Mg(C2H3O2)2, 2 KH2PO4, 25 NaHCO3, and 10 glucose or mannitol (final solution 290 mOsm/Kg H2O). For bicarbonate-free buffer, sodium N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonate (HEPES sodium salt) was used to replace HCO3-, and the buffer contains 1 mM acetazolamide (a cell-permeable inhibitor of carbonic anhydrase) and was gassed with 100% O2. Carbonic anhydrase is an enzyme that catalyzes the reversible reaction CO2+ H2O ↔ HCO3- + H+ and can therefore generate de novo HCO3- for transport across the cell membrane. HCO3--free buffer was composed of (in mM) 115 NaCl, 8 KCl, 1.25 CaCl2, 1.2 MgCl2, 2 KH2PO4, 25 HEPES sodium salt, 10 glucose or mannitol, pH 7.35 ± 0.02 (final solution 300 mOsm/Kg H2O). The sodium ions were replaced with choline in Na+-free solution. The ionic composition (in mM) of Na+-free buffer was 115 choline chloride, 8 KCl, 1.25 CaCl2, 1.2 MgCl2, 2 KH2PO4, 25 choline bicarbonate, 10 glucose or mannitol, pH 7.35 ± 0.02 (final solution 290 mOsm/Kg H2O). Tissues were exposed to these buffers and challenged with HCl at pH = 1.6 for the measurement of Isc as described.

Agonists and inhibitors Esophageal tissues mounted on Ussing Chambers were luminally or serosally treated with diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS, an inhibitor of anion transporters) (18), or 5(N-ethyl-N-isopropyl)-amiloride (EIPA, a selective blocker of Na+/H+ antiport) at varying concentrations for 30 min prior to HCl challenge, and the Isc was measured for 30 min post challenge. Other pharmacological reagents were used to investigate the role of CFTR channel in the mechanism of basal and acid-induced Isc. These studies include serosal addition of forskolin (an agonist for CFTR by stimulating cAMP-mediated chloride secretion) at 20 and 50 µM, and luminal addition of CFTRinh-172 (a specific inhibitor of CFTR) at 10 and 50 µM for 30 min prior to acid challenge. CFTRinh-172 is a cell-permeable inhibitor of CFTR that rapidly (within 2 min) reversed the increase of transepithelial ion fluxes induced by cAMP agonists in cell cultures and rodent intestinal tissues (38, 39). Therefore, in another set of experiment, escalating doses of CFTRinh-172 (1, 10 and 50 µM) was added to the luminal surface sequentially at 10 min intervals after acid exposure. Furthermore, serosal addition of carbachol at 10, 30, and 100 µM was used to test the involvement of the muscarinic receptor (21, 40, 41). To verify the involvement of capsaicinsenstivie afferent pathways, varying doses of capsaicin (0.03, 0.3, and 6 mg/ml) were added to the luminal or serosal chamber for 30 min and Isc was recorded. Some tissues were luminally pretreated with capsazepine (capsaicin sensitive TRPV-1 receptor antagonist) at 1 mM prior to acid challenge for the examination of Isc change (8). All reagents were purchased from Sigma Co. The functional

530 activities of these reagents, including forskolin, carbachol, and CFTRinh-172 were previously verified using muscle-stripped rat colonic tissues.

In vivo functional ablation of capsaicin-sensitive afferent nerve Chemical ablation of capsaicin-sensitive afferent nerve was achieved by subcutaneous injection of capsaicin (125 mg/kg for each rat in doses divided to 25 mg/kg in day 1, 50 mg/kg in day 2, and 50 mg/kg in day 3) (42, 43). The optimal effect of denervation was verified with a negative eye test by instillation of 0.2 mg/ml capsaicin onto the rat cornea. A positive eye test was observed in control rats with frequent wiping movements after instillation (42). The esophageal tissues in capsaicin-denervated rats were isolated for Ussing Chamber studies as described.

Statistics All data were presented as means ± SE. Statistical significance was tested by one way-ANOVA and Student-Newman-Keuls Methods. A P value < 0.05 was considered statistically significant. RESULTS

Rat and human esophageal tissues demonstrated transepithelial ion fluxes and lumen-negative potential differences The basal Isc of the middle and lower esophageal segments in Wistar rats were 5.03 ± 1.93 and 4.52 ± 0.40 µA/cm2 in the full thickness esophagus (Table 1); 0.44 ± 0.07 and 0.24 ± 0.08 µA/cm2 in the muscle-stripped esophagus (Table 2), respectively. Lumen-negative PD was demonstrated in the rat esophagus. PD values of -0.54 ± 0.16 mV and -0.66 ± 0.14 mV were seen in the middle and lower segments of full thickness esophagus, respectively (Table 1). Muscle stripped tissues displayed lesser PD of -0.11 ± 0.01 mV for the middle and -0.04 ± 0.01 mV for the lower esophagus (Table 2). Moreover, an Isc value of 5.91 ± 0.88 µA/cm2 and transmural PD of -0.98 ± 0.23 mV were recorded in human esophageal specimens (Table 3). The net transepithelial transport of ions were calculated by measuring the ion concentrations in the luminal and serosal buffer in the Ussing Chambers collected Table 1 The Isc and PD values of full thickness rat esophageal tissues luminally exposed to HCl. The middle and lower segments of the esophagus were either untreated (Control) or HCl challenged (Acid) for 15 min.

Esophageal segment

Rat (Middle)

Rat (Lower)

Control

Acid

Control

Acid

Isc (ȝA/cm )

5.03 ± 1.93

37.06 ± 3.18*

4.52 ± 0.40

28.79 ± 6.76*

PD (mV)

-0.54 ± 0.16

-3.01 ± 0.10*

-0.66 ± 0.14

-2.90 ± 0.53*

2

The Isc (short-circuit current) and PD (transepithelial potential difference) values are expressed as means ± SE; n=5/ per group. *P < 0.05, compared to their respective controls.

531 one hour after rat tissues were mounted. Results from the blood gas analyzers demonstrated that net absorption of Na+ and Cl-, and secretion of HCO3- and K+ occur in normal rat esophageal tissues (Table 4). Luminal challenge of hydrochloric acid induced a rapid increase of Isc in rat and human esophagus HCl luminal challenge at pH 1.6 stimulated a rapid 6- to 7-fold and 36- to 45fold increase of the Isc and the lumen-negative PD values in both middle and Table 2 The Isc and PD values of muscle-stripped rat esophageal tissues luminally exposed to HCl. The middle and lower segment of the esophagus were either untreated (Control) or HCl challenged (Acid) for 15 min.

Rat (Middle)

Esophageal segment

Rat (Lower)

Control

Acid

Control

Acid

Isc (ȝA/cm )

0.44 r 0.07

16.05 r 3.55*

0.24 r 0.08

10.81 r 4.33*

PD (mV)

-0.11 r 0.01

-3.08 r 0.46*

-0.04 r 0.01

-2.37 r 0.45*

2

Values are expressed as means ± SE; n=4-7/ per group. *P < 0.05, compared to their respective controls. Table 3 The Isc and PD values of human esophageal tissues luminally exposed to HCl. The human esophageal tissues containing intact mucosa and parts of submucosal regions were either untreated (Control) or HCl challenged (Acid) for 15 min.

Human esophagus

Esophageal segment

Control

Acid

Isc (ȝA/cm2)

5.91 r 0.88

19.48 r 4.56*

PD (mV)

-0.98 r 0.23

-2.14 r 0.30*

Values are expressed as means ± SE; n=6-8/ per group. *P < 0.05, compared to their respective controls. Table 4 Measurement of the ion concentrations in the luminal and serosal Krebs buffer after incubating with normal rat esophageal tissues for one hour. mM

[Lumimal]

[Serosal]

[Luminal-serosal]

Na+

144.00 ± 1.52

150.25 ± 0.65

-6.25 ± 1.71

K+

19.80 ± 0.47

16.93 ± 0.30

2.88 ± 0.37

142.50 ± 1.54

146.88 ± 0.52

-4.38 ± 3.00

23.75 ± 0.63

21.04 ± 0.55

2.71 ± 0.93

-

Cl

-

HCO3

Values are expressed as means ± SE, n=8/group.

532 lower segments, respectively, of the full thickness rat esophagus (Table 1-2 and Fig. 1A). The Isc increased immediately upon acid exposure, peaked at 10 min and maintained a plateau for 30 min (Fig. 1A). Similarly, in human surgical

A

control middle acid middle control lower acid lower

50

∆ Isc Isc (µA/cm (uA/cm22)

40

*

*

*

*

*

30

FIGURE20 1(a)

*

*

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∆ Isc (uA/cm (µA/cm22)) ßµIsc

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* *

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Fig. 1 Luminal challenge of hydrochloric acid induced significant increases of Isc in the rat and human esophageal tissues. (A) Both the middle and lower segments of the esophagus showed rapid rise of the Isc upon acid challenge (acid) compared to full thickness tissues (control). n= 8/group. *P < 0.05, compared to controls. (B) Acid challenge also induced a significant increase of Isc in the human esophageal epithelium. Data was obtained from six human surgical specimens. * P < 0.05, compared to controls.

Table 5 The change of Isc after acid challenge for 15 min in esophageal tissues bathed in ion substituted buffers. Buffer

ǻIsc (ȝA/cm2)

Normal Krebs

19.56 ± 1.52

+

Na -free HCO3--free Cl--free

33.51 ± 7.65 4.30 ± 0.94* 46.53 ± 17.82

Values were represented as mean ± SE; n=8/group. *P < 0.05, compared to values of normal Krebs.

533 esophageal specimens, an acute rise of Isc (3.3-fold to baseline) and an increment of lumen-negative PD value were also recorded in response of acid stimulation (Fig. 1B). In some of our experiments extending over 30 min post HCl challenge, the plateau of the increased Isc and PD values sustained for at least an hour in both rat and human esophageal tissues (data not shown). The effects of HCl challenge in the increase of Isc were not abolished by serosal addition of equal molar concentrations of choline chloride in both the middle and lower esophagus (data not shown), suggesting that chloride gradient may not play a role in the stimulation. The substitution of HCl with H2SO4 for challenges at pH 1.6 did not alter the acid-induced rise of Isc in both esophageal segments (data not shown). These results showed that exposure to hydrogen, but not chloride ion is the main trigger for the phenomenon. In addition, to investigate whether acute change in the luminal solute osmolarity upon HCl addition may contribute to the Isc rise, esophageal tissues were luminally challenged with 60 mM of chloline chloride but failed to show increase of Isc. Furthermore, both middle and lower segments of esophagus showed similar responses to acid challenges, and therefore, only results of the middle segments will be presented in the following sections. Acid-induced increase of Isc was abolished in bicarbonate-free buffers To investigate the component of ion(s) responsible for the increase of Isc upon acid challenge, ion substitution experiments were performed. The acid-induced increase of Isc was significantly diminished in tissues bathed in buffers lacking HCO3- ions. A 78 % decrease of the esophageal Isc change was seen at 15 min after acid challenge in HCO3--free buffers compared to that of normal Krebs buffer (Table 5). The absence of bicarbonate ions in the bathing solutions were confirmed using a blood gas analyzer (data not shown). On the other hand, the rise of Isc at 15 min after HCl exposure was not inhibited in tissues bathed with Na+- or Cl-- free buffers (Table 5). Pretreatment with DIDS or EIPA decreased the change of Isc upon acid challenges The involvement of ion transporters in the mechanism was evaluated by pharmacological blockage studies. The pretreatment of DIDS at the luminal or serosal side at 50 µM significantly diminished the rise of Isc induced by HCl (Fig. 2). The acid-induced change of Isc was also significantly inhibited by pretreatment with EIPA (50µM) (Fig. 3). The inhibitory effects of DIDS and EIPA on acid-induced increase of Isc were dose-dependent in the range of 10500 µM and 0.05-50 µM, respectively. Role of CFTR in acid-induced esophageal ion fluxes In order to investigate the involvement of CFTR channels in the mechanism of acid-induced rise of Isc in the rat esophageal tissues, we used a cAMP agonist

534 (forskolin) and a specific CFTR inhibitor (CFTRinh-172) for the studies. In our preliminary experiments, rat colonic tissues were utilized to verify the pharmacological efficiency of the reagents. We confirmed that serosal addition of forskolin at 20 µM significantly stimulated rapid and sustained increases of the Isc for longer than 30 min (29.62 ± 7.36 µA/cm2 at 15 min) in the rat colon. The forskolin-induced change of Isc was completely abolished by pretreatment of the colonic tissues with luminal addition of CFTRinh-172 at 50 µM. In rat esophagus, neither serosal forskolin (20-50 µM) nor luminal CFTRinh172 (10-50 µM) had any effect on the basal Isc (data not shown). Luminal addition of CFTRinh-172 did not significantly alter the level of acid-induced

control acid DIDS(luminal)+acid DIDS(serosal)+acid

50

22

∆ Isc ßµ Isc (µA/cm (uA/cm ))

40

*

30

*#

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*#

*

*

*

*#

*#

*#

*#

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control acid EIPA (luminal)+acid EIPA (serosal)+acid

50

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Fig. 2 Pretreatment with an inhibitor for anion transporters, DIDS, reduced the increase of Isc triggered by acid challenge in esophageal tissues. The acidinduced change of Isc was reduced by luminal or serosal addition of DIDS at 0.5 mM. * P < 0.05, compared to controls; # P < 0.05, compared to acid challenge. n=8/group.

*

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#

#

#

#

#

#

#

#

-10 0

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Fig. 3 Pretreatment with a selective NHE inhibitor, EIPA, abolished the acid-increased Isc in rat esophageal tissues. The acid-induced rise of Isc was diminished by either luminal or serosal addition of EIPA at 50 µM. * P < 0.05 compared to controls; # P < 0.05, compared to acid challenge. n=8/group.

535 increase of Isc. The rise of Isc was not attenuated by treatment of escalating doses of CFTRinh-172 after acid exposure at 10 min interval for a total length of 30 min (Fig. 4A), nor did pretreatment with the inhibitor 30 min prior to acid challenge had any effect (Fig. 4B). Involvement of nerve pathways in acid-induced rise of Isc To investigate the role of muscaric receptor in the mechanism, rat esophageal tissues were serosally treated with carbachol at 10-100 µM and the Isc was measured. The Isc recording after addition of carbachol (100 µM) for 30 min (1.31 ± 0.53 µA/cm2) was not different from that of baseline (1.64 ± 0.41 µA/cm2). The pharmacological agent used is functionally active since serosal addition of carbachol (30 µM) induced a rapid and transient increase of Isc (22.85 ± 3.80 µA/cm2) in rat colonic tissues. Capsaicin-sensitive afferent nerve pathways have been implicated in chemosensing of luminal acid and pain perception in the esophagus, stomach and duodenum (8, 31-34, 44). Therefore, we examined the role of capsaicin TRPV1 pathways in mediating acid-sensing and ion fluxes in the rat esophagus. Our results showed that direct challenge of varying doses of capsaicin (0.03, 0.3, and 6 mg/ml) onto the luminal or serosal sides of esophageal tissues failed to induce any change in the basal Isc of esophageal epithelium (Fig. 5A).

A

B

CFTRinh-172 alone CFTRinh-172 0 uM+acid CFTRinh-172 10 uM+acid CFTRinh-172 50 uM+acid

∆ Isc (uA/cm (µA/cm22) ßµIsc

60

40

*

*

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* *

0

0

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*

*

* *

* *

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time (min)

* *

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Fig. 4 CFTR is not involved in the mechanism of acid-induced rise of Isc. (A) Representative Isc reading of esophageal tissues treated with escalating doses of luminal CFTRinh-172 (1, 10, and 50 µM at 10 min intervals) after acid challenge. Luminal CFTRinh172 did not significantly attenuate acid-induced Isc. Three individual experiments were performed. (B) Treatment with CFTRinh-172 neither changed the basal Isc nor reduced the acidinduced rise of Isc. Luminal addition of CFTRinh-172 at 50 µM had no effect on the basal Isc of rat esophageal tissues (CFTRinh-172 alone). Pretreatment with CFTRinh-172 at 10 and 50 µM for 30 min did not reduce the acid-induced increase of Isc. n=8/group.

536 A

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∆ Isc Isc (uA/cm (µA/cm22))

30

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* 20

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-10

control

B

acid

capsaicin capsaicin CPZ (luminal) (serosal) + acid

50 control acid capsaicin (s.c.)+acid

22 ßµIsc )) ∆ Isc (uA/cm (µA/cm

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Fig. 5 Acid-induced increase of Isc is not mediated via capsaicin sensitive pathways. (A) Esophageal tissues were challenged with luminal or serosal addition of varying doses of capsaicin (0.03, 0.3, and 6 mg/ml) for 30 min. None of the dosage induces change in basal Isc. Data shown here represents the Isc values of rat esophageal tissues 15 min post-challenge with 6 mg/ml of capsaicin. Moreover, pretreatment with luminal capsazepine (CPZ, a TRPV-1 antagonist) at 1 mM failed to diminish the acidinduced Isc rise. (B) Functional ablation of capsaicin-sensitive afferent nerves in vivo did not alter the acid-induced Isc response in the esophagus. Animals were injected (s.c.) with capsaicin and a negative eye test was confirmed prior to ex vivo acid challenge studies. * P < 0.05, compared to controls (n=8/group).

Pretreatment with luminal capsazepine (a TRPV-1 antagonist) failed to diminish the acid-induced Isc rise (Fig. 5A). Moreover, in vivo functional ablation of capsaicin-sensitive afferent nerve by subcutaneous injection of capsaicin to animals did not alter the response of acid-induced increase of Isc in the esophagus (Fig. 5B). The denervation of capsaicin-sensitive afferent pathways in rats was verified by the presence of a negative eye test with corneal instillation of capsaicin. DISCUSSION

In the present study, we demonstrated rapid increase of Isc and lumen-negative transmural PD in rodent esophageal tissues upon luminal hydrochloric acid challenges. The acid-induced rise of Isc was abolished when tissues were bathed in bicarbonate-free buffer. DIDS- and EIPA- sensitive transporters played

537 important roles, whereas CFTR, capsaicin-sensitive and muscarinic-dependent pathways were not involved in the mechanism. The presence of basal Isc and lumen-negative PD were demonstrated in the middle and lower esophagus associated with net absorption of Na+ and Cl-, and secretion of HCO3- and K+ in rats. These results are in keeping with reports of other species, e.g. human, rabbit and opossum, of which active mucosal-toserosal transport of Na+ ions is partly responsible for the electrogenic transepithelial fluxes in the esophageal tissues (45 - 48). The challenge of luminal acid at pH 1.6 rapidly stimulated a seven-fold increase of Isc and increment of the lumen-negative PD. The acid-induced changes were significantly attenuated when tissues were bathed in HCO3--free, but not in Cl- or Na+-free Krebs buffer. Our findings indicated that epithelial HCO3- export, but not Na+ inward fluxes, may play a predominant role in the mechanism of acid-induced rise of Isc in the rat esophagus. The involvement of ion transporters in acid-induced Isc was supported by the inhibitory effects of DIDS and EIPA shown in our study. DIDS is a wellestablished pharmacological blocker for several anion transporters, including Cl/HCO3- exchangers, and SLC26 anion exchanger family, as well as Na+/Cl- cotransporters (49, 50), but is insensitive to CFTR (24). The results from our pharmacological blocking and ion substitution experiments suggested that DIDSsensitive HCO3- ion export is involved in the mechanism of acid-induced ion fluxes. We further demonstrated that pretreatment with a selective inhibitor to Na+/H+ exchanger (EIPA) significantly attenuated the increase of Isc upon acid stimulation (Fig. 4). NHE-1 isoform which is present in rat, rabbit and human esophageal epithelia (16, 17, 20, 51) was known to protect against intracellular acidification by extruding H+ out of the cell in exchange for Na+ to maintain a neutral pHi (17). Our results also suggested that NHE is involved in the mechanism of acid-induced change in Isc, possibly via the regulation of pHi to maintain cell viability in rat esophageal epithelium. In the rodent intestine, CFTR channels mediate the cAMP-dependent electrogenic Cl- and HCO3- secretion, in which luminal acid stimulation further increased this response (21, 23, 52). In our rat esophageal model, forskolin and CFTRinh-172 had no effect on the basal Isc (Fig. 4B), nor did treatment of CFTRinh-172 diminished the acid-induced rise of Isc (Fig. 4A). These results implied that CFTR may not be responsible for the electrogenic HCO3- export in the rat esophagus. A potential role of the recently identified apical anion exchanger, SLC26a6, warrants further studies (49). The physical barrier of esophageal stratified squamous epithelium connected by tight junctions is partly involved in the defense mechanism against gastric acid (2 - 5). Previous reports in rabbit esophageal tissues have demonstrated that acid challenge induce progressive changes of the lumen-negative transmural PD to a more positive value close to zero, as well as decline of tissue resistance, both as early signs (20 min post-acid challenge) of epithelial damages associated with

538 microscopic observation of dilated intercellular spaces at 30 min post challenge (3, 15, 53). A recent paper from the same group demonstrated that luminal acid exposure triggers an acute rise of Isc in rabbit esophagus, and attributed this phenomenon to the hydrogen influx via increased shunt permeability of the stratified epithelium (15). Moreover, previous studies in rat gastric and rabbit duodenal tissues have also demonstrated reversal of PD from negative to more positive values upon exposure to gastric mucosal breakers, e.g. low pH and chemicals, as indicators of barrier defects (54, 55). However, in our esophageal study, the acid-induced rise of Isc occurs immediately within seconds and kept a plateau for at least one hour. The lumen-negative transmural PD values become more negative after acid exposure and without decline over the period of one hour. Neither reduction of tissue conductance nor drop of serosal pH were observed in rat esophageal tissues during the acid exposure period in parallel with the absence of histological damages, suggesting that 60 mM of HCl triggers transepithelial ion fluxes but did not induce epithelial barrier injuries in the rat esophagus. Although we cannot rule out traces of hydrogen influx that may leak into the serosal side of epithelial cells inducing lesions or partially modulating the Isc in some cases, our results indicated that bicarbonate ion fluxes, and DIDSand EIPA-sensitive ion transporters play major roles in the mechanism of acidstimulated increase of Isc in rat esophagus. It is noteworthy that the critical balance between protective transepithelial bicarbonate ion fluxes and detrimental epithelial barrier defects upon luminal acid challenge may be responsible for the maintenance of esophageal homeostasis that determines the severity of pathological lesions. The mechanism of mucosal acidification inducing bicarbonate secretion in rat stomach and duodenum has been extensively studied by the group of Takeuchi K. The acid-induced HCO3- response in gastroduodenal mucosa was mediated by prostaglandin E and nitric oxide, as well as bradykinin receptors (34, 35, 56). In our esophageal study, pretreatment with capsazepine (capsaicin sensitive TRPV1 receptor antagonist) did not decrease the acid-induced Isc rise (Fig. 5A). This result is similar to that of duodenum and stomach in which the bicarbonate secretive effect upon luminal acidification is independent of the capsazepinesensitive TRPV1 (34, 57). Furthermore, a report showed that depending on the challenge dose of acid, bicarbonate secretion in the rat stomach is mediated by either capsaicin-sensitive or -insensitive pathway (57). The HCl dosage used in our esophageal study (60 mM) was much lower than the reported concentration required to activate capsaicin-sensitive afferent pathway in the duodenum (57). In the rat duodenum, luminal application of capsaicin increased the secretion of bicarbonate in a dose-dependent manner (34). In contrast, stimulation with capsaicin concentration as high as 6 mg/ml did not trigger a measurable change in transepithelial ion fluxes in the esophagus (Fig. 5a), suggesting a discrepancy of capsaicin-sensitive afferent pathway in different areas of the digestive tract. In addition, carbachol at a high dose up to 100 µM did not augment the basal Isc in

539 rat esophagus. The aforementioned results suggested that capsaicin-sensitive and muscarinic-dependent neural pathways were not involved in the mechanism of electrogenic ion fluxes in the rat esophagus. Taken together, although capsaicinsensitive afferent nerves and TRPV-1 receptor are critical for the transmission of pain sensation associated with GERD (29, 30), our data indicates that this pathway is not involved in the acid-induced esophageal ion fluxes. Our findings showed that acute luminal acid challenge triggers rapid increase of ion transport across rat and human esophageal epithelium. The acidinduced electrogenic transepithelial ion fluxes associated with bicarbonate export were dependent on DIDS- and EIPA-sensitive ion transporters, whereas CFTR channel and capsaicin-sensitive nerve pathway were not involved in the mechanism. This prompt increment of electrogenic ion flux upon HCl exposure may protect the esophageal epithelium against gastric acid injury, and aberrant regulation of the esophageal epithelial ion transport may in part contribute to the pathogenesis of GERD. Acknowledgements: The study was supported by grants from National Taiwan University Hospital (NTUHM24) and National Science Council (NSC95-2314-B-002-100), Taipei, Taiwan. REFERENCES 1. Orlando RC. The pathogenesis of gastroesophageal reflux disease: the relationship between epithelial defense, dysmotility, and acid exposure. Am J Gastroenterol 1997; 92(4 Suppl): 3S-5S. 2. Orlando RC. Pathophysiology of gastroesophageal refulx disease: offensive factors and tissue resistance. In: Orlando RC, editor. Gastroesophageal reflux disease. New York, NY: Marcel Dekker, Inc. 2000, pp. 165-192. 3. Carney CN, Orlando RC, Powell DW, Dotson MM. Morphologic alterations in early acidinduced epithelial injury of the rabbit esophagus. Lab Invest 1981; 45: 198-208. 4. Orlando RC. Mechanisms of acid damage to oesophageal epithelium: role of the paracellular pathway. J Intern Med Suppl 1990; 732: 53-57. 5. Orlando RC. Esophageal epithelial defense against acid injury. J Clin Gastroenterol 1991; 13 Suppl 2: S1-S5. 6. Abdulnour-Nakhoul S, Nakhoul NL, Orlando RC. Lumen-to-surface pH gradients in opossum and rabbit esophagi: role of submucosal glands. Am J Physiol Gastrointest Liver Physiol 2000; 278: G113-G120. 7. Abdulnour-Nakhoul S, Nakhoul NL, Wheeler SA, Wang P, Swenson ER, Orlando RC. HCO3secretion in the esophageal submucosal glands. Am J Physiol Gastrointest Liver Physiol 2005; 288: G736-G744. 8. Akiba Y, Guth PH, Engel E, Nastaskin I, Kaunitz JD. Acid-sensing pathways of rat duodenum. Am J Physiol 1999; 277(2 Pt 1): G268-G274. 9. Allen A, Flemstrom G. Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin. Am J Physiol Cell Physiol 2005; 288: C1-C19. 10. Hamilton BH, Orlando RC. In vivo alkaline secretion by mammalian esophagus. Gastroenterology 1989; 97: 640-648. 11. Meyers RL, Orlando RC. In vivo bicarbonate secretion by human esophagus. Gastroenterology 1992; 103: 1174-1178.

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542 50. Xu J, Henriksnas J, Barone S, et al. SLC26A9 is expressed in gastric surface epithelial cells, mediates Cl-/HCO3- exchange, and is inhibited by NH4+. Am J Physiol Cell Physiol 2005; 289: C493-C505. 51. Shallat S, Schmidt L, Reaka A, et al. NHE-1 isoform of the Na+/H+ antiport is expressed in the rat and rabbit esophagus. Gastroenterology 1995; 109: 1421-1428. 52. Clarke LL, Harline MC. Dual role of CFTR in cAMP-stimulated HCO3- secretion across murine duodenum. Am J Physiol 1998; 274(4 Pt 1): G718-G726. 53. Orlando RC, Powell DW, Carney CN. Pathophysiology of acute acid injury in rabbit esophageal epithelium. J Clin Invest 1981; 68: 286-293. 54. Vattay P, Feil W, Klimesch S, Wenzl E, Starlinger M, Schiessel R. Acid stimulated alkaline secretion in the rabbit duodenum is passive and correlates with mucosal damage. Gut 1988; 29: 284-290. 55. Takeuchi K, Ueshima K, Matsumoto J, Okabe S. Role of capsaicin-sensitive sensory nerves in acid-induced bicarbonate secretion in rat stomach. Dig Dis Sci 1992; 37: 737-743. 56. Aoi M, Aihara E, Nakashima M, Takeuchi K. Participation of prostaglandin E receptor EP4 subtype in duodenal bicarbonate secretion in rats. Am J Physiol Gastrointest Liver Physiol 2004; 287: G96-G103. 57. Aihara E, Hayashi M, Sasaki Y, Takeuchi K. Gastric HCO3- secretion induced by mucosal acidification: different mechanisms depending on acid concentration. Inflammopharmacology 2005; 13: 179-190. R e c e i v e d : January 7, 2008 A c c e p t e d : July 30, 2008 Author’s address: Linda C.H. Yu, Ph.D., Department of Physiology, National Taiwan University College of Medicine, No.1, Section 1, Jen-Ai Road, Taipei 100, Taiwan. Tel: 886-2-23123456 ext. 88237, Fax: 886-2-23964350; e-mail: [email protected]