AuslJ.Kxp.Biol.Med.Sci.. 63 (Pt. 2) 223-230 (1985)

©GASTRIC EMPTYING AND INTESTINAL TRANSIT IN BVFOMARINUS AND THE ACTIONS OF E PROSTAGLANDINS

by P. M. TAYLOR, M. J. TYLER AND D. J. C. SHEARMAN*

(From the Department of Zoology. University of Adelaide, Box 498, G.P.O. Adelaide,South Australia, 5001, and the 'Department of Medicine, University of Adelaide.)

(Accepted for publication December 3, 1984.)

Summary. Gastric emptying and intestinal transit have been studied in the anuraii Bufomarinus using Tc'^-labeiled chicken liver as (he test meal. Gaslric emplying was measuredas the proportion oT the test meal remaining in the siomach after 3 or 6 h. Intestinal transitwas calculated by the geometric centre method at (he same time intervals.

There was a high degree of variability in these parameters between individual animals,in both control and treated groups.

Both SC29333 (.Searle). a synthetic E prostaglandin, and prostaglandin E, given orallydelayed gastric emptying.

These results complement the finding that prostaglandin E, causes relaxation of anuranlongitudinal muscle in vitro, and lend further support to the suggestion thai prostagtandinE., may be responsible for the gut stasis associated with gastric brooding in ihe gastricbrooding frog Rheobatrachus situs.

INTRODUCTION

Our interest in gastrointestinal transit in the anuran Amphibia arises, inpart, from our studies following the discovery of the phenomenon of gastricbrooding in the frog Rheobatrachus silus (Corben, Ingram and Tyler, 1974;Tyler and Carter, 1981). Brooding of young in the stomach requires prolongedretention of gastric contents and appears to be associated with stasis of theentire gastrointestinal tract (Fanning, Tyler and Shearman, 1982). ProstaglandinE, is responsible for some of the changes in gastric physiology seen duringbrooding (Tyler et ai, 1983).

The endangered status of R. silus (Tyler, 1979) has necessitated the useof an alternative species of anuran to study phenomena associated with gastricbrooding (Tyler et al., 1983; de la Lande et ai, 1984). This substitution canbe justified by the inherent conservatism of the Anura, whose fundamentalstructure has remained unchanged throughout the entire period in which themammals evolved and diverged. Interspecific differences in anurans arguablyare of less significance than in more labile organisms.

Apart from early roentgenographic and manometric studies of gastricmotility (Roux and Balthazard, 1898; Patterson, I9I6) there are few data ongastrointestinal motility in the Amphibia. Rate of transit, particularly gastricemptying, is likely to be dependent on the nature of the food ingested. Many

224 P. M. TAYLOR, M. J. TYLER AND D. J. C. SHEARMAN

items of the anuran diet possess chitinous e.xoskeletons and remain in thestomach for long periods as observed in our laboratory where frogs are fedroutinely with the larvae of the beetle Tenebrio molitor In the present studya more digestible food was used to permit the investigation of thepharmacological effects on transit of a single^ orally administered dose ofprostaglandin.

Prostaglandin E; causes relaxation of longitudinal muscle in isolatedpreparations of anuran intestine (de Ia Lande et ai, 1984). There are no reportsof its effects on anuran intestinal motility in vivo.

Here we describe a study of gastrointestinal transit in Bufo marinus andthe effects of intragastric administration of prostaglandin E, on transit.

MATERlAt-S AND METHODS

Cane toads. Bufo marinus. were housed at 3 0 ^ in vivaria with a regular light regime of 12 h tightand 12 h darkness, and mainiained on a did of live larvae of Tenebrio molitor. Food was withheld for3-5 days prior to use of the subjects. For each experiment 12 toads were divided into two equal groups,approximately matched for weight, and used according to one of the following protocols:

(1) controls v, SC293.13 (Searle); 6 j*g in liver(2) controls v. SC29333: 30 ^g in liver(3) controls v. PGE, (Upjohn); 6 pg in liver(4) controls v. PGE, infusion into buccal cavity: 2-5 /tg/h in 0-5 ml water for I h prior to feeding

and the following 3 h.(5) controls v, PGE, infusion into buccal cavity; 25/ig/h in 0-5 ml water for ! h prior lo feeding and

the tollowing 3 h.

Coniroi animals from protocols 1-3 were fed liver with no added drug. In protocols 4 and 5 the controls

were infused with vehicle alone.Tc**"-labcIIed chicken liver, prepared according to the melhod of Meyer et al. (1976). was used in all

experiments. Preliminary couniing experiments with the chicken liver revealed uneven labelling; in (hrectrials the counts per gram weight of liver showed a standard deviation about the mean counts ranging from37''/o to 59''/o. The method of preparation described here yielded a much more even distribution of labelin the food; counts per pellet consistently showed a standard deviation about ihe mean of approximately10%. The liver was chopped finely into a slurry and suspended in gelatine. Pellets were punched from theresultant mould to standardise doses. Drugs were mixed with ihe chicken liver slurry immediately beforethe addition of gelatine in amounts calculated to produce the desired dose in the pellets. Some pellets fromeach mould were retained for use as couniing standards,

Al ihe Stan of an experiineni each toad was fed iwo chicken liver pellets {average weight 0-336 g,containing aboui 10" d.p.m.). with or without added drug, according to the proiocol and allocation locontrol or treatment group. Three loads from each group were sacrificed 3 h after feeding, ihe remainderafier 6 hand ihegastroiniesiinal iracls prepared for analysis. The abdomen was opened with a mid-veniralincision and ihe gut ligated at ihe oesophageo-gastric junction, ihc pylorus and ihe lower end of the rccium.Thegui wiih iis contents \vas ihen excised, freed of mesentery, and ihe inlcsline distal to thenyiorus ligatedinto five parts of equal length. This produced six parts in all; ihe siomach (1) and five segmenis of inieslinc;the si.\th part always included ihe entire reclum. Each segment and its contents were placed in a separatelabelled tube, made up to a standard volume with 7 M KOH. incubated overnight at 37" lo solubilise lissueund contents and counled on a Nuclear Chicago gamma counter sel for optimal counting of Tc*.

ResuUs were rejected from animals in which recovery differed by more ihan I5°'o from the mean standarddose. Total counis in each segment were expressed as a percentage of ihe standard dose. The proponionof counts in each segment was also used to calculate an index of transit using the geometric centre melhodof Milter, Galligan and Burks (1981). The geometric cenire represents a weighted mean of the distributionof the marker and is derived by calculating the sum ol ihe products of ihe fraction of ihc total countsin a segment and the segment number Using this method a value of 1 would indicate thai all the label

GASTRIC EMPTYING IN BUFO MARINUS 225

remained In ihe stomach and a value of 6 thai all the label was recovered from ihe last segment. ResuUsfrom treatment and control groups were compared using ihe Student's t test for unpaired data.

RESULTS

Recovery of the label in the gut segments generally was high with an overallaverage of 97-30'^o. Observation showed that major losses resulting inunacceptably low recoveries (<85'yo) were not due to rapid transit andelimination but to regurgitation of food or, infrequently, to loss of intestinalcontents at dissection. In early trials carcasses were monitored in a large volumecounter for residual Tc** activity. With only one exception carcasses containedless than 0-1% of the standard dose, and generally only background levels wererecorded.

Protocol

(1)SC293336>ig in food

% Standard Dose0 20 40 60 80

3 hrs

(21SC2933330)jg in food

f 3 hrs

6 hrB

(3)PGE26 >)g in food 6 firs

(4)PGE2 Infusion

(5)PGE2 infusion25

GUT I ,SEGMENT!

STOMACH INTESTINE

Fig. I. Representation of the passage of a labelled test meal through ihe alimentary canat of ihe toadBufo marinus. C control animals; T treated animals.

226 P. M. TAYIX)R, M. J. TYLER AND D. J. C. SHEARMAN

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GASTRIC EMPTYING IN BUFO MARINUS 227

Figure 1 illustrates the distribution of label in the gut for each protocol.In Table I values for the amount of label remaining in the stomach and thegeometric centre are given. The transit values exhibited wide variability whichis reflected in the high standard errors. However, comparison of mean valuesfrom control groups in protocols 1-3 (uninfused) reveals a high level ofconsistency which reinforces the idea that such variability is a normal featureof the process.

SC29333 at a dose of 6 Mg per toad had no effect on transit, but at 30 \L%per toad gastric emptying was significantly delayed and the geometric centrereduced at 3 h. Prostaglandin E, at 6 /ig per toad also appeared to slowtransit by delaying gastric emptying, but the results did not reach statisticalsignificance, possibly due to the small sample size. In the infusion protocolsPGE; at 25 /ig per hour appears to have delayed gastric emptying, but at 2-5^g per hour the infusions were without effect.

DISCUSSION

Early published methods of monitoring transit were invasive orunphysiologic, for example involving intubation or roentgenography inassociation with barium markers (review by Connell, 1978). Techniques forthe measurement of the passage of solid food through the gastrointestinal tractwithout involving the introduction of stimuli which might in themselves altermotility (Read et ai, 1983) have been developed using isotopic labelling offood and external monitoring of radioactivity. For accurate assessment of transitthe radionuclide label must not be readily dissociable from the food, nor mustit be absorbed or metabolised by the body. The Tc'̂ ^-labelled chicken liverdeveloped by Meyer et al. (1976) fulfils these requirements and currently isin routine cHnical use.

Studies of gastric emptying in humans using solid radiolabeiled mealsshow high individual variability. Domstad et ai (1980) reported a normal rangeof 40-85 min for the biologic half time of gastric emptying. Horowitz et ai(1982) showed that emptying of solids from the human stomach is characterisedby a lag period of variable duration followed by linear emptying. They gavevalues of 70 ±7 min (mean ± sem) for the 50% emptying time in a groupof 11 control subjects and retention at 100 min of 30 + 5% (mean ± sem).Using the same data, Collins et ai (1983) reported a significant variation (p<0-05) between subjects for the 50% emptying time of a solid meal. Suchvariability is comparable with that seen in the present study and we concludethat a wide normal range is a feature of the process. However, food was retainedin the stomach for a comparatively long time in the toads, despite the use ofa soft and finely chopped test meal. Variability in gastrointestinal motilitystudies in laboratory animals is a little lower, possibly because of the methodsof analysis employed (Jackson et ai, 1976) and the administration of liquidmeals, for which there is no lag period (Bertaccini, de Castiglione andScarpignato, 1981; Bertaccini and Scarpignato, 1982; Castro ^/oA, 1976; Jacksonet ai, 1976; Ruwart, Klepper and Rush, 1979). The use of a liquid meal inour studies was inappropriate as anurans do not drink.

228 P. M. TAYLOR, M. J. TYLER AND D. J. C. SHEARMAN

Oral or intragastric administration of food permits accurate measurementof gastric emptying but may invalidate assessment of intestinal transit unlessthe rate of gastric emptying is constant in the two groups being compared(Summers, Kent and Osborne, 1970; Ruwart et aL, 1979). Our results do notpermit this assumption. The delays in gastric emptying observed in some ofthe present studies may have masked changes in intestinal transit. We attemptedto circumvent the problem by placing food directly into the small intestinein the manner of other studies of intestinal transit (Summers et ai, 1970; Ruwartet ai, 1979; Castro et ai, 1976; Porreca and Burks, 1983; Stewart, Weisbrodtand Burks, 1978). Placement of a catheter into the duodenum by way of theupper gastrointestinal tract was difficult in small toads due to constriction ofthe pylorus and the exaggerated pyloric flexure. Instillation of a chicken liverslurry through the catheter often was difficult in conscious toads and frequentlywe found label in the stomach at dissection. No consistently successful techniquecould be devised.

The present results demonstrate that SC29333 (a synthetic PGE, withmethyl groups substituted to reduce degradation in vivo) and possibly PGE;can delay gastric emptying in B. marinus and may reduce intestinal transit.The failure of the high dose infusion of PGE, to cause statistically significantdelays is puzzling. Studies on the inhibition of acid secretion in the same animalsTaylor, Tyler and Shearman (1985) showed that PGH. was more potent thanSC29333. These findings are consistent with those from our studies with isolatedintestinal preparations from B. marinus in which PGE, caused relaxation oflongitudinal muscle. Nylander and Andersson (1975) found that oraladministration to human subjects of 140 mg dimethyl PGE, reduced themotor activity response to a subsequent meal in both the stomach and smallintestine, and Jackson et al. (1976) observed a dose-dependent delay in gastricemptying of a charcoal meal in mice given PGE, i.p. Konturek et ai (1982)reported an increase in the periodicity of the intestinal migrating complex andreduced intestinal motor activity in dogs given PGE, i.v. In an interestingclinical case study Book et ai (1979) provide evidence that raised levels of PGEsin serum and gastric juice may cause reduced motor activity in thegastrointestinal tract. However, in contradiction to these observations, studiesby Ruwart et al. (1978) in ileus rats, and by Misiewicz, Waller and Kiley (1969)and Nylander and Mattsson (1975) in humans, claim that the E prostaglandinsincreased the rate of gastric emptying and intestinal transit. Nompleggi et ai(1980) found enhanced gastric emptying of liquids in primates given15(s)-15-methyl PE, subcutaneously.

In conclusion, the E prostaglandins appear to cause retention of gastriccontents in Bufo marinus. The effect on intestinal transit is less certain. Thisaction is consistent with the phenomenon of gastric brooding seen inRheobatrachus. The level of PGE, secreted by the young in the stomach ofa brooding female is believed to be in the order of 4-78 x 10^ moles/day,which is comparable with the highest dose used here, 1-70 x 10*̂ moles/dayin an animal twice the weight. We consider it likely that the degree of distensionof the stomach, and possibly also the hormonal .status of the animal, may

GASTRIC EMPTYING IN BUFO MARINUS 229

affect the response to PGE,. Further studies are planned to test thesehypotheses.

Acknowledgements. This research was undertaken with the support of the Australian Research GrantsScheme. We thank Searle Ausiralia Ply. Lid. for the provision of SC29333 and Itie Department of NuclearMedicine. Instiiuie of Medical and Veterinary Science, Adelaide for (he provision of labelled chicken liver.Kerstin Lungershausen gave valuable help with the care of animals. Figure I was prepared by Ruth Hughes.

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