Mechanism of Deoxycholic AcidStimulation of the Rabbit Colon

S. J. SHIFF, R. D. SOLOWAY,and W. J. SNAPE, JR., Gastrointestinal Section,Department of Medicine, Hospital of the University of Pennsylvania,Philadelphia, Pennsylvania 19104

A B S T R A C T Previous studies showed that deoxy-cholic acid (DCA) stimulated migrating action poten-tial complexes (MAPC) in the colon. The aim of thisstudy was to clarify the mechanism of DCA-stimulatedcolonic motility.

Myoelectrical and contractile activity were mea-sured in New Zealand White rabbits from a loop con-structed in the proximal colon. During the control pe-riod, slow waves were present at a frequency of10.8±0.5 cycle/min and there were 1.5±0.5 MAPC/h. After adding DCA (16 mM) to the loop the slowwave activity was unchanged. However, MAPCin-creased to 15.1±2.4 MAPC/h (P < 0.001). MAPCac-tivity was not stimulated in the colonic smooth muscleoutside the loop. The intraluminal addition of procaineor tetrodotoxin to the colonic loop inhibited the DCA-stimulated increase in MAPCactivity (0.2±0.2 MAPC/h) (P < 0.005). Intravenous administration of atropineor phentolamine also inhibited MAPCactivity that hadbeen stimulated by DCA (P <0.005). Pretreatmentwith 6-hydroxydopamine also inhibited an increase inMAPCactivity. Propranolol, trimethaphan camsylate,or hexamethonium had no effect on DCAstimulationof MAPCactivity. Although the concentration of bilesalt increased in the mesenteric venous outflow fromthe colonic loop, the intravenous administration of bilesalt did not stimulate colonic MAPCactivity.

These studies suggest: (a) the action of DCA onsmooth muscle activity is a local phenomenon, (b) theincrease in MAPCactivity is dependent on intact cho-linergic and alpha adrenergic neurons, and (c) an in-crease in the concentration of bile salts in the serumis not associated with an increase in colonic MAPCactivity.

INTRODUCTIONRecent studies showed that bacterial toxins within theintestinal lumen increase intestinal secretion and alter

Received for publication 6 March 1981 and in revisedform 21 December 1981.

the motility pattern (1-4). The bacterial toxins stim-ulate migrating contractions that propulse the in-creased secretions through the intestinal tract (3, 4).Thus, in patients with infectious diarrhea, the increasein luminal fluid accumulation and the alteration inbowel motility may act together to cause severe diar-rhea.

Deoxycholic acid (DCA)' stimulates increased co-lonic contractility in patients with the irritable bowelsyndrome and reproduces their symptoms (5). DCAincreases the colonic mucosal secretion and stimulatesa propulsive migrating motor complex in the colon(5-9). The increase in mucosal secretion caused byDCA is related to an increase in intracellular cyclic3'5' AMPthat can be inhibited by the administrationof propranolol (10, 11). Similar to infectious diarrhea,the simultaneous presence of both secretory and motorabnormalities might lead to a greater degree of diar-rhea.

The mechanism of the migrating action potentialcomplex (MAPC) stimulated by DCAis presently un-clear. The purpose of this study was to determine themechanism of DCA-stimulated MAPCactivity. Clar-ification of the mechanism of the motility disorderassociated with a bile salt abnormality may help ther-apeutically in patients with bile acid associated gas-trointestinal motility dysfunction.

METHODSStudies were performed on 43 male New Zealand Whiterabbits weighing 2.5-3.8 kg. The rabbits were initially tran-quilized with Innovar (0.2 ml/kg intramuscular) (Pitman-Moore, Inc., Washington Crossing, NJ) and anesthetized withsodium pentobarbital (25 mg/kg) administered intrave-nously through the ear vein. A tracheostomy was performedin each animal and respiratory activity was controlled by asmall animal respirator (Harvard Apparatus Respirator, Har-vard Apparatus Co., Inc., S. Natick, MA).

The proximal colon was located through a midline ab-

'Abbreviations used in this paper: DCA, deoxycholicacid; MAPC, migrating action potential complexes.

J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/82/04/0985/08 $1.00Volume 69 April 1982 985-992

985

dominal incision. The temperature of the abdomen wasmaintained at 370C throughout the experiment by the useof a heat lamp. Beginning 2 cm from the ileocecal valve, a15-cm loop was constructed. Four bipolar silver-silver chlo-ride electrodes were sewn on the antimesenteric border ofthe serosa of the loop in contract with circular muscle. Insome experiments a fifth electrode was placed 6 cm distalto the loop (8).

A strain gauge (R. B. Products, Madison, WI) was sewnto the serosal surface between the second and the third elec-trodes. The strain gauge was shaped to accomodate the cur-vature of the colon. The strain gauge was oriented in thecircular direction on the antimesenteric wall of the colonwith sutures attached only to circular muscle. In some ex-periments a second strain gauge was sewn onto the loop. Italso was oriented in the circular direction.

A polyethylene tube (i.d. 0.030 in.) was inserted in theproximal end of the colonic loop through a small serosalpuncture and it was secured with a purse-string suture. Testsubstances were administered through the proximal tube. Asecond polyethylene tube (i.d. 0.250 in.) was inserted intothe distal end of the loop and it also was secured by a purse-string suture. The distal catheter permitted decompressionof the loop.

In studies where blood was to be collected from the ani-mals, a mesenteric vein draining the colonic loop was can-nulated using a Teflon angiocatheter (20 gauge), which wassecured by a suture. In two studies a mesenteric vein drainingthe distal colon, which was not part of the colonic loop andwas not exposed to DCA, was cannulated by the same tech-nique. Arterial blood was obtained by cannulating the rightfemoral artery with a polyethylene tube (i.d. 0.030 in.),which was secured by a purse-string suture. After the sur-gical procedure, the abdomen was covered with a clear plas-tic sheet allowing observation of the colonic loop during thestudy.

Each electrode was connected to a rectilinear recorder(Beckman Instruments, Inc., Fullerton, CA, R611 Dyno-graph) through AC couplers (9806A). Studies were per-formed with a time constant of 1.0 s and low pass filter of22 Hz. A grounding wire was placed in the subcutaneoustissue of the left hind limb.

Each strain gauge was connected to the rectilinear re-corder through a strain gauge coupler (9803). Respirationswere recorded by a pneumograph placed around the chestand connected to a pressure transducer (Statham P321A,Statham Instruments, Inc., Oxnard, CA).

All animals were allowed to stabilize for at least 1 h afterthe surgical preparation. After this period 3.0 cm2 of normalsaline was added to the loop. Myoelectrical and strain gaugerecordings were performed for a 1-h period. After the salinecontrol period, 3.0 cm3 of 16 mMdeoxycholic acid (SigmaChemical Co., St. Louis, MO) was added to the loop.Myoelectrical and strain gauge recordings were performedfor 1-4 h after the addition of the DCA. After stimulatingMAPCactivity by DCA, neural antagonists were adminis-tered. 3.0 cm3 of Procaine-HCl (1%) (Invenex, Inc., Hialeah,FL), or tetrodotoxin (40 mg/kg) (Calbiochem-Behring Corp.,American Hoechst Corp., San Diego, CA) were added in-traluminally through the proximal catheter. Atropine-sulf ate(0.1 mg/kg) (Vitarine, New York), phentolamine mesylate(1 mg/kg) (Ciba-Geigy Corp., Summit, NJ), or propranolol-hydrochloride (1 mg/kg) (Ayerst Laboratories, New York),were added intravenously through an ear vein in a 5-mininfusion. Trimethaphan camsylate was intravenously infusedcontinuously (4 mg/min). Hexamethonium (40 mg/kg)(Sigma Chemical Co.) was injected into two animals. Threeanimals were pretreated 48 h before the study with 6-hy-droxydopamine (50 mg/kg) (Sigma Chemical Co.) If more

than one drug was used in a day, the second drug was added1 h after MAPCactivity returned to preantagonist levels.

During the saline control period and during the additionof DCA to the colonic loop, blood was obtained from themesenteric vein leading from the colonic loop and from thefemoral artery and analyzed for plasma bile acids. In twoexperiments blood was also taken from a mesenteric veinthat was draining the colon 8-10 cm distal to the colonicloop. Plasma and intraluminal bile acid concentrations weredetermined by a modified method of Osuya et al. (12). Thetechnique in this study used a double beam spectrophoto-metric analysis (13). Plasma was treated with isopropanol toextract bile acids.

In three animals DCA was continuously perfused intra-venously through an ear vein at a rate of 21 gM/min for1 h. The amount of DCAinfused was calculated to raise thesystemic plasma bile acid concentration to levels comparableto those observed in the mesenteric venous outflow whenDCAwas given intraluminally. The DCAused for the in-travenous infusion was suspended in a 1% albumin-normalsaline solution to prevent hemolysis of the erythrocytes.During this period myoelectrical and strain gauge activitywere recorded and blood was collected from the femoralartery and mesenteric veins draining the loop. Blood sampleswere analyzed for plasma bile acid concentration. In addi-tion, intraluminal contents were collected from the distalcatheter of the loop and also analyzed for bile acid content.

Myoelectrical recordings were evaluated for slow wavefrequency in cycles per minute. Spike activity was measuredas the percentage of slow waves with superimposed spikepotentials. Migrating action potential complexes were de-fined as a continuous band of spike potential discharge last-ing for 2.5 s or longer and occurring on at least two conse-cutive electrode sites (8). The propagation velocity and theduration of the MAPCwere measured.

Statistical analysis was performed using the unpaired Stu-dent's t test.

RESULTS

Fig. 1 shows a myoelectrical recording from colonicloops of rabbits after adding normal saline (1A) ordeoxycholic acid (DCA) (1B) to the colonic loop. Fig.1A shows a record after the intraluminal infusion ofnormal saline. Slow wave activity is present at a fre-quency of 9.3 cycles/min. Many of the slow waveshave superimposed spike potentials. The strain gauges,monitoring circular muscle contractile activity, showno contractile response. Fig. lB shows a recordingfrom a colonic loop after adding DCA(16 mM) withinthe lumen of the colonic loop. Electrode E5 is placedon the distal colon outside the DCA-treated loop.MAPCare present, migrating aborad with a velocityof 17.6 mm/s. The MAPCoccurs only in the DCA-treated length of colon and the MAPCdoes not prop-agate outside the loop. Stain gauges show that a cir-cular muscle contraction occurred simultaneously withthe MAPCactivity. The MAPCnever occurred outsidethe colonic loop that had been treated with DCA.

Table I shows the indices of myoelectrical activityobtained from the colonic loops of the rabbits studied.The mean slow wave frequency was 10.8±0.5 cycles/

986 S. J. Shiff, R. D. Soloway, and W. J. Snape, Jr.

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FIGURE 1 Myoelectrical recording from colonic loops from rabbits. Electrode, El is orad andE4 is caudad in the loop. (A) Myoelectrical recording after adding 3.0 cm3 of normal saline tothe loop. No MAPCare present. Strain gauge (SG1 and SG2) are sewn at the level of E2 andE3. (B) Myoelectrical recording after adding 3.0 cm3 of 16 mMDCAto the loop. Electrode E5is 6 cm distal to the loop. 3 MAPCare present. The strain gauge (SG1) is sewn between E2and E3.

Mechanism of Deoxycholic Acid Stimulation of the Colon

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TABLE IMyoelectrical Characteristics of Rabbit Colon

Slow waves withSlow wave superimposedfrequency spike potentials

cpm %

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min after the addition of saline to the loop. Spike po-tentials were superimposed on the majority of the slowwaves after saline administration to the colonic loop(75.1±4.3%). The administration of 16-mM DCAdidnot significantly alter the slow wave frequency, or thepercentage of slow waves with spike potentials.

During the control period, after adding saline to theloops, 1.8±0.5 MAPC/h occurred within the loop.These MAPChad a propagation velocity of 9.7±1.3mm/s and a duration of 18.6±3.0 s. DCA increasedthe frequency of MAPCactivity to 15.1±2.4/h (P< 0.001). After adding DCA, the velocity of the MAPCincreased to 16.6±1.4 mm/s (P < 0.005) and the du-ration of the MAPC decreased to 12.1±1.0 s (P< 0.025).

Fig. 2 shows a myoelectrical recording from a co-lonic loop that had been stimulated by DCA. Procaine

was added intraluminally at the arrow. After addingDCA, MAPCare stimulated with concurrent migrat-ing contractions. The addition of procaine totallyabolished the MAPCactivity that had been stimulatedby DCA within 1 min. No MAPCactivity occurredafter adding procaine intraluminally.

Fig. 3 shows the number of MAPCafter adding DCAalone or after the simultaneous administration of theantagonists, procaine, intraluminally-or atropine,phentolamine, or propranolol, intravenously. DCAin-creased the MAPCactivity to 15.1±2.4 MAPC/h (P< 0.001). The intraluminal administration of procainereduced DCA-stimulated MAPC activity (0.2±0.2MAPC/h) (P < 0.005). In two animals intraluminaltetrodotoxin also abolished DCA-stimulated MAPCactivity. Atropine infusion reduced the MAPCfre-quency to 0.3±0.3 MAPC/h (P < 0.025). Phentolamine(1.0 mg/kg) an alpha adrenergic antagonist, similarlyreduced the MAPCfrequency to 0.3±0.3 MAPC/h(P <0.005). A lower concentration of phentolamine(0.25 mg/kg) also completely inhibited the MAPCac-tivity. Sympathetic denervation wth 6-hydroxydopa-mine prevented an increase in MAPCactivity afterDCA. However, after the administration of the betaadrenergic antagonist, propranolol, DCAcontinued tostimulate colonic MAPCactivity (9.8±1.4 MAPC/h)(P < 0.001).

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988 S. J. Shiff, R. D. Soloway, and W. J. Snape, Jr.

FIGURE 2 Myoelectrical recording from a colonic loop in which MAPCactivity had beeninduced with 16 mMDCA. Procaine added to the loop (t) abolished MAPCactivity.

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FIGURE 3 The number of MAPCper 1 h stimulated by 16 mMDCAwhen administered aloneand simultaneously with antagonists. All values compared for significance with the saline con-trol. The data is shown as a mean±SEM. Minimum of six animals was administered eachcompound.° P < 0.001.

Fig. 4 shows a myoelectrical recording after MAPCactivity has been stimulated by the intraluminal ad-ministration of 16 mMDCA. Trimethaphan camsylatewas infused intravenously at a rate of 4 mg/min. Thisdose of trimethaphan decreased the blood pressure,but had no effect on the MAPCactivity.

Fig. 5 shows the effect on MAPCactivity of DCAalone and the simultaneous administration of DCAandtrimethaphan camsylate. DCA increases the MAPCactivity from 1.2±0.7 MAPC/10 min to 3.5±0.6MAPC/10 min (P < 0.01). Ganglionic blockade withtrimethaphan camsylate did not block the DCAstim-ulation of MAPCactivity 2.5±1.3 MAPC/10 min (P> 0.05). Similar results were obtained after the ad-ministration of hexamethonium in two studies (2.4±0.1MAPC/10 min). Hexamethonium also decreased thesystemic blood pressure, but had no effect on DCA-stimulated MAPCactivity.

Fig. 6 shows the plasma bile acid concentration ob-tained from the mesenteric vein draining the colonicloop and from the femoral artery. After saline admin-istration to the colonic loop, the plasma bile acid con-centration was 13.3±1.7 ,uM from the mesenteric ve-nous outflow and 4.9±0.4 uM from the femoral artery.After adding 16 mMDCA into the colonic loop, the

plasma bile acid concentration in the mesenteric ve-nous blood flowing from the loops increased to 150±26.3MM(P < 0.001). The plasma bile acid concentrationin the femoral arterial blood did not change from basallevels after the administration of 16 mMDCA(5.2±1.2AM) (P > 0.05). Blood taken from a mesenteric veindraining the distal colon, not exposed to the intralu-minal administration of DCA, showed a plasma bileacid concentration of 4.1±0.1 MM. Thus, the level ofplasma bile acids was not changed in the venous out-flow from the colon not included in the DCA-treatedloop.

DCAwas infused intravenously to assess the effectof high intravascular concentrations of DCA. Fig. 7shows the effect of the intravenous infusion of DCAon plasma bile acid levels and the corresponding levelof MAPCactivity. The intravenous infusion of a 21-MM/min solution of DCA increased the plasma bileacid level to 214±85 ,uM in the mesenteric venousblood. Thus plasma concentration of the bile acid inthe mesenteric vein draining the colonic loop is similarto the concentration after instillation of DCAinto thecolonic loop. The level of plasma bile acids in the fem-oral artery is similar to the mesenteric venous level.The increase in serum DCAconcentration by the in-

Mechanism of Deoxycholic Acid Stimulation of the Colon 989

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FIGURE 4. Myoelectrical recording from a colonic loop of a rabbit. DCA-stimulated MAPCactivity is present. Trimethaphan camsylate (4.0 ml/min) decreased the blood pressure but didnot affect the MAPCactivity.

travenous administration of DCAdid not increase theintraluminal concentration of bile salts (12.7±1.7 AM)over control levels (8.1±2.8 AM) (P > 0.05). Despiteelevated plasma bile acid concentrations, there was no

increase in colonic MAPCactivity. Thus, stimulationof colonic MAPCactivity appears more dependent on

the intraluminal concentration of bile acids than theplasma levels of the bile acids.

DISCUSSION

High intraluminal concentrations of bile salts stimulatecolonic motor activity (5, 8, 9). In the rabbit, DCA

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stimulated MAPC activity in the proximal colon,which propulsed intraluminal contents through thecolonic loop (8). The increase in MAPCactivity is re-

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990 S. J. Shiff, R. D. Soloway, and W. J. Snape, Jr.

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lated to the concentration and the total duration of themucosal exposure to DCA (8). The purpose of thesestudies was to determine the mechanism throughwhich DCA stimulated the colonic smooth muscleMAPCactivity.

Mucosal contact by the DCAappears to be essentialfor the stimulation of colonic MAPCactivity. The in-crease in MAPCactivity occurred only within thesmooth muscle included in the colonic loop and didnot occur in the colon excluded from the loop con-taining DCA. In addition, intravenous infusion of DCAfailed to stimulate colonic MAPCactivity despite sim-ilar concentrations within the serum in the mesentericvein. Thus, the effect of DCAon colonic smooth mus-cle appears to be a local phenomenon and the receptorsthat are sensitive to DCA seem to be present in thewall of the colonic loop.

DCAappears to stimulate MAPCactivity within thecolonic loop through a neural mechanism. Procaine isa local anaesthetic that acts as an antagonist of local

neural reflexes (14, 15). Procaine blocks excitation ofthe neural membrane. The small diameter sensory af-ferent nerves are affected quickly (15). The intralu-minal administration of procaine inhibited DCA-stim-ulated MAPCactivity, despite the continued presenceof DCAwithin the loop. Procaine possibly inhibits theDCA stimulation of MAPCactivity by blocking theafferent neural receptors present within the mucosaor submucosa of the colonic loop or by blocking thetransmission of impulses through the myenteric plexus.Because procaine inhibited the generation of DCA-stimulated MAPCactivity, continuous stimulation ofthe neural receptors appears necessary for sustainedMAPC activity. Inhibition of the DCA-stimulatedMAPCactivity by the local administration of tetro-dotoxin confirms the importance of local neural re-flexes in the generation of the MAPCactivity.

Stimulation of colonic MAPCactivity by DCAap-pears to require intact cholinergic and alpha adren-ergic neural pathways. Both atropine, a muscarinicantagonist, and phentolamine, an alpha adrenergicantagonist, inhibit DCAstimulation of MAPCactivity.Beta adrenergic blockade with propranolol does notalter DCA-stimulated MAPC activity. Ganglionicblockade by trimethaphan or hexamethonium also hadno effect on DCAstimulation of MAPCactivity. Tri-methaphan and hexamethonium have their major an-tagonist effect on nicotinic-cholinergic synaptic junc-tions (16). The neural receptor antagonist drugs wereused at doses that blocked the action of the agonist.Thus, these data suggest that DCAstimulated a neuralreflex that requires intact muscarinic-cholinergic neu-rons and alpha adrenergic neurons, but does not re-quire a synapse through a nicotinic-cholinergic junc-tion.

These data suggest a complicated neural pathway.A muscarinic-cholinergic neuron may synapse with anadrenergic interneuron whose neurotransmission to apostganglionic neuron can be blocked with alpha ad-renergic antagonists (17). This mechanism is consistentwith the importance of muscarinic and alpha adren-ergic receptors in DCAstimulation of the colonic loopand with the lack of an effect by the blockade of nic-otinic ganglionic synapses.

DCAstimulation of colonic MAPCactivity differsfrom DCA stimulation of colonic mucosal secretion.DCAstimulates mucosal secretion through an increasein adenylate cyclase, which is mediated through thebeta adrenergic system (6, 10). Propranolol inhibits theincrease in colonic mucosal secretion, but it has noeffect on DCAstimulation of motor activity.

After demonstrating the importance of the neuralpathways for DCA stimulation of the colon, it wasnecessary to evaluate the direct effect of high serumlevels of DCA on the colonic smooth muscle. Whenthe serum levels of DCAwere increased to the level

Mechanism of Deoxycholic Acid Stimulation of the Colon

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observed in the mesenteric venous blood, there was noincrease in the number of MAPC. Despite the localincrease in serum and tissue levels of DCA, the bindingof DCAto local intraluminal neural receptors appearsto be most important.

These studies suggest that in addition to the knownsecretory response that occurs due to exposure to ex-cessive luminal concentrations of dihydroxy bile saltsin the colon, there is a locally initiated, neurally me-diated abnormal colonic motor pattern. This responsediffers from the secretory response because the motorresponse can be inhibited by muscarinic or alphaadrenergic blockade rather than beta adrenergicblockade.

ACKNOWLEDGMENTSThe authors thank Mrs. Eleanor Fayusal and Mrs. Jen-MeiYu for expert technical assistance. The authors thank GailBarnard for secretarial assistance.

These studies were supported by National Institutes ofHealth grant R01-AM-25396.

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992 S. J. Shiff, R. D. Soloway, and W. J. Snape, Jr.