δ-opioid receptors inhibit neurogenic intestinal secretion evoked by mast cell degranulation and type I hypersensitivity

Journal of Neuroimmunology 112 (2001) 89–96 www.elsevier.com / locate / jneuroin

d-opioid receptors inhibit neurogenic intestinal secretion evoked by mast cell degranulation and type I hypersensitivity Sutthasinee Poonyachoti, David R. Brown* Department of Veterinary PathoBiology, University of Minnesota Academic Health Center, 1988 Fitch Avenue, St. Paul, Minneapolis, MN 55108 -6010, USA Received 19 June 2000; received in revised form 28 August 2000; accepted 29 August 2000

Abstract Histamine and the mast cell degranulator, compound 48 / 80 produced elevations in short-circuit current, an electrical measure of active anion secretion, across porcine ileal mucosa sheets mounted in Ussing chambers. Luminally-applied b-lactoglobulin produced similar effects in mucosal sheets from cow’s milk-sensitized pigs. Their secretory effects were attenuated by blockers of H 1 -histamine receptors, neuronal conduction or epithelial Na 1 / K 1 / Cl 2 cotransport. The d-opioid agonist [D-Pen 2 ,D-Pen 5 ]enkephalin suppressed mucosal responses to these substances in a naltrindole-reversible manner. Furthermore, submucosal mast cells and d-opioid receptor-immunoreactive nerve fibers were observed in close juxtaposition. Intestinal neural pathways linking immediate hypersensitivity to secretory host defense appear to express inhibitory d-opioid receptors.  2001 Elsevier Science B.V. All rights reserved. Keywords: Pharmaconeuroimmunology; Food allergy; Enkephalin; Ileum; Pig

1. Introduction Immediate hypersensitivity reactions to foods, resulting in acute diarrhea and other gastrointestinal signs, are widespread, particularly among young children (Ahmed and Fuchs, 1997; Bischoff et al., 2000). Acute diarrhea and other gastrointestinal signs associated with antigen ingestion in sensitized individuals are thought to stem from the degranulation of mucosal mast cells (Stenton et al., 1998). Histamine, released from mucosal mast cells in the course of intestinal anaphylaxis, increases active anion secretion and short-circuit current (Isc ) across small intestinal and colonic mucosae through interactions with enteric submucosal neurons (Perdue and McKay, 1994). The profound antidiarrheal activity of opioids such as morphine, codeine and loperamide, which interact mainly Abbreviations: b-LG, beta-Lactoglobulin; CTOP, D-Phe-Cys-Tyr-DTrp-Orn-Thr-Orn-Thr-Pen-Thr-NH 2 ; DPDPE, [D-Pen 2 ,D-Pen 5 ]enkephalin; Gt , Tissue conductance; Isc , Short-circuit current; NTI, naltrindole hydrochloride; nor-BNI, nor-binaltorphimine; OR, opioid receptor; PBS, phosphate-buffered saline; PGP 9.5, protein gene product 9.5 *Corresponding author. Tel.: 11-612-624-0713; fax: 11-612-6250204. E-mail address: [email protected] (D.R. Brown).

with m-opioid receptors (OR) on myenteric neurons, has been attributed to their ability to inhibit intestinal propulsion (Schiller et al., 1982, 1984). In preparations of small intestinal mucosa from several animal species however, opioid agonists acting selectively on d-opioid receptors expressed by submucosal neurons decrease active anion secretion and Isc . (Dobbins et al., 1980; Kachur and Miller, 1982; Sheldon et al., 1990). Enkephalins are the putative endogenous ligands for these receptors and the enkephalin degradation inhibitor, acetorphan, has been shown to possess antidiarrheal efficacy (Baumer et al., 1992; Roge et al., 1993). In this study, we tested the hypothesis that opioids and mast cell products released in the course of an allergic response to milk, a common mucosal allergen, act through convergent neural circuits to influence active ion transport in the ileum. It has been estimated in rat ileum that 50% of mast cells are in close proximity to submucosal nerve fibers (Williams et al., 1995). The porcine ileum was employed as a biomedical model because it is anatomically, functionally, and pathophysiologically similar to the human small bowel. Furthermore, isolated mucosa–submucosa preparations from porcine ileum respond to histamine and possess neuronal d-opioid receptors (Brown and

0165-5728 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0165-5728( 00 )00387-8

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Treder, 1989; Quito and Brown, 1991; Brown et al., 1998). The results of this investigation suggest that the major mast cell product, histamine, and perhaps other mast cell mediators increase anion secretion through an atropineresistant neural pathway that expresses inhibitory d-opioid receptors.

2. Materials and methods

2.1. Animals Yorkshire pigs of each sex and weighing between 14 and 20 kg were obtained from the University of Minnesota Swine Facility (Rosemount, MN). Pigs were housed under conventional conditions and given access to standard, nonmedicated feed (Rosemount Feed Mill, Rosemount, MN) and water ad libitum.

2.4. Tissue preparation Pigs received nonmedicated pig feed ad libitum and were not fasted prior to sacrifice. The pigs were initially sedated with an intramuscular injection of tiletamine hydrochloride–zolazepam (Telazol  ; 8 mg / kg, Fort Dodge Laboratories, Fort Dodge, IA), in combination with xylazine (8 mg / kg). The animals were euthanized by barbiturate overdose (Beuthanasia-D  , 30 mg / lb, i.v., Schering-Plough Animal Health Corp., Kenilworth, NJ) in accordance with approved University of Minnesota Animal Care Committee protocols. A segment of ileum 10–20 cm in length was removed and opened longitudinally along the antimesenteric border. Ileal segments were placed in icecold oxygenated Ringer–bicarbonate solution modified to approximate the composition of porcine extracellular fluid containing 10 mM glucose (ionic composition in mM: Na 1 , 148.5; K 1 , 6.3; Cl 2 , 139.7; Mg 21 , 0.7; Ca 21 , 3.0; 2 2 HCO 2 3 , 19.6; HPO 4 , 1.3; H 2 PO 4 , 0.3; pH 7.4).

2.5. Measurement of mucosal ion transport 2.2. Drugs [D-Pen 2 , D-Pen 5 ]-enkephalin (DPDPE) and D -Phe-CysTyr-D-Trp-Orn-Thr-Orn-Thr-Pen-Thr-NH 2 (CTOP) were obtained from Peninsula Laboratories, Inc. (Belmont, CA) and dissolved in 0.01 M acetic acid with 0.1% bovine serum albumin. The solubilized peptides were aliquoted at stock concentrations of 1–10 mM and stored until use at 2208C. Naltrindole hydrochloride (NTI) was purchased from Research Biochemicals Inc. (Natick, MA). nor-Binaltorphimine (nor-BNI; Takemori and Portoghese, 1992) was a gift from Dr. Philip S. Portoghese (Department of Medicinal Chemistry, University of Minnesota School of Pharmacy, Minneapolis, MN). All other drugs and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

2.3. Sensitization to milk proteins Nine pigs were anesthesized with Telazol  (10 mg / kg, i.m.; combination of tiletamine HCl and zolazepram HCl; Fort Dodge Laboratories, Fort Dodge, IA). Sodium bicarbonate (3.33% w / v in 0.8% saline) was administered by gavage through a 40 cm, 18 FR single use catheter (Rusch, Duluth, GA) in a volume of 30 ml to neutralize gastric acid. Fifteen min later cholera holotoxin, a permissive oral adjuvant, was introduced into the stomach at a dose of 2 mg after the method of Foss and Murtaugh (1999). Animals were given free access to standard feed and pasteurized cow’s milk, which was substituted for water for a period of 2 weeks. Milk was withdrawn and water was reintroduced to the animals at least 3 days before all experiments.

The ileum was stripped of its serosa and underlying smooth muscle layers by blunt dissection and the remaining mucosa–submucosa preparation was mounted between two lucite Ussing-type half chambers (Jim’s Instrument Manufacturing Co., Iowa City, IA) having a flux area of 2.0 cm 2 . Both sides of each mucosal sheet were bathed in the modified Ringer–HCO 2 3 solution which was maintained at pH 7.4 and 39.18C (porcine core temperature) and oxygenated continuously with 5% CO 2 in O 2 . In addition, 10 mM mannitol and D-glucose were present in luminal and contraluminal bathing solutions, respectively. The short-circuit current (Isc ) across the tissues, a measure of net ion transport, was monitored continuously by an automatic voltage clamp (Model TR100, JWT Engineering, Overland Park, KS). After the baseline Isc stabilized, the circuit was opened before and after drug administration to measure potential difference (mV); tissue conductance (Gt in mS / cm 2 ) was calculated according to Ohm’s law. Peak elevations in Isc relative to the baseline Isc were determined after the contraluminal addition of histamine (3 or 10 mM) or compound 48 / 80 (10 ng / ml). In mucosal sheets from milk-sensitized pigs, b-lactoglobulin (b-LG) or ovalbumin was added to either the luminal or contraluminal bathing media at a concentration of 300 mg / ml. In some experiments, tissues were pretreated with saxitoxin (STX; 0.1 mM); diphenhydramine (DPH; 0.1 or 10 mM); cimetidine (0.1 mM); atropine (0.1 mM); furosemide (FS; 10 mM); DPDPE (0.1 mM) or morphine (0.1 mM). Each substance was added to the contraluminal bathing medium 5 min before the contraluminal addition of histamine or 48 / 80, or before luminal challenge with b-LG. In some experiments, an opioid antagonist selective for either d-(NTI), k-(nor-BNI) or m-(CTOP) opioid

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receptors was added at a concentration of 0.1 mM to the contraluminal aspect of mucosal sheets 5 min prior to DPDPE addition.

2.6. Immunohistochemistry and histological staining Muscle-stripped preparations of ileal mucosa–submucosa from three pigs, identical to those used in pharmacological experiments, were cut in blocks of 1 to 2 cm 2 and fixed for 2 h at 48C in a 2% paraformaldehyde solution in phosphate buffered saline (PBS; pH 7.4). Tissues were cryoprotected in graded (10–30%) concentrations of sucrose in PBS, embedded in TissueTek O.C.T compound (Baxter Healthcare Corp., McGaw Park, IL), and frozen. Transverse sections (10 mm) of frozen tissues were thawmounted on Superfrost-plusE slides (Fisher Scientific, Pittsburgh, PA) and stored at 2208C. Ileal sections from each animal were incubated with rabbit anti-human protein gene product 9.5 antiserum (PGP 9.5; Chemicon International, Inc., Temecula, CA) as a general neuronal marker at a dilution of 1:250 for 1 h or rabbit anti-mouse d-OR antiserum (DOR-442; gift of Dr. Robert P. Elde, Department of Neuroscience, University of Minnesota Medical School, Minneapolis, MN) at a dilution of 1:1000 for 24 h (Brown et al., 1998). Immunoreactivity was detected using a rabbit IgG Histostain-SPE kit (Zymed Laboratories, Inc., So. San Francisco, CA) following the manufacturer’s protocol for the ABC-peroxidase technique. Control experiments included the omission of the primary d-OR antibody from the staining protocol or its preabsorption with a relevant peptide corresponding to the N-terminal epitope in murine d-OR that was detected by the antiserum. This peptide was incubated with antireceptor antiserum overnight at 48C. After centrifugation, the supernatant was used in place of the primary antibody in the staining protocol. Preincubation of anti-d-OR antiserum with 30-fold molar excess of the peptide completely eliminated the ability of the anti-d-OR antibody to detect immunoreactive neural elements. Mucosal mast cells in the tissue sections were subsequently stained with 0.1% Alcian blue 8GX (pH 0.3) for 1 h. In some sections, the presence of connective tissue mast cells was assessed by safranin O staining; tissues were stained with 0.5% dye at pH 1.0 for 30 s. Sections were coverslipped with a glycerol–gelatin slide-mounting medium composed of 59% (w / v) glycerol, 1% phenol and 0.9% gelatin (Sigma Chemical Co.). Tissue sections were examined under brightfield microscopy and photomicrographs were obtained using an Olympus microscope attached to a Spot camera (Spot Camera Diagnostic Instruments, Ann Arbor, MI). Images were digitized using Spot software (version 2.2) and further processed using an Adobe Photoshop (version 4.0) software program.

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2.7. Data analysis Peak changes in Isc and Gt were determined as the mean6S.E. relative to their baseline values in n tissues from at least 3 pigs. Comparisons between a control and treatment mean were made by two-tailed paired or unpaired t-tests. Comparisons between a control mean and multiple treatment means were made by one-way analysis of variance (ANOVA) followed by a Dunnett or Tukey multiple comparison test when appropriate. A P value of 0.05 or less was chosen as the limit for statistical significance.

3. Results

3.1. Compound 48 /80 -induced mucosal transport Short-circuit current (Isc ) and Gt attained stable baseline levels in mucosal sheets within 30–40 min after tissues were mounted (mean Isc 5251.063.3 mA / cm 2 and Gt 5 25.161.0 mS / cm 2 ; n5117 tissues from 30 to 40 pigs). These parameters remained unaffected in tissues treated on their contraluminal aspect with either the neuronal conduction blocker STX (0.1 mM), the H 1 -histamine receptor blocker DPH (10 mM) or the Na 1 / K 1 / Cl 2 cotransport blocker furosemide (10 mM). The mast cell degranulating agent, compound 48 / 80, increased both Isc and Gt at a contraluminal concentration of 10 ng / ml. Its effects on Isc (Fig. 1) but not Gt (Table 1), were significantly reduced in tissues pretreated with STX, DPH or furosemide. To verify

Fig. 1. Histograms illustrating mucosal Isc responses to the mast cell degranulator, compound 48 / 80 (10 ng / ml, contraluminal aspect) in the absence and presence of saxitoxin (STX; 0.1 mM); diphenhydramine (DPH; 10 mM); furosemide (FS; 10 mM); or DPDPE (DP, 0.1 mM) in the absence and presence of 0.1 mM naltrindole (NTI / DP). Data represent the mean6S.E. of 48 / 80-induced changes in Isc obtained in 5–9 tissues from 4 to 5 pigs. Differences between mucosal responses to 48 / 80 in the absence (Control) and presence of each inhibitor are indicated as **P, 0.01, Dunnett’s t-test. In addition, the mean changes in Isc produced by 48 / 80 in tissues pretreated wih DPDPE (DP) or naltrindole plus DPDPE (NTI / DP) were significantly different from each other (P,0.01, Tukey’s test).

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Table 1 Effects of inhibitor pretreatment on changes in tissue conductance (DGt ) produced by compound 48 / 80 and b-lactoglobulin Condition

DGt (mS / cm 2 )a

n tissues (n pigs)

Compound 48 / 80 (10 ng / ml) alone Saxitoxin, 0.1 mM / compound 48 / 80 Diphenhydramine, 10 mM / compound 48 / 80 Furosemide, 10 mM / compound 48 / 80 DPDPE, 0.1 mM / compound 48 / 80 b-Lactoglobulin (300 mg / ml) alone b Saxitoxin, 0.1 mM / b-lactoglobulin b Diphenhydramine, 0.1 mM / b-lactoglobulin b Furosemide, 10 mM / b-lactoglobulin b DPDPE, 0.1 mM / b-lactoglobulin b

5.660.5 6.161.0 7.360.8 6.961.0 9.064.6 2.960.9 5.961.1 4.661.4 20.260.4 3.762.0

34 5 5 4 11 14 4 4 3 12

(30) (4) (4) (4) (9) (9) (4) (4) (3) (9)

a

Mean6S.E. change relative to baseline Gt prior to addition of 48 / 80 or b-lactoglobulin. Both 48 / 80 and b-lactoglobulin significantly increased Gt relative to baseline values (P,0.05 for both agents relative to pre-drug baseline Gt , paired t test). However, their abilities to change Gt were not significantly reduced (P.0.05, Dunnett t-test) in tissues pretreated with inhibitory drugs or DPDPE. b Responses in tissues from milk-sensitized pigs.

that the action of 10 mM DPH was limited to H 1 -histamine receptor blockade and that it did not include blockade of muscarinic cholinergic receptors, the effects of the cholinergic agonist carbachol were compared in untreated tissues and tissues pretreated with DPH. Diphenhydramine did not significantly alter carbachol-induced elevations in Isc (DIsc after 10 mM carbachol in absence and presence of DPH534.466.7 and 31.465.9 mA / cm 2 , P.0.05, unpaired t-test, n515 and 10 tissues from 5 pigs, respectively). The selective d-OR agonist DPDPE, at a contraluminal concentration of 0.1 mM, had no significant effect on Isc or Gt , but reduced 48 / 80-evoked elevations in Isc by 49% (Fig. 1). DPDPE had no effect on 48 / 80-induced increases in Gt (Table 1). The inhibitory effects of DPDPE on 48 / 80-induced ion transport were reversed in tissues pretreated with the selective d-OR antagonist NTI (Fig. 1). Naltrindole alone did not significantly alter mucosal electrical parameters (DIsc and DGt . after 0.1 mM NTI52 8.862.1 mA / cm 2 and 22.261.7 mS / cm 2 , respectively; P.0.05 for each parameter relative to baseline values, paired t test, n519 tissues from 5 pigs). As indicated above, STX alone decreased mucosal Isc responses to 48 / 80 by 8666%, respectively. In the presence of STX, DPDPE had no significant additional inhibitory effect on 48 / 80-induced elevations in Isc (9063% reduction; n53 tissues from 3 pigs).

3.2. Histamine-induced mucosal transport Because the action of compound 48 / 80 was inhibited by the antihistamine DPH, the secretory actions of histamine and their susceptibility to opioid inhibition were next examined. At contraluminal bath concentration of 3 mM, histamine rapidly increased Isc , but unlike compound 48 / 80, had no significant effect on Gt . STX, at a contraluminal concentration of 0.1 mM, abolished the effects of 3 mM histamine on Isc (Fig. 2, top). To examine the involvement of submucosal cholinergic neurons in the effects of his-

tamine, some tissues were pretreated with the muscarinic cholinergic antagonist atropine prior to histamine addition. Mucosal Isc responses to 10 mM carbachol, but not 10 mM histamine were abolished after atropine treatment (Table 2).Mucosal Isc responses to 3 mM histamine were significantly reduced in tissues pretreated with 0.1 mM DPH, but remained unaltered in the presence of the H 2 -histamine antagonist cimetidine at an equivalent concentration (Fig. 2, top). Cimetidine alone had no effect on baseline Isc or Gt .After its contraluminal administration at 10 mM, furosemide significantly reduced Isc elevations produced by histamine (Fig. 2, top). DPDPE significantly reduced mucosal Isc responses to 3 mM histamine when administered alone or in tissues pretreated with either the selective m-OR antagonist CTOP or the k-OR antagonist nor-BNI (Fig. 2, bottom). Neither antagonist altered baseline Isc or Gt (data not shown). In the presence of NTI, however, DPDPE did not significantly reduce histamine-evoked increases in Isc (Fig. 2, bottom).

3.3. b -LG-induced transport in ileal mucosa from milksensitized pigs In ileal mucosa sheets from milk-fed pigs, mean Isc and Gt were 23.366.4 mA / cm 2 and 27.462.4 mS / cm 2 , respectively (n559 tissues from 9 pigs). The luminal addition of the major milk protein b-LG (300 mg / ml) rapidly increased Isc (Fig. 4) and Gt (Table 1). When applied to the contraluminal aspect of mucosal sheets, b-LG did not significantly alter Isc or Gt . To verify that mucosal responses were specific for the milk antigen b-LG, mucosal responses to ovalbumin were also measured. Addition of ovalbumin to either the luminal or contraluminal bathing media produced no significant changes in Isc (Fig. 3) or Gt . Although STX, DPH or furosemide alone had no effect on baseline Isc or b-LGinduced changes in Gt (Table 1) in milk-sensitized tissues, each blocker significantly reduced mucosal Isc responses to luminal b-LG challenge (Fig. 4). Contraluminal addition

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Fig. 2. Top. Histograms illustrating mucosal Isc responses to 3 mM histamine (contraluminal administration) in the absence (Control) and presence of saxitoxin (STX; 0.1 mM), diphenhydramine (DPH; 0.1 mM), cimetidine (CM; 0.1 mM) or furosemide (FS; 10 mM). Bottom. Mucosal responses to 3 mM histamine in the absence (Control) and presence (DP) of 0.1 mM DPDPE. Five min before DPDPE addition, some tissues were contraluminally treated with one of the following selective opioid receptor antagonists at a concentration of 0.1 mM: naltrindole (NTI / DP; d-OR), CTOP (CT / DP; m-OR) or nor-BNI (BNI / DP; k-OR). Data represent the mean6S.E. of histamine-induced changes in Isc obtained in 5–10 tissues from 4–5 pigs. Significant differences between mucosal responses to histamine in the absence (Control) and presence of each inhibitor are indicated as **P,0.01, Dunnett’s t-test. In addition, the mean changes in Isc produced by histamine in tissues pretreated wih DPDPE (DP) or naltrindole plus DPDPE (NTI / DP) were significantly different from each other (P,0.05, Tukey’s test).

of DPDPE had no significant effect on Isc or Gt in milksensitized tissues (DIsc and DGt . after DPDPE53.361.9 mA / cm 2 and 22.366.6 mS / cm 2 , respectively; P.0.05, respectively for each parameter relative to baseline values; paired t-test; n512 tissues from 9 pigs). It also did not alter b-LG-evoked changes in Gt (Table 1). However, DPDPE inhibited b-LG-evoked changes in Isc in milksensitized tissues and its effects were reversed by NTI (Fig. 4). In contrast to DPDPE, the mixed m-OR agonist morphine, at a contraluminal bath concentration of 0.1 Table 2 Effects of atropine pretreatment on mucosal responses to carbachol and histamine Condition a

DIsc (mA / cm 2 )

Carbachol, 10 mM Atropine, 0.1 mM / carbachol Histamine, 10 mM Atropine, 0.1 mM / histamine

48.865.6 060* 75.367.0 77.064.5

a n56 tissues from 4 to 5 pigs. *P,0.05 vs. atropine-untreated condition, unpaired t-test.

Fig. 3. Representative chart records of Isc responses of milk-sensitized mucosal sheets to either 300 mg / ml b-lactoglobulin (b-LG) or 300 mg / ml ovalbumin (OVA) applied to the either luminal or contraluminal aspect of mucosal sheets. Tracings are representative of mucosal responses to b-LG or OVA in 1 tissue from each of 4 milk-fed pigs. Mean changes in Isc produced by luminal or contraluminal b-LG were 25.462.9 and and 23.7561.0 mA / cm 2 , respectively. Mean changes in Isc produced by luminal or contraluminal ovalbumin were 2.860.3 and 0.260.1 mA / cm 2 , respectively.

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Fig. 4. Histogram illustrating mucosal Isc responses to the luminal addition of b-lactoglobulin in the absence (Control) and presence of saxitoxin (STX; 0.1 mM); diphenhydramine (DPH; 0.1 mM); cimetidine (CM; 0.1 mM); furosemide (FS; 10 mM); morphine (MOR, 0.1 mM); or DPDPE (DP; 0.1 mM) added to the contraluminal aspect of tissues. In addition, some tissues were contraluminally pretreated with 0.1 mM naltrindole 5 min before DPDPE addition (NTI / DP). Data represent the mean6S.E. of b-LG-induced changes in Isc obtained in 3–5 tissues from 3 to 4 milk-sensitized pigs. Significant differences between mucosal responses to b-LG in the absence and presence of each inhibitor are indicated as **P,0.01, Dunnett’s t-test. In addition, the mean changes in Isc produced by b-LG in milk-sensitized tissues pretreated wih DPDPE (DP) or naltrindole plus DPDPE (NTI / DP) were significantly different from each other (P,0.01, Tukey’s test).

mM, had no effect on mucosal electrical parameters alone and it did not attenuate mucosal responses to b-LG (Fig. 4).

3.4. Relationship of mast cells to enteric neurons in porcine ileum Five or six mast cells could be visualized by Alcian blue staining in each 10 mm thick section of porcine ileum (n55–6 tissue sections from each of 3 pigs). Mast cells were localized in the region of the crypts or in the submucosa in close proximity to PGP 9.5- or d-ORimmunoreactive nerve fibers (Fig. 5). No connective tissue mast cells were detected by safranin O staining (data not shown).

3.5. Discussion Histamine and compound 48 / 80, which evokes the noncytotoxic release of histamine and other preformed mediators from mast cells, rapidly increased Isc after their contraluminal addition to mucosa–submucosa sheets from porcine ileum. Similarly, luminal exposure to the milk antigen b-LG was associated with Isc elevations in mucosal sheets from milk-sensitized pigs. In accordance with previous reports, it is likely that histamine interacts primarily with H 1 -histamine receptors to elevate Isc because most of its action was inhibited by the classical antihistamine DPH, administered at a concentration that blocked H 1 -histamine receptors without affecting muscarinic cholinergic receptors (Brown and Miller, 1991). As shown previously in porcine distal colon, the effect of histamine on Isc in porcine ileum was not mediated by H 2 -histamine receptors because it was preserved in tissues pretreated with the H 2 -histamine receptor antagonist cimetidine (Traynor et al., 1993). The elevations in Isc

Fig. 5. Digitized photomicrographs illustrating mast cells in the porcine ileal mucosa stained with Alcian blue and either (A) rabbit anti-human PGP-9.5 antiserum or (B) rabbit anti-mouse d-OR antiserum and visualized by the ABC-peroxidase technique. Single arrows point to mucosal mast cells and double arrows (in A and B) indicate immunoreactive nerve fibers in the submucosa. An adjacent section (C) serving as a preabsorption control for d-OR immunoreactivity shows an absence of specific d-OR immunoreactivity in neurons and nerve fibers. Asterisks indicate non-specific staining. OSP, outer submucous plexus; V, villus / crypt. Bar in A corresponds to a length of 10 mm in A, 20 mm in B and 500 mm in C.

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produced by 48 / 80 and b-LG were also attenuated by DPH and it is likely that they were occurred secondary to mast cell degranulation and histamine release. The prominent histaminergic component in the mucosal response to b-LG in milk-sensitized tissues is suggestive of an immunoglobulin E-mediated hypersensitivity reaction. Increases in Gt produced by 48 / 80 and b-LG, on the other hand, were probably not mediated by histamine because of their insensitivity to DPH, but might be evoked by other mast cell products, such as tryptase, eicosanoids and cytokines (White, 1999). Saxitoxin markedly attenuated mucosal Isc responses to histamine, 48 / 80, and b-LG, a result indicating that histamine-induced Isc elevations are mediated by submucosal neurons expressing STX-sensitive Na 1 channels. Because the action of histamine was resistant to atropine, it appears that the neural circuits stimulated by histamine do not contain muscarinic cholinergic synapses. In previous studies of histamine action on ion transport in small and large intestinal mucosa preparations from other species, the effects of histamine on Isc were similarly reduced after neuronal conduction blockade (Brown and Miller, 1991; Perdue and McKay, 1994). The increases in Gt produced by compound 48 / 80 or b-LG were not affected by STX, a result suggesting that they might be attributable to direct interactions of 48 / 80 with epithelial tight junctions and the release of mast cell mediators other than histamine that are capable of affecting tissue conductance through non-neural mechanisms (Berin et al., 1998).The transient nature of histamine-, 48 / 80- and b-LG-induced changes in Isc precluded direct analyses of the ion fluxes underlying these responses. By inhibiting Na 1 / K 1 / Cl 2 cotransport, loop diuretics such as furosemide reduce basolateral chloride entry into enterocytes and impair active chloride secretion (Haas, 1994). Furosemide had no effect on baseline Isc in the porcine ileal mucosa. However, it significantly inhibited Isc responses to all three agents, and we interpret this result to indicate that the histamine-induced Isc is associated with active anion secretion. An effect of b-LG on ion transport after its contraluminal addition to milk-sensitized tissues was not observed, a result suggesting that the luminal route was more accesssible for antigen–antibody interactions. Food antigens have been shown previously to stimulate intestinal secretion after their luminal application to sensitized rats and humans (Crowe et al., 1990; Santos et al., 1999). The specificity of b-LG action is supported by the lack of effect of ovalbumin on Isc or Gt when this antigen was administered to either side of milk-sensitized mucosal sheets. Based on its pattern of pharmacological sensitivity to STX, DPH and furosemide, it is likely that b-LG, acting through an IgE-mediated mechanism in milk-sensitized tissues, provokes mast cell release of histamine, which in turn interacts with submucosal neurons mediating active anion secretion. Indeed, mast cells in the porcine ileum were detected predominately in the submucosa, and some cells were in close proximity to submucosal neurons. Enkephalins and their derivatives have proabsorptive or

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antisecretory effects in the small intestine of many species through interactions with d-opioid receptors (Quock et al., 1999). In the porcine ileal mucosa, the selective d-opioid agonist DPDPE increases net salt absorption by tetrodotoxin- and naloxone-sensitive mechanisms and blunts Isc elevations evoked by transmural electrical stimulation of submucosal nerves (Quito and Brown, 1991). In the present study, DPDPE inhibited mucosal Isc responses to histamine, compound 48 / 80 and b-LG. Its antisecretory effects were likely to be mediated by submucosal neurons as DPDPE had no significant suppressant effect on mucosal responses to 48 / 80 in tissues pretreated with STX. Naltrindole, a highly-selective d-opioid receptor blocker, attenuated or prevented the inhibitory effects of DPDPE on mucosal responses to 48 / 80, histamine and b-LG. In contrast, antagonists acting selectively at k- or m-ORs did not significantly inhibit the antisecretory action of DPDPE. Moreover, the mixed m- and k-OR agonist morphine did not affect mucosal responses to b-LG in sensitized tissues. These results are in agreement with our previous finding that the selective k-OR agonist U-50,488H is 25-fold less potent than DPDPE in suppressing Isc . Responses to electrical transmural stimulation in the porcine distal jejunal mucosa (Quito and Brown, 1991). Although morphine and several other opioid ligands can promote histamine release from mast cells (Barke and Hough, 1993), this did not occur in the present study; unlike histamine and 48 / 80, neither morphine nor DPDPE elevated Isc . The functional relationship between mast cell products and opioid-sensitive neural pathways in the intestinal submucosa is underscored by histochemical results demonstrating that mast cells are in close proximity to nerve fibers expressing d-opioid receptors. Moreover, DPDPE has recently been found to attenuate active secretion in the isolated porcine ileal mucosa induced by the activation of type 2 proteinase-activated receptors, a target of mast cell tryptase (Green et al., 2000). Based on these functional and anatomical findings, we conclude that DPDPE interacts with d-opioid receptors present on submucosal neurons to decrease electrogenic anion secretion that is associated with an immediate hypersensitivity reaction in the small intestine.The present results suggest that the submucosal neural pathways mediating mast cell-mediated anion secretion are subject to modulation by d-opioid receptors. By inhibiting activity in these neural circuits, d-opioid receptor agonists may be effective in alleviating acute diarrhea associated with immediate hypersensitivity reactions. On the other hand, such drugs may impair the ability of the small intestine to mount an adequate secretory defense against luminal antigens. These possibilities remain to be further explored.

Acknowledgements The authors thank Drs. Mary Pampusch (Department of Animal Science, University of Minnesota), Anjali Kulkar-

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ni-Narla and Dennis Foss (Department of Veterinary PathoBiology, University of Minnesota) for excellent technical assistance or advice during this study. We are also grateful for gifts of nor-BNI and anti-d-opioid receptor antiserum from Drs. Philip Portoghese (Department of Medicinal Chemistry, University of Minnesota) and Robert P. Elde (Department of Neuroscience, University of Minnesota), respectively. This study was funded by NIH grant DA-10200. S.P. was supported by a Royal Thai Government fellowship.

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