Endothelins promote egg albumin-induced intestinal anaphylaxis in rats

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Endothelins Promote Egg Albumin–Induced Intestinal Anaphylaxis in Rats TAKEHARU SHIGEMATSU,* SOICHIRO MIURA,‡ MASAHIKO HIROKAWA,* RYOTA HOKARI,* HAJIME HIGUCHI,* YOSHIKAZU TSUZUKI,* HIROYUKI KIMURA,* RURI C. NAKATSUMI,* HIROSHI SERIZAWA,* HIDETSUGU SAITO,* and HIROMASA ISHII* *Department of Internal Medicine, School of Medicine, Keio University, Tokyo; and ‡Second Department of Internal Medicine, National Defense Medical College, Saitama, Japan

Background & Aims: The basic mechanisms of food allergies are still unknown. The aims of this study were to investigate whether endothelins (ETs) in the intestinal mucosa are involved in the pathogenesis of intestinal anaphylaxis. Methods: Sprague–Dawley rats were sensitized to chicken egg albumin (EA) by intraperitoneal injection. Fourteen days after sensitization, EA was administered in the jejunal segments to induce intestinal anaphylaxis. Net water outflux and histamine release into loops and serum concentrations of rat mast cell protease II (RMCP-II) were determined. ET-1 and ET-3 concentrations in the jejunal mucosa were determined, and expression of the corresponding messenger RNAs was examined by competitive polymerase chain reaction. Results: In sensitized animals, challenge with intraluminal antigen caused a significant increase in net water outflux and histamine release together with an elevation of serum RMCP-II concentrations. Mucosal concentrations of ET-1 and ET-3 and expression of their messenger RNAs were significantly increased in sensitized animals after EA challenge. Treatment with an ETA-receptor antagonist, but not an ETB-receptor antagonist, attenuated the increase in net water outflux, histamine release, and serum RMCP-II concentrations in rats with EA-induced intestinal anaphylaxis. Conclusions: Release of ETs in the intestinal mucosa increased in sensitized animals after EA challenge. ETs may play a significant role in the development of intestinal anaphylaxis via an ETA receptor.

he clinical manifestations of allergic reactions from food intolerance may be localized to the gut and thus may include intestinal discomfort, nausea, vomiting, and diarrhea. Most of these reactions seem to be related to type I immediate immunoglobulin (Ig) E–dependent hypersensitivity involving degranulation of mast cells, which is associated with the release of mediators such as histamine, serotonin, and eicosanoids.1,2 These mediators alter the permeability, intestinal transport, and motility of the digestive tract. Although many pathophysiological

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changes in the intestinal mucosa have been reported using a variety of animal models of intestinal anaphylaxis,3,4 the underlying mechanisms that give rise to the development of intestinal anaphylaxis are not fully understood. Endothelin (ET), a 21–amino acid peptide, is a potent endothelium-derived contracting factor.5 Currently, three ET isopeptides (ET-1, ET-2, and ET-3) and two distinct ET receptors with different affinities for ET-1 and ET-3 have been identified. ET-1 has a greater affinity than ET-3 for the ETA receptor, whereas these two peptides show similar binding to the ETB receptor. These receptors, originally identified in vascular tissues, have also been found in many nonvascular tissues, including heart, kidney, brain, and liver.6,7 In keeping with the wide distribution of ET receptors, ETs elicit a wide variety of biological effects, including positive inotropy and chronotropy, inhibition of sodium reabsorption, neuroendocrine functions,6,8 and a growth-promoting activity toward various cell types.9 Several recent reports have stressed that the gastrointestinal tract is a major target of ETs. We have shown that ET-1 plays a significant role in endotoxin-induced microcirculatory damage in the rat small intestine, suggesting a potential role for ET-1 in intestinal mucosal injury.10 However, there is little information about the exact source and target of ETs in the intestinal mucosa under physiological and pathological conditions. Recently, increased expression of ET in airway epithelium and markedly elevated ET concentrations in bronchoalveolar lavage fluid have been detected in patients with symptomatic asthma.11,12 Increased plasma concentrations of ET have also been observed during anaphylacAbbreviations used in this paper: BSA, bovine serum albumin; EA, egg albumin; ET, endothelin; FCS, fetal calf serum; PCR, polymerase chain reaction; RBL, rat basophilic leukemia; RMCP-II, rat mast cell protease II; RT, reverse transcription. r 1998 by the American Gastroenterological Association 0016-5085/98/$3.00

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tic shock in guinea pigs.13 These observations indicate that ET may be synthesized and released during type I (anaphylactic) immune reactions. The objective of this study was to determine temporal changes in ET-1 and ET-3 concentrations in rat intestinal mucosa and their possible relationship to the pathogenesis of anaphylaxis-induced intestinal response. These issues were addressed using a specific ETA-receptor antagonist, BQ-123,14 an ETA/ETB–receptor antagonist, bosentan,15 and an ETB-receptor antagonist, BQ-788.16 Our results show the possible participation of ET in anaphylaxis-induced intestinal disturbances.

Materials and Methods

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Histamine concentrations in the effluent collected were measured by a radioimmunoassay using monoclonal antibodies (Immunotech, Marseilles, France).22

Measurement of Rat Mast Cell Protease II Concentrations Samples (500 µL) were collected from portal venous blood, and serum concentrations of the specific mucosal mast cell protease enzyme rat mast cell protease II (RMCP-II) were assayed using an enzyme-linked immunosorbent assay kit (Moredun Animal Health Ltd., Edinburgh, Scotland). RMCP-II concentrations were also determined in the culture supernatant of rat basophilic leukemia (RBL)-2H3 cells after addition of ET-1 to the medium.

Animal Model Sprague–Dawley rats weighing 100–150 g were sensitized by intraperitoneal injection of 10 µg of chicken egg albumin (EA) (grade V; Sigma Chemical Co., St. Louis, MO) and 10 mg of aluminum hydroxide (1.0 mL of alum solution) as an adjuvant.17 Control rats were injected with material prepared in the same manner without antigen. Specific IgE titers were determined by passive cutaneous anaphylaxis.18 Sensitized animals had serum titers of 1:$64, whereas control animals had no detectable anti-EA antibody. Heat treatment of serum from sensitized rats abolished reactivity caused by passive cutaneous anaphylaxis. Animals were studied on day 14 after the sensitization after fasting for 24 hours.

Intestinal Perfusion Animals were anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg) and surgically prepared for intestinal perfusion.19,20 A 20-cm segment of proximal jejunum, starting 10 cm distal to the ligament of Treitz, was cannulated at the proximal and distal ends and perfused in situ at 0.15 mL/min. The perfusate contained 140 mmol/L Na1, 10 mmol/L K1, 120 mmol/L Cl2, 30 mmol/L HCO32, and 5 g/L of unlabeled polyethylene glycol 4000 plus 10 µCi/L of [14C]polyethylene glycol 4000 as the nonabsorbable marker, with an osmolality of 300 mOsm and a pH of 7.4 at 37°C. Samples of perfusate were collected over consecutive 15-minute periods from the distal cannula into chilled containers (4°C). For a 1-hour equilibration period, antigen-free basal solution was perfused. The perfused intestinal loop was either challenged by adding 10 µg/mL of EA to the perfusate or sham challenged by adding 10 µg/mL of bovine serum albumin (BSA; Sigma fraction V) to the perfusate. Net H2O flux was calculated using standard formulas.21 14C radioactivity was measured with a Beckman LS-9800 liquid scintillation system.

Measurement of Histamine Release Animals prepared as described above underwent infusions with buffer solutions at a constant rate (9 mL/h).

ET Determination ET concentrations in tissue homogenates and plasma were determined by an enzyme immunoassay kit using a monoclonal antibody against ET-1 (Takeda Pharmaceutical Co. Ltd., Osaka, Japan) or a polyclonal antibody against ET-3 (IBL Co., Gunma, Japan).23 Samples of venous blood (500 µL) were collected in vacuum tubes and centrifuged at 1000g for 10 minutes. The plasma obtained was stored at 270°C in polypropylene tubes containing aprotinin and ethylenediaminetetraacetic acid (EDTA) at final concentrations of 300 KIU/mL and 2 mg/mL, respectively. The plasma was extracted using Sep-pak C-18 cartridges (Waters Assoc., Milford, MA), and the extracts were dissolved in 250 µL of assay buffer (0.02 mol/L phosphate buffer, pH 7, containing 10% Block Ace, 0.4 mol/L NaCl, and 2 mmol/L EDTA). For measurement of ET concentrations in intestinal mucosa, the small intestine was removed and the mucosal layers were separated, immediately immersed in liquid nitrogen, and stored at 280°C. The mucosa was homogenized at 4°C in 10 volumes of 1 mol/L acetic acid containing pepstatin (10 µg/mL) for 1 minute and then boiled in a water bath for 10 minutes to inactivate proteases. The samples were centrifuged at 10,000g for 30 minutes at 4°C. The supernatant was applied to Sep-pak C-18 cartridges and eluted with 4 mL of 4% acetic acid/86% ethanol. The eluted samples were then dried under nitrogen and dissolved in 250 µL of the assay buffer. Samples in 100-µL aliquots were then added to each well of a 96-well microplate coated with an antibody directed against ET-1 or ET-3 and incubated at 4°C for 24 hours. After washing with phosphate-buffered saline (PBS), the plate was reacted with 100 µL of anti-ET (15–21) Fab8–horseradish peroxidase diluted 1:400 in incubation buffer (0.02 mol/L phosphate buffer, pH 7, containing 1% BSA, 0.4 mol/L NaCl, and 2 mmol/L EDTA) at 4°C for 24 hours. After three washes with PBS, the activity of bound enzyme was measured with an enzyme-linked immunosorbent assay reader (NJ-2000; Inter Med, Tokyo, Japan) using o-phenylenediamine as a chromogen. Concentrations of protein in the mucosal sample were measured by the method of Lowry et al.24

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RNA Extraction and Polymerase Chain Reaction Amplification Total RNA was extracted from the intestinal mucosa by the acid guanidinium thiocyanate–phenol chloroform extraction method. In the case of RBL-2H3 cells, total RNA was isolated from cells by RNAzol (Biotcx, Houston, TX). Cells were lysed with 1.0 mL of RNAzol per dish. The concentration of the extracted RNA was calculated by measuring the optical density at 260 nm. The ratio of the optical density at 260 nm to that at 280 nm was always higher than 1.9. The integrity of RNA was assessed by the 28S and 18S RNA bands on agarose gel electrophoresis. Aliquots containing 5 µg each of RNA were reversetranscribed using the reverse transcription (RT)–polymerase chain reaction (PCR) kit from Stratagene (La Jolla, CA). Briefly, 5 µg of RNA in 38 µL of diethyl pyrocarbonate–treated water were mixed with 0.3 µg of oligodeoxythymidine, heated at 65°C for 5 minutes, and then cooled slowly at room temperature. The following reagents were added to the tubes: 5 µL of 103 synthesis buffer (final concentration in mmol/L, 10 Tris-HCl, pH 8.3, 50 KCl, 1.5 MgCl2), 1 µL of RNase Block Ribonuclease Inhibitor (40 U/µL), 2 µL of 100 mmol/L deoxynucleoside triphosphates (dNTPs), and 1 µL of 50 U/µL Moloney murine leukemia virus reverse transcriptase. The reaction mixture was incubated for 1 hour at 37°C, and the reaction was terminated by incubating the tube at 90°C for 5 minutes and on ice for 10 minutes. The tube was stored at 280°C for use in the PCR, which was performed using the Takara Taq kit (recombinant Taq DNA polymerase; Takara Biochemicals, Tokyo), with rat-specific primers prepared on a DNA synthesizer (Sawady Technology, Tokyo). The ET-1 antisense primer, bases 675–699, was 58AAGATCCCAGCCAGCATGGAGAGCG-38; the ET-1 sense primer, bases 157–181, was 58-CGTTGCTCCTGCTCCTCCTTGATGG-38; the ET-3 antisense primer, bases 479–499, was 58-GCTGGTGGACTTTATCTGTCC-38; and the ET-3 sense primer, bases 23–42, was 58-TTCTCGGGCTCACAGTGACC38. The complementary DNA (cDNA) amplification products were predicted to be 477 base pairs (bp) in length for ET-3 and 543 bp for ET-1. To carry out the PCR, 2 µL of RT products were added to the PCR master mix, containing 103 PCR reaction buffer, which was diluted to have the following final composition: 10 mmol/L Tris–HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 2.5 U of recombinant Taq DNA polymerase, 50 pmol each of primers, and 200 µmol/L of dNTPs. The tubes were placed in a Programmed Tempcontrol System (Applied Biosystems Japan Co., Tokyo). The PCR products were size-fractionated by agarose gel electrophoresis. After electrophoresis and ethidium bromide staining, DNA bands were visualized with an ultraviolet transilluminator. For ET receptors, PCR was performed with the following specific primers: the ETA-receptor antisense primer, bases 384–403, was 58-GGAGATCAATGACCACGTAG-38; the ETA-receptor sense primer, bases 215–5, was 58-CAGATCCACATTAAGATGGG-38; the ETB-receptor antisense primer,

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bases 967–986, was 58-AGGACCAGGCAGAATACTGT-38; the ETB-receptor sense primer, bases 918–937, was 58GCAGGATTGCCTTGAATGACC-38. The cDNA amplification products were predicted to be 418 bp in length for the ETA receptor and 900 bp for the ETB receptor. Competitive PCR was performed using the PCR MIMIC Construction Kit (Clontech Laboratories Inc., Palo Alto, CA). Nonhomologous internal standard DNA fragments called PCR MIMICs were constructed for use in competitive PCR amplification for quantification of target messenger RNA (mRNA) levels. PCR MIMIC consists of a heterologous DNA fragment with primer templates that are recognized by a pair of gene-specific primers. In this study, we designed the PCR MIMIC so that the PCR MIMIC product would be 340 bp in size. Serial dilutions of PCR MIMICs were added to PCR amplification reactions containing constant amounts of the experimental cDNA samples.

Pharmacological Inhibition Studies We evaluated the effects of BQ-123 (donated by Banyu Pharmaceutical Co.), bosentan (donated by Roche, Basel, Switzerland), and BQ-788 (purchased from CalbiochemNovabiochem AG, La¨ufelfingen, Switzerland) in rats with EA-induced intestinal anaphylaxis. BQ-123 (0.1 mg · kg21 · min21) was continuously infused from the mesenteric artery by an infusion pump from 1 hour before the preparation until the end of experiments. As a control, PBS alone was given at the same dose. The dose of 0.1 mg · kg21 · min21 was chosen because with this dose we had previously obtained significant protective effects in changes of microvascular hemodynamics in rat small intestine induced by endotoxin treatment.10 Bosentan (30 mg/kg) was orally administered 1 hour before perfusion, and an additional intravenous injection of bosentan (10 mg/kg) was performed 15 minutes before the perfusion. This dose of bosentan was shown to antagonize against the pressure action of large doses of ET-1 (0.3 nmol/kg) in pithed rats.15 BQ-788 (10 nmol · kg21 · min21) was administered intravenously from 15 minutes before the preparation until the end of experiments, according to the previous report.25 For in vitro studies using the mast cell line RBL-2H3, BQ-123 (100 nmol/L) and bosentan (10 µmol/L) were added to the culture media along with ET.

Mast Cell Lines and Stimulation Experiments An RBL cell line, RBL-2H3, was obtained from Human Science (Osaka, Japan) and maintained in Eagle minimum essential medium, 20% fetal calf serum (FCS), and 4 mmol/L L-glutamine. For stimulation experiments, ET-1 (1029 mol/L; American Peptide Co., Sunnyvale, CA) was added to culture medium containing 5% FCS. Culture supernatant was collected at intervals for 12 hours for determination of RMCP-II concentration. RBL-2H3 cells were also stimulated by IgE. Rat IgE (Chemicon International Inc.) was added to culture media containing 5% FCS at the concentration of 0.8 µg/mL. After 1

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Figure 1. Time-course changes of ET-1 concentrations in the jejunal loops after EA challenge. Rats were sensitized by intraperitoneal injection of 10 µg of chicken EA and 10 mg of aluminum hydroxide as adjuvant (sensitized). Control rats were injected with material prepared in the same manner without antigen (control). Fourteen days later, intestinal loops were either challenged by adding 10 µg/mL EA to the perfusate or sham challenged by adding 10 µg/mL BSA to the perfusate. ET-1 concentrations of intestinal mucosa were determined by enzyme immunoassay. d, Sensitized 1 EA; j, sensitized 1 BSA; s, control 1 EA. Values are expressed as means 6 SEM of six experiments. *P , 0.05 vs. control 1 EA; †P , 0.05 vs. sensitized 1 BSA.

and 2 hours, total RNA was extracted from cells and expression of ET-1 mRNA was determined by RT-PCR.

Statistics All results are expressed as means 6 SEM. Differences between groups were evaluated by one-way analysis of variance (ANOVA) and Fisher’s post hoc test. Statistical significance was set at P , 0.05.

Results Figure 1 shows the time-course changes of ET-1 concentration in the jejunal loops after the challenge with EA. EA challenge in sensitized animals induced a significant increase in ET-1 concentration at 15 minutes and further increased the ET-1 levels in a time-dependent manner, producing a greater than threefold increase at 1 hour and a greater than fourfold increase 2 hours later. On the other hand, the ET-1 concentration in the intestine was not significantly changed after infusion of BSA solution in either the control animals or the sensitized animals. Infusion of EA into the loops of control animals did not cause any significant alteration in ET-1 concentrations. Figure 2 illustrates the time-course changes of ET-3 concentration in the jejunal mucosa after EA challenge. Similarly, EA challenge in sensitized animals induced a significant time-dependent increase in ET-3 concentration in the jejunal loops and ET-3 reached a significantly increased level at 30 minutes. Again, ET-3 concentration in the intestine was not significantly changed after the

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Figure 2. Time-course changes of ET-3 concentrations in the jejunal loops after EA challenge. Rats were sensitized by chicken EA. Fourteen days later, intestinal loops were challenged by adding EA or BSA to the perfusate. ET-3 concentrations of intestinal mucosa were determined by enzyme immunoassay. d, Sensitized 1 EA; j, sensitized 1 BSA; s, control 1 EA. Values are expressed as means 6 SEM of six experiments. *P , 0.05 vs. control 1 EA; †P , 0.05 vs. sensitized 1 BSA.

infusion of buffer alone, and EA challenge to the control animals did not cause any significant alteration in ET-3 concentrations. Next, we examined the increase in endothelin expression in the intestinal mucosa in the anaphylactic state at the transcriptional level. Figure 3 compares ET-1 mRNA expression in control animals and in sensitized animals after 1 hour of EA challenge into the jejunal loop. ET-1 mRNA expression is reflected by a 543-bp band in this figure. Expression of ET-1 mRNA was increased after EA challenge in sensitized animals compared with control animals. This increase began to reach a significant level at 30 minutes, but ET-1 mRNA expression was suppressed at 2 hours (data not shown). Similarly, expression of ET-3 mRNA was also increased in sensitized animals with EA challenge compared with control animals at 1 hour (Figure 4). Figure 5 shows the serum concentration of RMCP-II in portal venous blood in intestinal anaphylaxis. The serum concentration of RMCP-II showed a significant increase at 30 minutes and reached the maximum level at 1 hour in sensitized animals compared with control animals challenged with EA or sensitized animals challenged with BSA solution. BQ-123 and bosentan both significantly attenuated the increase in RMCP-II concentrations in sensitized animals challenged with EA. On the other hand, BQ-788 did not significantly attenuate the EAinduced increase in RMCP-II levels. Figure 6 illustrates the time-course changes in net water flux in jejunal loops. A significant net water outflux was induced starting at 15 minutes in sensitized animals challenged with EA, but not in either control animals or sensitized animals challenged with BSA

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Figure 3. ET-1 mRNA expression in control animals and in sensitized animals after 1 hour of EA challenge into the jejunal loop as determined by competitive RT-PCR. A specific band for ET-1 mRNA is expressed at 543 bp. The PCR MIMIC product was designed at 340 bp. Expression of ET-1 mRNA was increased in sensitized animals challenged with EA compared with control animals. HaeIII: molecular weight standard, FX-174RF DNA-HaeIII digest.

solution (Figure 6A). Pretreatment with BQ-123 or bosentan significantly attenuated net water outflux caused by anaphylaxis in the jejunal mucosa, whereas BQ-788 did not significantly inhibit the EA-induced net water outflux (Figure 6B). Figure 7 shows the time-course changes in the amounts of histamine release in intestinal perfusates. The hista-

mine concentrations in the loops were significantly increased in parallel with the values of net water outflux in sensitized animals challenged with EA infusion compared with either control animals or sensitized animals challenged with BSA solution (Figure 7A). BQ-123 and bosentan both inhibited the increase in histamine release induced by intestinal anaphylaxis. In contrast, BQ-788

Figure 4. ET-3 mRNA expression in control animals and in sensitized animals after 1 hour of EA challenge to the jejunal loop as determined by competitive RT-PCR. A specific band for ET-3 mRNA is expressed at 477 bp. The PCR MIMIC product is at 340 bp. Expression of ET-3 mRNA was increased in sensitized animals challenged with EA compared with control animals.

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Figure 5. Serum concentrations of RMCP-II in portal venous blood in intestinal anaphylaxis after EA challenge. Fourteen days after sensitization with EA, intestinal loops were challenged by EA or BSA. RMCP-II concentration was determined by enzyme-linked immunosorbent assay. In some experiments, sensitized animals were treated with the ETA-receptor antagonist BQ-123 (0.1 mg · kg21 · min21 intravenously [IV]), the ETA/ETB–receptor antagonist bosentan (30 mg/kg per os and 10 mg/kg IV), or the ETB-receptor antagonist BQ-788 (10 nmol · kg21 · min21 IV) before EA challenge. Values are expressed as means 6 SEM of six experiments. *P , 0.05 vs. control 1 EA; †P , 0.05 vs. sensitized 1 BSA; #P , 0.05 vs. sensitized 1 EA.

did not significantly reduce the EA-induced histamine release (Figure 7B). Because enhanced ET release in the intestinal mucosa was shown to be a crucial step in the development of intestinal anaphylaxis, we investigated whether a mast cell line, RBL-2H3, could respond to accumulated ETs. RBL-2H3 cells expressed ETA-receptor mRNA but not ETB-receptor mRNA as determined by RT-PCR (Figure 8). Treatment with ET-1 significantly increased the RMCP-II concentrations in the culture media in a time-dependent manner (Figure 9). BQ-123 and bosentan both significantly attenuated the ET-1–induced increase in RMCP-II release. Figure 10 shows ET-1 mRNA expression in RBL-2H3 cells after stimulation with rat IgE. RBL-2H3 cells showed the specific expression of ET-1 mRNA as shown at 543 bp. Increased expression of ET-1 was observed both 1 and 2 hours after IgE administration.

Discussion In this study, a significant increase in the mucosal concentrations of ET-1 and ET-3 and increased expression of ET-1 and ET-3 mRNAs were observed in the intestinal mucosa of sensitized animals after antigen challenge. Treatment with the ETA-receptor antagonists, but not the ETB-receptor antagonist, significantly attenuated EA-induced increases in net water outflux, histamine release into the loops, and elevation of serum RMCP-II concentrations in animals with intestinal anaphylaxis,

Figure 6. Time-course changes in net water flux in jejunal loops in intestinal anaphylaxis after EA challenge. (A) Rats were sensitized with EA or injected with alum alone. Fourteen days later, intestinal loops were either challenged by EA or BSA. (B) In some experiments, sensitized animals were treated with BQ-123; 0.1 mg · kg21 · min21 IV), bosentan (30 mg/kg per os and 10 mg/kg IV), or BQ-788 (10 nmol · kg21 · min21 IV) before EA challenge. Values are expressed as means 6 SEM of six experiments. *P , 0.05 vs. control 1 EA; †P , 0.05 vs. sensitized 1 BSA; #P , 0.05 vs. sensitized 1 EA.

suggesting that ET synthesis is induced in the intestinal mucosa by antigen challenge and that this enhanced ET production may play a significant role in the development of intestinal anaphylaxis. In particular, attenuation of serum RMCP-II concentrations by ETA-receptor antagonists indicates a possible participation of ET in the degranulation of mast cells in intestinal anaphylaxis. Recently, Yamamura et al.26 reported that cultured mouse bone marrow–derived mast cells are capable of releasing not only histamine but also leukotriene C4 in response to ET-1 stimulation via ETA receptors. They also found that ET-1 is one of the most potent histaminereleasing factors in mouse peritoneal mast cells discov-

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Figure 8. ETA- and ETB-receptor mRNA expression in the rat mucosal mast cell line RBL-2H3 as determined by RT-PCR. A specific band for ETA-receptor mRNA was expressed at 418 bp, but there was no ETB-receptor mRNA expressed in this cell line.

Figure 7. Time-course changes in histamine release in jejunal loops in intestinal anaphylaxis after EA challenge. Histamine concentrations in the effluent collected were determined by radioimmunoassay. (A) Rats were sensitized with EA or injected with alum alone (control). Fourteen days later, intestinal loops were challenged by EA or BSA. (B) In some experiments, sensitized animals were treated with BQ-123, bosentan, or BQ-788 before EA challenge. Values are expressed as means 6 SEM of six experiments. *P , 0.05 vs. control 1 EA; †P , 0.05 vs. sensitized 1 BSA; #P , 0.05 vs. sensitized 1 EA.

ered to date. In the present study, we showed that RBL cells, which are homologous to mucosal mast cells,27 have ETA-type receptors and that these cells released RMCP-II in response to ET-1. Our in vitro observations suggest that ETA receptors are associated with the release of RMCP-II existing in rat mucosal mast cells. These data suggest the possibility that large-scale release of vasoactive substances from mucosal mast cells is induced by ETs during anaphylaxis in rat small intestine. It should be noted that in this study, bosentan and BQ-123 both significantly attenuated net water outflux caused by anaphylaxis in jejunal mucosa, indicating a possible participation of ETs in changes in intestinal transport via ETA receptors. Alterations of intestinal transport (enhanced secretion and/or decreased absorption) are an important mechanism in the pathophysiology of diarrhea in food allergy.1 Previous experiments have

implicated that stimulation of ion transport by the intestinal epithelium during intestinal anaphylaxis is caused by a combination of the effects of mast cell mediators and neurotransmitters such as substance P.17 Hypersecretory responses to secretagogues in the sensitized tissues have been reported by Barrett et al.28 in the colonic epithelial cell line T84 after coculture with intact cells or lysates of the rat mucosal mast cell–like cell line RBL-2H3. ETs may be involved in intestinal secretion through their effects on release of mast cell mediators or neurotransmitters in intestinal anaphylaxis. However, it is also conceivable that ETs directly affect ion transport in

Figure 9. Effect of ET-1 on RMCP-II release into the culture supernatant of the mucosal mast cell line RBL-2H3 in vitro, and the attenuating effects of BQ-123 and bosentan. ET-1 (1029 mol/L) was added to the culture medium containing 5% FCS. At different time intervals (1–12 hours), culture supernatant was collected and RMCP-II concentrations were determined by enzyme-linked immunosorbent assay. BQ-123 (100 nmol/L) and bosentan (10 µmol/L) were added to culture media along with ET. Values are expressed as means 6 SEM of six experiments. *P , 0.05 vs. control; #P , 0.05 vs. ET-1 alone.

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Figure 10. Effect of IgE administration on ET-1 mRNA expression in the rat mucosal mast cell line RBL-2H3 as determined by RT-PCR. A specific band for ET-1 mRNA is expressed at 543 bp. Rat IgE was added to culture media containing 5% FCS at a concentration of 0.8 µg/mL. After 1 and 2 hours, expression of ET-1 mRNA was increased compared with controls (0 h).

the intestine. Roden et al.29 have reported that ET-1 stimulates Cl2 and K1 secretion in the descending colon of rabbit. More recently, Hosokawa et al.30 showed that ETA and ETB receptors regulate Na1 and Cl2 transport by different mechanisms in the colonic mucosa of rats, although the contribution of ET receptors to the regulation of ion transport in the small intestine remains to be elucidated. We found increased production of ET-1 and ET-3 in the intestinal mucosa of sensitized animals after antigen challenge. Although the exact source of ET is not known in this anaphylactic model, there are several possible cellular sources, such as vascular endothelial cells, vascular smooth muscle cells, mast cells, and intestinal epithelial cells.1 In anaphylaxis, mast cells themselves may become a source of ETs. Ehrenreich et al.31 showed by a combination of high-performance liquid chromatography and a radioimmunoassay specific for ET-1 that primary murine bone marrow mast cells as well as various mast cell lines contain and secrete immunoreactive ET-1. In this study, we showed that mRNA expression of ET-1 was stimulated in RBL cells after administration of IgE, suggesting that the elevated levels of IgE in situ may induce ET-1 production in anaphylactic state. Local accumulation of ETs in turn could represent an important component of their pathophysiological action in intestinal anaphylaxis, allowing them to act as cytokine-like factors via autocrine/paracrine mechanisms. We have recently shown32 that ET-1 is expressed and released from

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the monolayers of the nontransformed rat intestinal epithelial cell lines. We further showed that IL-2, a potent cytokine presumably secreted from lymphocyte and macrophage populations, stimulates ET-1 release from intestinal cells. These findings suggest that intestinal epithelial cells are another important source of ETs in anaphylaxis and that endothelin can facilitate coordinated response by epithelial cells and cellular constituents of the mucosal immune system in the intestinal mucosa. The observed elevation in ET concentrations in the intestinal mucosa is not a consequence of circulatory disturbances or of respiratory insufficiency. In our experimental model, systemic arterial blood pressure showed no significant changes during the experiments. The mean ET-1 concentration in the serum of animals sensitized with EA was 3.9 6 2.0 pg/mL, which was not significantly different from the control values of 1.8 6 1.3 pg/mL, suggesting that the increased synthesis and release of ETs were confined to the small intestine during intestinal anaphylaxis. In this study, we found that concentrations of both ET-1 and ET-3 were increased in the intestinal mucosa. The difference in the potential roles of these two peptides in the development of intestinal anaphylaxis remains unknown. However, in this study we found that the mucosal concentration of ET-1 is greater than that of ET-3 in anaphylactic animals and also showed that a rat mucosal mast cell–like cell line only expresses the ETA receptor. Because ET-3 shows a lower binding affinity for the ETA receptor than ET-1, ET-1 is likely to play a more significant role than ET-3 in mast cell–mediated responses in intestinal anaphylaxis.

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Received September 19, 1997. Accepted April 13, 1998. Address requests for reprints to: Hiromasa Ishii, M.D., Professor of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Fax: (81) 3-33569654. Supported in part by grants from the Ministry of Education, Science and Culture of Japan and from Keio University, and by Funds for Comprehensive Research on Long-term Chronic Disease from the Ministry of Welfare of Japan. The authors thank Dr. Mitsuo Yano (Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd.) for the generous donation of BQ-123 and Roche for the generous donation of bosentan.