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Am J Physiol Gastrointest Liver Physiol 293: G850-G856, 2007. First published August 16, 2007; doi:10.1152/ajpgi.00277.2007
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NEUROREGULATION AND MOTILITY

Mast cell-mediated long-lasting increases in excitability of vagal C fibers in guinea pig esophagus

Shaoyong Yu,1 Marian Kollarik,2 Ann Ouyang,1 Allen C. Myers,2 and Bradley J. Undem2

1Pennsylvania State University, College of Medicine, Hershey, Pennsylvania and 2Johns Hopkins School of Medicine, Baltimore, Maryland

Submitted 18 June 2007 ; accepted in final form 14 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Several esophageal pathologies are associated with an increased number of mast cells in the esophageal wall. We addressed the hypothesis that activation of esophageal mast cells leads to an increase in the excitability of local sensory C fibers. Guinea pigs were actively sensitized to ovalbumin. The mast cells in the esophagus were selectively activated ex vivo by superfusion with ovalbumin. Action potential discharge in guinea pig vagal nodose esophageal C-fiber nerve endings was monitored in the isolated (ex vivo) vagally innervated esophagus by extracellular recordings. Ovalbumin activated esophageal mast cells, leading to the rapid release of ~20% of the tissue histamine stores. This was associated with a consistent and significant increase in excitability of the nodose C fibers as reflected in a two- to threefold increase in action potential discharge frequency evoked by mechanical (increases in intraluminal pressure) stimulation. The increase in excitability persisted unchanged for at least 90 min (longest time period tested) after ovalbumin was washed from the tissue. This effect could be prevented by the histamine H1 receptor antagonist pyrilamine, but once the increase in excitability occurred, it persisted in the nominal absence of histamine and could not be reversed even with large concentrations of the histamine receptor antagonist. In conclusion, activation of esophageal mast cells leads to a pronounced and long-lived increase in nociceptive C-fiber excitability such that any sensation or reflex evoked via the vagal nociceptors will likely be enhanced. The effect is initiated by histamine acting via H1 receptor activation and maintained in the absence of the initiating stimulus.

histamine; visceral pain; allergy; nociceptor

MAST CELLS ARE PRESENT IN the esophagus, and their numbers are consistently elevated in esophageal pathologies including eosinophilic esophagitis, gastroesophageal reflux disease associated with food allergy, Barrett's esophagus, and squamous cell carcinoma of the esophagus (3, 14, 16). Beyond the histological assessment of mast cell numbers (7, 22), however, there is relatively little information on the physiological consequence of mast cell activation in the esophagus. In other tissues mast cell activation has been shown to modulate the activity of autonomic (18, 22), enteric (4, 13, 17, 19), and sensory nerves (8, 9). On the basis of the location in the esophageal wall (7, 22), we hypothesize that sensory nociceptive nerves will likely be within the sphere of influence of mediators released from mucosal mast cells.

Sensory nerves that provide the organism with a sense of injury or impending injury are referred to as nociceptors. Generally these sensory fibers are unmyelinated capsaicin-sensitive C fibers. They are considered polymodal in that many types of stimuli can elicit action potential discharge, with the common feature of the stimuli being that they are associated with tissue inflammation or are potentially damaging to the tissue. The mechanically induced activation of nociceptors in the esophagus, for example, is distinguished from the low-threshold mechanosensitive A fibers (tension receptors) in that the action potential discharge frequency continues to increase in the nociceptors as the intraluminal pressure extends into the noxious range. On the basis of these criteria, the guinea pig esophagus is innervated by at least three separate types of nociceptive-like sensory nerves. There are two types of vagal nociceptive C fibers, one with cell bodies situated in the jugular ganglion, the other with cell bodies situated in the nodose ganglion (29). In addition there are spinal nociceptors innervating the esophagus with cell bodies situated in the dorsal root ganglion (20, 21).

In this study we specifically address the hypothesis that activation of esophageal mast cells modulates the excitability of nodose nociceptive C fibers in the guinea pig esophagus. We take advantage of the immediate hypersensitivity reaction to selectively activate the tissue mast cells with an antigen challenge ex vivo. The data support the conclusion that mast cell activation does not overtly evoke action potential discharge in these nerves but causes a substantial and persistent increase in their responsiveness to mechanical and chemical activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Male Hartley guinea pigs (Hilltop Laboratory Animals, Scottsdale, PA) weighing 100–300 g were used. All experiments were approved by the Johns Hopkins Animal Care and Use Committee and Pennsylvania State University Animal Care and Use Committee.

Sensitization and allergen challenge. Guinea pigs were sensitized with three intraperitoneal injections of ovalbumin (OVA, 10 mg/kg in 0.9% saline) every 48 h. Three weeks after the last injection, guinea pigs were killed by CO2 inhalation and exsanguinations and the tissue was harvested. Antigen challenge was performed by using OVA (10 µg/ml) diluted in Krebs bicarbonate solution [KBS, composed of (in mM) 118 NaCl, 5.4 KCl, 1.0 NaH2PO4, 1.2 MgSO4, 1.9 CaCl2, 25.0 NaHCO3, and 11.1 dextrose, and gassed with 95% O2-5% CO2].

Histamine release assay. Esophageal specimens (80–120 mg) were equilibrated in oxygenated KBS (37°C) for 60 min, with the buffer replaced at 15-min intervals. Following this equilibration period the tissue were treated with buffer vehicle for 20 min, at which time the supernatant solution was obtained for spontaneous release analysis. The specimens were then treated with OVA (10 µg/ml) for 20 min to measure the OVA-induced histamine release. The supernatant from all samples was collected and stored at –70°C. The tissue pellet was triturated in the presence of 0.4 N perchloric acid to obtain the remaining tissue content of histamine. Histamine was assayed by the automated fluorometric assay as previously described (24).

In separate experiments the kinetics of histamine release was measured from the isolated whole esophagus in a preparation similar to that used for extracellular recordings. The whole esophagus was constantly superfused with oxygenated KBS (37°C) at the rate 2 ml/min for a 60-min equilibration period. After the equilibration the superfusate was collected for analysis in 1-min intervals. After two samples were obtained for spontaneous release analysis, OVA (10 µg/ml) was added to the superfusion solution, and the superfusate was collected for an additional 30 min.

Immunofluorescent-labeling of mast cell tryptase. The dissected esophagus from sensitized animals was immersed in the KBS-containing antigen (OVA, 10 µg/ml) for 20 min to induce antigen-mediated mast cell degranulation. This was compared with control tissues exposed to Krebs buffer solution without antigen.

The esophageal tissues were first fixed in 4% formaldehyde in phosphate-buffered saline (PBS) overnight at 4°C. The tissues were then rinsed in PBS, cryoprotected with 18% sucrose in PBS for 18–24 h. Afterward, the tissues were covered with optimum cutting temperature mounting medium and frozen on dry ice. The tissues were cut in serial sections of 12-µm thickness on a cryostat, collected on silane-coated slides, and air dried for 30 min.

Sections were incubated with blocking solution containing 1% bovine serum albumin (BSA), 10% normal goat serum, and 0.1% Tween 20 in PBS for 60 min. The tissues were incubated 12–24 h at 4°C with primary antibody in PBS containing 0.1% Triton X-100 (TX) and 1% BSA (PBS-TX-BSA). The primary antibody was polyclonal rabbit antiserum to tryptase (diluted 1:200; Chemicon International, Temecula, CA). Slides were then washed in PBS-TX-BSA and incubated with goat anti-rabbit labeled with Alexa 488 (diluted 1:100, Molecular Probe) in PBS-TX-BSA for 2 h at room temperature. The sections were then rinsed with PBS and with glycerin buffered with phosphate to pH 8.6, coverslipped. The slides were analyzed and counted by a conventional epifluorescence microscopy (Olympus DX60) with the filters set to allow visualization of Alexa 488 (excitation filter 450–480 nm, barrier filter 500–515 nm).

Extracellular single fiber recordings from the vagal afferent nerves innervating the esophagus. The isolated, vagally innervated, perfused esophagus was set up as previously described (29). The esophagus and trachea were dissected with intact bilateral extrinsic vagal innervation (including jugular and nodose ganglia). The tissue was pinned in a small Sylgard-lined Perspex chamber filled with Krebs solution (35°C) containing indomethacin (3 µM). The chamber had two compartments: the esophagus with attached trachea (to support the recurrent laryngeal nerves) and the vagus were pinned in the tissue compartment, and the rostral aspect of the vagus nerves including the nodose and jugular ganglia were pinned in the recording compartment. The two compartments were separated by a silicone grease plug and were separately superfused with KBS (pH 7.4; 35°C; 4–6 ml/min). Polyethylene tubing was inserted 3–5 mm into the cranial and caudal esophagus and secured for perfusion.

The pressure in the fluid (KBS)-filled esophagus was measured with a differential pressure transducer connected in series to the esophagus and recorded simultaneously with neural activity by the chart recorder (TA240S, Gould, Valley View, OH).

Isobaric esophageal distension for 20 s with an intraluminal pressure of 10–100 mmHg separated by at least 60 s was used to determine the distension pressure-nerve activity relationship of an esophageal afferent fiber. Distension with a pressure of 10 or 60 mmHg (20 s) was routinely used to assess the viability and mechanical responsiveness of an afferent fiber during experiments.

Extracellular recordings were performed as described before (29). An aluminosilicate glass microelectrode (pulled with a Flaming-Brown micropipette puller, Sutter Instrument, Novato, CA) and filled with 3 M sodium chloride (electrode resistance 2 M{Omega}). The electrode was placed into an electrode holder connected directly to the headstage (A-M Systems, Everett, WA). A return electrode of silver-silver chloride wire and earthed silver-silver chloride pellet were placed in the perfusion fluid of the recording compartment. The recorded signal was amplified (Microelectrode AC amplifier 1800, A-M Systems) and filtered (low cutoff 0.3 kHz; high cutoff 1 kHz), and the resultant activity was displayed on an oscilloscope (TDS 340, Tektronix, Beaverton, OR) and the chart recorder. The data were stored and analyzed on a Macintosh computer using the software TheNerveOfIt (sampling frequency 33 kHz; PHOCIS, Baltimore, MD) and further processed by using spreadsheet software (Microsoft Excel 98).

To identify a vagal afferent nerve fiber projecting to the esophagus, the recording electrode was positioned in the nodose ganglion (left or right) via a micromanipulator. A distension-sensitive unit was identified when esophageal distension (with a rapid increase in intraluminal pressure to 100 mmHg for 5 s) evoked action potential discharge. The serosal surface of the esophagus was then searched with a punctate mechanical probe (Von Frey hair, 1 mN, filament diameter < 0.5 mm) applied to the tissue, with a firm probe (outside diameter ~1 mm) having already been inserted into the esophageal lumen. A mechanosensitive receptive field was located when the punctate stimulus evoked discharge of action potentials with waveforms identical to the action potentials evoked by distension. The receptive field was then stimulated electrically (pulse duration = 1 ms, frequency = 1 Hz) with a concentric electrode inserted into the esophagus with the tip positioned at the site of the mechanosensitive receptive field. The initial voltage (100 V) was gradually reduced to the lowest voltage (threshold voltage) at which each stimulation pulse was followed by a single action potential (30–90 V for most afferent nerve fibers recorded). The waveforms of the electrically evoked action potentials were identical to those induced by distension and the punctate mechanical stimulus. Conduction time was measured as the time between the stimulation pulse and the action potential (visualized by oscilloscope). Variability of conduction time (during the train stimulation, 10 Hz, 10 s) <3 ms indicated direct electrical stimulation (indirect activation of a unit by electrically evoked muscle contractions was readily discernable by high variability >20 ms of conduction time). Conduction velocity was calculated by dividing the length of the approximated nerve pathway by conduction time. The nerve fiber was considered a C fiber if it conducted action potentials at <1 m/s.

The chemicals diluted in KBS solution were delivered to the esophagus in the external perfusate for 10–20 min. The nerve activity (action potential discharge) was monitored continuously and analyzed in 1-s bins (yielding the number of action potentials in each second, Hz). The peak frequency (Hz) was defined as the maximal frequency of action potential discharge. The response to the particular stimulus was considered positive when the stimulus evoked action potential discharge with a peak frequency of at least 3 Hz (in the fibers with no baseline activity) or a peak frequency at least three times the frequency of baseline activity. The time to onset of the response was defined as the time elapsed between addition of the drug to the tissue to the onset of the action potential discharge. The agonists capsaicin and {alpha},beta-methylene-ATP were used at the end of each experiment at their maximally effective concentration of 1 and 30 µM, respectively. The stock solutions of capsaicin (10 mM in ethanol), pyrilamine (10 mM in water), and {alpha},beta-methylene-ATP (10 mM in water), were stored at –20°C and diluted in KBS to final concentration on the day of use. All drugs were purchased from Sigma-Aldrich.

Evaluation of esophageal tissue mechanics. The effect of histamine on the relationship between tension in the esophageal wall and the esophageal diameter was evaluated using tissue bath experiments. Two parallel steel wires were inserted into the 2–3 mm long (L, axial length) circular piece (ring) of esophagus, one wire stationary and the other connected to a calibrated micromanipulator and force transducer. The tissue was placed in KBS (35°C). The distance between the wires (dw) was positioned (tolerance of ±0.005 mm) to distend the esophageal ring with a force (F) equivalent to calculated tension in the esophageal wall at pressure 5, 10, and 30 mmHg for 20 s each. This protocol was repeated after 30 min of incubation with histamine (100 µM). Because the esophageal ring is stretched its internal circumference (ci) equals two times the distance between the wires (ci = 2 x dw). An intraesophageal pressure that would cause the same degree of distention as the force applied to distend the ring was calculated from the equation: pressure = force/(internal circumference x axial length of the ring), P = F/(ci x L). Data are presented as the relationship between the pressure (P) and esophageal diameter (d, obtained by dividing internal circumference by {pi}, d = ci/{pi}).

Data analysis. Histamine release was expressed either as nanograms per milliliter per minute or as the percentage of the total histamine content of the tissue. Total numbers of tryptase-positive mast cells were counted per cross section of the esophagus. The distension-evoked nerve response was quantified as the peak frequency of the action potential discharge presented as means ± SE. For statistical analysis of the change in the overall mechanical response we used the area under the curve (AUC) calculated using standard geometrical formulas with the resultant formula (the units are omitted): AUC = 20 x (pf10+pf30) + 30 x (pf30+pf60) where pf10, pf30, and pf60 are the peak frequencies at the distention pressures 10, 30, and 60, respectively, and the coefficients 20 and 30 refer to difference between tested pressure points used (i.e., 20 mmHg = 30–10 mmHg). The areas under the curve were then compared by paired t-test in paired experiments and one-way ANOVA for repeated measurements. The response to chemical agents was quantified as the total number of action potentials discharged in response to a drug after the spontaneous activity (if present) was subtracted. The chemical response is presented as means ± SE and compared by paired t-test. For all experiments the significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Esophageal mast cell activation. A histological assessment revealed that mast cells are consistently situated in the lamina propria of the guinea pig esophagus (Fig. 1). When the esophagus of actively sensitized animals was exposed ex vivo to 10 µg/ml of OVA (x30 min), the number of mast cells per cross section under high-powered field (x200) identified by use of anti-tryptase antibody decreased from 40 ± 5 to 4 ± 2 (P < 0.001, n = 4). This degree of decrease in mast cell number is consistent with activation and degranulation of nearly all the tissue mast cells (Fig. 1).


Figure 1
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  		Fig. 1. Histological assessment of the mast cells in the guinea pig esophagus. The mast cells were stained by anti-tryptase antibody. A: section from control tissue. B: section from tissue exposed to ovalbumin (OVA; 10 µg/ml, 30 min). C: quantification of the tryptase-positive cells. LP, lamina propria; EP, epithelium.

 
Histamine release was also quantified as an assessment of mast cell activation. We found that, in control animals, the esophagus contains 4.9 ± 0.5 µg histamine/g tissue wet wt; for comparison the guinea pig trachea contains ~10 µg/g wet wt (25). Exposing the sensitized esophagus to 10 µg/ml OVA resulted in the release of 17 ± 1% (n = 6) of the total tissue stores of histamine.

Nodose C-fiber excitability. The conduction velocity of the C fibers evaluated averaged 0.7 ± 0.02, n = 75. Exposing the actively sensitized esophagus to OVA (ex vivo) was seldom associated with overt action potential discharge in nodose C fibers (n = 9). In four of nine fibers there was a slight increase in baseline activity, but this did not increase beyond 0.5 Hz.

As we previously reported (29), nodose C-fiber can be reproducibly stimulated by step increases in intraluminal pressure. The action potential discharge frequency increased in a graded fashion with step increases in intraluminal pressure of 10, 30, and 60 mmHg (see representative trace in Fig. 2A). Exposing the actively sensitized esophagus to OVA (30 min) caused a two- to threefold increase in the peak frequency and total number of action potentials in response to increases of intraluminal pressure (Fig. 2). In tissues obtained from nonsensitized (control) animals, OVA had no effect on the response of the C fibers to increases in intraluminal pressure (Fig. 2).


Figure 2
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  		Fig. 2. Activation of the mast cells increases mechanical excitability of vagal nociceptive C fibers in the guinea pig esophagus. A: representative traces of the extracellular single nerve fiber recordings from an isolated innervated esophagus. In the OVA-sensitized guinea pigs superfusion with OVA (10 µg/ml, 30 min) enhanced the C-fiber response (top traces) to esophageal distention with defined pressure (bottom traces). B: activity expressed as a total number of action potentials induced by 20 s distention to defined pressure in OVA-sensitized (n = 9) and naive (n = 6) guinea pigs. C: activity expressed as a peak frequency of the action potential discharge induced by 20-s distention to defined pressure in OVA-sensitized (n = 9) and naive (n = 6) guinea pigs. **P < 0.01, ***P < 0.001; the responses were compared by analyzing the area under the curve (see MATERIALS AND METHODS).

 
Next, the duration of the OVA-induced increase in nodose C-fiber responsiveness was evaluated. Washing the tissue free of OVA for 30, 60, and 90 min (longest time period evaluated) failed to reverse the OVA-induced effect (Fig. 3). We also measured the time course of the antigen-induced histamine release from the tissues. The histamine reached a peak within 2 min of OVA exposure then rapidly declined to near basal levels by 20–30 min (Fig. 3).


Figure 3
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  		Fig. 3. Activation of mast cells induces persistent increase of the esophageal C-fiber mechanical excitability that outlasts histamine release. The time course of the OVA-induced histamine release from the esophageal tissue (n = 3) and the time course of the increase of the mechanical sensitivity (n = 5; shown as the fold increase in the peak action potential discharge evoked by distention with pressure 60 mmHg). OVA (10 µg/ml) was superfused over the tissue for 30 min starting at time 0 min (*P < 0.05, ANOVA).

 
Pretreating the tissue with the competitive histamine H1 receptor antagonist pyrilamine (1 µM) for 30 min prevented the OVA-induced increase in nodose C-fiber responsiveness (Fig. 4A). By contrast, exposure to the same dose of pyrilamine for 30 min after the OVA-induced effect was established had no effect on the response (i.e., failed to reverse the OVA-induced effect) (Fig. 4B).


Figure 4
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  		Fig. 4. The selective histamine H1 receptor antagonist pyrilamine prevents OVA-induced increase of the C-fiber excitability but does not reverse the effect once established. A: pretreatment with pyrilamine (1 µM, for 30 min prior to OVA) prevents the OVA-induced increase of the C-fiber mechanical excitability in the OVA-sensitized guinea pigs (n = 9, not significant). OVA (10 µg/ml) was superfused for 30 min in the continuous presence of pyrilamine. B: pyrilamine is ineffective to reverse the increase of the C-fiber excitability induced by OVA. Once the potentiation effect occurred, esophageal vagal C-fiber responses to distension were not different, *P < 0.05, n = 5, ANOVA.

 
Histamine (100 µM) mimicked the effect of OVA in that it failed to consistently activate the nodose C fibers, but it caused a substantial increase in the responsiveness of the C fibers to mechanical stimulation (Fig. 5A). As with the OVA-induced responses, this histamine-induced effect was prevented by pretreating the tissue with pyrilamine (Fig. 5B). In another series of experiments a 10-fold lower concentration of histamine elicited a less consistent increase in the pressure-C-fiber response that failed to reach statistical significance (n = 11, P = 0.09).


Figure 5
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  		Fig. 5. Histamine acting on the histamine H1 receptor mimics the OVA-induced increase of the C-fiber excitability. A: histamine-induced (100 µM, 30 min) increase of the C-fiber excitability (n = 7, **P < 0.01). B: pretreatment with the selective H1 receptor antagonist pyrilamine (1 µM, 30 min) prevented the effect of histamine on the C-fiber excitability (n = 7, not significant). Histamine (100 µM) was superfused for 30 min in the continuous presence of pyrilamine. C: pretreatment with histamine (100 µM, 30 min) did not affect force-distention relationships of the esophageal tissue (n = 9, not significant), indicating that the histamine-induced changes in mechanical responsiveness of C fibers are not due to changes in esophageal mechanics.

 
Also consistent with the antigen-induced response, once the histamine-induced increase in C-fiber responsiveness was established, it was not reversible upon perfusing the tissue with histamine-free solution for 60 min (Fig. 6). Moreover, following the hour of perfusing the tissue with histamine-free buffer solution, an additional 30-min treatment with pyrilamine failed to reverse the histamine-induced response (Fig. 6).


Figure 6
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  		Fig. 6. The selective histamine H1 receptor antagonist pyrilamine does not reverse histamine-induced increase of C-fiber excitability once the increase is established. Histamine (100 µM, 30 min)-induced increase of the C-fiber excitability was not reversed by 30 min of wash with normal buffer. The enhanced excitability was not reversed with additional 30 min of superfusion with pyrilamine (1 µM) (n = 5, **P < 0.01, ANOVA).

 
Large mechanical forces have been shown to stimulate C fibers. Whereas histamine can contract esophageal smooth muscle, we evaluated whether the C-fiber response could have been elicited indirectly secondary to an increase in intraluminal pressure. Histamine did not influence the stress-strain relationship of the mechanical effect evoked by increasing intraluminal pressure (Fig. 5C). Moreover, methacholine, another agonist that contracts esophageal smooth muscle, failed to influence the nodose C-fiber response to increases in intraluminal pressure. No difference in the pressure-response curves of peak frequency of action potential discharge in response to 10, 30, and 60 mmHg was observed when carried out in the absence or presence of methacholine (10 µM; n = 5, P > 0.1, data not shown).

Nodose C fibers are reproducibly activated by the P2X agonist {alpha},beta-methylene ATP (29). As with the response to mechanical stimulation, OVA (30 min) significantly increased the C fibers response to stimulation with {alpha},beta-methylene ATP (Fig. 7).


Figure 7
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  		Fig. 7. Activation of mast cells increases the C-fiber excitability to chemical stimuli. The response to the P2X receptor selective agonist {alpha},beta-methylene-ATP (30 µM, 10 min) was obtained before and 30 min following the superfusion with OVA (10 µg/ml). The data represent means ± SE of 6 experiments. *P < 0.05.

 
Nodose neurons also project low-threshold mechanosensors (tension receptors) to the guinea pig esophagus. These fibers conduct action potentials in the A{delta} range (29). We evaluated the effect of histamine (100 µM) on the response of tension receptors to esophageal distension caused by a 10-mmHg increase in perfusion pressure. Unlike the C fibers, histamine had no effect on the response of the A{delta} fibers. The peak action potential discharge caused by the distension averaged 31 ± 5 Hz and 34 ± 3 Hz before and after histamine, respectively (P > 0.1, n = 4, conduction velocity = 3.2 ± 0.3 m/s).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The data support the hypothesis that activation of mast cells in the esophageal mucosa leads to long-lasting increases in the excitability of nearby vagal nociceptive C fibers. The increase in excitability is expressed by a substantial increase in the peak frequency of action potential discharge per given amount of activating stimulus (e.g., increases in intraluminal pressure or exposure to a purinergic agonist). The antigen-induced excitability increase requires the activation of histamine H1 receptors, but once the increased excitability is established it is enduring and no longer requires the presence of the initiating mediator(s).

Mast cells in various tissues, including the gastrointestinal tract, are commonly situated near nerves (28). Several studies have demonstrated that activation of tissue mast cells leads to profound physiological and phenotypic changes in nearby nerves (1, 2, 23, 28). The characteristics of the neuronal effect evoked upon mast cell activation are dependent on the nerve type and species studied. Antigenic activation of mast cells leads to long-lasting potentiation of parasympathetic synaptic transmission (15), and it has been associated with increases and decreases in transmission through the sympathetic and enteric nervous system (17, 27, 28). Relatively few studies have directly assessed the effect of mast cell activation on visceral nociceptive C fibers. In rats, antigenic activation of mast cells leads to action potential discharge in jejunal C fibers, due mainly to the effects of histamine and 5-HT interacting with H1 and 5-HT3 receptors, respectively (6).

The present study presents the new evidence for an effect of mast cell activation on esophageal afferent fibers. The observation that mast cell activation failed to overtly evoke action potential discharge in the C fibers but substantially increased the responsiveness to mechanical stimulation is similar to results obtained in trachea vagal afferent A{delta} fibers (18), but the mechanism and mediators appear to be different. The lack of overt activation of the C fibers in the present study contrasts with aforementioned effect of antigen activated mast cells on rat jejunal C fibers (6). There are several explanations for this difference. First, our studies were carried out ex vivo, where the background activity is largely diminished. In in vivo studies an increase in excitability may lead to action potential discharge by enhancing the response of the fiber to "background" physiological activity. Second, the jejunal C fibers likely represent mainly neural crest-derived dorsal root ganglion neurons, whereas our study focused specifically on the placodally derived nodose C fibers. We have previously shown that the placodal C fibers in the esophagus respond differently to various mediators compared with neural crest-derived esophageal vagal C fibers (29). Thirdly, there may be differences between C fibers in the lower gut compared with the esophagus. Finally, different species were used in the two studies. It may be of particular relevance in this regard that rat and mouse mast cells contain relatively large amounts of 5-HT compared with guinea pig (and human) mast cells (11, 12).

The results support the conclusion that histamine and its activation of H1 receptors are responsible for initiating the antigen-induced increase in nodose C-fiber excitability. Pretreating the tissue with the selective histamine H1 receptor antagonist pyrilamine completely prevented the antigen-induced response. Moreover, exogenously applied histamine mimicked the antigen-induced response, which was prevented by pyrilamine pretreatment.

When the time course of the antigen response is considered, however, the results are somewhat paradoxical. Once the antigen-induced increase in C-fiber excitability reached its peak (sometime within the first 30 min of antigen exposure), it remained unchanged for at least 90 min (longest time period evaluated) after washing of the tissue with fresh buffer solution. If histamine is the sole mediator, one might predict that the antigen-effect would be short lived; in our perfusion system, we found that the antigen-induced histamine release peaked within the first 5 min and was essentially complete within the first 30 min of exposure. Surprisingly, after the antigen-induced response or histamine-induced response was established, prolonged treatment with fresh buffer solution containing a relatively large concentration (1,000-fold greater than its Kd value) of pyrilamine was ineffective at reversing the response.

Considered together, the results are consistent with at least two logical scenarios that are at present difficult to experimentally differentiate. First, histamine H1 receptor activation may lead to events within the C-fiber terminals that somehow long outlast the actual H1 receptor stimulation by histamine. For example, there may be some phenotypic change or some persistent phosphorylation of an ion channel leading to an increase in excitability. More needs to be known about the mechanism by which histamine increases nodose C-fiber excitability before this hypothesis can be thoroughly investigated. We have found that histamine can inhibit certain potassium currents leading to an increase the input resistance and membrane depolarization in guinea pig nodose ganglion neurons, but these effects were rapidly reversible upon removal of histamine (26). In somatosensory sensory fibers, phospholipase Cbeta has been implicated in histamine H1 receptor signaling, but phospholipase activation via G protein-coupled receptors is unlikely to persist for hours beyond receptor activation (5). Nevertheless, circumstantial support for this scenario is found by the recent studies of Kayssi et al. (8). They noted that exposing colonic nociceptive neurons for a brief 3 min to a protease-activated receptor (PAR)-2 activating peptide increased the neuron's excitability, and this persisted for at least 60 min after the stimulus was washed from the cells. The PAR-2, like the histamine H1 receptor, is a G protein-coupled receptor often linked to the Gq pathway.

The other scenario consistent with our results is that the histamine released from mast cells interacts with H1 receptors on other cell types in the tissue, resulting the release of a secondary mediator that, in turn, causes the long-lasting increase in C-fiber excitability.

It is unlikely that the effect of histamine or antigen on the C fiber's response to intraluminal pressure is secondary to effects on the stress-strain relationships caused by smooth muscle contractions. First, histamine had no effect on the tissue distension caused by the increases in intraluminal pressure that were studied. Second, methacholine, another agonist that contracts esophageal smooth muscle, did not increase the C fiber's response to increases in intraluminal pressure. Third, the antigen-induced increase in excitability was also observed when the stimulus was chemical ({alpha},beta-methylene ATP) rather than mechanical.

In the present study we took advantage of the immediate hypersensitivity reaction to selectively activate mast cells with antigen. This may have direct relevance to esophageal disorders associated with allergy. It should be kept in mind, however, that mast cells can be activated by endogenous stimuli other than allergens. Our results indicate that if such a stimulus is present in inflamed esophageal tissue, it may lead to a long-lasting enhancement in nociceptive nerve activity and in the consequential effects this has on sensations and esophageal reflex activity.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by the National Institutes of Health, Bethesda, MD.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. J. Undem, JHU Asthma Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (e-mail: bundem{at}jhmi.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.


   REFERENCES
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 ABSTRACT
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