1 Department of Biology and 2 Departments of Physiology and Medicine, Queen's University, Kingston, Ontario, Canada K7L 5G2
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ABSTRACT |
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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, -lactoglobulin (
-Lg; 5 µM), evoked large
dilations, which became completely desensitized.
-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
-Lg, demonstrated significant
reductions of the dispersed and intact granule areas compared with
preparations not exposed to
-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
-Lg (5 µM); the other preparation received the drugs
in reverse order. Prior treatment with capsaicin or
-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
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INTRODUCTION |
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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, -lactoglobulin (
-Lg).
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METHODS |
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Guinea pigs (500-700 g) were sensitized to the cow's milk protein
-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
-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
PGF2; 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
-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
-Lg; the other received
-Lg followed by
capsaicin. Repeated applications to preparations were separated by a
10-min washout period. Vasodilations produced by
-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 -Lg (6) and suggest that 5 µM
-Lg gives relatively consistent responses (11). In this study, when
none of the preparations from an animal responded to
-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 -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
-Lg, and, in the other,
-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
PGF2
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-11Statistics
Data are expressed as means ± SE. Data from the paired capsaicin and ![]() |
RESULTS |
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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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Characterization of -Lg-Evoked Vasodilations
Vasodilator responses.
Superfusion of the cow's milk protein, -Lg (5 µM), evoked a large
dilation (74 ± 5%; n = 13) in
81% (13/16) of milk-sensitized preparations preconstricted with 300 nM
PGF2
(Fig.
2). Dilations were not maintained in the
continued presence of
-Lg (5 µM); all preparations became
completely desensitized after a 2-min or less exposure to
-Lg.
Repeat application of
-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
-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
-Lg.
Therefore, all preparations from one animal were studied within this
time frame.
-Lg (5 µM) had no effect on the resting outside
diameter of arterioles in preparations that were not preconstricted
with PGF2
(n = 3). The resting or preconstricted
arteriolar diameter in preparations from control animals was not
altered by
-Lg (5 µM) (n = 3).
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Actions of the H1 antagonist, pyrilamine,
on -Lg-evoked vasodilations.
The possibility that the
-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
-Lg alone, whereas the other preparation received
-Lg and
pyrilamine (1 µM). In all animals in which
-Lg evoked dilations in
the control preparation (n = 6),
-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
PGF2
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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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 PGF2 (300 nM) was applied. This increase, however, was <7% greater than the
vasoconstrictor response to
PGF2
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
-Lg-evoked vasodilator response was inhibited by 85%
compared with controls (n = 4, P < 0.05) (Fig.
4).
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Electron microscopy.
The mean areas of both dispersed and intact granules in mast cells from
milk-sensitized preparations superfused with 5 µM -Lg were
significantly reduced compared with preparations that had not been
exposed to
-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
-Lg (Fig. 5).
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Examination of Nerve-Mast Cell Interactions
Capsaicin and -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.
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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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DISCUSSION |
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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, -Lg. First, the
-Lg-evoked dilation desensitized during the initial superfusion and a repeat application of
-Lg had no effect after the initial application of
-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
-Lg acts selectively on the mast cell. This
specificity was also supported by studies that demonstrated that
-Lg
had no effect in preparations that were not milk sensitized. Electron
microscopic studies further characterized the effect of
-Lg on
sensitized mast cells, demonstrating a significant decrease in mean
area of both dispersed and intact granules. These data suggest that
-Lg exposure releases preformed mediators such as histamine (2, 7).
This deduction is supported by our pharmacological data that
demonstrated that
-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
-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 -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
-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 -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
-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 -Lg stimulation did not reduce the capsaicin-evoked
response and, similarly, stimulation of capsaicin-sensitive nerves did
not reduce the
-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.
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ACKNOWLEDGEMENTS |
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We thank Margaret Bolton and Gail Pringle for technical assistance.
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FOOTNOTES |
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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.
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