1 Division of Gastroenterology
and 4 Department of Surgery, Previous studies
indicated that the peptide substance P (SP) causes
Cl
genistein; histamine; short-circuit current; neurokinin 1 receptor
SUBSTANCE P (SP), an 11-amino acid neuropeptide (11),
is present in enteric nerves and sensory neurons of the small and large
intestine (13, 18, 58). An increasing body of evidence indicates that
SP is involved in the pathophysiology of intestinal secretion and
inflammation in animals and humans. Administration of SP-receptor
antagonists in rats reduced secretory and inflammatory changes in
animal models of acute and chronic intestinal inflammation (8, 9, 33,
36, 40). Furthermore, SP immunoreactivity and SP binding are increased
in colon of patients with inflammatory bowel disease (25, 32).
Interestingly, mice genetically deficient in the SP neurokinin 1 (NK1) receptor are almost
protected from the secretory and inflammatory changes mediated by
Clostridium difficile toxin A (10),
providing direct evidence for the importance of these receptors in
intestinal secretion and inflammation.
Several electrophysiological studies demonstrate that SP induces
secretion in animal intestine. For example, serosal application of SP
causes a rapid increase of short-circuit current
(Isc) in pig
(7, 38), guinea pig (21, 23), and mouse (53) small intestine, and
guinea pig (29) and dog colon (42) mounted in Ussing chamber. These
studies (7, 21, 23, 29, 38, 42, 53) also provide pharmacological
evidence that enteric nerves and mast cells may be involved in these
SP-mediated responses, in agreement with previous findings showing a
SP-mucosal mast cell interaction in vivo and in vitro (47, 53, 57).
Morphological studies also indicate that mast cells are in intimate
contact with nerves in rat small intestine (50) and human colon (49). However, the effects of SP on human colonic mucosa have not been investigated.
We studied the effects of SP on human colonic mucosal electrophysiology
in vitro using Ussing chambers. Participation of enteric nerves and
mast cells and the involvement of the secretagogues histamine and PGs
in SP-mediated changes in electrical parameters were also examined.
Furthermore, using a specific antibody directed against the COOH
terminus of the human SP receptor, we provide direct
immunohistochemical evidence for the presence of SP receptors on human
colonic mucosal nerves. Because tyrosine phosphorylation has been
demonstrated to be involved in SP-mediated signal transduction in
nonepithelial cells in vitro (30), the effect of the tyrosine kinase
inhibitor genistein on SP-induced colonic responses was also investigated.
Materials
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-dependent secretion in
animal colonic mucosa. We investigated the effects of SP in human
colonic mucosa mounted in Ussing chamber. Drugs for pharmacological
characterization of SP-induced responses were applied 30 min before SP.
Serosal, but not luminal, administration of SP
(10
8 to
10
6 M) induced a rapid,
monophasic concentration and
Cl
-dependent,
bumetanide-sensitive short-circuit current
(Isc) increase, which was inhibited by the SP neurokinin 1 (NK1)-receptor antagonist CP-96345, the neuronal blocker TTX, the mast cell stabilizer
lodoxamide, the histamine 1-receptor antagonist pyrilamine, and the PG
synthesis inhibitor indomethacin. SP caused TTX- and
lodoxamide-sensitive histamine release from colonic mucosa. Two-photon
microscopy revealed NK1
(SP)-receptor immunoreactivity on nerve cells. The tyrosine kinase
inhibitor genistein concentration dependently blocked SP-induced Isc increase
without impairing forskolin- and carbachol-mediated Isc increase. We
conclude that SP stimulates
Cl
-dependent secretion in
human colon by a pathway(s) involving mucosal nerves, mast cells, and
the mast cell product histamine. Our results also indicate that
tyrosine kinases may be involved in this SP-induced response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Methods
Ussing chamber experiments.
In this study a total of 104 individual specimens of tumor-free
left-sided colon was used. After removal of the seromuscular layer by
blunt dissection, two to six mucosal sheets from each specimen,
measuring 5-10 cm2, were
vertically mounted in Ussing chambers (DCTSYS, Precision Instrument
Design; 1.0 cm2 surface area), as
previously described (15, 19, 43). Luminal and serosal sides were
bathed at 37°C in 8 ml of nutrient buffer containing (in mM) 122.0 NaCl, 2.0 CaCl2, 1.3 MgSO4, 5.0 KCl, 20.0 glucose, and
25.0 NaHCO3 (pH 7.45 when gassed
with 95% O2-5% CO2). When
Cl-free buffer was used,
Cl
was replaced by an
equimolar concentration of isethionate (42, 53). Potential difference
(PD) and Isc were
continuously measured and recorded every 1-10 min. Luminal and
serosal solutions were connected via Ag-AgCl electrodes to a
voltage-current clamp (model VCC600, Physiological Instruments).
Resistance was calculated using Ohm's law from the open circuit PD and
the
Isc.
PD values were given in millivolts (lumen negative),
Isc values in
microamperes per square centimeter, and resistance in ohms times square
centimeter. PD and resistance values were corrected for
the junctional potentials (<0.1 mV) between luminal and serosal
solutions and the buffer resistance, respectively, as previously
described (43). Drug-induced Isc and
resistance responses are presented as changes from values before drug
administration and given as
Isc and
resistance, respectively. Baseline values for
Isc and
resistance were 82 ± 10 µA/cm2 and 101 ± 16
· cm2,
respectively (n = 72). The protocol
for use of human tissues was approved by the Ethics Committee of Beth
Israel Deaconess Medical Center and University Clinic of Vienna.
Measurements of epithelial permeability.
Epithelial permeability to
[3H]mannitol was
determined as previously described (43). After an equilibration period
of 30 min,
[3H]mannitol (26.4 Ci/mmol; DuPont NEN, Boston, MA) was added to 8 ml of serosal buffer at
a final concentration of 0.32 nM. Luminal aliquots of 200 µl were
taken for scintillation counting using 5 ml of "Quicksave A"
scintillation fluid (Zinser, Maidenhead, UK) and replaced with 200 µl
of fresh buffer to eliminate a transepithelial solute gradient. The
radioactivity of
[3H]mannitol in the
luminal fluid was measured in counts per min (cpm/200 µl) and was
determined for two subsequent 30-min periods before and after
administration of serosal
106 M SP.
Determination of relative paracellular resistance.
To determine the separate contribution of the paracellular and
transcellular pathway to the epithelial resistance response during
SP-induced Isc
increase, we used the approach of Yonath and Civan (60) and Parkos et
al. (37). Conductance, the reciprocal of transepithelial resistance,
was plotted against
Isc for each of
the three phases of SP-induced
Isc changes:
phase 1, period of increase of
Isc 0-15 min
after administration of SP; phase 2,
period of decrease of
Isc 15-30
min after; phase 3, period of further
Isc decrease
30-45 min after. By regression analysis, the value for conductance
at the x-axis intercept (where
Isc would equal
zero) was determined, and the reciprocal of this value was used to
ascertain the relative paracellular resistance during each of the three
phases [resistance
( · cm2) = 1/conductance (mS/cm2) × 1,000].
Experimental Design
After 30 min of baseline incubation, colonic tissues were incubated under serosal presence or absence of 10Pharmacological blockade of SP effects.
Thirty minutes before serosal administration of 106 M
SP, human colonic tissues were exposed serosally to either the
muscarinic-receptor antagonist atropine
(10
5 to
10
7 M) (38, 53) or to the
nicotinic-receptor antagonist hexamethonium (10
4 to
10
7 M), the neurotoxin TTX
(10
5 to
10
8 M) (3), the mast cell
stabilizer lodoxamide (10
5
to 10
8 M) (28), the
histamine 1 (H1)-receptor
antagonist pyrilamine (10
6
to 10
8 M) (53, 54), the
H2-receptor antagonist ranitidine
(10
5 to
10
7 M) (53), the PG
synthesis inhibitor indomethacin
(10
5 to
10
8 M) (42, 54), the
inhibitor of the
Na+-K+-2Cl
cotransporter bumetanide
(10
5 to
10
7 M) (54), the
Na+-K+-ATPase
inhibitor ouabain (10
6 M,
10
7 M) (35), or the
K+-channel blocker charybdotoxin
(10
6 M,
10
7 M) (44).
Effect of drugs on basal human colonic electrophysiology.
Incubation of tissues with
106 M of indomethacin for
30 min or genistein for 60 min caused a 25%
(P < 0.05 vs. baseline,
n = 6) and 30%
(P < 0.01 vs. baseline,
n = 8) decrease of human colonic
Isc,
respectively. Serosal presence of ouabain for 30 min caused a
statistically significant decrease of colonic
Isc (baseline vs.
30 min of ouabain: 78 ± 6.1 vs. 26.4 ± 4.8 µA/cm2;
P < 0.01, n = 6), without changing
transepithelial resistance. None of the other drugs used in this study
had an effect on basal electrophysiological values (data not shown).
Human colonic mucosa displayed stable transepithelial resistance over
the 2-h incubation, indicating excellent tissue viability (data not shown).
Histamine assay.
Histamine release from human colonic mucosa was measured by a
commercially available ELISA assay (enzyme immunoassay kit ref. 1153, Immunotech, Westbrook, ME). Four human colonic explants from a single
individual were mounted in Ussing chambers in parallel and incubated
with either serosal buffer alone or buffer containing 106 M of either TTX or
lodoxamide 30 min before serosal administration of
10
6 M of SP. The fourth
tissue was incubated with buffer alone and received vehicle instead of
SP (n = 6, quadruplicate). Histamine (pg · ml
1 · cm
2)
was determined in serosal aliquots (50 µl) taken before and 5 and 10 min after administration of serosal SP. All samples were stored at
70°C for no more than 3 days before histamine measurements. Histamine concentration was also determined in luminal aliquots taken
before and 10 min after SP administration
(n = 3).
Histology. After Ussing chamber experiments colonic tissues were processed for light microscopy as previously described (15, 43). None of the colonic tissues used showed any signs of inflammation or malignancy. Furthermore, neither SP nor any of the drugs used caused morphological changes in human colon (data not shown).
SP-receptor antiserum. Antiserum generated against a peptide representing the last 15 amino acids of the human SP receptor COOH terminus was prepared by Immuno-Dynamics (La Jolla, CA) according to the m-maleim-idobenzoyl-N-hydroxysuccimide coupling method described by Kitigawa and Aikawa (27) and characterized by ELISA. This antiserum immunoprecipitated photoaffinity-labeled SP receptors expressed in Chinese hamster ovary cells transfected with the human SP receptor (31).
Immunofluorescent labeling. Freshly frozen mucosal preparations of human colon were cut (5 µm) and fixed in 4% paraformaldehyde. Sections were washed in Tris-buffered saline (TBS, pH 7.5) and incubated with blocking solution (TBS, pH 7.5, containing 50 mM ammonium chloride, 1% normal donkey serum, and 3% BSA) for 1 h at room temperature. Sections were next incubated for 1 h with 1:200 dilution of either the NK1-receptor antiserum or with rabbit preimmune serum at room temperature. In some experiments the NK1-receptor antiserum (1:200 dilution) was preincubated overnight at room temperature with the COOH-terminal 15-amino acid peptide used to generate the antiserum before addition to the sections. For immunofluorescent labeling of nerve cells, sections were incubated for 1 h with a mouse monoclonal IgG1 antibody directed against rat neurofilament polypeptide (1:50 dilution; NCL-NF200, Novocastra Laboratories, Newcastle upon Tyne, UK). All dilutions were made in blocking buffer. The sections were then washed in TBS and incubated for 1 h at room temperature with FITC-conjugated anti-rabbit antibody or tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-mouse IgG antibody (all Jackson ImmunoResearch Laboratories, West Grove, PA) (1:50 dilution). After being washed in TBS, sections were mounted in antibleach solution (10% PBS, 90% glycerol, containing 1 mg/ml n-propyl gallate), examined, and photographed using a laser confocal microscope (Zeiss, Thornwood, NY) or a two-photon scanning microscope.
Two-photon fluorescence microscopy. The two-photon scanning system was adapted to a Zeiss Axiovert 110 microscope as previously described (34, 59). The excitation source is a Ti-Sapphire laser (Mira 900, Coherent, Palo Alto, CA) tuned to 730 nm. The typical power incident on the sample is <5 mW. The samples were imaged with a Zeiss ×40 fluor (1.2 numerical aperture, oil) objective. For the colocalization experiments, the FITC staining was imaged with a filter combination consisting of a 35-nm wide band-pass filter center at 535 nm, a 500-nm long-pass filter, and a BG-39 infrared filter; the TRITC staining was imaged with a filter combination consisting of a 600-nm long-pass filter and two BG-39 Schott glass infrared filters. The filter combination is chosen to minimize interference between the two color channels (FITC, green; TRITC, red). We have further obtained an image of whole tissue fluorescence (see Fig. 6D) including FITC and TRITC fluorescence and tissue autofluorescence using a combination of two BG-39 Schott glass filters, as described previously (59).
Statistical Analysis
All data are expressed as means ± SE, and probabilities were regarded as significant when they reached a 95% level of confidence (P < 0.05) using Student's t-test for paired and unpaired observations. ![]() |
RESULTS |
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SP Effects on Colonic Electrophysiology
The effects of different concentrations of SP on human colonic electrophysiology were compared using Isc, transepithelial PD, and electrical resistance. Serosal administration of 10
|
Serosal SP exposure (106 M)
caused a rapid, transient
Isc increase and
resistance decrease, which peaked after 10 min (Fig. 1,
A and
C; P < 0.001 vs. controls) and returned toward baseline values after 40 min. Luminal addition of
10
6 M SP did not cause
changes in electrical parameters (n = 6, data not shown).
As shown in Fig. 2, preincubation of
tissues with 106,
10
7, and
10
8 M of the
NK1-receptor antagonist CP-96345
concentration dependently inhibited SP
(10
6 M)-induced
Isc and
resistance changes by 80, 60, and 20%, respectively, whereas
administration of 10
9 M had
no effect. In contrast, the inactive enantiomer of the NK1-receptor antagonist CP-96344
(10
6 M) did not have an
effect on SP-induced changes in electrical parameters. These results
indicate that SP induces
Isc increase by
acting on NK1 receptors in human
colon.
|
Effect of SP on Paracellular Epithelial Resistance
To determine whether the SP-induced resistance decrease is due to an increase in transcellular or paracellular epithelial conductance, we assessed the effect of serosal SP on transepithelial [3H]mannitol flux and relative paracellular resistance of human colon. SP (10Ionic Basis of SP-Induced Isc Increase
Our results showed that the SP-induced Isc increase was associated with a decrease of colonic PD, indicating an increase of negative charges on the luminal side that could be attributed to either enhanced movement of positive charges from the luminal to the serosal side of the mucosa (i.e., Na+) (4, 22, 45) or movement of negative charges into the lumen (i.e., ClEffect of TTX
Because previous in vitro studies showed that SP-induced secretion in guinea pig and dog colon was inhibited by pretreatment with the neuronal blocker TTX (29, 42), we investigated the effect of neuronal blockers on SP-induced changes in electrical parameters in human colonic mucosa. We found that the SP-induced Isc increase was inhibited by 12.4 ± 3.6% (P < 0.05), 41 ± 2.1% (P < 0.01), 84 ± 1.5% (P < 0.001), and 98 ± 1.2% (P < 0.001) after preincubation of the colonic mucosa with 10Involvement of Mast Cells and Histamine
Several studies indicate that mast cells and histamine participate in SP-mediated ion secretion in small and large intestine (29, 33, 53). Thus we tested the effect of different concentrations of the mast cell stabilizer lodoxamide and the H1- and H2-receptor antagonists pyrilamine and ranitidine, respectively, on SP-induced Isc increase. We found that the SP-induced Isc increase was inhibited by 97.4 ± 1.0% (P < 0.001), 73.2 ± 3.1% (P < 0.01), and 38.6 ± 4.5% (P < 0.05) after 10SP-Induced Histamine Release
The results with the H1-receptor antagonist pyrilamine and the mast cell-stabilizer lodoxamide indicated that histamine is involved in the mediation of SP-induced Isc increase. We therefore tested whether SP was able to induce histamine release from human colonic mucosa mounted in Ussing chamber. As shown in Fig. 3, serosal administration of 10
|
Effect of Indomethacin
Mast cells, fibroblasts, and inflammatory cells of the lamina propria are major sources of PGs, which are potent stimulators of intestinal secretion (1, 5, 13, 22). Thus we tested the concentration-dependent effect of the PG synthesis inhibitor indomethacin on SP-induced Isc increase. We found that the SP-induced Isc increase was inhibited by 71 ± 2.0% (P < 0.001), 48 ± 2.5% (P < 0.01), 22 ± 2.3% (P < 0.05), and 3.0 ± 1.2% (P > 0.05) after preincubation of the colonic mucosa with 10Effect of Genistein
Because tyrosine phosphorylation has been demonstrated to be involved in SP-mediated signal transduction in human astrocytoma cells in vitro (30), we investigated the effect of the tyrosine kinase inhibitor genistein on SP-induced changes in electrical parameters. Serosal administration of genistein before and during SP exposure concentration dependently inhibited SP-induced Isc increase and resistance decrease. Although SP-induced Isc increase was completely blocked by 10
|
Distribution of NK1 (SP)-Receptors in Human Colonic Mucosa
Using an antibody directed against the COOH terminus of the SP receptor, we determined the distribution of SP receptor in human colonic mucosa by confocal microscopy. As shown in Fig. 5A, SP-receptor immunoreactivity was abundant in cells of the colonic lamina propria but was not observed in the epithelial cell layer. Preincubation of the SP-receptor antiserum with an excess of the COOH-terminal 15-amino acid peptide of the SP receptor before incubation with the colonic sections showed almost complete disappearance of staining (Fig 5B). Furthermore, tissues exposed to control rabbit antiserum showed very little immunoreactivity (Fig. 5C). SP-receptor immunoreactivity on nerve cells was determined using double-staining and two-photon microscopy (Fig. 6). Sections of human colonic mucosa were stained with SP-receptor antiserum (Fig. 6A) or anti-neurofilament antibody (Fig. 6B) and imaged with a two-photon fluorescence microscope. Marked area in Fig. 6D, showing whole tissue fluorescence, corresponds to the areas shown at higher magnification in Fig. 6, A-C. As shown in Fig. 6C, the merged images show colocalization of SP receptor on lamina propria nerve cells underlying the epithelium.
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DISCUSSION |
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The findings of this study indicate that SP induces
Cl-dependent secretory
response in normal human colon in vitro, which is mediated via
NK1 receptors, mucosal nerves and
mast cells, and the mast cell product histamine. Furthermore, a
tyrosine kinase(s) may be involved in the mediation of SP-induced
secretion. Our evidence that SP induces a
Cl
-dependent
Isc increase is
in agreement with data obtained in guinea pig (29) and dog colon (42)
and guinea pig (21, 23) and mouse (53) small intestine in vitro. We
understand that ion flux studies using radiolabeled
22Na+
and
36Cl
represent a more accurate method to measure transepithelial ion movement. However, because for radiosafety reasons we cannot perform these experiments in our laboratory, we used indirect pharmacological studies to assess the ionic basis of the SP-induced
Isc increase.
In the human colon SP-induced Isc increase was inhibited by the specific neuronal blocker TTX, which is in keeping with data obtained in animal small and large intestine in vitro (7, 21, 23, 29, 38, 42, 53). In contrast, the nicotinic and muscarinic ACh-receptor antagonists hexamethonium and atropine, respectively, did not inhibit SP-induced secretion, in agreement with results obtained in dog colon (42). However, Kuwahara and Cooke (29) previously showed that atropine inhibits SP-induced secretion in guinea pig colon. These different responses could be attributed to species differences in tissues used in our study and those of Kuwahara and Cooke (29). Together, our data suggest that in human colonic mucosa ACh-containing neurons are not involved in the mediation of SP-induced neuronal reflexes.
The mast cell "stabilizer" lodoxamide and the H1-receptor antagonist pyrilamine profoundly inhibited SP-induced changes in electrical parameters in human colon. SP caused a lodoxamide-sensitive release of histamine from human colonic mucosa (Fig. 3). Wang et al. (53) showed that mast cell-deficient mice exhibit a reduced ileal secretory response to SP, and Kuwahara and Cooke (29) showed that pyrilamine inhibited SP-induced Isc increase in guinea pig colon in vitro. Using mast cell-deficient mice, we showed that intestinal secretion and inflammation mediated by C. difficile toxin A involves a SP mast cell-dependent pathway (57). The fact that histamine directly stimulates colonic secretion via H1 receptors is well established (22, 24, 54, 56). We also found that, on an equimolar basis, the H2-receptor antagonist ranitidine caused a smaller but significant inhibition of SP-induced Isc increase in human colon compared with the effect of the H1-receptor antagonist pyrilamine. Frieling and co-workers (16) showed that in the guinea pig colon histamine acts on enteric nerves via H2 receptors. Taken together findings in this and the studies discussed above (16, 22, 24, 29, 53, 54, 56) indicate that in normal human colon SP-induced Isc increase is mediated via histamine acting on H1 receptors on colonic epithelial cells and probably on H2 receptors on nerve cells.
Our results indicate that PGs are involved in the mediation of
SP-induced colonic secretion in line with previous findings in guinea
pig (29) and dog (42) colon in vitro. Although the cellular sources of
PGs in our experimental system cannot be defined, it is well
established that they can be released from lamina propria cells,
including basophils, fibroblasts, and mast cells (5, 13, 58).
Furthermore, there is evidence that histamine induces increased
synthesis of PGE2 in guinea pig
colon (54), and PGE2 itself causes
a transient Cl secretory
response in human and rat colon in vitro (46, 55). Using a coculture
system, Berschneider and Powell (2) demonstrated that fibroblasts
amplify histamine-induced secretion in T84 cell monolayers and that
this effect is inhibited by indomethacin.
Results in this paper indicate that SP induces changes in electrical parameters by acting on NK1 receptors on lamina propria nerve cells of human colonic mucosa (Fig. 2), in keeping with studies in guinea pig (29) and canine (42) colon. Other laboratories also provided evidence that several cell types in the animal colonic mucosa may express SP receptors, including nerves (6, 39), endothelial cells (39), and mast cells (47). In contrast, Cooke et al. (14) showed that luminal administration of the SP analog [Sar9, Met (O2)11]SP causes an Isc increase in guinea pig colon in vitro. Recent studies demonstrate expression of mRNA for NK1 (SP) receptor in dog crypt colonocytes (26) and in isolated guinea pig colonic epithelial cells (14). Different ligands (SP vs. SP analog), experimental approaches (immunohistochemistry vs. in situ hybridization), and species (human vs. guinea pig and dog colon) may account for the differences observed in this study and the study of Cooke et al. (14) and Khan et al. (26).
We report here that genistein inhibited SP-induced Isc increase in the human colon (Fig. 4A), indicating that tyrosine kinases are involved in this response. Several studies also indicate participation of tyrosine kinases in the signal transduction pathway following binding of SP to its receptor in nonepithelial cells (20, 30). We also show that genistein blocked the SP-mediated Isc increase without impairing secretory responses to forskolin and carbachol (Fig. 4B). Thus genistein may inhibit tyrosine kinases required for SP-activated signal transduction and does not alter secretagogue-stimulated secretion from colonic epithelial cells.
Our results indicate that nerve cells, mast cells, and histamine are
involved in the secretory effects of SP. Because mast cells represent
the major source of colonic histamine (5, 17), we conclude that in the
human colon mast cells release histamine on SP-induced nerve cell
stimulation. However, based on our results we cannot determine whether
SP directly activates mast cells to release histamine. This would seem
unlikely because previous studies showed that SP used at 30 µM failed
to stimulate release of histamine from human intestinal mucosal mast
cell preparations (12). According to the findings obtained in this and
previous studies discussed above, the following sequence of events
appears to mediate the SP effects in normal human colon. When
NK1 receptor binding occurs, SP
activates nerve cells to release a noncholinergic mediator(s), which in
turn stimulates mast cells to release histamine and probably PGs. These
secretagogues in turn directly activate
Cl secretion from colonic
epithelial cells (2, 24, 46, 55, 56). However, involvement of mediators
released from other cells of the colonic lamina propria, including
endothelial cells, basophils, and fibroblasts, in the mediation of
these SP effects cannot be excluded (2, 5).
In conclusion, our findings indicate that in the normal, noninflamed human colon SP responses are processed and amplified via "cross talk" between enteric nerves and mast cells, lamina propria immune cells, and fibroblasts and mediators (histamine, PGs) released from these cells. Our results are in keeping with the notion that enteric nerves and mast cells act as a functional unit in sensing, processing, and transducing SP-induced stimuli to epithelial cells and cells of the lamina propria that result in the modulation of colonic secretion. In addition, results in this paper may be relevant to the participation of SP in the pathophysiology of human colonic secretory disorders (25, 32).
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ACKNOWLEDGEMENTS |
---|
This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47343 (C. Pothoulakis) and DK-42061 (J. B. Matthews) and by grants from the "Jubiläumsfonds der Österreichischen Nationalbank" and the "Anton Dreher-Gedächtnisschenkung des medizinischen Dekanates der Universität Wien." M. Riegler was supported by a fellowship from Max Kade Foundation, New York, I. Castagliuolo by a Research Fellowship Award from the Crohn's and Colitis Foundation of America, and P. T. C. So by Natural Science Foundation Grant MCB-960 4382 and DuPont Educational Aid Program.
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FOOTNOTES |
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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 and other correspondence: C. Pothoulakis, Div. of Gastroenterology, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (E-mail: cpothoul{at}caregroup.harvard.edu).
Received 29 October 1998; accepted in final form 12 March 1999.
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