The mechanisms of diarrhea in Asiatic cholera have been studied extensively. Cyclic AMP, 5-hydroxytryptamine, prostaglandins, and the function of neuronal structures have been implicated in the pathogenesis of cholera. To elucidate the role of the different isoforms (COX-1 and COX-2) of cyclooxygenase in cholera toxin (CT)-induced fluid secretion and intraluminal prostaglandin E2 (PGE2) release in the rat jejunum in vivo, the effects of the COX-2 inhibitors NS-398 ([N-(2-cyclohexaloxy-4-nitrophenyl)methanesulfonamide]) and DFU [5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone], and of the COX-1 inhibitor SC-560, were studied. Net fluid transport was measured gravimetrically and PGE2 by radioimmunoassay. COX-1 and COX-2 mRNA expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and COX-2 protein by Western blot analysis in mucosal scrapings. CT caused profuse net fluid secretion in all control rats. The COX-2 inhibitors NS-398 and DFU, but not the COX-1 inhibitor SC-560 or dexamethasone, dose-dependently inhibited CT-induced fluid secretion and PGE2 release. RT-PCR showed expression of COX-1 and of COX-2 mRNA in control rats. CT did not induce an increase and dexamethasone did not reduce COX-2 mRNA, whereas lipopolysaccharide caused a marked induction of COX-2 mRNA, which was inhibited by dexamethasone. A weak band of COX-2 protein was observed in controls; however, CT enhanced COX-2 levels, which remained unaffected by dexamethasone. It can be assumed that post-transcriptional modulation is responsible for CT-induced increase in COX-2 protein. COX-1 does not seem to be involved. Therefore, PGE2 produced by COX-2 seems to be responsible for the profuse fluid secretion induced by CT, and COX-2 appears to be a specific target for the treatment of Asiatic cholera.
Cholera, caused by a Gram-negative bacterium, Vibrio cholerae, is commonly considered to be dependent on a cAMP-mediated active secretory mechanism. Several other mediators have, however, been implicated in the mediation of CT-induced intestinal fluid secretion. In 1970, an increase in 5-HT levels in the blood of choleraic rabbits was demonstrated (Bhide et al., 1970). CT administered into the duodenums of rabbits caused severe degranulation of enterochromaffin cells as revealed by electron microscopy (Osaka et al., 1975). Since these observations, substantial evidence has accumulated to prove the involvement of 5-HT in the genesis of CT-induced fluid and electrolyte secretion (Cassuto et al., 1982; Nilsson et al., 1983; Beubler et al., 1989, Turvill et al., 1998). It has been furthermore suggested that CT may cause diarrhea by stimulating prostaglandin (PG) synthesis (Beubler et al., 1989). This concept is supported by the observation that CT is apparently associated with increased local PG synthesis (Bedwani et al., 1975; Speelmann et al., 1985) and that cyclooxygenase inhibitors impair the secretory effect of CT (Wald et al., 1977). The finding that indomethacin in some studies inhibits CT-induced secretion but not mucosal cAMP accumulation (Wald et al., 1977; Beubler et al., 1986) favors the notion that PGs may play a primary role in the secretory mechanism. On the other hand, PGE2 has been shown to be an important intermediate in the transduction mechanism, which leads to 5-HT-induced intestinal secretion (Beubler et al., 1986). From these experiments, a hypothesis was proposed that CT stimulates an apical receptor on the enterochromaffin cells and that serotonin released by the stimulus may cause PG formation, which mediates the diarrheogenic action of CT. However, the isoenzymes of cyclooxygenase, COX-1, or COX-2 involved in the relevant PG synthesis has not been determined. COX-1 is considered the constitutive form, being expressed in most tissues and cells (O'Neill and Ford-Hutchinson, 1993) and COX-2, the inducible form that is increased by inflammatory stimuli (Kujubu et al., 1991) and mitogenic stimuli (Raz et al., 1989) as well as in tumors (Eberhart et al., 1994) and ulcerated gastric tissue (Mizuno et al., 1997; Schmassmann et al., 1998). However, the constitutive expression of COX-2 was demonstrated in several tissues, including the brain, kidney, female reproductive tract, and other organs. Recently, constitutive COX-2 mRNA was identified in rat jejunal mucosa (Beubler et al., 1999) and constitutive COX-2 protein was demonstrated in mouse colon (MacNaughton et al., 2000). The aim of the present study was to elucidate the role of the different isoforms of cyclooxygenase, COX-1 and COX-2, in CT-induced fluid secretion by using selective COX-1 and COX-2 inhibitors, and by determining mRNA for COX-1 and COX-2, and COX-2 protein levels in mucosal scrapings of the rat jejunum.
Materials and Methods
Preparation of Animals.
Female Sprague-Dawley rats (180 ± 20 g body weight; obtained from Himberg, Institut für Labortierkunde und -genetik, Universität Wien, Medizinische Fakultät, Austria) were used in this study. They were maintained on a standard laboratory diet and deprived of food for 18 h before the experiments but had free access to water. The rats were anesthetized with sodium pentobarbitone (60 mg/kg i.p.), the abdomen was opened via a midline incision, and a polyethylene catheter (PE60) was placed in the jejunum ∼5 cm distal to the flexura duodenojejunalis and fixed by ligation. The second ligation was made ∼20 cm distal to the first ligation. The loop was carefully rinsed with 20 ml of body-warm saline and the distal ligation was tied off. Intact blood flow to the jejunum is maintained by this preparation. The jejunal loop was then returned to the abdominal cavity, and the whole preparation was allowed to rest for 1 h under a heat lamp to preserve body temperature. Anesthesia was maintained by s.c. injection of sodium pentobarbitone (20 mg/kg).
One hour after the preparation, during which the fluid left from rinsing was absorbed, either 2.0 ml of Tyrode's solution or Tyrode's solution plus CT (0.5 μg/ml; Sigma, Vienna, Austria) was instilled into the jejunal loop and the exposure lasted for 4 h. The COX inhibitors or vehicle were administered twice s.c., the first administration immediately before the start of the preparation and the second administration 2 h after instillation of Tyrode's solution or Tyrode's solution plus CT. Dexamethasone was administered s.c. 24 h and 1 h before the start of the experiment.
Determination of Net Fluid Transport.
Net fluid transfer rates were determined gravimetrically 4 h after instillation of Tyrode's solution or Tyrode's solution plus CT. The catheter was removed, the proximal ligation tied off, and the jejunal loop quickly withdrawn and weighed. Net fluid transport was expressed as milliliters per gram (ml/g) wet weight of jejunum. Net absorption was indicated by a negative value and net secretion by a positive value.
Determination of Prostaglandin E2.
Prostaglandin E2 was determined in aliquots of jejunal fluid obtained 4 h after Tyrode or Tyrode plus CT exposure. To minimize PGE2 release due to mechanical stimulation, the gut wall was punctured with a hypodermic needle and the fluid carefully withdrawn into a 1-ml syringe. Prostaglandin E2was determined by radioimmunoassay as described previously (Jobke et al. 1973, Schuligoi et al. 1998) using [5,6,8,11,12,14,15(N)-3H]-PGE2(Amersham, Little Chalfont, UK) as tracer and synthetic PGE2 (Sigma) as standard.
Treatment of Rats with LPS.
A separate set of experiments was performed to examine the effect of Escherichia coli LPS (Sigma) on COX mRNA expression. Rats were deprived of food for 18 h before the experiments but had free access to water. Three hours after treatment with LPS (5 mg/kg i.p.) or 0.9% NaCl, rats were sacrificed with an overdose of pentobarbital. The jejunum was taken out, and scrapings of the mucosa were used for RNA extraction.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
RT-PCR was performed as described previously (Amann et al., 1999) with total RNA from the mucosa of the jejunum (scrapings were taken immediately after the determination of net fluid transfer rates and frozen in liquid nitrogen). The RNA was extracted using Trizol (Life Technologies, Lofer, Austria) and after treatment with RNase-free DNase I (Roche, Vienna, Austria) to remove contaminating DNA, RNA was purified using a Nucleo-Spin Kit (Machery and Nagel, Düren, Germany). Reverse transcription of 0.8 μg RNA was performed with avian myoblastosis virus reverse transcriptase, and an oligo(dT)15 primer (Promega, Mannheim, Germany). For COX-1, COX-2 and glyceraldehyde-3 phosphate dehydrogenase (GAPDH), specific primers were used to amplify the reverse-transcribed products. For COX 1, 1 μl, for COX-2, 3 μl and for GAPDH, 1 μl of the reverse- transcribed product were used for PCR. PCR was carried out using 2.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates (Sigma), 20 pmol primers and 0.25 U of Taqpolymerase (Promega). Samples were denatured for 2 min at 95°C and specific cDNA amplified using a Stratagene RoboCycler (Stratagene, La Jolla, CA) cycling program: 95°C for 1 min, 72°C for 2 min. Final extension time was 4 min at 72°C. A total of 33 cycles were performed for COX-1 and COX-2 and 25 for GAPDH amplification. The yield of the amplified product was tested to be in the linear range for amount of input of the reverse-transcribed product and PCR cycle number and optimal conditions were chosen. The PCR products were separated by electrophoresis in an agarose gel stained with ethidium bromide. Specific primers for COX-1 and COX-2 (Beiche et al., 1998) yielded an amplification product of 447 bp and 279 bp, respectively, and for GAPDH (obtained from CLONTECH, Heidelberg, Germany) a 450-bp fragment was obtained. GAPDH was used as an internal reference to verify similar levels of RNA used for reverse transcription.
The ethidium bromide-stained bands were visualized under UV light with a Gel Doc 2000 system (Bio-Rad, Vienna, Austria), and the optical density of the bands was analyzed with Quantity One software (Bio-Rad). As a marker, a Precision Molecular Mass Standard (Bio-Rad) was used (Fig. 4). This marker shows 200 to 1000 bp and the respective concentration of DNA. For quantification, concentration curves were calculated and the amplified bands measured in relation to the concentration of the standard. The ratios of COX/GAPDH were calculated.
Western Immunoblot Analysis on COX-2.
Tissue samples of rat jejunal mucosa were dissolved in RIPA lysis buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS, Sigma) containing protease inhibitors [100 μg/ml phenylmethylsulfonyl fluoride (Sigma), aprotinin 30 μg/ml, and 100 mM sodium orthovanadate (10 μl/ml)]. Tissue homogenates were prepared by disrupting the tissues by repeated vortexing and boiling for 10 min. Samples were centrifuged at 15,000g for 20 min at 4°C to obtain clear lysate. The supernatant was transferred to a new microcentrifuge tube, and the remaining pellet was dissolved in 500 μl of SDS-loading buffer. Protein concentration of supernatant was determined using a BCA protein determination kit (Pierce, Rockford, IL). Likewise, we determined the protein concentration before loading the pellet dissolved in SDS by performing sample dilutions of 1:2 and 1:10, then by testing with a BCA protein assay kit. Equal amounts of protein from supernatant and pellet were loaded onto an SDS-12% polyacrylamide gel and electrophoresed according to the manufacturer's instructions (Bio-Rad). After electrophoresis, samples were transferred to nitrocellulose membranes. Once transfer was complete, membranes were briefly rinsed in 1× TBS (Tris-buffered saline) and blocked in 3% gelatin [in 1× TTBS (TBS + 0.05% Tween 20)] overnight at room temperature. After blocking, the gelatin was removed and the membrane washed twice for 10 min in 1× TTBS, followed by incubation in primary antibody (1 μl/ml) in 1% gelatin (in 1× TTBS) for 2 h. The polyclonal antibodies to COX-2 were made to a region, which was conserved between mouse, rat, and human, and were obtained from Santa Cruz Biotechnology, Santa Cruz, CA (catalog number SC1745). After incubation, the membrane was rinsed twice with 1× TTBS for 10 min each followed by incubation with appropriate alkaline phosphatase (AP)-conjugated secondary antibody (diluted 1:5000) (Santa Cruz Biotechnology) in 1% gelatin for 1 h. After incubation, the membrane was rinsed three times with 1× TTBS for 10 min each followed by a final wash in 1× TBS for 10 min. After washes, AP substrate (Bio-Rad) was applied to the membrane according to the manufacturer's instructions.
NS-398 ([N-(2-cyclohexaloxy-4-nitrophenyl) methanesulfonamide]) (Gilroy et al., 1998) was obtained from Cayman Chemical (Ann Arbor, MI) and DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H-furanone) (Riendeau et al., 1997) was a generous gift from Dr. A. W. Ford-Hutchinson (Merck-Frosst Canada, Montreal, Canada). COX-1 inhibitor SC-560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol] (Smith et al., 1998) was kindly provided by Dr. R. A. Marks (Searle, Skokie, IL). Stock solutions (10 mg/ml) were prepared in dimethylsulfoxide (DMSO):ethanol (1:4). Dilutions were made in DMSO:ethanol (1:9). Dexamethasone (dissolved in 0.9% NaCl) and indomethacin (dissolved in 2% sodium carbonate solution, and diluted with Sorensen buffer, pH 7.2) were obtained from Sigma.
Values were calculated as means ± S.E.M. Statistical analysis was performed using Student'st test, one-way analysis of variance, or Kruskal-Wallis one-way analysis of variance, when appropriate, using Dunnett's or Dunn's post-test, respectively (SigmaStat statistical software, Jandel Scientific, Erkrath, Germany). P < 0.05 was considered statistically significant.
CT and Fluid Transfer Rates—Effects of Cyclooxygenase Inhibitors and Dexamethasone.
The selective COX-2 inhibitors NS-398 and DFU were without an effect on fluid absorption in control rats (Figs. 1 and 2); however the CT-induced fluid secretion was dose dependently inhibited (Figs. 1 and2).
The selective COX-1 inhibitor SC-560 had no effect on enhanced fluid absorption at a dose of 10 mg/kg in control rats. In contrast to selective COX-2 inhibitors, SC-560, even at the highest dose of 30 mg/kg, did not influence the CT-induced fluid secretion (CT: 2.323 ± 0.028, n = 30 versus 2.287 ± 0.109,n = 12; CT plus SC-560).
The nonselective COX-1 and COX-2 inhibitor indomethacin was without any effect on fluid absorption in controls (Tyrode's solution: −1.39 ± 0.10, n = 35 versus −1.71 ± 0.06,n = 9, Tyrode's solution plus indomethacin) and only partially, although significantly, inhibited CT-induced fluid secretion (CT: 2.323 ± 0.028, n = 30 versus 0.624 ± 0.147, n = 9, CT plus indomethacin, 20 mg/kg s.c.).
Pretreatment of rats with dexamethasone (2 × 1 mg/kg within 24 h) did not affect the fluid absorption in untreated controls (Tyrode's solution: −1.39 ± 0.10, n = 35 versus −2.065 ± 0.08, n = 12, Tyrode's solution plus dexamethasone); nor did it influence the CT-induced fluid secretion (CT: 3.269 ± 0.413, n = 13 versus 3.665 ± 0.446, n = 14, CT plus dexamethasone).
CT-Induced PGE2 Biosynthesis—Effects of Cyclooxygenase Inhibitors and Dexamethasone.
CT (0.5 μg/ml, 4 h) significantly enhanced intraluminal PGE2 release about 3-fold compared with controls (Fig.3). The selective COX-2 inhibitors NS-398 and DFU, which did not have any effect on PGE2synthesis in controls, did significantly inhibit the CT-induced increase in PGE2 release (Fig. 3). The selective COX-1 inhibitor SC-560, as well as dexamethasone, did not influence the PGE2 synthesis in control rats or rats treated with CT (Fig. 3).
Effect of CT and LPS on COX-1 and COX-2 mRNA Expression in Jejunal Mucosa.
COX-1 and COX-2 mRNA expression was determined using RT-PCR. COX mRNA levels were normalized to GAPDH to correct for variations in RNA concentration.
COX-1 mRNA expression was detected in jejunal mucosa (COX-1/GAPDH: 48.1 ± 12.7) and was not influenced by exposure of the jejunal mucosa to CT (0.5 μg/ml intraluminal, 4 h, COX-1/GAPDH: 31.4 ± 7.5). Dexamethasone also had no effect on the relative signal intensities of COX-1/GAPDH (data not shown). COX-2 mRNA expression was observed in the jejunal mucosa of control rats (n = 4). Treatment of rats with CT (0.5 μg/ml intraluminal, 4 h) did not cause an increase of COX-2 mRNA expression. (Fig. 4). Dexamethasone pretreatment of rats (1 mg/kg twice within 24 h) had no effect on COX-2 mRNA expression in controls and also did not influence COX-2 mRNA expression in CT-treated mucosa (Fig. 4A), an observation which argues against an induction of COX-2 mRNA by CT.
In contrast, treatment of rats with E. coli LPS caused a marked induction of COX-2 mRNA expression in the jejunal mucosa, an effect that was completely inhibited by pretreatment of rats with dexamethasone (Fig. 4B).
Effect of CT on COX-2 Protein Levels.
COX-2 protein levels were determined using Western blot analysis. In the supernatant fraction of the jejunal mucosa of untreated rats, the 72- to 74-kDa protein band, which specifically reacts with COX-2 polyclonal antibody, was either absent or only weakly detected (Fig.5, lanes 1–4). All of the animals challenged with CT (0.5 μg/ml, 4 h) clearly showed a COX-2 protein band (Fig. 5, lanes 5–8), which was increased by 3- to 5-fold compared with control animals. Pretreatment of the rats with dexamethasone (1 mg/kg, twice within 24 h) did not significantly alter COX-2 protein levels in CT-treated rats (Fig. 5, lanes 9–12). In addition to these experiments performed in vivo, we have also shown that CT up-regulated COX-2 antigen levels (3- to 5-fold) in a murine macrophage cell line RAW264.7 in vitro. Untreated macrophages did not exhibit COX-2 corresponding band on Western blots (data not shown). Both for in vitro and in vivo experiments, we have used appropriate negative and positive controls in our Western blot analysis. The positive controls included LPS-treated rat mucosal intestinal tissue as well as LPS-induced macrophages, which exhibited a strong 72- to 74-kDa band. Our negative control included normal rat intestinal tissues and cell lysates from untreated macrophages, which did not react with the antibodies to COX-2.
It has been shown repeatedly that the release of PGs is causally involved in the secretory response to CT (for references, see Beubler et al., 1989 and Peterson et al., 1999). It is now well established that cyclooxygenase, the hemoprotein that catalyzes the formation of PGs, prostacyclins, and thromboxanes, exists in two isoforms. The concept was that the constitutive isoform COX-1 is responsible for the production of PGs involved in physiological functions, and the inducible COX-2 is expressed in response to inflammatory or mitogenic stimuli. In the meanwhile, however, several groups have reported the constitutive expression of COX-2 in several organs and tissues (see Introduction).
The present results show that CT-induced fluid secretion as well as PGE2 formation are dose-dependently, and almost completely, inhibited by the selective COX-2 inhibitors NS-398 and DFU. The COX-1 inhibitor SC-560 did not affect either CT-induced fluid secretion or CT-induced PGE2 synthesis. NS-398, DFU and SC-560 were used at doses previously shown to be selective for inhibition of COX-2 and COX-1, respectively (Futaki et al., 1993;Riendau et al. 1997; Smith et al., 1998; Wallace et al., 2000). Therefore it seems that CT-induced PGE2 formation and the resulting fluid secretion are mediated primarily via COX-2. The nonselective COX inhibitor indomethacin, as shown before (Beubler et al., 1990), only partially reduces the secretory response, and it has been demonstrated previously that indomethacin inhibits PGE2 formation (Beubler and Horina, 1990).
In this study we have shown the constitutive expression of both COX-1 and COX-2 mRNA in the rat jejunal mucosa. CT had no effect on COX-1 mRNA expression and also did not influence COX-2 mRNA expression. Treatment of rats with dexamethasone, known to interfere with COX-2 induction by transcriptional and post-transcriptional mechanisms (Newton et al., 1998), did not change the expression of COX-2 mRNA in control rats, just as it had no effect on the COX-2 mRNA expression of CT-treated rats. In contrast, LPS caused a marked induction of COX-2 mRNA expression, and as expected, this increase was totally blocked by pretreatment of rats with dexamethasone. These results suggest that COX-2 mRNA is constitutively expressed in the rat jejunum and CT did not induce COX-2 mRNA, in contrast to LPS, that is known to induce COX-2 expression in a variety of models (Herschman, 1996). A different picture was reflected when COX-2 antigen levels were analyzed by Western blotting. Although in controls, most of the samples analyzed did not show COX-2 antigen with some samples exhibiting a weak signal plausibly indicating low grade inflammation in animals, CT treatment of the jejunal mucosa caused an increase of COX-2 protein, which was not influenced by treatment of rats with dexamethasone, arguing against induction of COX-2 transcription. Studies from our laboratory and from others have indicated the specificity of the COX-2 antibodies used and have shown that these antibodies did not react with COX-1 (Muller-Decker et al., 1998, 1999; Murakami et al., 1998). Therefore, our data suggest a post-transcriptional effect of CT on COX-2. Post-transcriptional regulation of COX-2 protein has also been postulated in colon carcinogenesis (Dixon et al., 2000). It can be speculated that CT interferes with translational regulation of COX-2 or inhibits the degradation of COX-2 protein. Constitutive COX-2 expression has been shown in a variety of tissues and in this aspect, interestingly, MacNaughton et al. (2000) have demonstrated that COX-2 is constitutively expressed in the mouse colon. The finding that dexamethasone did not affect the CT-induced PGE2formation and fluid secretion further substantiates the concept of a constitutive expression of COX-2. However, since we used total mucosal scrapings of the jejunum, the results concerning mRNA and protein are averaged and we cannot exclude that changes in mRNA and/or protein levels may occur in discrete subpopulations of cells. The mechanism of CT-induced PGE2 synthesis is poorly understood. However, it is known that CT increases phospholipase A2 activity, probably by induction of phospholipase A2-activating protein mRNA (Peterson et al., 1996). Phospholipase A2specifically hydrolizes fatty acids from phospholipids and constitutes a central point of control for PG biosynthesis. The present data suggest that arachidonic acid is metabolized via COX-2 to PGE2 and that selective inhibition of COX-2 significantly inhibits CT-induced PGE2biosynthesis and that results in reduced fluid secretion.
The isoform of cyclooxygenase, COX-1 seems not to be involved in the mediation of CT-induced fluid secretion. The specific COX-1 inhibitor SC-560 did not affect CT- induced fluid secretion and PGE2 formation, despite near maximal inhibition of platelet thromboxane B2 biosynthesis (data not shown). Indomethacin, which inhibits both COX-1 and COX-2, only partially reduces the secretory response, as shown before (Beubler and Horina, 1990), although via its COX-2 effect, complete inhibition would have been expected. Also in human cholera, results obtained with indomethacin are not so clear. In experiments performed in Bangladesh (Rabbani and Butler, 1985), indomethacin was not able to inhibit fluid loss; however, Van Loon et al. (1992) described a significant inhibition of PGE2 and net fluid secretion in humans with indomethacin.
It may be speculated that indomethacin, due to its inhibitory effect on COX-1, is more toxic at the dose used than the pure COX-2-inhibiting compounds NS-398 and DFU. Although indomethacin does inhibit PGE2 overflow into the gut lumen like NS-398 and DFU, this does not reflect inhibition of PGE2formation in the tissue. In the case of indomethacin, this inhibition may be incomplete, as already stated by Rabbani and Butler (1985).
Similar to our data, NS-398 significantly blocked PGE2 formation and the secretory response induced by arachidonic acid in mouse colon, suggesting that a substantial portion of arachidonic acid-induced secretion was mediated through a COX-2-dependent mechanism, whereas the response to arachidonic acid was also blocked by pretreatment with the selective COX-1 inhibitor SC-560, suggesting the involvement of both isoenzymes in this kind of stimulation (MacNaughton et al., 2000). In contrast, CT seems to operate selectively through COX-2.
Since arachidonic acid uses both isoenzymes, and CT operates only via COX-2, it may be speculated that COX isoenzymes are activated in specific intracellular locations or in specific cell types to produce the final common secretagogue, PGE2.
There is no doubt that the enteric nervous system is also involved in the mediation of CT- or prostaglandin-induced secretion (Cassuto et al., 1981; Jodal et al., 1993). CT may bind directly to neurons in the myenteric plexus (Jiang et al., 1993) or stimulate neurons via 5-HT3 receptors after having released 5-HT from enterochromaffin cells (Beubler and Horina, 1990). An important role is also played by 5-HT in the mediation of CT-induced mucin secretion, using primarily 5-HT4 receptors (Moore et al., 1996). On the other hand, 5-HT binds to receptors on cells of the mucosa, presumably 5-HT2 receptors (Beubler and Horina 1990), leading to the initiation of arachidonic acid metabolism and release of PGs (Beubler et al., 1989; Siriwardena et al., 1993). To summarize these observations, it can be stated that CT uses 5-HT, PGs, the enteric nervous system, and possibly also cyclic AMP to exert its effect; however, the exact nature of the cascade of events leading to CT-induced fluid secretion remains speculative.
In conclusion, it has been demonstrated that CT, besides other mediators, uses PGE2 to exert its tremendous secretory effect. In the case of CT, the PGs released are solely produced via COX-2. Since no induction of COX-2 mRNA was observed in total mucosal scrapings of the jejunum, and dexamethasone was without an effect on COX-2 mRNA or COX-2 protein levels, it can be assumed that post-transcriptional modulation is responsible for the CT-induced increase in COX-2 protein levels. COX-1 seems not to be involved in CT-induced fluid secretion, a finding that distinguishes COX-2 as a new and specific target for the treatment of Asiatic cholera.
We thank Sabine Dirnberger, Martina Ofner and Hans Hosbein, University of Graz, Graz, Austria, and Kristine Kuhl, University of Galveston, Galveston, Texas, for technical assistance, as well as Irmgard Russa for preparing the manuscript.
Send reprint requests to: Prof. Dr. Eckhard Beubler, Department of Experimental and Clinical Pharmacology, Karl-Franzens-University of Graz, Universitätsplatz 4, A-8010 Graz, Austria. E-mail:
This study was supported by the Austrian Scientific Research Funds (No. P 12158-MED and P 13512-MED), the Franz Lanyar Stiftung and a grant from the Crohn's and Colitis Foundation of America.
Parts of this study were presented at the meeting of the American Gastroenterological Association in the spring of 1999 in Orlando, Florida and in the Proceedings of the Fifth Workshop of the Intestinal Mucosa Function Group of the German Society of Gastroenterology, in press.
- cholera toxin
- prostaglandin, COX, cyclooxygenase
- reverse transcription-polymerase chain reaction
- glyceraldehyde-3-phosphate dehydrogenase
- Tris-buffered saline
- alkaline phosphatase, bp, base pair
- The American Society for Pharmacology and Experimental Therapeutics