• View in gallery

    Mean ± SEM Isc over time in mucosa obtained from the proximal (black circles) and distal (white circles) portions of the colon of 9 dogs and incubated in Ringer's solution with (A) or without (B) 1μM indomethacin. Time 0 was the start of incubation.

  • View in gallery

    Photomicrograph of a section of stripped canine colonic mucosa before (control) and after the mucosa was mounted in an Ussing chamber for 3 or 4 hours. Notice the intact muscularis mucosa and submucosa adherent to each stripped mucosa. There were minimal changes after 3 hours, compared with results for control tissues, except that some surface cells lacked nuclei and there was slight transmural infiltration of cells (white arrow). After 4 hours, there was slightly more transmural infiltration of cells (white arrow) and sloughing at the surface, goblet cells were larger, and glands were wider (black arrows). H&E stain; bar = 20 μm.

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Evaluation of the secretory response to endogenous and exogenous prostaglandins in mucosa from the proximal and distal portions of the colon of dogs

Mayuko Omori DVM, PhD1, Richard C. Hill VetMB, PhD2, Karen C. Scott PhD3, and Guy D. Lester DVM, PhD4
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  • 1 Department of Small Animal Clinical Sciences, University of Florida, Gainesville, FL 32610.
  • | 2 Department of Small Animal Clinical Sciences, University of Florida, Gainesville, FL 32610.
  • | 3 Department of Small Animal Clinical Sciences, University of Florida, Gainesville, FL 32610.
  • | 4 Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL 32610.

Abstract

Objective—To compare secretory responses to prostaglandin (PG) E2 in mucosa obtained from the proximal and distal portions of the colon of dogs.

Sample—Colonic mucosa from cadavers of 18 clinically normal adult dogs.

Procedures—Short-circuit current (ISC) and maximum change in ISC (ΔIsc) in response to administration of 1μM PGE2 were measured across mucosa obtained from the proximal and distal portions of the colon. Responses were evaluated in mucosa (n = 6 dogs) incubated in Ussing chambers with or without 1 mM amiloride or without chloride in the Ringer's bathing solution. Responses were also evaluated in mucosa (n = 9 dogs) incubated with or without pretreatment with 1 μM indomethacin, with or without amiloride in the subsequent bathing solution. Histologic changes in mucosa from 3 dogs were assessed over time.

Results—ISC and ΔISC were significantly reduced when chloride was removed from, but not when amiloride was added to, the bathing solution and were significantly reduced after pretreatment with indomethacin. The ΔISC was significantly greater in mucosa from the distal portion of the colon than in the proximal portion of the colon. Histologic changes after incubation for 3 hours were minimal.

Conclusions and Clinical Relevance—ISC and ΔISC resulted from electrogenic chloride secretion. Chloride secretion was reduced when release of PGs was prevented by indomethacin and was induced by administration of PGE2. Chloride secretion in response to PGE2 was greater in mucosa from the distal portion of the colon than in mucosa from the proximal portion of the colon.

Abstract

Objective—To compare secretory responses to prostaglandin (PG) E2 in mucosa obtained from the proximal and distal portions of the colon of dogs.

Sample—Colonic mucosa from cadavers of 18 clinically normal adult dogs.

Procedures—Short-circuit current (ISC) and maximum change in ISC (ΔIsc) in response to administration of 1μM PGE2 were measured across mucosa obtained from the proximal and distal portions of the colon. Responses were evaluated in mucosa (n = 6 dogs) incubated in Ussing chambers with or without 1 mM amiloride or without chloride in the Ringer's bathing solution. Responses were also evaluated in mucosa (n = 9 dogs) incubated with or without pretreatment with 1 μM indomethacin, with or without amiloride in the subsequent bathing solution. Histologic changes in mucosa from 3 dogs were assessed over time.

Results—ISC and ΔISC were significantly reduced when chloride was removed from, but not when amiloride was added to, the bathing solution and were significantly reduced after pretreatment with indomethacin. The ΔISC was significantly greater in mucosa from the distal portion of the colon than in the proximal portion of the colon. Histologic changes after incubation for 3 hours were minimal.

Conclusions and Clinical Relevance—ISC and ΔISC resulted from electrogenic chloride secretion. Chloride secretion was reduced when release of PGs was prevented by indomethacin and was induced by administration of PGE2. Chloride secretion in response to PGE2 was greater in mucosa from the distal portion of the colon than in mucosa from the proximal portion of the colon.

Prostaglandins are lipid molecules synthesized by cyclooxygenase that affect the response of mammalian colonic mucosa to disease.1–4 Higher concentrations of PGs are found in inflamed5 and cancerous4,6 colonic tissue than in surrounding normal tissue. Prostaglandins stimulate secretion in the colon, which can result in diarrhea.7 Prostaglandin E2 is a primary secretagogue and is probably the principal mediator among PGs in the mammalian colon.8,9 Nonsteroidal anti-inflammatory drugs, such as indomethacin, block the synthesis of PGs by inhibiting cyclooxygenase. Indomethacin can reduce PGE2-mediated secretory diarrhea caused by rectal villous adenoma in humans10 and can inhibit PGE2-induced colorectal carcinogenesis in rats11 and in human cells in vitro.12

It has been suggested in some reports13,14 that there are regional differences in ion transport within the large intestine of mammals; however, studies15–22 of canine colonic mucosa have exclusively used the proximal portion of the colon. Therefore, the purpose of the study reported here was to determine ex vivo whether there are regional differences in ion transport and secretory response to exogenous PGE2 and indomethacin in canine colonic mucosa.

Materials and Methods

Sample population—Colons were excised from cadavers of 18 mixed-breed dogs (10 males and 8 females). The first 6 cadavers were mixed-breed hound dogs that had been euthanatized at the end of an unrelated study. The remaining 12 cadavers were obtained from a local animal control facility. In each instance, dogs had been euthanatized by IV injection of pentobarbitone sodium. All dogs had been fed a commercial maintenance diet formulated for dogs and appeared to be healthy. The study was approved by the Institutional Care and Use Committee of the University of Florida.

Tissue collection and preparation—Cadavers were transported in ice to our laboratory. The colon (from the ileocolic junction to the point at which the colon enters the pelvic inlet) was excised within 30 minutes after the dogs were euthanatized. Each colon was incised along its antimesenteric border, washed, and pinned with the mucosa side down on a layer of wax in a dish containing Ringer's solution. The muscle layers were then carefully separated from the mucosa, and sheets of mucosa from the proximal and distal portion of the colon of each dog were mounted in Ussing chambers.

Six to 8 Ussing chambersa were used for each experiment. Each Ussing chamber was composed of a polymethyl methacrylate chamber and a glass water-jacketed 30-mL reservoir chamber containing Ringer's solution. A sheet of colonic mucosa was stretched flat between the 2 halves of each polymethyl methacrylate chamber so that a 1.34-cm2 area was exposed to the bathing solution. Each half chamber was connected to a voltage current clamp systemb by an agar-calomel electrode bridge and an agar-silver-silver chloride electrode bridge. Open-circuit transmural PD across the agarcalomel bridge was measured continuously, and an Isc was applied across the agar-silver-silver chloride bridge at intervals to nullify this PD.

The final concentrations of solutes in the Ringer's solution were 149mM Na+, 6mM K+, 3mM Ca2+, 0.7mM Mg2+, 140mM Cl, 1.6mM PO42−, 20mM HCO3, and 10mM glucose. Chloride-free Ringer's solution was made by substituting gluconate for chloride. Each solution was continuously aerated with 95% O2-5% CO2 and maintained at 37°C in each Ussing chamber. The pH was stabilized at 7.4, and osmolarity was 300 mOsm/L. Amiloride was dissolved in deionized water, and PGE2c and indomethacin were dissolved in 95% ethanol and diluted with deionized water. All chemicals, except for PGE2, were obtained from the same source.d

After the Isc had stabilized, PGE2 (final concentration, 1μM) was added to the mucosal bathing solution. The baseline value for Isc and PD and the response to PGE2 (ΔIsc and ΔPD) were measured.

Experimental design—Three experiments were conducted.

Experiment 1

Experiment 1 used colons from 6 cadavers. Three sheets of mucosa from the proximal portion of each colon and 3 sheets of mucosa from the distal portion of each colon were mounted in 6 Ussing chambers. After 60 to 80 minutes, when Isc had stabilized, pairs of mucosa from the proximal and distal portions of the colon were not treated, treated with 1mM amiloride in the mucosal bathing solution, or treated with a chloride-free bathing solution; subsequently, PGE2 was added to each bathing solution.

Experiment 2

Experiment 2 used colons from 9 cadavers. Each colon was divided longitudinally into 2 pieces. One piece was washed and the mucosa dissected in ice-cold Ringer's solution that contained 1μM indomethacin, and the other piece was washed and the mucosa dissected in ice-cold Ringer's solution without indomethacin. The duration of this preincubation period was approximately 30 minutes. Two sheets of mucosa from the proximal portion of each piece of colon and 2 sheets of mucosa from the distal portion of each piece of colon were mounted in 8 Ussing chambers that contained Ringer's solution at 37°C without indomethacin. After incubation for 3 hours, when Isc had stabilized, amiloride (final concentration, 1mM) was added to the mucosal bathing solution of one of each pair of sheets of mucosa; PGE2 subsequently was added to each bathing solution.

Experiment 3

Experiment 3 used colons from 3 cadavers. Each colon was divided longitudinally into 2 pieces. Mucosa was dissected from 1 piece of colon in ice-cold Ringer's solution that contained 1μM indomethacin, and mucosa was dissected from the other piece of colon in Ringer's solution without indomethacin. Sheets of mucosa from each piece of colon were then fixed in neutral-buffered 10% formalin, either immediately or after they had been mounted in Ussing chambers for 3 hours, 4 hours, or > 5 hours (4 hours was the maximum duration of incubation for experiments 1 and 2). A 5-μm-thick cross section of each sheet of mucosa was stained with H&E stain and examined via a light microscope.

Statistical analysis—Electrical variables (Isc, AUC of Isc and PD, and ΔIsc and APD) were reported as the mean sc± SEM. The AUC of Isc was calculated by summing the areas of the rhomboids below adjacent data points, assuming a straight linear relationship between adjacent data points. Results were compared via a multi-factor ANOVA with location (distal portion vs proximal portion) and treatment as factors by use of a statistical computer program.23,e Post hoc comparisons were performed with a Bonferroni correction. Values of P < 0.05 were considered significant.

Results

Experiment 1—When mucosa was incubated in bathing solution that contained chloride, ΔIsc was significantly greater for the distal portion than for the proximal portion of the colon (Table 1). When gluconate replaced chloride in the bathing solution, Isc and the response to PGE2 decreased significantly in both the proximal and distal portions of the colon. There was no evidence of an effect of amiloride on Isc, PD, ΔIsc, or ΔPD.

Table 1—

Mean ± SEM Isc and PD for canine colonic mucosa incubated in Ussing chambers in Ringer's solution with or without chloride or 1 mM amiloride and the maximum ΔIsc and ΔPD in response to 1μM PGE2.

Colon locationTreatmentIsc(μA/cm2)PD (mV)ΔIsc (μA/cm2)ΔPD (mV)
Proximal portionChloride without amiloride54 ± 188.0 ± 6.04 ± 31.0 ± 0.3
 With chloride and amiloride63 ± 435.0 ± 3.06 ± 40.2 ± 0.2
 Without chloride or amiloride8 ± 6*1.0 ± 1.0*−3 ± 90
Distal portionChloride without amiloride52 ± 264.0 ± 2.024 ± 151.0 ± 1.0
 With chloride and amiloride48 ± 184.0 ± 2.023 ± 311.0 ± 1.0
 Without chloride or amiloride5 ± 6*1.0 ± 1.0*0 ± 1*1.0 ± 4.0

Values reported represent results for colonic tissues from 6 dogs.

Within a column within a colon location, value differs significantly (P < 0.05) from the value for Ringer's solution that contained chloride.

Within a treatment group, value differs significantly (P < 0.05) from the corresponding value for the proximal portion of the colon.

Experiment 2—The Isc of the mucosa from both the proximal and distal portions of the colon increased less when the colonic mucosa was prepared in Ringer's solution that contained indomethacin than when tissue was not preincubated with indomethacin (Figure 1). The AUC of Isc during the first 180 minutes was significantly (P = 0.002) greater in untreated mucosa than in mucosa treated with indomethacin, and Isc at 180 minutes was significantly (P < 0.001) greater in untreated mucosa from the distal portion of the colon than in mucosa from the distal portion of the colon that was preincubated with indomethacin (Table 2). The AUC and Isc at 180 minutes differed, but not significantly (P = 0.07 and P = 0.06, respectively), between mucosa from the proximal and distal portions of the colon. The ΔIsc in response to exogenous PGE2 was significantly (P < 0.001) greater in mucosa from the distal portion of the colon than in mucosa from the proximal portion of the colon and was significantly (P = 0.001) greater in mucosa from both the proximal and distal portions of the colon when tissue was preincubated with indomethacin than when the tissues were untreated (Table 3). There was no evidence of an effect of amiloride on Isc, PD, ΔIsc, or ΔPD.

Figure 1—
Figure 1—

Mean ± SEM Isc over time in mucosa obtained from the proximal (black circles) and distal (white circles) portions of the colon of 9 dogs and incubated in Ringer's solution with (A) or without (B) 1μM indomethacin. Time 0 was the start of incubation.

Citation: American Journal of Veterinary Research 72, 3; 10.2460/ajvr.72.3.404

Table 2—

Mean ± SEM AUC of Isc and Isc of canine colonic mucosa incubated with or without 1μM indomethacin.

 With indomethacinWithout indomethacin  
VariableProximal portion of colonDistal portion of colonProximal portion of colonDistal portion of colon
AUC of Isc (mA·min/cm2)*4.0 ± 1.95.9 ± 2.95.5 ± 3.59.9 ± 4.6
Isc(μA/cmsc2) at 180 min16 ± 1225 ± 16a37 ± 33a69 ± 32b

Values reported represent results for colonic tissues from 9 dogs.

The AUC of Isc (from 0 to 180 minutes) was calculated on the basis of an assumption of a straight linear relationship between adjacent data points; values were significantly (P = 0.002) less in mucosa treated with indomethacin.

Values with different superscript letters differ significantly (P = 0.01).

Table 3—

Mean ± SEM maximum ΔIsc and ΔPD in colonic mucosa preincubated in Ussing chambers in Ringer's solution with or without 1 μM indomethacin or 1 mM amiloride and then incubated with 1 μM PGE2.

ColonTreatmentΔIsc (μA/cm2)ΔPD (mV)
Proximal portionWithout indomethacin or amiloride7 ± 130.1 ± 0.3
 Without indomethacin but with amiloride3 ± 40.2 ± 0.3
 With indomethacin but without amiloride16 ± 221.0 ± 0.6
 With indomethacin and amiloride19 ± 250.1 ± 0.3
Distal portionWithout indomethacin or amiloride31 ± 38*1.0 ± 1.0
 Without indomethacin but with amiloride22 ± 14*2.0 ± 1.0
 With indomethacin but without amiloride77 ± 27*3.0 ± 2.0
 With indomethacin and amiloride63 ± 35*2.0 ± 1.0

Values reported represent results for colonic tissues from 9 dogs.

Value differs significantly (P < 0.01) from the corresponding value for the proximal portion of the colon.

Value differs significantly (P < 0.01) from the corresponding value for the distal portion of the colon that was not incubated with indomethacin

Experiment 3—Mucosal Isc and PD remained constant during the first 4 hours after tissue was mounted in Ussing chambers, but transmucosal PD increased slightly and Isc decreased gradually when mucosa was mounted for > 4 hours. Mucosal height of the transverse section of tissues removed from chambers after 3 (1.2 to 2.3 μm; mean, 1.8 μm) or 4 (1.2 to 2.3 μm; mean, 1.8 μm) hours was similar to that of unmounted control tissue (1.7 to 2.5 μm; mean, 2.1 μm). Histologically, an intact muscularis mucosa and submucosa were adherent to each stripped mucosa (Figure 2). There was minimal sloughing of surface cells and no changes in goblet cells in mucosa after 3 hours, compared with results for control tissues, regardless of colonic location or indomethacin treatment. However, some surface cells lacked nuclei, and there was slight transmural infiltration of cells. The tissue removed after 4 hours had slightly more transmural infiltration of cells and sloughing at the surface; in addition, the glands were wider and the goblet cells were larger. Tissue that had been mounted for > 5 hours had a necrotic appearance, and mucosal architecture was severely disrupted.

Figure 2—
Figure 2—

Photomicrograph of a section of stripped canine colonic mucosa before (control) and after the mucosa was mounted in an Ussing chamber for 3 or 4 hours. Notice the intact muscularis mucosa and submucosa adherent to each stripped mucosa. There were minimal changes after 3 hours, compared with results for control tissues, except that some surface cells lacked nuclei and there was slight transmural infiltration of cells (white arrow). After 4 hours, there was slightly more transmural infiltration of cells (white arrow) and sloughing at the surface, goblet cells were larger, and glands were wider (black arrows). H&E stain; bar = 20 μm.

Citation: American Journal of Veterinary Research 72, 3; 10.2460/ajvr.72.3.404

Discussion

The colonic mucosa absorbs and secretes water and ions.24,25 The Isc represents net active ion transport and serves as a measure of colonic mucosal function ex vivo.26,27 In the study reported here, removal of chloride from the bathing solution almost completely abolished the Isc and ΔIsc in both the proximal and distal portions of the colon of dogs. Thus, baseline electrogenic ion transport and the electrogenic secretion of ions in response to PGE2 appear to be almost entirely associated with electrogenic chloride secretion in both the proximal and distal portions of the canine colon.

Addition of a low dose (10−4 to 10−5M) of amiloride to the mucosal bathing solution inhibits electrogenic sodium transport, and addition of a high dose (10−3M) of amiloride inhibits electrically silent sodium transport.13,28 The lack of any change in Isc in response to the addition of a high dose of amiloride before or after the addition of PGE2 suggests that sodium transport is electrically silent in both the proximal and distal portions of the canine colon. This contrasts with results obtained in other species by use of Ussing chambers.13,14,29 For example, sodium transport in rabbits is electrically silent in the proximal portion of the colon but is electrogenic in the distal portion of the colon.13,29 In humans, sodium transport is electrogenic in the distal portion of the colon (amiloride completely abolishes the Isc) and is both electrogenic and electroneutral in the proximal portion of the colon (amiloride decreases Isc by approximately 43%).13 In rats, sodium transport is electrically silent throughout the colon,14 but increased concentrations of aldosterone in the bloodstream caused by long-term salt depletion can induce amiloride-sensitive electrogenic sodium absorption.30,31 Lack of salt restriction may explain the reason that amiloride did not affect ΔIsc in colonic mucosa of dogs in the present study. It is extremely unusual to restrict salt in clinically norscmal dogs because most commercial foods formulated for dogs contain adequate amounts of salt. Nevertheless, the effect of amiloride on radiolabeled sodium transport in colonic mucosa from salt-restricted and salt-replete dogs must be measured before the effects of amiloride on transport in canine colonic mucosa can be fully evaluated. Regional differences in dose responses to PGs and other lipid mediators also need to be evaluated.

In the study reported here, we found that both Isc and ΔIsc were greater in the distal portion than in the proximal portion of the colon. These differences were more apparent when the tissue was preincubated with indomethacin. Other investigators reported13 that Isc was much greater in the distal portion than in the proximal portion of the colon in humans who had undergone resection of the colon for carcinoma or diverticular disease; however, NSAIDs were not used to control the production of endogenous prostanoids in these diseased tissues. Furthermore, electrogenic sodium transport was an important component of the Isc in the human colonic mucosa, whereas it was not an important component in the canine colonic mucosa in the present study.

A regional difference in the large intestinal secretory response has also been detected in rabbits.32,33 In those studies, PAF elicited an increase in permeability in mucosa from both the cecum and distal portion of the colon but elicited a biphasic increase in Isc only in the mucosa from the distal portion of the colon of rabbits. However, PAF acts indirectly by stimulating the release of multiple secretagogues, including PGs. In addition, PAF also induced a sustained increase in the permeability in rabbit colon,32,33 whereas the increase in permeability induced by exogenous PGE2 was only transient in the dog colonic mucosa of the present study.

Analysis of results of the study reported here revealed that Isc was smaller and more stable when colonic mucosa was treated with indomethacin. Because indomethacin inhibits PG synthesis, the high Isc across untreated mucosa must have been attributable to electrogenic chloride secretion induced by endogenous PGs synthesized in response to tissue manipulation. Other investigators have also reported that the Isc of mucosa from the proximal portion of the canine colon is lower when treated with indomethacin17 and that indomethacin decreases the Isc in porcine ileum by converting electrogenic chloride secretion to electroneutral sodium and chloride absorption.34

Structural integrity of the mucosa must be maintained to allow physiologically normal ion transport. The mucosa used in the present study was grossly normal before it was mounted in the Ussing chambers. Bathing solutions were isotonic and maintained at physiologic pH to preserve tissue integrity during each experiment. Histologic examination revealed that surface cells were largely preserved in tissues mounted in chambers for up to 4 hours. The dose of indomethacin used in the present study also appeared to cause no tissue damage. Loss of mucosal cells became severe after > 5 hours in Ussing chambers, but the experiments were completed before sloughing of cells would have caused a problem. It did take longer for the Isc to stabilize in tissues used for experiment 2. We speculate that this delay was a result of tissues being preincubated in ice-cold Ringer's solution because the Isc is temperature sensitive.35

Prostaglandin E2 induces colonic secretion directly by stimulating colonocytes or indirectly by stimulating submucosal nerves.36 Neurologic control of secretion can be eliminated by removing the submucosa and its plexus from the mucosa or by pretreating a mucosa-submucosal preparation with tetrodotoxin.36 However, in our experience, a mucosal preparation free of submucosa typically loses mucosal structural integrity while mounted in the Ussing chambers, and tetrodotoxin was not used. Therefore, this experiment did not distinguish between a direct and a neurally mediated response to PGE2. Other authors have reported that blockade of nerve activity with tetrodotoxin or atropine does not alter the response to PGE2 in the colon of dogs,17 rats,36 or guinea pigs.37 Nevertheless, further investigation of regional differences in the neural control of secretion is warranted.

We concluded that baseline electrogenic ion transport and the electrogenic secretion of ions in response to PGE2 appear to be almost entirely associated with electrogenic chloride secretion in both the proximal and distal portions of the canine colon. There was no evidence of electrogenic sodium transport in either the proximal or distal portion of the canine colon. Electrogenic chloride secretion in response to endogenous PGs released during manipulation could explain the higher Isc in the tissues collected without indomethacin. The electrogenic secretory response to endogenous and exogenous PGE2 was much greater in the distal portion than in the proximal portion of the canine colon.

ABBREVIATIONS

AUC

Area under the curve

ΔISC

Change in short-circuit current

ΔPD

Change in potential difference

ISC

Short-circuit current

PAF

Platelet-activating factor

PD

Potential difference

PG

Prostaglandin

a.

Model U-9500, MRA International, Naples, Fla.

b.

Model DVC1000, World Precision Instruments Inc, Sarasota, Fla.

c.

BIOMOL Research Laboratories, Plymouth Meeting, Pa.

d.

Sigma Chemical Co, St Louis, Mo.

e.

SAS, version 6.12, SAS Institute Inc, Cary NC.

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Contributor Notes

Dr. Omori's present address is 12634 Carmel Country Rd 117, San Diego, CA 92130.

Dr. Lester's present address is Division of Health Sciences, School of Veterinary and Biomedical Sciences, Murdoch University, Perth, WA 6150, Australia.

Dr. Hill was supported in part by the Waltham Centre for Pet Nutrition.

Published as University of Florida College of Veterinary Medicine Journal Series No. 657.

Address correspondence to Dr. Hill (hillr@ufl.edu).