Gut 1996; 38: 53-58

In vitro contractile effects of short chain fattyacids in the rat terminal ileum

C Cherbut, A-C Aube, H M Blottiere, P Pacaud, C Scarpignato, J-P Galmiche

AbstractShort chain fatty acids (SCFAs), producedin the gut by bacterial fermentation ofcar-bohydrates, change intestinal motility bymechanisms as yet unknown. This studyexamined the mechanism(s) of action ofSCFAs on contractility using isolated ratterminal ileum segments and isolated ilealsmooth muscle celis. Strip contractionswere recorded under isometric conditions.Intraceliular calcium concentration([Ca2+1i) was measured in single cellsloaded with indo-1 penta-acetoxymethylester (indo-1 AM). SCFAs (10-9 to 10-2molIl) induced concentration dependentcontractions. The effect was not differentamong the individual SCFAs. Exogenousacids (namely tartaric and citric acids)caused similar responses as SCFAs,whereas sodium acetate had no effect. Thecontraction was not blocked by tetrodo-toxin, atropine or hexamethonium, show-ing that it was not mediated through acholinergic pathway. Moreover, removalof the mucosa or addition of procaine (alocal anaesthetic) to the bath did notchange the SCFA induced contraction,while verapamil (a calcium-channelantagonist) completely suppressed it. Inaddition, application of SCFAs to isolatedileal myocytes evoked peaks in [Ca2+]iinhibited by D 600 (a blocker of voltagedependent calcium channels). Takentogether, these results suggest that thecontractile response stimulated by SCFAsin the rat terminal ileum could result froman acid sensitive calcium dependent myo-genic mechanism.(Gut 1996; 38: 53-58)

Keywords: rat, ileum, motility, short chain fatty acids,intestinal myocytes.

Short chain fatty acids (SCFAs) are producedby bacterial anaerobic fermentation of carbo-hydrates in the forestomach of ruminants andin the hindgut of monogastric mammals. Theyaccumulate in concentrations up to 150mmol/1 in the human colon, where theyrepresent the major organic anions. Acetate,propionate, and butyrate account for morethan 85% of formed SCFAs and are producedin nearly constant molar ratio 60:25:15.1These acids are also present in other parts ofthe gut, but their concentration seldomexceeds 1 to 5 mmolIl in the small intestineand stomach.2 3 SCFAs are quickly absorbedby intestinal mucosa,4 are readily metabolisedby intestinal epithelium and liver,5 6 exhibit

various physiological effects, and may be impli-cated in some gastrointestinal disorders.Among their biological actions, SCFAsstimulate water and electrolyte absorption,7potentiate proliferation of epithelial cells andcolonocytes,8 are a differentiating agent invarious cancer cell lines in vitro,9 and influencegastrointestinal motility.'0SCFAs could inhibit motility of the rat

colon,"1 whereas they stimulate contractions inthe terminal ileum of dogs and humans.'2 13Moreover, they shorten stomach to caecumtransit time in the rat,'4 ileal emptying in thedog,'5 and increase parietal tone of the humanileum.16 Intravenous injection of SCFAs alsoelicits ileal contractions in anaesthetised rats.'7It has been suggested that motor effects ofSCFAs may protect the ileum against colonic-ileal reflux.13The mechanism(s) of the action of SCFAs

in the ileum are as yet unknown. SCFAsinfluence ileal motility by local reflexes, whichdo not require systemic control, and whichare not abolished by either adrenergic orcholinergic blockage.'4 18 However, neither thespecificity of action of individual SCFAs, northe role of luminal pH are clearly established.Furthermore, it is not defined whether SCFAsact through the enteric plexus or directly onthe intestinal smooth muscle cells.

In this study we investigated the effect ofindividual SCFAs on contractility of isolatedileal segments. In addition, the action ofSCFAs on calcium movements in isolated ilealsmooth muscle cells was investigated to gainnew insights on their mechanism(s) of action.

Methods

MEASUREMENTS OF MUSCLE CONTRACTION

Animals and apparatusMale Wistar rats (250-300 g) were killed bycervical dislocation. One to 1.5 cm long seg-ments of the terminal ileum (2 cm before theileocaecal valve) were quickly removed, openedalong the mesenteric border, and cleaned ofintraluminal contents in a Krebs-bicarbonatesolution (pH 7.4) composed of (mmol/l): 128NaCl, 4.5 KC1, 2.5 CaCl2, 118 MgSO4, 1.18KH2PO4, 125 NaHCO3, 5.55 D-Glucose.Either whole thickness ileal strips (1 cmX 3 mmin the longitudinal axe) or strips whose mucosawas scraped off, were prepared, then suspendedunder 1 g tension in a 10 ml organ bathcontaining continuously oxygenated (5/oCO2, 95% 02) Krebs-bicarbonate solution.Preparations were allowed to equilibrate for 60minutes. Isometric longitudinal mechanical

Human NutritionResearch Centre,INRA, Laboratory ofNutrition and AppliedTechnology, Nantes,FranceC CherbutH M Blottiere

Laboratory ofDigestive Function andNutrition, NorthHospital, Nantes,FranceA-C AubeC ScarpignatoJ-P Galmiche

Laboratory ofPhysiology, UniversityofBordeaux, FranceP Pacaud

Correspondence to:Dr C Cherbut, INRA, BP1627, 44316 Nantes cedex03, France.

Accepted for publication30 June 1995

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ig

2 min

* 0

Acetic acid Sodium acetate10-3 mol/l 10-3 mol/l

Acetycholine10- mol/l

Figure 1: Representative tracing showing the contractions evoked by application ofacetylcholine (10-6 mol/l), acetic acid (10-3 mol) or sodium acetate (10-3 moll) to ratisolated ileal strips recorded under isometric conditions.

activity of the segments was then recordedusing a force transducer (Basile no 7005,Comerio, VA, Italy) as previously detailed.'9 Atthe beginning ofeach experiment, acetylcholinechloride (Ach, 10-6 mol/l) was applied ascontrol. Tested substances were then appliedon each preparation with repeated washings of20 to 30 minutes between each concentration.Viability of each preparation was confirmed atthe end of each experience by control ofspontaneous mechanical activity and responseto Ach. Results are expressed as percentage ofmaximal response recorded with Ach.

Experimental designIn the first series of experiments, individualSCFAs: acetic acid, propionic acid, butyricacid, and a mixture of the three SCFAs (acetic60%, propionic 25%, butyric 15%) thatrepresents the ratio of individual SCFAs foundin the rat large intestine20 were applied to thebath in non-cumulative increasing concentra-tions (10-9 to 10-2 moml). Their effects werecompared with a sodium salt, sodium acetatesolution, applied to the bath in non-cumulativeincreasing concentrations (10-7 to 10-2 molI).

In the second series of experiments, theeffect ofSCFAs was compared with two exoge-nous organic (namely tartaric acid and citricacid) and inorganic (namely hydrochloric acid)acids applied in non-cumulative increasingconcentrations (10-9 to 10-2 mol/l).

Finally, we investigated the effect of agentseither affecting neural transmission or blockingendogenous receptors involved in the physio-logical control of gastrointestinal motility onthe motor action of SCFAs. The drugs usedwere procaine hydrochloride (4-10-4 mol/l),tetrodotoxin (TTX, 10-6 molll), atropine sul-phate (10-6 mol/l), hexamethonium bromide(10-4 mol/l), naloxone hydrochloride (10-5mol/), and [D-Argl, D-Trp7,9, Leull]-sub-stance P (spantide, 10-5 mol/l). In addition,blockade of cyclooxygenase by indomethacin(10-6 mol/l) was tested to rule out any interfer-ence of endogenous postraglandins. We alsoevaluated the involvement of extracellular cal-cium in the SCFA induced response by testing

the effect of verapamil hydrochloride (10-6molll). These drugs were added three minutes(atropine, hexamethonium, verapamil), fiveminutes (naloxone, spantide, procaine, TTX),or 20 minutes (indomethacin) before applica-tion of acetic acid (10-3 mol/l). The concen-tration of these drugs was based on their abilityto antagonise more than 80% of the responseproduced by their respective agonist in concen-tration of 10-6 mol/l.

All the tested substances were applied involume not exceeding 1% of the bath volume,pH of the bath solution being measured aftereach application.

MEASUREMENTS OF INTRACELLULAR CALCIUM([ca2+ I)

Cell preparationMale Wistar rats (250-300 g) were killed bycervical dislocation and the terminal ileumremoved. Smooth muscle cells were isolatedfrom the ileum longitudinal muscle using themethod of Bitar et al,21 with little modifica-tions. The muscle was cut in small pieces andincubated at 37°C for three successive 30minute periods in Ca2+ free phosphate buffersolution (PBS) containing 2% bovine serumalbumin, 0a 1% collagenase, and 0a 1% soy-bean trypsin inhibitor. The pieces were thenwashed in enzyme free PBS and cells allowedto disperse under gentle trituration withpipette.

Calcium measurementsSmooth muscle cells were loaded with1 ,moll-indo-1 penta-acetoxymethyl ester(indo- 1 AM) by incubation in PBS solution for25 minutes at room temperature. [Ca2+]i wasestimated from Indo-1 fluorescence asdescribed in detail by Loirand et al.22 [Ca2+]iwas estimated from fluorescence ratio(405/480 nm) using a calibration for indo-1determined within cells. It was measuredbefore and after application of acetic acid(10-3 mol/1) to the isolated smooth musclecells, with or without addition ofD 600 (10-5molIl) to the bath. D 600 is a blcwker ofvoltagedependent calcium channels.

ANALYSIS OF DATAAll data are presented as mean (SEM).Significance among data was tested by usingone way analysis of variance (ANOVA),followed by Scheffe F test. EC50 and 950/o con-fidence intervals were calculated from theregression lines of the concentration responsecurves of each individual acid. All the calcula-tions were performed using the StatView SE+Graphics (Abacus concepts, CA, USA) run-ning on a Mac computer.

CHEMICALSSCFAs, drugs, and chemicals were purchasedfrom Sigma Chemical (L'Isle d'AbeauChesnes, St Quentin Fallavier Cedex, France),

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< 800

a)Mo 60 TCLa)

E 40

Eo 20

0-10 -8 -6 -4 -2

log concentration (mol/1)Figure 2: Concentration response curves for the effect ofacetylcholine (Ach) and a mixture ofSCFAs on ratisolated terminal ileum obtained under isometric recordingconditions. Each point represents the mean of the valuesobtainedfor 12 measurements. Vertical bars are standarderrors of the mean.

and enzymes were purchased from INC(Orsay, France).

Results

EFFECTS OF SCFAS, SODIUM ACETATE, ANDEXOGENOUS ACIDSWhole thickness segments of the rat terminalileum showed regular spontaneous phasiccontractions. Application of SCFAs evoked atonic contraction that was not sustained butfaded rapidly to reach the base line in 43A4(10.7) s, regardless of the concentration tested(Fig 1). The occurrence of the effect was con-centration dependent with a threshold concen-tration of about 0.1 p.mol/l and a mean EC50 of64 pimol/l (95% confidence intervals: 2, 59Rmol/l). The maximal amplitude of thecontraction was registered after application ofSCFAs at the concentration of 10 mmol/, andrepresented about 40%/o of the maximalresponse elicited by Ach (10-3 moml (Fig 2)).There was no significant difference among thefour different SCFA solutions (Table). Bothorganic (weak) and inorganic (strong) acidsinduced concentration dependent contractionssimilar to those produced by SCFAs (Table),whereas sodium acetate had no effect (Fig 1),regardless of the concentration (10-7 to

Effective concentrations (EC50, ,umol/l) causing 50% of themaximal contractile response of isolated ileal strips todifferent SCFAs and exogenous acids

95% Confidence intervals(,umo1')

EC50(g,mot/l) Lower Upper

SCFAsAcetic 81 1 77Propionic 59 3 30Butyric 67 3 85Mixture 50 1 43

Exogenous acidsOrganic

Tartaric 20 2 26Citric 17 0-8 26

InorganicChlorhydric 73 3 87

10-2 mol/l). Increasing concentrations ofSCFAs, added to the bath, provoked a step-wise decrease of pH of the bathing solution,which dropped by about 1.5 unit after reachingthe concentration of 10-2 mol/l (Fig 3). Inmuscle strips whose mucosa had beenremoved, application of SCFAs also evoked acontraction that was virtually identical to theresponse elicited in the whole ileum prepara-tion (Fig 4).

EFFECTS OF DRUGS ON SCFA INDUCEDCONTRACTIONThe SCFA induced contraction was not inhib-ited by hexamethonium (blocker of nicotinicreceptors), spantide (substance P antagonist),naloxone (opiate receptor blocker), andindomethacin (cyclooxygenase inhibitor).Muscarinic blockade with atropine reducedthe response, though not significantly(p=0-291 (Fig 5)). Moreover, neither pro-caine, a local anaesthetic, nor TTX, whichblocks nervous transmission, affected thecontraction pattern. On the contrary, a cal-cium channel antagonist, verapamil, was ableto completely abolish the SCFA induced con-traction (Fig 5).

EFFECTS OF SCFAS ON INTRACELLULARCALCIUMBath application of acetic acid (10-3 mol/1) onisolated ileal smooth muscle cells increased[Ca2+]i from a resting concentration of 124 (5)nmol/ to a peak value of 277 (7) nmol/1(p<0.01). Addition of D 600 (a blocker ofvoltage dependent calcium channels, 10-5mol/l) into the bath significantly inhibited theSCFA induced response, the incremental[Ca2+]i being reduced to 29 (3) nmolIl (Fig 6).

DiscussionIt is well known that SCFAs are able to stimu-late ileal motility in vivo. In conscious animals,infusion of SCFAs into the ileal lumen induces

8

7.5-

6.5-

6- - - 0-10 8 -6 -4 -2 o

log (SCFAs)Figure 3: Effect of increasing concentrations ofSCFAs onpH of the bathing solution. Each point represents the meanof the values obtainedfor 48 measurements. Vertical barsare standard errors of the mean. *p<0. 05.

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0

ci0C0.C0

4-

Eto

E0

1._0

F-|WholeEM Mucos

25 H

o-5

Figure 4: Contractile resp10-3 moll) of rat ileal stimucosa (n =8). Each col;values obtainedfor 12 mistandard errors of the met

bursts of phasicmigrate aborally,'2in the dog'5 as wellthe rat.'4 The stimithe ileum is alsosubjects.'3 In thesstimulation dependand was maximal alin the colon (100-1

In this investigatifirst time that appliacidic form, evokes

TT

30 r

T

15i

o

Figure 5: Contractile responses to acetic acid (10-3 molApretreated with Krebs' solution (KBS), atropine sulphathexamethonium bromide (Hxm, 10-4 molWl), spantide (10-5 moW), tetrodotoxin (TTX, 10-6 mobll), procaine Pindomethacin (Ind, 10-6 molil), and verapamil hydrochcolumn represents the mean of the values obtainedfor eigstandard errors of the mean. *p<0001.

thickness strip contraction in the rat terminal ileum in vitro,a free strip an effect that was not blocked by atropine or

TTX, but was abolished by blocking voltagedependent calcium channels. Although the

NS motility of isolated strips could representT %//////// stationary, propulsive or retropulsive patterns,

the in vivo effects seen suggest that the SCFAinduced contractions are of propulsive type.

Conversely from Kamath et al,12 we tested'physiological' concentrations of SCFAs,

NS which ranged from 1 gmo1/l to 10 mmoUl.These concentrations reflect the amounts ofSCFAs measured in the peripheral blood23 andileum of humans2 3 as well as ileal contents ofrats (Cherbut, unpublished results). Althoughin vivo studies have shown the potency ofSCFAs to be inversely related to the chain

log (acetic acid) length, acetic acid being more potent than5onses to acetic acid (10-5 and butyric one,12 in our in vitro experimental con-rips either with (n= 12) or without ditions the contractile activity of the different!umn represents the mean of the SCFAs was virtually the same. The differenceeasurements. Vertical bars arean. NS: non-significant. of potency among the individual SCFAs seen

in vivo might reflect a difference in theirepithelial metabolism: enterocytes indeed oxi-

contractions that rapidly dise butyrate preferentially than acetate.5 Itand shorten ileal emptying could be therefore speculated that, as SCFAsas orocaecal transit time in were applied to the mucosal side in the dogulatory effect of SCFAs in ileum, butyrate was trapped within theseen in healthy human epithelium, while acetate was excreted into the

'e studies, the degree of mucosal circulation. A smaller amount ofled on acid concentration butyrate may have thus reached the smootht the concentrations found muscle, explaining its lower potency compared50 mmol/l). with acetate. This cannot of course occur inLon, we have shown for the vitro, as both mucosal and serosal sides werecations of SCFAs, in their bathed with the same butyrate solution.a concentration dependent While SCFAs stimulate ileal motility, they do

not affect the motor pattern of the upper smallintestine. In vivo, either intraduodenal24 orintravenous'7 administration of SCFAs did not

T 1 1 change both duodenojejunal motility and transittime. Similarly, motility of isolated jejunalsegments in vitro was not affected by SCFAs.25

Conflicting results have been reported con-cerning the SCFA effect on the large intestine.Indeed, while Yajima26 reported a contracting

:_ activity of SCFAs on isolated rat colon,Squires et al" actually found an inhibitoryaction of these compounds at physiologicalconcentrations. In agreement with the second

KBS Spa Nal of these results, infusion of SCFAs into the ratcolon significantly decreased myoelectricalactivity in vivo (Cherbut et al, unpublishedresults). As expected, suppression of SCFAconcentration by oral administration of anti-biotics, is followed by a stimulation of colonicactivity.27 Conversely from consistent resultsobtained in the ileum, the contrasting effectseen in the colon could rely on the experimen-tal model adopted - that is, strips versus wholepreparation. It is, however, worth mentioningthat the results obtained in the whole colonicpreparation (that used by Squires et al), whichis more relevant to the in vivo motility, are in

KBS Ind Ver good agreement with the inhibitory effects seenapplied to rat isolated ileum in the conscious rat. Taken together, these

e (Atr, 10-6 mol/l), findings suggest that regional differences in theSpa, 10-5 molil), naloxone (Nal, response to intraluminal SCFAs may exist

'lyoride(Ver(Po-6 Mo.0 moo), along the gastrointestinal tract.

rht measurements. Vertical bars are The presence of chemoreceptors sensitive toSCFAs was first proposed by Kamath et al 13 to

30 r

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XEE'4-0

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Ileal motor effects ofSCFAs 57

500 A0EC 400

0e 300

0)

0 -3E 100 Acetic acid 10- mol/Icc

o0 12 24 36 48 60Time (s)

^500 BB0EE

400

0'P 300

o20000

Acetic acid 10- mol/I._

0 12 24 36 48 60Time (s)

Figure 6: Representative tracing showing the effect of aceticacid (10-3 molJl) applied to isolated ileal smooth musclecells on intracellular cakium concentration. The bathingsolution did not contain (A) or contained (B) D 600 (10,umol/l) to inhibit calcium entry through voltage, dependentcalcium channels.

explain the effect of these compounds on ilealmotility. Such receptors, detected in theepithelium of the ruminant reticulorumen, arelocalised close to sensory vagal fibres.28 Similarreceptors may exist in the rat colonicmucosa.26 However, such a receptorial mech-anism could not explain the ileal motor effectof SCFAs seen in vitro. Indeed, neither theapplication of procaine, nor the absence ofmucosa changed the contractile response toSCFAs in the ileal strips.

In our experimental conditions, TTX,atropine or hexamethonium, failed to affect themotor stimulating activity of SCFAs, suggest-ing that cholinergic pathways are not involved.This is consistent with results obtained in dogswhere adrenergic, cholinergic or serotoninergicblockade do not affect the ileal stimulatoryeffect of SCFAs.'8 In addition, neitherindomethacin nor naloxone affected the invitro ileal response to SCFAs. These data arein agreement with results obtained inhumans'3 but differ from those of dog experi-ments,'8 where indomethacin stimulated ilealmotility and naloxone inhibited the contrac-tions evoked by SCFAs.

It was recently shown that SCFAs canrelease peptide YY from endocrine cells. Thisis the case with isolated and perfused rabbitcolon29 and of in situ colon (Roze, personalcommunication) where instillation of SCFAsincreases circulating concentrations of peptideYY. As peptide YY plays an importantparacrine part in the regulation of gastro-intestinal motility,30 it could participate in theileal motor action of SCFAs. Blood concentra-tions of peptide YY are unchanged, however,after a bolus of SGFAs is injected into the

human ileum.13 In any event, a SCFA inducedrelease of peptide YY or other peptides fromthe epithelium cannot explain the contractileresponse of the isolated ileum, as removal ofthe mucosa containing L cells did not changethe response. All these data, taken together,strongly suggest that SCFAs could act directlyon the ileal smooth muscle without involvingany kind of neural pathway or paracrine regu-lation.

Contractions induced by SCFAs in theisolated terminal ileum were completelysuppressed when the calcium channel antagon-ist, verapamil, was added to the bath.Furthermore, application of acetic acid onisolated ileal smooth muscle cells induced atwofold increase in [Ca2+]i, an effect that wassignificantly inhibited by D 600, a blocker ofvoltage dependent calcium channels. Themyogenic effect of SCFAs could therefore relyon the entry of extracellular calcium into thecytoplasm.The similarity of action of the different

SCFAs and of exogenous acids (both organicand inorganic) as well as the lack of effect ofsodium acetate are consistent with the ideathat these compounds affect ileal motilitythrough pH changes. Reduction of intra-cellular pH (pHi) might be responsible for the[Ca2+]i movements. Indeed, the addition ofweak acids to isolated vascular and uterinesmooth muscles induces a concentrationdependent decrease of the pHi,31 which isassociated with smooth muscle relaxation.What we saw, however, after addition ofSCFAs to ileal strips was a contraction. This isnot surprising considering the interactionsbetween the H+ ions and Ca2+ in the cyto-plasm and their subsequent effect on contrac-tion in the intestinal smooth muscle are notcompletely understood and may differ fromthose operating in the vascular and uterinemuscle cells.

In summary, results of this study show thatSCFAs, which are produced in the gastro-intestinal lumen by bacterial fermentation ofcarbohydrates, stimulate ileal motility in a con-centration dependent fashion. The SCFAinduced contraction seems to entail neither aneural pathway nor paracrine regulation. Acalcium dependent myogenic mechanismcould be responsible for this motor effect.A C Aube is supported by a fellowship from Eridania Beghin-Say, Paris, France.

1 Cummings JH, Macfarlane GT. Control and consequencesof bacterial fermentation in the human colon. J App!Bacteriol 1991; 70: 443-59.

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3 Hoverstad T, Bjorneklett A, Fausa 0, Midvedt T. Short-chain fatty acids in the small-bowel bacterial overgrowthsyndrome. ScandJ3 Gastroenterol 1985; 20: 492-9.

4 Binder HJ, Sandle GI, Rajendran VM. Colonic fluid andelectrolyte transport in health and disease. In: Phillips SF,Pemberton JH, Shorter RG, eds. The large intestine:physiology, physiopathology and disease. New York: RavenPress, 1991: 141-68.

5 Roediger WE. Cellular metabolism of short chain fatty acidsin colonic epithelial cells. In: Roche AF, ed. Short-chainfatty acids: metabolism and clinical importance. Report of thetenth Ross conference on medical research. Columbus.OH: Ross.Laboratories, 1991: 67-70.

6 Demigne C, Remesy C. Hepatic metabolism of short chainfatty acids. In: Roche AF, ed. Short-chain fatty adids:

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7 Binder HJ, Metha P. Short-chain fatty acids stimulate activesodium and chloride absorption in vitro in the rat distalcolon. Gastroenterology 1989; 96: 989-96.

8 Sakata T. Effects of short chain fatty acids on epithelial cellproliferation and mucus release in the intestine. In: RocheAF, ed. Short-chain fatty acids: metabolism and clinicalimportance. Report of the tenth Ross conference onmedical research. Columbus, OH: Ross Laboratories,1991: 63-6.

9 Kruh J, Defer N, Tichonicky L. Molecular and cellulareffects of sodium butyrate. In: Roche AF, ed. Short-chainfatty acids: metabolism and clinical importance. Report of thetenth Ross conference on medical research. Columbus,OH: Ross Laboratories, 1991: 45-9.

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11 Squires PE, Rumsey RDE, Edwards CA, Read NW. Effectof short-chain fatty acids on contractile activity and fluidflow in rat colon in vitro. Am J Physiol 1992; 262:G813-7.

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13 Kamath PS, Phillips SF, O'Connor MK, Brown ML,Zinsmeister AR. Short-Chain fatty acids stimulate ilealmotility in humans. Gastroenterology 1988; 95: 1496-502.

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22 Loirand G, Gregoire G, Pacuad P. Photoreleased inositol1,4,5-triphosphate-induced response in single smoothmuscle cells of rat portal vein. J Physiol 1994; 479: 41-52.

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26 Yajima T. Contractile effect of short-chain fatty acids on theisolated colon of the rat. JPhysiol 1985; 368: 667-78.

27 Cherbut C, Ferre JP, Corpet DE, Ruckebusch Y, Delort-Laval J. Alterations of intestinal microflora by antibiotics.Effects on fecal excretion, transit time, and colonic motil-ity in rats. Dig Dis Sci 1991; 36: 1729-34.

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31 Taggart M, Austin C, Wray S. A comparison of the effectsof intracellular and extracellular pH on contraction inisolated rat portal vein. J Physiol 1994; 475: 285-92.

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