Cellular pathways of mast cell- and capsaicin-sensitive nerve-evoked ileal submucosal arteriolar dilations

L. Atwood1, C. James1, G. P. Morris1, and S. Vanner2

1 Department of Biology and 2 Departments of Physiology and Medicine, Queen's University, Kingston, Ontario, Canada K7L 5G2

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

This study characterized mast cell- and capsaicin-sensitive sensory nerve vasodilator mechanisms regulating submucosal arterioles in the guinea pig ileum. The outside diameter of arterioles in in vitro submucosal preparations from milk-sensitized guinea pigs was monitored using videomicroscopy. Superfusion of the cow's milk protein, beta -lactoglobulin (beta -Lg; 5 µM), evoked large dilations, which became completely desensitized. beta -Lg-evoked dilations were blocked by pyrilamine or NG-monomethyl-L-arginine plus indomethacin but not by TTX. Electron microscopic studies revealed that mast cells, in preparations receiving beta -Lg, demonstrated significant reductions of the dispersed and intact granule areas compared with preparations not exposed to beta -Lg. Paired experiments were conducted to determine if capsaicin-sensitive, nerve-evoked responses involved mast cell degranulation. One preparation received capsaicin (200 nM) followed by beta -Lg (5 µM); the other preparation received the drugs in reverse order. Prior treatment with capsaicin or beta -Lg had no effect on subsequent dilations evoked by the alternate treatment. Electron microscopy showed that nerve-arteriole associations were 10 times closer than nerve-mast cell associations. Mast cell numbers were not increased by milk sensitization. These findings suggest that mast cell- and capsaicin-sensitive nerve-evoked vasodilator mechanisms act independently in a model in which mast cell numbers are not increased.

histamine; substance P; blood flow

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

MAST CELLS ARE IMPORTANT modulators of blood flow within the small intestine and elsewhere within the gastrointestinal tract (24). These cells are found predominantly in the submucosa and lamina propria in close association with submucosal arterioles, the major resistance vessels within the intestine. Classically, the mast cell is recognized as a principal mediator of immediate hypersensitivity reactions (7, 24) and more recently as playing an important role in the vascular response during chronic inflammation (9, 10, 15). Despite the obvious importance of this effector system, the underlying pathways that lead to activation of mast cells, and the cellular pathways that subsequently activate vasodilator mechanisms, are not completely understood.

The mast cell contains multiple putative vasodilator substances that may be released following mast cell activation (2). Some substances, such as histamine or 5-hydroxytryptamine (5-HT), are stored in preformed granules, whereas others, such as arachidonic acid metabolites, are produced de novo. In vitro studies of exogenously applied mast cell mediators have identified several vasodilator pathways (14, 20). For example, histamine has been shown to stimulate endothelial cells to release nitric oxide and possibly prostacyclin (3). Other substances, such as arachidonic acid metabolites, appear to act through endothelium-dependent pathways (14). In addition to direct activation of the arteriole, some mast cell mediators may also act through neural pathways. Histamine, 5-HT, and arachidonic acid metabolites have been shown to activate submucosal neurons (5). A subpopulation of the neurons that innervate submucosal arterioles causes vasodilation by releasing ACh from nerve terminals (19). Given the multiple cellular pathways and putative mast cell mediators, it seems likely that the vasodilator actions of mast cells could involve several cellular pathways.

The ultimate role that mast cell-evoked vasodilation plays in the regulation of mucosal blood flow will also depend on the nature of the cellular pathways that lead to activation of these cells. In addition to the classical role of mast cells in the immune-mediated, immediate hypersensitivity reactions, there is growing morphological and functional evidence that nerves within the intestine may activate mast cells (8, 15-17, 21, 28, 29). A number of morphological studies have drawn attention to the close association between mast cells and nerves, implying a communication between these two cell types. Ultrastructural studies have suggested that membrane-to-membrane contacts may actually exist between mast cells and nerves (22). The precise subsets of nerves that may be involved in this interaction are not completely understood, but evidence suggests that extrinsic sensory (capsaicin-sensitive) nerves are involved (16, 22, 29). These nerves can be immunohistochemically identified by the colocalization of substance P and calcitonin gene-related product (CGRP) (11), and studies have shown that these nerve fibers are in close contact with mast cells (17, 22). This association appears to also exist in inflamed intestine from patients with inflammatory bowel disease (9, 32). Despite morphological and functional data suggesting that capsaicin-sensitive nerves innervate mast cells, this relationship remains poorly defined (15).

Our recent in vitro studies in the guinea pig ileum have shown that activation of capsaicin-sensitive nerves evokes dilation of submucosal arterioles (25) and that these arterioles are also dilated by exogenous application of putative mast cell mediators (3). The purpose of the present study was to determine the cellular pathways involved in mast cell vasodilator responses in the intestine and to examine whether capsaicin-sensitive nerves can activate this pathway. A milk-sensitized guinea pig model was used to enable mast cells to be selectively activated with the milk protein, beta -lactoglobulin (beta -Lg).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Guinea pigs (500-700 g) were sensitized to the cow's milk protein beta -Lg by feeding them cow's milk (2%) ad libitum and dry guinea pig food supplemented with vitamin C for 21 days. Water was excluded to ensure that the guinea pigs drank milk. After the 21-day period, animals were fed a beta -Lg-free diet, water, and vitamin C supplements for 3 days. Control animals were age matched. Animals were killed by cervical dislocation, carotid transection was performed, and submucosal preparations were dissected from distal ileum as previously described (25).

Videomicroscopic Studies

Submucosal preparations were pinned in a small organ bath (0.5 ml) and bathed with a physiological saline solution containing (in mM) 126 NaCl, 2.5 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 5 KCl, 25 NaHCO3, and 11 glucose, which was gassed with 95% O2-5% CO2 at 35-36°C. All drugs were added to the bath by superfusion. In a small series of experiments, preparations were pinned in a modified bath that was divided into two chambers by a small Plexiglas divider placed over the tissue, as previously described (28). Silicone gel placed between the divider and the tissue prevented communication of solutions between the chambers. Each chamber was superfused separately with the physiological saline solution.

The outside diameter of arterioles was monitored using a computer-assisted videomicroscopy system (Diamtrak), as previously described (18). Briefly, a PCVision Imaging Technology frame-grabber board (Infrasea; Richmond, BC, Canada) installed in an IBM-compatible PC/AT computer was used to digitize television images of the arteriole, and the Diamtrak software enabled the distance between the walls of the arteriole to be measured. The resolution was ~1 µm, and the sampling rate was 15 Hz. Measurements were stored using a computer-assisted data acquisition system (Axotape; Axon Instruments, Foster City, CA).

Vasodilator responses were examined by first constricting vessels 80-95% (maximum) with 300 nM PGF2alpha ; maximal constriction of these final resistance vessels causes complete occlusion of the lumen. Extrinsic sensory nerves were stimulated by superfusion of capsaicin (200 nM). Our previous studies (25) showed that 200 nM capsaicin evokes a near-maximal vasodilator response that results from selective activation of extrinsic (capsaicin-sensitive) sensory nerves. Responses to superfusion of capsaicin and the milk protein beta -Lg became desensitized to repeated applications. Therefore, studies that examined the interactions of mast cells and capsaicin-sensitive nerves were conducted by simultaneously examining two preparations from the same animal, using two experimental setups. One preparation received capsaicin followed by beta -Lg; the other received beta -Lg followed by capsaicin. Repeated applications to preparations were separated by a 10-min washout period. Vasodilations produced by beta -Lg and capsaicin returned to baseline in the continued presence of the agonist. These dilations were quantitated by measuring the amplitude and duration at one-half the amplitude of the response and expressing the product of the two in micrometers per second. All other agonists evoked continuous vasodilations for the duration of their application. These responses were expressed as a percentage of the maximal response to muscarine, as previously described (25). In some experiments, a small segment of the preparation, which included the site on the arteriole monitored by videomicroscopy, was excised and fixed for subsequent histological evaluation (see Procedures for Light and Electron Microscopy).

Previous studies have shown that 20-25% of guinea pigs fed cow's milk do not become sensitized to beta -Lg (6) and suggest that 5 µM beta -Lg gives relatively consistent responses (11). In this study, when none of the preparations from an animal responded to beta -Lg, all preparations from that animal were excluded from analysis.

Procedures for Light and Electron Microscopy

To determine whether sensitization to milk protein affected mast cell numbers, two submucosal segments were dissected from 2-cm long, full-thickness segments of ileum from five milk-sensitized guinea pigs and six control animals. The submucosa was pinned as a flat sheet, on a plate of Sylgard gel, with the mucosal surface facing up. The segments from each animal were then fixed for 2 h at room temperature in Carnoy's fixative (60% absolute ethanol, 30% chloroform, and 10% glacial acetic acid) and stored in 70% alcohol. The tissue was subsequently hydrated and stained for 30 min with Alcian blue and counterstained for 45 s with safranin (23). Submucosal preparations were then dehydrated and placed on glass microscope slides, and coverslips were affixed with Permount.

To determine the effects of beta -Lg on mast cell morphology, tissues were studied from two groups of milk-sensitized animals (n = 5 animals/group). In one group, the superfusion fluid did not contain beta -Lg, and, in the other, beta -Lg (5 µM) was added to the superfusion fluid and the resulting change in outside diameter was recorded (see Videomicroscopic Studies). Submucosal preparations from each animal were then fixed for 2 h in 2.5% glutaraldehyde, buffered to pH 7.2 with sodium cacodylate, and postfixed for 1 h in cacodylate-buffered 1% osmium tetroxide. After routine processing, tissue blocks were embedded in Epon-araldite. Semithin sections (0.5-1.0 µm thick) were cut perpendicularly to the arteriole and placed on glass slides. The arteriole was identified with a light microscope on sections stained with toluidine blue, the plastic block was trimmed to include the arteriole and surrounding tissues, and ultrathin sections were cut, mounted on copper grids, and stained by conventional methods. To determine whether exposure to capsaicin affected numbers of stainable mast cells, four submucosal preparations were dissected from the ileum of each of six milk-sensitized guinea pigs. The submucosal preparations were pinned on two Sylgard plates and placed in 500-ml beakers containing 100 ml of Krebs solution. The tissues were bubbled with oxygen at 35°C for a 20-min stabilization period. After stabilization, all four preparations were exposed to 300 nM PGF2alpha for 30 s to preconstrict the microvasculature. The control group for capsaicin was then exposed to the vehicle (Tween 80 stock solution) for 5 min. The experimental group was exposed to 200 nM capsaicin dissolved in the vehicle for 5 min. Both sets of tissues were then rinsed with fresh Krebs solution and fixed for histological examination of Alcian blue/safranin-stained mast cells, as described above.

Mast Cell Counts, Stereology, and Substance P Immunohistochemistry

Stained mast cells were counted for each segment from a field of view (×40 objective; field diameter of 430 µm) centered on the largest branching arteriole in the segment. Only those mast cells completely enclosed by the field were counted. Slides were coded before counting to avoid experimenter bias.

Ultrathin sections were viewed using a Zeiss EM 10 CR transmission electron microscope. All grids were coded to avoid observer bias. Grids were searched systematically, and all mast cells were photographed. The nuclear and cytoplasmic areas and the areas of intact (homogeneous, electron-dense matrix) and dispersed (granular and less dense matrix) granules were calculated using the point counting method of Weibel (30). To ensure a homogeneous study population and to exclude the possibility of data bias due to peripheral sections possibly containing different numbers of granules, data were calculated and reported for all mast cells that had a nuclear area >30% of the total area of the cell. Results are expressed as area covered by each compartment and are reported as means ± SE. Distances of closest approach between mast cells, nerves, and blood vessels (arterioles and venules) were measured using micrographs that contained at least two of these structures, and the presence or absence of an intervening fibroblast process was noted.

Substance P immunoreactivity was determined using standard immunohistochemical techniques (11, 24). The antiserum was substance P (rat, 1:500; Fitzgerald), and the labeled secondary antibody was 5-[(4,6-dichlorotriazine-2-yl)amino]-fluorescein goat anti-rat.

Drugs

The following drugs were used: capsaicin, histamine, pyrilamine maleate, indomethacin, 9,11-dideoxy-11alpha ,9alpha -epoxymethanoprostaglandin F2alpha (PGF2alpha ), TTX, 5-HT, bradykinin, prostacyclin, PGE1 (Sigma, St. Louis, MO), and NG-monomethyl-L-arginine (L-NMMA) (Calbiochem, San Diego, CA). Capsaicin was dissolved in Tween 80, alcohol, and saline (10:10:80% vol).

Statistics

Data are expressed as means ± SE. Data from the paired capsaicin and beta -Lg stimulation experiments were analyzed using a paired t-test.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The outside diameter of submucosal arterioles examined in this study ranged from 44 to 96 µm (n = 99). When submucosal preparations were pinned in the organ bath, arterioles were actively stretched, which maintains an optimum length-tension relationship (27). Arterioles in preparations from control and milk-sensitized guinea pigs did not develop spontaneous resting tone.

Mast cells could be readily identified in whole mount submucosal preparations under the light microscope following exposure to Alcian blue (Fig. 1). These cells were distributed throughout the connective tissue but were found in increased numbers along blood vessels. Capsaicin-sensitive sensory nerves fibers, identified by their substance P immunoreactivity (12, 25), have dense projections along the arterioles and are in close association with mast cells. Counts of mast cell numbers per field of view demonstrated that mast cell numbers were not increased in preparations from milk-sensitized guinea pigs compared with control animals (53.4 ± 3.2 vs. 58.0 ± 8.7 cells, respectively; n = 11 preparations).


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Fig. 1.   Light micrographs showing close relationship of mast cells, capsaicin-sensitive nerves, and submucosal arterioles. A: arrows designate mast cells stained with Alcian blue-safranin closely apposed to a submucosal arteriole (a). B: substance P immunoreactivity delineates capsaicin-sensitive nerves (arrows) traveling in close association with a submucosal arteriole. Ganglia contain intrinsic immunoreactive submucosal neurons in addition to nerve terminals of capsaicin-sensitive nerves.

Characterization of beta -Lg-Evoked Vasodilations

Vasodilator responses. Superfusion of the cow's milk protein, beta -Lg (5 µM), evoked a large dilation (74 ± 5%; n = 13) in 81% (13/16) of milk-sensitized preparations preconstricted with 300 nM PGF2alpha (Fig. 2). Dilations were not maintained in the continued presence of beta -Lg (5 µM); all preparations became completely desensitized after a 2-min or less exposure to beta -Lg. Repeat application of beta -Lg (5 µM; n = 5) had no effect (Fig. 2) following a washout period of up to 20 min. Consequently, in all subsequent experiments with beta -Lg, only single applications were examined in each preparation. In addition, preliminary studies demonstrated that preparations dissected and oxygenated at room temperature for periods >1 h usually did not respond to beta -Lg. Therefore, all preparations from one animal were studied within this time frame. beta -Lg (5 µM) had no effect on the resting outside diameter of arterioles in preparations that were not preconstricted with PGF2alpha (n = 3). The resting or preconstricted arteriolar diameter in preparations from control animals was not altered by beta -Lg (5 µM) (n = 3).


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Fig. 2.   beta -Lactoglobulin (beta -Lg) superfusion dilates submucosal arterioles from milk-sensitized animals. A: representative trace showing that superfusion of beta -Lg (5 µM) evokes a large dilation of the submucosal arteriole. This response desensitizes during continued superfusion of beta -Lg. Arteriole was preconstricted with PGF2alpha (300 nM), which causes a 80-95% maximal constriction. B: after a 10-min washout, reapplication of beta -Lg has no effect. Arteriole was preconstricted as in A. Resting outside diameter was 96 µm.

The possibility that beta -Lg-evoked dilations were in part mediated by release of mast cell mediators that activated submucosal vasodilator neurons (19) was examined by comparing the magnitude of beta -Lg-evoked responses in the presence and absence of 1 µM TTX. Control beta -Lg-evoked dilations (mean area = 1,040 ± 522 µm · s, n = 3) were not significantly different from beta -Lg responses elicited with TTX in the bath (mean area = 1,254 ± 1,001 µm · s, n = 3).

Actions of the H1 antagonist, pyrilamine, on beta -Lg-evoked vasodilations. The possibility that the beta -Lg-evoked dilations were mediated by histamine released from mast cells was examined by studying paired preparations from the same animals (n = 8); one preparation received beta -Lg alone, whereas the other preparation received beta -Lg and pyrilamine (1 µM). In all animals in which beta -Lg evoked dilations in the control preparation (n = 6), beta -Lg had no effect in the presence of pyrilamine in the other preparation (Fig. 3). The selectivity of pyrilamine for histamine receptors was examined by studying the effects of this antagonist on other putative mast cell mediators (2). Arterioles preconstricted with PGF2alpha were dilated by 5-HT (EC50 = 1 µM; n = 5), PGE1 (EC50 = 60 nM; n = 5), PGI2 (EC50 = 20 nM; n = 5), and bradykinin (EC50 = 3 nM; n = 3). Dose-response curves obtained in the presence and absence of pyrilamine (1 µM) were not significantly different (Fig. 3).


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Fig. 3.   Vasodilations evoked by superfusion of beta -Lg are blocked by the H1 antagonist pyrilamine. A: representative traces showing a control vasodilation evoked by superfusion of beta -Lg (5 µM). In a separate preparation from the same animal, beta -Lg has no effect in the presence of pyrilamine (1 µM). Submucosal arterioles in both preparations were preconstricted as described in Fig. 2. Outside diameters were 80 and 90 µm. B: vasodilator dose responses for putative mast cell mediators 5-hydroxytryptamine (5-HT), PGE1, PGI2, and bradykinin are not altered by pyrilamine. Each point represents mean ± SE for 3 or more preparations. , Control responses. , Responses obtained in the presence of pyrilamine. All drugs were added in a noncumulative fashion.

Effects of L-NMMA and indomethacin. The nitric oxide synthase inhibitor L-NMMA (300 µM) had no effect on the resting diameter of the arterioles, as previously described (3). These studies and others (19) also demonstrated that L-NMMA inhibited endogenous release of nitric oxide from this preparation. The blockade of the dilative effect of endogenous nitric oxide release by L-NMMA resulted in enhanced preconstriction when PGF2alpha (300 nM) was applied. This increase, however, was <7% greater than the vasoconstrictor response to PGF2alpha alone and therefore did not significantly affect the measurement of vasodilator responses. After a 5-min incubation period with L-NMMA (300 µM), the mean area of the beta -Lg-evoked vasodilator response was inhibited by 85% compared with controls (n = 4, P < 0.05) (Fig. 4).


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Fig. 4.   Summary of the results examining the effects of NG-monomethyl-L-arginine (L-NMMA) alone or combined with indomethacin. Compared with control beta -Lg-evoked (5 µM) vasodilator responses (solid bar), preincubation with L-NMMA (300 µM) alone inhibited beta -Lg-evoked responses by 85%. When L-NMMA (300 µM) and indomethacin (10 µM) were combined, beta -Lg-evoked dilations were completely blocked (n = 4 for each group). * P < 0.05.

The cyclooxygenase inhibitor indomethacin (10 µM) also did not affect the resting diameter of the arteriole, as previously described (3). When indomethacin (10 µM) was combined with L-NMMA (300 µM) during the 5-min incubation period, beta -Lg (5 µM)-evoked dilations were completely blocked (n = 4) (Fig. 4).

Electron microscopy. The mean areas of both dispersed and intact granules in mast cells from milk-sensitized preparations superfused with 5 µM beta -Lg were significantly reduced compared with preparations that had not been exposed to beta -Lg (Fig. 5) (P < 0.05 and P < 0.01, respectively). Mean nuclear and cytoplasmic areas of mast cells were not altered by exposure to beta -Lg (Fig. 5).


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Fig. 5.   beta -Lg exposure decreases the mean area of both dispersed and intact mast cell granules. A: representative electron micrograph of a mast cell depicting the electron-dense intact granules (ig) and the electron lucent dispersed granules (dg). n, Nucleus. Magnification = ×6,300. B: summary of mean areas of cellular components of mast cells in electron micrographs of control (n = 18) and beta -Lg-treated preparations (n = 10). Mean areas of both intact (* P < 0.01) and dispersed (* P < 0.05) granules were decreased following exposure to beta -Lg.

Examination of Nerve-Mast Cell Interactions

Capsaicin and beta -Lg-evoked vasodilator responses. Paired experiments were conducted to determine if stimulation of capsaicin-sensitive sensory nerves evoked a vasodilator response that was mediated in part through the activation of mast cells and subsequent release of their vasodilator mediators. Capsaicin-sensitive nerves were stimulated by superfusion of capsaicin (200 nM). Our previous studies have shown that 200 nM capsaicin selectively activates these nerves (28) and does not act directly on the mast cell to cause release of mast cell vasoactive mediators.

In this experimental series, one of the paired preparations was first superfused with capsaicin (200 nM), whereas the other preparation was superfused with beta -Lg (5 µM). After a 10-min washout period, the paradigm was reversed; the preparation initially treated with capsaicin received beta -Lg, whereas the other preparation, initially treated with beta -Lg, received capsaicin. Preliminary studies also demonstrated that responses to beta -Lg may be lost over time periods >45 min. For this reason, in studies in which beta -Lg was applied following capsaicin, only paired preparations in which beta -Lg elicited a response could be included for data analyses. The magnitude of capsaicin-evoked dilations did not differ regardless of whether or not the preparation had first received beta -Lg (n = 9; Fig. 6). Similarly, prior treatment with capsaicin did not affect beta -Lg responses (n = 9; Fig. 6). In a separate series of experiments, possible capsaicin-sensitive nerve-mast cell interactions were further studied by examining the effects of the H1 antagonist pyrilamine (1 µM). Vasodilator responses evoked by superfusion of capsaicin (200 nM) (86 ± 12%; n = 5) were not significantly different compared with those evoked by capsaicin (200 nM) with pyrilamine (1 µM) in the bath (79 ± 6%; n = 5). The effects of capsaicin stimulation on mast cell numbers were also examined in light microscopic histological studies. Mast cell numbers in preparations superfused with capsaicin (mean = 37.8 ± 3.6, n = 5) did not differ significantly from control preparations (mean = 30.8 ± 8.2, n = 5).


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Fig. 6.   Vasodilations evoked by beta -Lg or capsaicin are not altered by prior activation of capsaicin-sensitive nerves or mast cells, respectively. A: representative traces of vasodilation of a submucosal arteriole evoked by superfusion of capsaicin (200 nM). After a 10-min washout, beta -Lg (5 µM) superfusion elicits a typical vasodilator response. Arteriole was preconstricted as described in Fig. 2. Resting outside diameter was 70 µm. B: representative traces of vasodilation of a submucosal arteriole evoked by superfusion of beta -Lg (5 µM). After a 10-min washout period, capsaicin (200 nM) was applied. Arteriole was preconstricted as described in Fig. 2. Resting outside diameter was 90 µm. C: summary of mean peak dilations evoked by beta -Lg or capsaicin and following prior treatment with either capsaicin or beta -Lg, respectively. Prior activation of capsaicin-sensitive nerves did not alter responses evoked by beta -Lg-stimulated mast cell degranulation. Similarly, prior beta -Lg-stimulated mast cell degranulation did not alter dilations evoked by stimulation of capsaicin-sensitive nerves. Each bar represents mean ± SE of 9 preparations.

The possibility that mast cell mediators activate capsaicin-sensitive nerves was further examined by comparing the vasodilator actions of the mast cell mediator histamine and capsaicin using a double-chamber bath (Fig. 7). This preparation enables capsaicin-sensitive nerves to be stimulated in the proximal chamber and vasodilator responses to be recorded in the distal chamber. Capsaicin-sensitive nerves travel along the arteriole (see Fig. 1) between the two chambers, and our previous denervation studies have shown that capsaicin-evoked dilations result from selective activation of capsaicin-sensitive nerves (26). Superfusion of supramaximal concentrations (3) of histamine (30 µM) into the proximal chamber did not elicit a dilative response in the opposite chamber (n = 4). However, after a 10-min washout of the histamine, application of capsaicin (2 µM; n = 4) into the proximal chamber elicited a large vasodilator response in the distal chamber (Fig. 7).


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Fig. 7.   Mast cell mediator histamine does not activate capsaicin-sensitive nerves. Top: schematic drawing of a submucosal preparation in a double-chamber bath. Proximal and distal segments of the arteriole are chemically isolated by a Plexiglas divider (vertical bar). The short parallel bars bracketing the distal branch point of the arteriole designate the site of videomicroscopy recording. Each chamber is superfused by separate flow pipettes (pipettes A and B). Capsaicin-sensitive nerves travel along submucosal arteriole as shown in Fig. 1. Bottom: representative traces from a single preparation. Arterioles in the recording chamber were preconstricted with PGF2alpha (300 nM) superfused through pipette B. Resting outside diameter was 50 µm. Superfusion of a supramaximal concentration of histamine (30 µM) through pipette A did not elicit any dilation. After a 10-min washout period, superfusion of capsaicin (2 µM) through pipette A elicited a large vasodilation.

Electron microscopic assessment of arteriole, nerve, and mast cell relationships. Morphological studies were combined with functional studies by identifying the region of arterioles monitored by videomicroscopy and following completion of the functional studies excising these segments and preparing them for subsequent electron microscopic study (see METHODS). Measurements of distances between arterioles and nerves, nerves and mast cells, and mast cells and arterioles were made from electron micrographs (n = 50; Fig. 8). These measurements demonstrated that nerves and arterioles in these preparations were much more closely associated than nerves and mast cells (Fig. 8). Previous studies suggest that fibroblasts may play an important amplification role in cell-cell signaling (12). In the present study, 94% (n = 17) of mast cell-arteriole associations were separated by a fibroblast process, whereas only 44% (n = 9) of mast cell-venule associations exhibited a similar finding.


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Fig. 8.   Nerves are much more closely associated with arterioles than with mast cells. A: representative electron micrograph of a submucosal arteriole showing the relationship of nerve (n) and arteriole (a). B: representative electron micrograph of a submucosal arteriole showing the relationship of mast cell (m) and nerves (n). Magnification for A and B = ×4,000. C: summary of mean distances between mast cells and nerves (mright-arrown), mast cells and arterioles (mright-arrowa), and nerves and arterioles (nright-arrowa). Mean distance between nerves and arteriolar wall was ~10 times less than that between nerves and mast cells.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Submucosal arterioles, the major resistance vessels controlling mucosal blood flow within the intestine, are regulated by multiple effector systems. This study examined two cellular pathways involved in this control: mast cell degranulation and capsaicin-sensitive nerves. Mast cell degranulation evoked a histamine-dependent vasodilation of submucosal arterioles, and activation of capsaicin-sensitive nerves released neurotransmitter(s), which also dilated these vessels. These immune and neurally mediated responses, however, appear to act through parallel and independent pathways.

Several fundamental observations were made concerning the actions of the milk protein, beta -Lg. First, the beta -Lg-evoked dilation desensitized during the initial superfusion and a repeat application of beta -Lg had no effect after the initial application of beta -Lg to the preparation was washed out for up to 20 min. This likely reflects that the first application of antigen (5 µM) was sufficient to occupy all antibody sites and resulted in maximal release of mast cell mediators and/or was irreversibly bound to the antibody. These findings indirectly support that beta -Lg acts selectively on the mast cell. This specificity was also supported by studies that demonstrated that beta -Lg had no effect in preparations that were not milk sensitized. Electron microscopic studies further characterized the effect of beta -Lg on sensitized mast cells, demonstrating a significant decrease in mean area of both dispersed and intact granules. These data suggest that beta -Lg exposure releases preformed mediators such as histamine (2, 7). This deduction is supported by our pharmacological data that demonstrated that beta -Lg-evoked dilations were blocked by the H1 antagonist pyrilamine (Fig. 3). They do not, however, exclude that membrane-derived mediators and/or cytokines may also be released (15). Together, these data provide strong evidence for the specificity of beta -Lg as a probe to activate sensitized mast cells in this preparation.

A number of preformed and membrane-derived mast cell mediators were candidates (2, 14, 20) for the mast cell-dependent vasodilations observed in this study. Although exogenous application of any one of multiple mediators, including 5-HT, bradykinin, PGE1, and PGI2, in addition to histamine, dilated submucosal arterioles, the mast cell-dependent dilations were completely blocked by the H1 antagonist, pyrilamine (see Fig. 3). This inhibitor appears to be selective for the histamine receptor because the vasodilator dose-response curves (see Fig. 3) were not altered by pyrilamine. Furthermore, previous studies have shown that histamine-evoked dilations in this preparation are mediated by H1 receptors located on the arteriole (3). In these studies, exogenous application of 6 µM histamine caused maximal dilations and hence the beta -Lg-evoked maximal dilations observed in the present study suggest that histamine concentrations at the H1 receptor were at least in the micromolar range. In addition to these direct actions of histamine on the blood vessel, there is also evidence that mast cell degranulation can activate submucosal neurons (5). Although submucosal cholinergic vasodilator neurons comprise a subpopulation of submucosal neurons (18), these neurons did not appear to play a significant role in the mast cell-mediated vasodilation observed in the present study because beta -Lg-evoked dilations were not inhibited by TTX. When these studies are taken together, they suggest that the mast cell-mediated vasodilator responses were mediated almost exclusively by release of preformed granules of histamine, which activated H1 receptors on the arterioles.

The role of arteriolar endothelium in the mast cell-mediated dilations was also examined using inhibitors of nitric oxide synthase and cyclooxygenase. The beta -Lg-evoked responses were inhibited by the nitric oxide synthase inhibitor L-NMMA and completely blocked when a combination of L-NMMA and indomethacin was given. These findings suggest that both nitric oxide and prostacyclin mediate the mast cell-evoked dilations. These vasodilator substances appear to originate from the endothelium, rather than the mast cell, because beta -Lg-evoked responses are completely blocked by the histamine antagonist. This finding is in agreement with our previous studies (3), which suggested that histamine activates H1 receptors on submucosal arterioles, resulting in the release of nitric oxide and prostacyclin from endothelium. In these studies, indomethacin alone had no effect, suggesting a possible interaction between these two second messenger systems.

There are both morphological and functional data from other studies to suggest that nerves and mast cells function as a homeostatic unit (17). Anatomic investigations of the spatial associations of mast cells and enteric nerves have shown that these cell types are frequently found in close association (21, 22). In some cases, these cells may make membrane-to-membrane contact (1, 22), although this may be dependent on the tissue and species examined (9, 31). The associations have been described for peptidergic nerves colocalizing substance P-CGRP; nerve fibers chemically coding these peptides originate from cell bodies in the dorsal root ganglia (12). Such associations have led to speculation concerning functional connections between capsaicin-sensitive sensory nerves and mast cells. This concept has been supported by functional studies examining the effects of capsaicin, the neurochemical probe for capsaicin-sensitive nerves (16, 25, 28), and the actions of the putative neurotransmitter substance P. Several in vivo inflammatory models have demonstrated that capsaicin-evoked degranulation of mast cells increases both gastric mucosal and intestinal serosal blood flow (16, 28). These studies imply that capsaicin-sensitive nerves release neuropeptides that in turn cause degranulation of mast cells. In a mouse model, substance P-evoked ion transport was mediated in part by a mast cell-dependent pathway and appeared to involve activation of neurokinin (NK1) receptors on the mast cell, with ensuing release of histamine (29). Despite this evidence, the results of the current study suggest that significant capsaicin-sensitive nerve-mast cell connections do not exist in our model. In our study, we demonstrated that degranulation of mast cells by beta -Lg stimulation did not reduce the capsaicin-evoked response and, similarly, stimulation of capsaicin-sensitive nerves did not reduce the beta -Lg response (see Fig. 6). Furthermore, pharmacological studies demonstrated that capsaicin-sensitive nerve-evoked responses were not inhibited by pyrilamine, suggesting that the putative mast cell-mediator histamine was not involved in this response. We also demonstrated that histamine did not activate the capsaicin-sensitive nerves that mediated the vasodilator response (see Fig. 7). This finding suggested that mast cells did not activate the capsaicin-sensitive nerves. There may be several possibilities that explain the lack of capsaicin-sensitive nerve-mast cell interactions in our study compared with others (15). First, in contrast to the inflammatory models in which mastocyctosis occurs (16, 28), the mast cell numbers were not increased in this study. Our measurement of cellular associations supports this hypothesis, demonstrating that nerves and blood vessels were much more closely apposed than mast cells and nerves. This may suggest that a critical relationship between nerves and mast cells only exists in the setting of a significant increase in mast cell numbers. Alternatively, or in addition, it is possible that in the inflammatory milieu capsaicin-sensitive nerves are altered, i.e., increased release of neuropeptides occurs, or mast cell properties are altered resulting in a functional connection between these two cellular pathways.

The control of mucosal blood flow is critical to the maintenance of electrolyte transport and the maintenance of mucosal integrity. The present study suggests that the release of both vasoactive mediators from mast cells and neuropeptides from capsaicin-sensitive nerves is important for this regulation and that, at least in some noninflammed tissues, these are poised to act independently of each other.

    ACKNOWLEDGEMENTS

We thank Margaret Bolton and Gail Pringle for technical assistance.

    FOOTNOTES

This study was supported by the Medical Research Council (S. Vanner) and the National Sciences and Engineering Research Council (G. P. Morris). L. Atwood was supported by an Ontario Graduate Scholarship.

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. §1734 solely to indicate this fact.

Address for reprint requests: S. Vanner, Hotel Dieu Hospital, 166 Brock St., Kingston, ON, Canada K7L 5G2.

Received 11 March 1998; accepted in final form 28 July 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(5):G1063-G1072
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