Department of Physiology and Cell Biology, Ohio State University, College of Medicine and Public Health, Columbus, Ohio 43210
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ABSTRACT |
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Enteric neuroimmune interactions in
gastrointestinal hypersensitivity responses involve antigen detection
by mast cells, mast cell degranulation, release of chemical mediators,
and modulatory actions of the mediators on the enteric nervous system
(ENS). Electrophysiological methods were used to investigate electrical and synaptic behavior of neurons in the stomach and small intestine during exposure to -lactoglobulin in guinea pigs sensitized to cow's milk. Application of
-lactoglobulin to sensitized
preparations depolarized the membrane potential and increased neuronal
excitability in small intestinal neurons but not in gastric neurons.
Effects on membrane potential and excitability in the small intestine were suppressed by the mast cell stabilizing drug ketotifen, the histamine H2 receptor antagonist cimetidine, the
cyclooxygenase inhibitor piroxicam, and the 5-lipoxygenase inhibitor
caffeic acid. Unlike small intestinal ganglion cells, gastric myenteric neurons did not respond to histamine applied exogenously. Antigenic exposure suppressed noradrenergic inhibitory neurotransmission in the
small intestinal submucosal plexus. The histamine H3
receptor antagonist thioperamide and piroxicam, but not caffeic acid,
prevented the allergic suppression of noradrenergic inhibitory
neurotransmission. Antigenic stimulation of neuronal excitability and
suppression of synaptic transmission occurred only in milk-sensitized
animals. Results suggest that signaling between mast cells and the ENS underlies intestinal, but not gastric, anaphylactic responses associated with food allergies. Histamine, prostaglandins, and leukotrienes are paracrine signals in the communication pathway from
mast cells to the small intestinal ENS.
food allergy; enteric nervous system; enteric immune system; anaphylaxis; mast cells
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INTRODUCTION |
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INGESTION OF FOREIGN ANTIGENS in the form of food or pathogens sensitizes the enteric immune/inflammatory system. Subsequent exposures to the sensitizing antigen are detected by enteric mast cells stimulated to degranulate and release a variety of biologically active chemical substances. Antigen-evoked mast cell degranulation in the small and large intestine starts an immediate (type I) hypersensitivity reaction characterized by mucosal hypersecretion (1, 17, 34) and strong contractions of the musculature (3). Application of tetrodotoxin or atropine in vitro suppresses the secretory and motor responses. Blockade by tetrodotoxin or atropine implicates participation of the enteric nervous system (ENS) in the hypersensitivity responses (2, 3, 17).
Results of earlier electrophysiological studies in colonic submucosal neurons in antigen-sensitized guinea pigs demonstrated paracrine signaling between mast cells and the ENS (10, 12). Characteristics of this signaling supported the hypothesis that enteric immunoneural communication is a meaningful defensive event that results in adaptive behavior of the bowel in response to circumstances within the lumen that are threatening to the functional integrity of the whole animal (35-37). The immunoneural defensive system integrates specialized memory and sensory functions of enteric mast cells with capacity of the ENS for interpretation of paracrine signals and programming of intestinal secretory and motor behavior that is adaptive for the well being of the animal.
Immune detection of a sensitizing antigen by enteric mast cells starts a cascade of signaling events beginning with transfer of paracrine information to neural networks in the ENS. The neural networks interpret the paracrine signal as a threat to the integrity of the bowel and respond by "calling-up" from their program library a specific program that coordinates stimulated mucosal secretion and powerful propulsive motility to quickly clear the antigenic threat from the intestinal lumen (35-37). Symptomatic side effects of operation of the immunoneural defense program are watery diarrhea, fecal urgency, and abdominal pain (35).
Several kinds of immune/inflammatory cells are potential sources of
paracrine signals to the ENS. Among these are lymphocytes, macrophages,
polymorphonuclear leukocytes, and mast cells. Most is known about
signaling between mast cells and the elements of the local neural
networks. Paracrine communication between mucosal mast cells and the
colonic submucosal plexus was identified as a contributing factor in
earlier studies of type I hypersensitivity reactions associated with
sensitization to milk in the guinea pig model (6, 7, 17, 36,
37). Antigen cross-linking of immunoglobulin IgE or IgG
antibodies attached at FcRI receptors on the surfaces of mast cells
activates degranulation and the release of a variety of mediators
(6, 21). Paracrine-mediated excitation of submucosal
secretomotor neurons by mast cell products accounts for stimulation of
secretion on exposure of the sensitized bowel to the sensitizing
antigen (6, 17, 26).
Histamine is implicated as a significant messenger in communication
between mast cells and the enteric neural networks.
Electrophysiological studies found that when the guinea pig colon was
sensitized to the parasite Trichinella spiralis or to cow's
milk, reexposure to T. spiralis somatic antigen or
-lactoglobulin evoked a dramatic increase in neuronal firing that
mimicked slow synaptic excitation (10, 12). Excitatory
response to the antigen was significantly reduced but not abolished by
the histamine H2 receptor antagonist cimetidine. Antagonism
by cimetidine suggested that the increased neuronal excitability was
due, in part, to release of histamine. Experimental application of
exogenous histamine to enteric neurons simulates the effects of
degranulation of mast cells seen in the colon of milk-sensitized guinea
pigs (9, 23, 33). Signaling functions of histamine appear
to be related exclusively to mast cells and immunoneural communication,
because histamine is not found in enteric neurons and is unlikely to
function as a neurotransmitter in the ENS (25).
Microelectrode recording from single neurons in the enteric microcircuits revealed that suppression of fast nicotinic synaptic transmission occurred when the sensitized intestine was exposed to the sensitizing antigen (10, 12). Pretreatment with the histamine H3 receptor antagonist burimamide reduced but did not abolish this effect (10, 12). Like the excitatory responses in the cell somas of enteric neurons, suppression of fast nicotinic synaptic transmission seemed also to be mediated partly by mast cell degranulation and release of histamine.
Mucosal mast cells release substances apart from histamine that could potentially influence enteric neurophysiology. Included among these are serotonin, prostaglandins, leukotrienes, cytokines, and possibly others (7, 21). Each of these putative mediators mimics some of the actions of antigenic stimulation when applied to neurons in the submucosal plexus of guinea pig colon (8, 11, 13, 16, 39). Other mast cell mediators are assumed to be coreleased and act on the enteric neurons during antigen exposure. This assumption is on the basis of observations that histamine antagonists often suppress significantly, but do not abolish, the neural effects of antigen exposure in the sensitized guinea pig colon.
Histamine acts at presynaptic inhibitory receptors on sympathetic postganglionic axons to suppress the release of norepinephrine in submucosal ganglia (20). Norepinephrine is an inhibitory neurotransmitter that evokes slow inhibitory PSPs (IPSPs) when released from sympathetic neurons and membrane hyperpolarization when experimentally applied to secretomotor neurons in the submucosal plexus. Release of norepinephrine is a mechanism by which the sympathetic nervous system can exert "braking" action on the excitability of secretomotor neurons and therefore on neurogenic mucosal secretion of H2O, electrolytes and mucus. Release of histamine during exposure to a sensitizing antigen is predicted to stimulate secretion both by excitation of histamine H2 receptors on secretomotor neurons and removal of the braking action of the sympathetic nervous system. Histamine removes sympathetic braking action by activation of presynaptic H3 inhibitory receptors at norepinephrine release sites on sympathetic postganglionic axons.
Studies (10, 12) of communication between the mucosal
immune system and the ENS in sensitized animals are, so far, limited to
the colonic submucosal plexus. An aim of the present study was to
extend investigation of immunoneural communication to the myenteric
plexus of the gastric corpus and antrum and to the submucosal plexus of
the small bowel. An allied aim was to identify specific mediators other
than histamine that might be released on antigen challenge and be
involved in immunoneural signaling. A third aim was to investigate
effects of -lactoglobulin exposure on noradrenergic inhibitory
neurotransmission in small intestinal submucosal neurons of
milk-sensitized guinea pigs.
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MATERIALS AND METHODS |
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Sensitization procedure.
The general protocol was to induce gastrointestinal hypersensitivity to
milk protein by feeding cow's milk to one group of guinea pigs in
parallel with an age-matched, nonsensitized control group. Two-week-old
male albino guinea pigs of the Hartley strain were used. The
experimental group was sensitized by ingestion of cow's milk in place
of drinking water over a 3-wk period, as described by others (1,
6, 10). Tap water replaced milk for 1-3 days before the
animals were killed and set up for electrophysiological recording from
the enteric neurons during exposure to -lactoglobulin in vitro.
Nonsensitized animals drank only tap water during the 3 wk and were
never exposed to milk protein in the diet. Both groups of animals were
fed standard guinea pig chow. All protocols were reviewed and approved
by the Ohio State University Laboratory Animal Care and Use Committee.
Tissue preparation.
After 3 wk of milk exposure, preparations of the myenteric plexus from
the gastric corpus and antrum or the submucosal plexus of the ileum
were prepared by microdissection and setup in perfused tissue chambers
for neuronal electrophysiological recording. Electrophysiological behavior of the neurons in the milk-sensitized and nonsensitized tissues was recorded, followed by recording of the effects of application of -lactoglobulin. Specificity of the effects of the
milk antigen was tested by application of ovalbumin, which served as an
unrelated antigen.
Electrophysiological recording.
Our methods for intracellular recording from the myenteric and
submucosal plexuses are described in detail elsewhere (27, 28,
31, 32, 40). Transmembrane electrical potentials were recorded
with conventional "sharp" microelectrodes filled with 2% biocytin
in 2 M KCl containing 0.05 M Tris buffer (pH 7.4). Resistances of the
electrodes ranged between 80 and 120 M. The preamplifier (model
M767; World Precision Instruments, Sarasota, FL) was equipped with a
bridge circuit for intraneuronal injection of electrical current.
Constant current rectangular pulses were driven by a stimulator (model
SD9; Grass Instrument Division, Astro-Med, Warwick, RI). Electrometer
output was amplified and observed on an oscilloscope (model 5113;
Tektronics, Beaverton, OR). Synaptic potentials were evoked by focal
electrical stimulation of interganglionic fiber tracts with electrodes
made from 20-µm-diameter Teflon-insulated platinum wire connected
through model SIN5 stimulus-isolation units to model S48 stimulators
(Grass Instrument Division, Astro-Med). Fast excitatory postsynaptic
potentials (EPSPs) were recorded with an expanded time scale and
displayed through a real-time digital oscilloscope (model TDS210;
Tektronics) and output to a laserjet printer. Chart records were made
on Astro-Med thermal recorders. Amplitudes of action potentials on some
records were blunted by the low-frequency response of the recorders.
All data were recorded on videotape for later analysis.
Immunohistochemical methods. At the end of each recording session, the marker dye biocytin was injected into the impaled neurons from the recording electrodes by the passage of hyperpolarizing current (0.5 nA for 10-30 min). The anal end of the preparations was marked, and the tissue was transferred into a disposable chamber filled with fixative containing 4% formaldehyde plus 15% of a saturated solution of picric acid and kept at 4°C overnight. The preparations were cleared in three changes of dimethyl sulfoxide and three 10-min washes with PBS. After they were cleared, the preparations were reacted with fluorescein streptavidin (1:100) for 1 h and examined under a Nikon Eclipse E600 fluorescent microscope with appropriate filters.
Neurochemical coding of the neurons that responded to antigenic stimulation was determined by first reacting the preparations with streptavidin coupled to Texas Red to reveal biocytin fluorescence and assess neuronal morphology. They were then processed for immunohistochemical localization of vasoactive intestinal peptide (VIP) and choline acetyltransferase (ChAT) immunoreactivity. For VIP localization, rabbit anti-VIP (1:250) (code IHC7161, Peninsula, Belmont, CA) was used; for ChAT, goat anti-ChAT (1:100) (code AB144P, Chemicon, Temecula, CA) was used. The preparations were then incubated with secondary antibodies labeled with fluorescein. Fluorescent labeling was examined under a Nikon Eclipse E600 fluorescent microscope equipped with appropriated filters and a SPOT-2 chilled color and black and white digital camera (Diagnostic Instruments, Sterling Heights, MI).Drug application.
Actions of pharmacological agents and -lactoglobulin antigen were
studied by pressure microejection or by application in the superfusion
solution. Micropipettes (10-µm-tip-diameter) manipulated with the tip
close to the impaled neurons were used to microeject the substances.
Pressure pulses of nitrogen with predetermined force and duration were
applied to the micropipettes through electronically controlled solenoid
valves. The concentrations of antagonists selected for the work were
based on pilot studies and the literature as follows: cimetidine
(9), BRL 24934 (11), thioperamide (20), piroxicam (15), MDL 72222 (41).
Chemicals.
Chemical agents used and sources were as follows: acetylcholine,
anti-guinea pig IgG, biocytin, cimetidine, compound 48/80, ketotifen,
-lactoglobulin, ovalbumin, piroxicam and pyrilamine were obtained
from Sigma (St. Louis, MO). PGE2, leukotriene
C4 (LTC4) and caffeic acid were from Cayman
Chemicals (Ann Arbor, MI). Thioperamide and MDL 72222 were from Tocris
Cookson (Ballwin, MO). BRL 24924 was from SmithKline Beecham
(Betchworth, UK). Histamine, R-
-methylhistamine and norepinephrine
were from RBI (Matick, MA). Fluorescein and Texas Red straptavidin were
from Vector (Burlingame, CA). VIP and ChAT antiserum were from Chemicon
(Temecula, CA).
Data analysis. Data are expressed as means ± SE; n-values refer to the number of neurons. Concentration-response relationships were constructed using the following least-squares fitting routine: V = Vmax/[1 + (EC50/C)nH], where V is the observed response, EC50 is the concentration that induces the half-maximal response, and nH is apparent Hill coefficient. The graphs were drawn by averaging results from all experiments and fitting to a single concentration-response curve by using Sigma Plot software (SPSS, Chicago, IL). Paired or unpaired Student's t-test was used to determine statistical significance. P values of <0.05 were considered statistically significant.
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RESULTS |
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Gastric neurons.
Results were obtained for 29 gastric myenteric neurons consisting of 16 in the corpus and 13 in the antrum from 10 guinea pigs that were
sensitized by ingestion of cow's milk. Results for nonsensitized
H2O controls were obtained for 24 neurons consisting of 10 neurons in the corpus and 14 in the antrum from 10 animals. Neurons
were classified electrophysiologically as gastric I, gastric II,
gastric III, or AH-type according to criteria described previously (27, 28, 31, 32). Ratios for the percentages of each of the specific classes of gastric neurons found in milk-sensitized preparations relative to nonsensitized preparations were: 1)
31:33% for gastric I, 2) 48:42% for gastric II, and
3) 21:25% for gastric III. No AH-type neurons were found in
either milk-sensitized or nonsensitized preparations. Mean resting
membrane potentials for the three types of gastric neurons in
milk-sensitized guinea pigs were not significantly different from the
controls (Table 1). No spontaneous
discharge of action potentials was found in either the milk-sensitized
preparations or the nonsensitized controls.
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Small intestinal submucosal neurons.
Results were obtained for 54 ileal submucosal neurons from 28 guinea
pigs sensitized by cow's milk and compared with 40 ileal submucosal
neurons from 24 milk-free animals. The submucosal neurons were
classified electrophysiologically as S- and AH-type according to the
criteria of Mihara (22). Ratios of percentages of neuronal types impaled in milk-sensitized relative to nonsensitized preparations were 89:85% for S-type and 11:15% for the AH-type. The mean resting membrane potential for S- and AH-type neurons in the milk-sensitized ileum was not significantly different from the controls (Table 1).
Spontaneous discharge of action potentials was found in neurons from
both sensitized and nonsensitized preparations. Incidence of
spontaneous discharge was highest in the milk-sensitized preparations with 29.6% (16 of 54) of the neurons firing spontaneously compared with the occurrence of spontaneous discharge in 7.5% (3 of 40) of the
neurons in nonsensitized preparations. For preparations from
milk-sensitized animals, application of -lactoglobulin (0.1-10 µM) in the superfusion solution evoked a concentration-dependent depolarization of the membrane potential in 41 of 48 S-type and 6 of 6 AH-type neurons (Fig. 2).
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Neurochemical identification.
Immunofluorescence was used to help assess the functional identity of
the neurons. Data on immunoreactivity for VIP and ChAT were obtained
for 23 submucosal neurons with electrophysiological responses to
antigen challenge. Data on immunoreactivity for VIP and CHAT were
obtained for 23 submucosal ganglion cells. Ten S-type neurons with
Dogiel type I morphology were examined for VIP immunoreactivity. Immunoreactivity for VIP was expressed by 4 of the 10 neurons (Fig. 4,
A-C). Thirteen
S-type neurons with Dogiel type I morphology were tested for ChAT
immunoreactivity. Immunoreactivity for ChAT was expressed in 7 of the
13 neurons (Fig. 4, D-F).
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Mast cell stabilization.
Ketotifen was used as a pharmacological tool to investigate further the
involvement of mast cell degranulation in the neuronal responses of
sensitized preparations to application of -lactoglobulin. Ketotifen
is a drug with anti-allergic and anti-inflammatory properties. Its
mechanism of action is stabilization of mast cell membranes and
prevention of release of histamine and other mediators (14, 18).
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Pharmacology of antigen-evoked responses.
Histaminergic and serotonergic receptor antagonists and drugs that
suppress prostaglandin and leukotriene synthesis were used to test the
hypothesis that histamine, prostaglandins, and leukotrienes were
paracrine mediators of -lactoglobulin-evoked neuronal responses found in the milk-sensitized preparations. Application of cimetidine, a
histamine H2 receptor antagonist, significantly reduced the depolarizing responses evoked by
-lactoglobulin in sensitized preparations (Fig. 6). The membrane
potential in 10 neurons was depolarized by 13.7 ± 2.0 mV after
the addition of 5 µM
-lactoglobulin to the superfusion solution.
The presence of 10 µM cimetidine reduced the depolarization to
44.2 ± 4.8% of the control values (P < 0.001).
Application of the H1 receptor antagonist pyrilamine (10 µM) or the H3 receptor antagonist thioperamide (10 µM)
had no effect on the depolarizing responses to 5 µM
-lactoglobulin in six neurons (P > 0.05).
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Histamine, PGE2, and LTC4.
Results obtained with histamine H2 receptor antagonist and
drugs expected to suppress prostaglandin or leukotriene synthesis suggested that endogenous histamine, prostaglandins, and leukotrienes may partially mediate the excitatory responses to antigen challenge in
small intestinal neurons from milk-sensitized guinea pigs. To qualify
as mediators, the actions of histamine, prostaglandins, and
leukotrienes should mimic the effects -lactoglobulin when they are
applied to the neurons of sensitized preparations. Application of
histamine (10 µM), PGE2 (100 nM), and LTC4
(100 nM) evoked membrane depolarization and enhanced excitability in
the same neurons that responded to antigen challenge (Fig.
7). Histamine, PGE2, and
LTC4 also evoked membrane depolarization and enhanced excitability in neurons of the nonsensitized small intestine, whereas
-lactoglobulin was without effect (Fig. 7). In contrast to the small
intestine, neither histamine in 21 neurons, PGE2 in 15 neurons, nor LTC4 in 12 neurons evoked depolarizing
responses when applied to gastric neurons in milk-sensitized or
nonsensitized preparations (data not shown).
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Sympathetic neurotransmission.
Short-train electrical stimulation (20 Hz, 0.2 s or less) applied
to the interganglionic fiber tracts evoked slow inhibitory postsynaptic
potentials (IPSPs) in most of the submucosal neurons from
milk-sensitized and nonsensitized guinea pig small intestine. Past
experience suggested that the IPSPs were mediated by the release of
norepinephrine from sympathetic nerve terminals and its action at
2-adrenergic receptors (20, 24).
Application of 5 µM
-lactoglobulin in the superfusion solution
reversibly suppressed the IPSPs by 60.7 ± 3.4% of control in 14 of 17 neurons from sensitized preparations (Fig.
8A). No suppression of IPSPs occurred on application of the antigen to preparations from
nonsensitized animals.
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DISCUSSION |
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Guinea pigs in the present study developed an allergic reaction to
milk protein that was manifest in the ENS of the small intestine
without coincident signs of food allergy in the ENS of the stomach. In
this respect, the stomach differed from the ENS of the guinea pig colon
that, like the small bowel, develops allergic responses to milk
protein(10). Absence of antigenic sensitization in the
stomach was unexpected, because mast cells occupy the gastric mucosa.
No expression of functional receptors for histamine, prostaglandins, or
leukotrienes by gastric enteric neurons was the most plausible explanation for failure to find an allergic reaction in the stomach. Absence of expression of receptors for the mast cell mediators was
reflected by lack of any action of exogenously applied histamine, PGE2, or LTC4. This contrasts with the ENS of
the small and large intestine where exposure to any one of these three
mediators depolarizes the resting membrane potential and augments
neuronal excitability. Aside from failure of expression of receptors
for the mast cell mediators, the possibility that gastric mast cells do
not express FcRI receptors for sensitizing antibodies to
-lactoglobulin cannot be ruled-out as part of an explanation for
failure to find signs of allergic sensitization in the stomach.
Involvement of mast cells. Several immune/inflammatory cell types including polymorphonuclear leukocytes, lymphocytes, macrophages, and dendrocytes in addition to mast cells are present at all levels of the alimentary canal and are often found in close association with neural elements of the ENS (29, 30). Histoanatomical and immunophysiological observations suggest that cells of the enteric immune system are strategically positioned to establish a first line of defense against foreign invasion at a vulnerable interface between the body and the outside environment. The cell type involved in antigenic responses in antigen-sensitized guinea pigs in the present and in earlier studies (10, 12) appears to be mast cells that synthesize and store histamine and other paracrine mediators. Mast cell involvement is supported by the finding, in the present study that application of a mast cell degranulating agent (i.e., compound 48/80) or anti-IgG evoked neuronal electrical behavior in milk-sensitized preparations that mimicked the effects of antigenic stimulation. Moreover, ketotifen, which is a mast cell stabilizing drug, attenuated responses to antigen challenge in milk-sensitized preparations.
Mast cell mediators.
Microdissected preparations in the present study consisted of myenteric
or submucosal plexus, mast cells, and other cellular elements in the
connective tissue matrix. Exposure of the sensitized mast cells to
-lactoglobulin was expected to trigger degranulation and release of
a variety of mediators including histamine, prostaglandins, leukotrienes, cytokines, nitric oxide, and proteases, all of which are
known either to be stored in cytoplasmic granules or to be newly
synthesized on antigenic stimulation. Observations in the present study
are consistent with previous work (10) that identified histamine as one of the mediators that stimulate neuronal excitability. Mast cells are most strongly implicated as the source of endogenous histamine released by antigenic stimulation, because histamine is not
generally found in enteric neurons (25). Elevated levels of endogenous histamine available for action at receptors on submucosal neurons account for part of the excitatory responses found in milk-sensitized small intestine after challenge with
-lactoglobulin. Experimental application of histamine to enteric neurons of both normal and milk-sensitized guinea pigs resulted in membrane
depolarization and augmentation of excitability that resembled the
effects of antigenic challenge. Most evidence suggests that the
excitatory histaminergic receptors on enteric neurons of the guinea pig
belong to the H2 receptor subtype (9, 23).
Evidence from the present study pointed to the H2 receptor
as the involved subtype, because cimetidine, but not pyrilamine or
thioperamide, suppressed antigen-evoked responses in the
milk-sensitized preparations.
Submucosal plexus.
Neural networks in the submucosal plexus are an important part of the
neurophysiological control of intestinal secretion. These networks are
synaptically "wired" with neurons that can be differentiated
according to their electrical and synaptic behavior, morphology, and
neurochemistry. Our immunohistochemical results revealed that the
submucosal neurons with elevated excitability during antigen exposure
also expressed immunoreactivity for VIP or ChAT. Most evidence suggests
that neurons with VIP immunoreactivity and slow IPSPs in the guinea pig
submucosal plexus are secretomotor neurons (4). This
population of secretomotor neurons innervates the intestinal crypts of
Lieberkühn and releases VIP as a neurotransmitter, which
stimulates the secretion of H2O, electrolytes, and mucus. Submucosal neurons with ChAT immunoreactivity are identified as either
a second population of secretomotor neurons or as interneurons (4). Results of the present study indicate that
secretomotor neurons were among those neurons with elevated
excitability during antigen exposure in the sensitized preparations.
Elevation of secretomotor neuronal excitability would be expected to
enhance mucosal secretion. Excitation of the secretomotor neurons may be the neural correlate of the enhanced mucosal secretion reported to
occur in guinea pigs sensitized to cow's milk and later challenged with -lactoglobulin (17, 34). Augmented stimulation of
secretomotor neurons can be implicated in the secretory diarrhea
associated with food allergy (35, 37).
Sympathetic neurotransmission.
Electrical stimulation of sympathetic postganglionic axons in the
submucosal plexus evokes slow IPSPs in the cell bodies of secretomotor
neurons (20, 38). The slow IPSPs are mediated by release
of norepinephrine and its action at 2-adrenoceptors on
the secretomotor neurons (24, 38). Aside from augmented neuronal excitability, antigenic exposure in milk-sensitized
preparations resulted in inhibition of sympathetic neurotransmission as
revealed by suppression of stimulus-evoked slow IPSPs. On the other
hand, we found that antigen exposure did not suppress slow IPSP-like responses evoked by micropressure "puffs" of norepinephrine.
Failure of antigen exposure to suppress the IPSP-like responses to
norepinephrine, while suppressing slow noradrenergic IPSPs in the same
neuron, satisfies criteria for presynaptic inhibition of norepinephrine release from the sympathetic nerve terminals. Presynaptic inhibition at
slow noradrenergic synapses is a common occurrence in the enteric microcircuits during exposure to inflammatory mediators, such as
bradykinin (15), interleukin-1
, interleukin-6
(39), and enterotoxins (38). Presynaptic
inhibition is also one of the actions of histamine on enteric neurons.
The presynaptic histaminergic receptor behaves pharmacologically like
the histamine H3 subtype and is selectively blocked by the
H3 receptor antagonist thioperamide (20).
PGE2, but not LTC4, also acts at presynaptic
inhibitory receptors to suppress noradrenergic IPSPs (16).
Our finding that the 5-lipoxygenase inhibitor, caffeic acid, did not
block
-lactoglobulin-evoked suppression of the slow IPSPs suggests that unlike histamine or prostaglandins, release of leukotrienes does
not affect noradrenergic neurotransmission.
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ACKNOWLEDGEMENTS |
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National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-37238 and DK-57075 and a generous gift from Evelyn Walker, Palo Alto, CA, supported this study.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. D. Wood, Dept. of Physiology and Cell Biology, 304 Hamilton Hall, 1645 Neil Ave., Ohio State University, Columbus, OH 43210 (E-mail wood.13{at}osu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
September 11, 2002;10.1152/ajpgi.00241.2002
Received 21 June 2002; accepted in final form 6 September 2002.
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