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Vol. 293, Issue 2, 539-544, May 2000
Gastrointestinal Research Group and Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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The relative contributions of cyclooxygenase (COX)-1 and COX-2 in mediating prostaglandin (PG)-dependent chloride secretion were investigated in segments of mouse colon mounted in Ussing-type diffusion chambers. COX-2 mRNA and protein were constitutively expressed as shown by reverse transcription-polymerase chain reaction and Western immunoblot, respectively. COX-2 immunoreactivity was detected immunohistochemically in cells lying subjacent to the crypt epithelial cells. In segments of colon mounted in Ussing chambers, arachidonic acid caused a concentration-dependent increase in short-circuit current that was blocked by piroxicam, the COX-2 inhibitor NS-398, and the COX-1 inhibitor SC-560. Exposure to the PG-dependent secretagogue, bradykinin, also caused an increase in short-circuit current that was not blocked by piroxicam or SC-560, and only by the highest dose of NS-398. When incubated in the presence of 10 µM arachidonic acid, segments of mouse colon produced both PGE2 and PGD2. Synthesis of PGE2 but not PGD2 was blocked by NS-398 and SC-560. These data demonstrate that both COX-1 and COX-2 are constitutively expressed in the mouse colon, and both contribute to PG-dependent electrolyte transport.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Prostaglandins
(PGs) are produced from free arachidonic acid through the catalytic
activity of two cyclooxygenase (COX) enzymes. Until recently, COX-1 was
considered the constitutive isoform, being expressed in almost all
tissues, whereas COX-2 was considered inducible, because its expression
could be triggered by inflammatory cytokines such as interleukin-1
and tumor necrosis factor-
. This paradigm led to the belief that
COX-1, which is found in abundance in the gastrointestinal tract,
produced PGs that were cytoprotective and that subserved physiological
functions such as control of mucosal blood flow and electrolyte
secretion. In contrast, COX-2-derived PGs were considered deleterious
because they were found at sites of inflammation (where COX-2 was
induced) and were hence thought to trigger, participate in, or
exacerbate the inflammatory response. This dogma has provided the
impetus for the recent development of selective COX-2 inhibitors that would inhibit inflammatory COX-2 and spare cytoprotective COX-1 (Masferrer et al., 1994
; Hawkey, 1999). Traditional nonsteroidal anti-inflammatory drugs are relatively nonselective and their chronic
use in conditions such as rheumatoid arthritis is associated with a
high incidence of serious adverse gastrointestinal events (Wallace and
Granger, 1992).
Recently, however, evidence has mounted that clouds the "COX-1 is
good, COX-2 is bad" paradigm. Several groups have reported constitutive expression of COX-2 in numerous tissues, including the
kidney (Harris et al., 1999), female reproductive tract (Slater et al.,
1994) and fetal membranes (Slater et al., 1999), central nervous system
(Yasojima et al., 1999), and vascular endothelium (McAdam et al.,
1999). We have shown in a carageenan-induced rat paw edema model that
COX-2 inhibitors only exhibited anti-inflammatory effects at
concentrations that have been shown to also inhibit COX-1 (Wallace et
al., 1998). In addition, it has recently been shown that COX-2-derived
PGs may exhibit anti-inflammatory properties (Gilroy et al., 1999). A
further complicating observation is the demonstration that COX-1 can be
induced under certain circumstances (Jun et al., 1999). Given these
recent data, it is clear that the physiological roles of the
cyclooxygenases need to be reevaluated.
In the intestinal tract, secretion of chloride ions and water into the
lumen is considered, along with low epithelial permeability, part of a
primary host defense mechanism preventing the translocation of luminal
bacteria, bacterial products, and antigens into the mucosa (Wood,
1993). Permeability defects have been implicated in relapse of
inflammatory bowel disease (Wyatt et al., 1993) and secretory function
is suppressed in inflamed intestine (Goldhill et al., 1993; MacNaughton
et al., 1998). It has long been known that the response of the
epithelium to numerous secretagogues is regulated by PGs.
PGE2, probably derived from subepithelial myofibroblasts, is a "final common mediator" of secretagogues encompassing various lipid mediators, cytokines, oxygen radicals, amines, and enzymes (Hinterleitner and Powell, 1991). It acts both
directly on the enterocyte and via submucosal secretomotor neurons
(Dekkers et al., 1997). In contrast, PGD2 is
antisecretory, functioning to block secretion stimulated by submucosal
secretomotor neurons of the enteric nervous system (Goerg et al.,
1992). The balance between PGE2 and
PGD2 is important in the regulation of epithelial
electrolyte transport (Keenan and Rangachari, 1989). However, the COX
isoform responsible for production of PGs involved in intestinal
secretion is not known. Given the role of PGs in regulating this
important host defense mechanism, and the increasing interest in
selective COX-2 inhibitors, we sought to determine the relative roles
of COX-1- and COX-2-derived PGs in mediating PG-dependent chloride
secretion with a mouse model.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Animals
Male C57Bl/6 mice (4-6 weeks of age) were obtained from Charles River (Montreal, Quebec, Canada) and were housed in wire-bottomed cages under constant temperature (22°C) and photoperiod (12-h light/dark cycle). They were allowed unrestricted access to standard mouse chow and water. Mice were allowed to acclimatize to these conditions for at least 5 days before inclusion in an experiment. All procedures involving animals were approved by the University of Calgary Animal Care Committee and were conducted according to the guidelines of the Canadian Council on Animal Care.
COX-1 and COX-2 Expression and Localization
Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Mice were euthanized by cervical dislocation. Colons were removed and rinsed in cold (4°C) Krebs buffer. Segments of tissue (50-100 mg) were placed in 1.0 ml TRIzol reagent (Life Technologies, Grand Island, NY) and homogenized with a polytron homogenizer (Brinkmann, Mississauga, Ontario, Canada). After homogenization, samples were left at room temperature for 5 min and then chloroform was added (0.2 ml/1 ml TRIzol). Samples were shaken by hand for 15 s, left at room temperature for 2 to 3 min, and then centrifuged at 12,000g for 30 min at 4°C. A 500-µl aliquot of the aqueous phase was placed in a microfuge tube and isopropyl alcohol (0.5 ml/1 ml TRIzol) was added to precipitate the RNA. The tubes were then centrifuged at 12,000g for 20 min at 4°C to pellet the RNA. The supernatant was removed and the RNA was washed with 75% ethanol, vortexed, and centrifuged again. The ethanol was removed and the RNA pellet was air-dried before dissolving in RNase-free water. The ratio of RNA/DNA and the tRNA concentration of the sample were determined with a GeneQuant II nucleic acid analyzer (Pharmacia Biotech, Baie d'Urfé, Quebec, Canada).
The extracted RNA was then reverse transcribed by adding 2.0 µg of RNA per sample to a reaction mixture that contained 2 µl of 10× PCR buffer, 2 µl of mixed nucleotide triphosphates (10 mM), 2 µl of random nucleotide hexamers (N6; 900 pmol/µl), 0.5 µl of RNA guard (27 U), 11 µl of double-distilled water, and 1.5 µl of Superscript reverse transcriptase (300 U). Samples were placed in a thermocycler (Barnstead Thermolyne, Chicago, IL) and reverse transcribed at 42°C for 50 min. For PCR, 2 µl of the reverse-transcribed cDNA was placed in a tube that contained 2 µl of 2 mM mixed nucleotide triphosphates, 5 µl of 10× PCR buffer, and 35 µl of double-distilled water. Two microliters of each of the 5' and 3' primers (Table 1) were added for COX-1 and COX-2 and GAPDH. GAPDH was used as an internal control. Taq polymerase was added with a hot start to reduce nonspecific binding. The samples were cycled as follows: 94°C for 1 min, 55°C for 30 s, and 72°C for 1 min. Optimal amplification was achieved at 32 cycles for COX-1 and COX-2 and 22 cycles for GAPDH. The PCR products were run on a 1% agarose gel containing ethidium bromide. After separation the bands were visualized under UV light with a Gel Doc 2000 (Bio-Rad, Mississauga, Ontario, Canada), and analyzed with Quantity One software (Bio-Rad).
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Western Immunoblot. Mice were euthanized by cervical dislocation. Colons were removed and rinsed in PBS consisting of 137 mM NaCl, 8.16 mM Na2HPO4, 1.47 mM KH2PO4, and 3.22 mM KCl. Samples were homogenized in 1 ml of suspension buffer (0.1 M NaCl; 0.01 M Tris-HCl, pH 7.6; 0.001 M EDTA, pH 8.0; 1 µg/ml aprotinin; 100 µg/ml phenylmethylsulfonyl fluoride; and 1% Triton X-100) and centrifuged at 12,000g for 5 min at room temperature. Half of the supernatant was removed and added to an equal volume of sample buffer (catalog no. S-3401; Sigma Chemical Co., Mississauga, Ontario, Canada). The other half of the sample was used to determine protein concentration with a protein assay kit (Bio-Rad). Samples were boiled for 10 min and the DNA was sheared by sonication. Samples were then centrifuged at 12,000g for 5 min at room temperature and the supernatant run on an 8% acrylamide gel in a mini trans-blot electrophoretic transfer cell (Bio-Rad). Protein was transferred to nitrocellulose overnight at 30 V and 4°C. After transfer, the membrane was blocked in 5% milk for 1 h and washed briefly in PBS-Tween. The COX-1 or COX-2 primary antibodies (goat anti-mouse; Santa Cruz Biotechnologies, Santa Cruz, CA) were diluted 1:75 in 1% milk/PBS-Tween. The membrane was placed in the primary antibody for 1.5 h, and then washed five times in PBS-Tween. The secondary antibody (rabbit anti-goat IgG conjugated to horseradish peroxidase; Santa Cruz Biotechnologies) was diluted 1:10,000 in 1% milk/PBS-Tween. The membrane was placed in the secondary antibody for 1 h, and then washed five times in PBS-Tween. Luminol reagent (Santa Cruz Biotechnologies) was applied to the membrane for 2 min. The membrane was then exposed to X-ray film to visualize the proteins. The X-ray film was analyzed with a GS-710 calibrated imaging densitometer (Bio-Rad) and Quantity One software (Bio-Rad).
Immunohistochemistry. Segments of mouse colon were removed and fixed in Zamboni's fixative overnight. They were then washed in PBS (three times) and then cryoprotected in PBS containing 20% (w/v) sucrose. Sections (12-14 µm) were cut with a cryostat (Microm GmbH, Heidelberg, Germany) and placed onto poly(D-lysine)- coated microscope slides. Sections were washed three times in PBS and were incubated with a rabbit anti-COX-2 primary antibody at 4°C overnight. Sections were then rinsed with PBS and incubated with donkey anti-rabbit IgG CY3-conjugated secondary antibody for 90 min. They were then washed three times in PBS, coverslipped, and viewed under epifluorescence with a Zeiss Axioplan microscope. Images were captured and digitized with a charge-coupled device video camera and Northern Exposure imaging software (Carsen Vision, Edmonton, Alberta, Canada).
Electrolyte Transport Studies In Vitro
Mice were sacrificed by cervical dislocation and segments of colon removed and gently flushed with cold Krebs' buffer (4°C). The segments were opened longitudinally along the mesenteric border and mounted on standard Ussing-type diffusion chambers (Navicyte Inc., Sparks, NV). Tissues were bathed on both the mucosal and serosal surfaces with Krebs' buffer containing 10 mM D-glucose. The bathing solutions were maintained at 37°C and pH 7.4 and were aerated with 5% CO2, 95% O2. The transepithelial potential difference was maintained at zero volts by applying a short-circuit current (Isc) with a voltage clamp apparatus (EVC4000; World Precision Instruments, Sarasota, FL). The Isc was taken as the measure of net active electrolyte transport by the colonic epithelium. Tissues were allowed to equilibrate with respect to Isc for 15 to 20 min. Drugs were applied to the serosal bath.
Materials
Routine chemicals were obtained from BDH Chemicals (Toronto, Ontario, Canada). NS-398 was obtained from Research Biochemicals Inc. (Natick, MA). SC-560 was a generous donation from Dr. Frank Degner, Boehringer-Ingelheim GmbH. Piroxicam was obtained from Sigma Chemical Co. Other chemicals were obtained as indicated in the text.
Statistical Analysis
Data are expressed as the mean ± SE. Comparisons among groups were made by one-way ANOVA with post hoc Newman-Kuels test with Instat version 3.00 (GraphPad Software, San Diego, CA). A P value of <.05 was considered to be significant.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Expression of COX-2 in Mouse Colon.
COX-1 and COX-2 mRNA
expression was observed in full-thickness segments of mouse colon
(n = 3), as demonstrated by RT-PCR (Fig.
1). Western blot analysis revealed COX-1
and COX-2 proteins in full-thickness segments of mouse colon
(n = 3; Fig. 1).
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Prostaglandin-Dependent Secretion.
Concentration-response
relationships were established for the PG-dependent secretagogues
bradykinin (1 nM-1 µM; Musch et al., 1983) and arachidonic acid
(1-100 mM; Field et al., 1984). Both caused concentration-dependent
increases in Isc in unstripped segments of mouse
colon mounted in Ussing chambers (Fig.
3). The response to arachidonic acid
occurred within 1 min, peaked within 5 min, and either remained
elevated or returned to baseline only slowly. The
Isc remained elevated above baseline for the
duration of the experiment. The response to bradykinin also began
within 1 min, peaked within 3 min, and then returned to baseline over the next 5 min.
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; Smith et al., 1998). A high dose (100 µM) of the nonselective
inhibitor piroxicam was used to block both COX-1 and COX-2. The
selective COX-2 inhibitor NS-398 caused a concentration-dependent inhibition of the response to arachidonic acid that was almost complete
at 30 µM (Figs. 4 and
5). The secretory response to arachidonic acid also was blocked by the selective COX-1 inhibitor SC-560 at
nanomolar concentrations and by the nonselective COX inhibitor piroxicam (Fig. 5).
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Prostaglandin Synthesis.
To determine the degree to which the
COX inhibitors blocked PG synthesis, further experiments were conducted
in which whole-thickness segments of mouse colon were incubated at
37°C in Krebs buffer containing 10 µM arachidonic acid or 10 nM
bradykinin plus piroxicam (100 µM), NS-398 (1-30 µM), or SC-560
(0.1-1 µM). These concentrations of arachidonic acid and bradykinin
stimulated chloride secretion in Ussing chamber studies (see above).
Controls were incubated with vehicle, arachidonic acid, or bradykinin
only. After 10 min, the tissues were removed and processed for
measurement of PGs as previously described (Wallace et al., 1990).
Tissue levels of PGE2 and
PGD2 were determined with commercial
enzyme-linked immunosorbent assay kits (Cayman Chemical, Ann Arbor,
MI). The levels of PG synthesis in the presence of the inhibitors were expressed as a percentage of the maximal response, in the presence of
arachidonic acid or bradykinin, to take interassay variation into
account. In the presence of arachidonic acid, piroxicam almost completely blocked PGE2 synthesis (Fig.
6). NS-398 caused a
concentration-dependent inhibition of PGE2 that
was almost complete at 30 µM. SC-560 also reduced
PGE2 synthesis in a dose-dependent manner, but
did not cause more than an ~55% inhibition (Fig. 6). In the same
tissues, PGD2 synthesis was significantly
inhibited by 100 µM piroxicam but not by NS-398 (1-30 µM) or
SC-560 (0.1-1 µM; Fig. 6). Bradykinin did not stimulate
PGE2 or PGD2 synthesis
compared with unstimulated controls (Fig.
7). NS-398 (30 µM) and SC-560 (300 nM)
reduced PGE2 synthesis in the presence of
bradykinin. Neither inhibitor significantly affected
PGD2 synthesis. Piroxicam induced a significant reduction in PG synthesis compared with controls (Fig. 7).
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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In this study, we have demonstrated the constitutive expression of
both COX-1 and COX-2 in the mouse colon, and have provided evidence
that both COX-1 and COX-2 contribute to PG-dependent secretion in
vitro. It has now been widely demonstrated that COX-2 is constitutively
expressed in a variety of tissues, including brain, kidney, female
reproductive tract, fetal tissues, and vascular endothelium. As
expected, COX-1 was constitutively expressed in mouse colon. However,
we now demonstrate constitutive COX-2 expression in mouse colon. In
each mouse studied, COX-2 mRNA and protein were expressed. COX-2
expression was not due to ongoing inflammation because none of the mice
exhibited clinical, macroscopic, or microscopic signs of colonic
inflammation. Of note, we also have shown constitutive colonic
expression of the inducible isoform of nitric oxide synthase in the
absence of inflammation (MacNaughton et al., 1998). Based on our
immunohistochemical data, it appears that COX-2 is expressed primarily
in cells lying subjacent to the crypt enterocytes. The subepithelial
myofibroblasts have long been known to be a source of PGs that regulate
epithelial function (Hinterleitner and Powell, 1991) and are localized
to this region of the mucosa. However, the isoform of COX present in
these cells, and which is responsible for the production of secretory
PGE2, is not known. Our data are suggestive of,
but not proof of, subepithelial myofibroblasts as the cellular source
of COX-2-derived PGs regulating epithelial secretory function.
Both arachidonic acid and bradykinin caused concentration-dependent
increases in short-circuit current in segments of mouse colon mounted
in Ussing chambers. Both of these secretagogues have been reported to
stimulate chloride secretion through the synthesis of PGs (Musch et
al., 1983; Field et al., 1984). Pretreatment of the tissue with the
nonselective COX inhibitor piroxicam almost abolished the response to
arachidonic acid, but failed to significantly inhibit the response to
bradykinin. The response to arachidonic acid was significantly and
concentration-dependently blocked by the COX-2 inhibitor NS-398,
suggesting that a substantial portion of arachidonic acid-mediated
secretion was through a COX-2-dependent mechanism. Similarly, the
response to arachidonic acid was reduced by pretreatment with the
selective COX-1 inhibitor SC-560. That the response could be blocked by
both selective COX-1 and COX-2 inhibitors was curious. The most obvious
explanation would be that one or both of these inhibitors were acting
nonselectively. However, care was taken to use concentrations of NS-398
and SC-560 that have previously been shown to be selective for COX-2
and COX-1, respectively (Futaki et al., 1994; Smith et al., 1998). NS-398 at 100 µM did not block the activity of COX-1 isolated from
sheep seminal vesicles, whereas the IC50 of
NS-398 against COX-2 isolated from sheep placenta was 3.8 µM (Futaki
et al., 1994). Although action against isolated, purified enzyme may
not equate to activity in tissue, 30 µM NS-398, the highest
concentration used herein, has been reported to be a selective COX-2
inhibitor in rat aortic organ culture (Hamilton et al., 1999). SC-560
demonstrated a 1000-fold higher selectivity for human recombinant COX-1
than for human recombinant COX-2 in vitro (Smith et al., 1998).
The lack of effect of COX inhibition on the secretory response to
bradykinin was unexpected, given the previous reports of the
PG-dependence of this response (Musch et al., 1983; Field et al.,
1984). A possible explanation may be that bradykinin was added to the
tissue in the presence of arachidonic acid. The availability of
arachidonic acid has been shown to significantly affect the ability of
COX-2 inhibitors to block COX-2 (Hamilton et al., 1999). In our study,
only the highest dose of NS-398 (30 µM) significantly blocked the
response to bradykinin. However, the ability of the COX inhibitors to
block the response to arachidonic acid itself in these preparations
argues against an effect of arachidonic acid on their ability to block
the subsequent response to bradykinin. It may be that in the mouse
colon, there is a substantial PG-independent component to the secretory
response, and this is confirmed by the experiments showing a lack of
effect of bradykinin on PG synthesis, at a concentration that elicited
a change in Isc. Indeed, it has been reported
that there is a substantial neural component to the response to
bradykinin in rat (Perkins et al., 1988) and dog (Rangachari et al.,
1993) colons. Our data support a PG-independent response to bradykinin
in mouse colon, however, determining the exact mechanism of action was
beyond the scope of this study.
To further characterize the isoforms of COX present in the mouse colon, experiments were conducted in which isolated, full-thickness segments of colon were incubated in the presence of arachidonic acid or bradykinin and either NS-398, SC-560, or piroxicam, with subsequent measurement of tissue levels of PGE2 and PGD2. Arachidonic acid induced PG synthesis. NS-398 caused a concentration-dependent decrease in the synthesis of PGE2, with almost complete inhibition at 30 µM. Similarly, piroxicam at 100 µM abolished PGE2 synthesis. SC-560 only reduced PGE2 synthesis to ~55% of control. This is interesting in that these same doses of SC-560 almost abolished arachidonic acid-induced chloride secretion. These data suggest that there may be a PG-independent inhibitory effect of SC-560 on arachidonic acid-induced chloride secretion in this in vitro model. Interestingly, bradykinin, at a dose that stimulated chloride secretion, did not stimulate PG synthesis. This supports our contention (see above) that bradykinin-induced secretion in the mouse colon is dependent on mechanisms other than PGE2, and represents a significant species difference compared with guinea pig and rabbit.
Although NS-398 and SC-560 inhibited PGE2
synthesis, they did not significantly block the synthesis of
PGD2. The synthesis of PGD2
was, however, almost completely blocked by 100 µM piroxicam. The
differential effect of the COX inhibitors on PGE2
and PGD2 synthesis was not expected because both
COX-1 and COX-2 catalyze the production of PGH2,
the precursor for both PGE2 and
PGD2. The results suggest that the effect of
NS-398 in particular may be a combination of inhibition of COX-2 and a
further downstream step in the synthesis of PGE2
from PGH2. However, that NS-398 did not affect
the chloride secretory response to serosally applied PGE2 (data not shown) argues against this
conclusion. Further studies will be required to determine the exact
mechanism of action in this regard. That these compounds can block
PGE2 synthesis, although leaving
PGD2 synthesis relatively untouched, has
important physiological implications for mucosal function.
PGE2 and PGD2 have
important interactions regulating colonic secretion (Keenan and
Rangachari, 1989). In particular, PGD2 is clearly
antisecretory (Goerg et al., 1992), so a shift in the balance of
eicosanoids in favor of PGD2 would lead to a
suppression of secretion.
Constitutive expression of COX-2 in the intestinal mucosa, and its
ability to produce secretory PGs, argues for an important role of this
isoform of COX in the host's response to infections. Recently, Beubler
et al. (1999) demonstrated that NS-398 and DFU (another COX-2
inhibitor), given at doses that are selective for COX-2, could block
the large secretory response to cholera toxin in rat jejunum in vivo.
Interestingly, the effect was not altered by pretreatment with
dexamethasone, suggesting that constitutive, and not induced, COX-2
mediated the response to cholera toxin (Beubler et al., 1999). Given
the importance of the secretory response as part of the mucosal host
defense mechanism (Wood, 1993), the effects of COX-2-inhibitors on
eicosanoid balance could have important clinical implications.
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Acknowledgments |
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We thank Dr. Keith Sharkey, University of Calgary, for assistance with immunohistochemistry.
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Footnotes |
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Accepted for publication January 12, 2000.
Received for publication August 2, 1999.
1 This study was supported by a New Investigator Establishment grant from the Canadian Association of Gastroenterology and the Medical Research Council of Canada.
2 W.K.M. is an Alberta Heritage Foundation for Medical Research Scholar.
Send reprint requests to: Wallace MacNaughton, Ph.D., Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1. E-mail: wmacnaug{at}ucalgary.ca
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Abbreviations |
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PG, prostaglandin; COX, cyclooxygenase; RT-PCR, reverse transcription-polymerase chain reaction; Isc, short-circuit current; IR, immunoreactivity.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Isc) of isolated segments of mouse colon to
serosal application of arachidonic acid (
) or bradykinin (
). Both
caused a concentration-dependent increase in short-circuit current.
, PGE2; 
, PGD2. *P < .05, ***P < .001.