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Am J Physiol Gastrointest Liver Physiol 292: G429-G437, 2007. First published September 14, 2006; doi:10.1152/ajpgi.00376.2006
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The peristaltic reflex induced by short-chain fatty acids is mediated by sequential release of 5-HT and neuronal CGRP but not BDNF

J. R. Grider and B. E. Piland

Departments of Physiology and Internal Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia

Submitted 12 August 2006 ; accepted in final form 13 September 2006

ABSTRACT

Short-chain fatty acids (SCFAs) accelerate colonic transit. This study examined whether this action was mediated by activation of the peristaltic reflex. SCFAs (acetate, butyrate, or propionate) were applied to the central compartment of a three-compartment flat-sheet preparation of the rat middle to distal colon. The release of serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and CGRP was measured in all three compartments. Ascending contraction and descending relaxation were measured in the orad and caudad compartments. The addition of SCFAs at physiological to supraphysiological concentrations (0.5–100 mM) to the central compartment elicited concentration-dependent ascending contraction and descending relaxation (EC50 ~5 mM). At this concentration, SCFAs induced an 8- to 11-fold increase in 5-HT release and a 2- to 3-fold increase in CGRP release in the central compartment only. They had no effect on BDNF release. CGRP release was inhibited by a 5-HT4 but not a 5-HT3 receptor antagonist. Ascending contraction and descending relaxation were also inhibited by 5-HT4 and by CGRP receptor antagonists added to the central compartment. 5-HT and CGRP release, as well as ascending contraction and descending relaxation induced by mechanical stimulation of the mucosa (2–8 strokes), were significantly augmented by 1 mM acetate. Acetate (1 mM) also doubled propulsive velocity in isolated whole segments of the guinea pig colon. In conclusion, chemical stimulation of the mucosa by SCFAs triggers a peristaltic reflex mediated by the release of 5-HT from mucosal cells and activation of 5-HT4 receptors on sensory CGRP-containing nerve terminals. This SCFA-induced peristaltic pathway augments the peristaltic reflex elicited by mechanical stimulation of the mucosa.

enteric nervous system; neurotrophins; neuropeptides; colon; gastrointestinal motility; calcitonin gene-related peptide; brain-derived neurotrophic factor; serotonin

THE SHORT-CHAIN FATTY ACIDS (SCFA), acetate, propionate, and butyrate, produced by a bacterial fermentation of dietary fiber in the colon, are an important source of energy for colonic epithelial cells (4, 8, 9, 34). These anions regulate various colonic functions, including colonic electrolyte transport and motor activity and the growth, proliferation, and differentiation of colonic mucosal cells (4, 6, 7, 9, 18, 24, 35, 36, 39). Their concentration in the colon of most mammals, including humans, varies with the nature and availability of the substrate, the type and amount of microflora present in the colon, the pH, the rate of transit of luminal contents, and the site of measurement within the colon (1, 4, 10, 37, 38). In general, the molar ratio for acetate, propionate, and butyrate is 60:20:20. In the rat, concentrations are highest in the proximal colon and cecum (e.g., range of 8–40 mM for individual SCFAs and range of 70–140 mM for total SCFAs) and decrease caudally to their lowest concentrations in the distal colon (e.g., range of 1–20 mM for individual SCFAs and range of 10–70 mM for total SCFAs) (1). Conversely, the pH is lowest in the proximal colon and increases caudally. This general pattern of distally decreasing concentrations of SCFAs and increasing pH holds true for most mammals including the human, although absolute values vary with species and diet (4, 37, 38).

Intraluminal exposure of the intestinal and colon to SCFAs has been reported to cause both an increase and decrease in motility as judged by contractile activity or transit (2, 5–7, 11, 20, 21, 24, 28, 29, 33, 36, 39). The difference in the reported effects is likely due to the concentration of SCFAs, the region of colon and type of preparation, and the method of determining effects on motility. The mechanism of action is generally considered to be neurally mediated by either extrinsic or intrinsic afferent neurons leading ultimately to stimulation of myenteric cholinergic neurons (5, 7, 11, 21, 24, 33, 36). Paracrine agents such as serotonin [5-hydroxytryptamine (5-HT)] and prostaglandins have been implicated in the local effects (5, 11, 21, 29), whereas the distal motility effects, know as the ileocolonic break, are mediated at least in part by release of the hormone peptide YY (PYY) and its interaction with neural pathways (5, 7, 25–27). Thus, there is strong evidence that SCFAs have a considerable role in regulating the propulsive activity of the gut, although their effects on the individual components of the underlying peristaltic reflex have not been fully elucidated.

Previous studies (12, 13, 15) have shown that mechanical stimulation of the mucosa triggers an increase of 5-HT release from enterochromaffin cells and initiates peristaltic activity. The release of 5-HT activates 5-HT4 receptors located on the terminals of CGRP-containing sensory neurons that relay the stimulus via interneurons to excitatory motor neurons orad and inhibitory motor neurons caudad to the site of stimulation (12, 31). A recent study (16) has shown that mechanical stimulation of the mucosa also elicits the release of the neurotrophin brain-derived neurotrophic factor (BDNF), which acts to augment the release of 5-HT and CGRP and thereby augment the peristaltic reflex. It is not clear if this same mechanism is responsible for mediating the response to SCFAs, although several studies (24, 29) have indicated that removal of the mucosa eliminates the response to SCFAs.

The present study was designed to examine whether chemical stimulation of the mucosa by SCFAs stimulates 5-HT release and activates the same peristaltic pathway. A flat-sheet three-compartment preparation of the rat colon was used that enabled measurements of 5-HT, BDNF, and CGRP release in response to the mucosal application of acetate, propionate, and butyrate, together with the measurement of mechanical responses orad (ascending contraction) and caudad (descending relaxation) to the site of stimulation. All three SCFAs stimulated 5-HT and CGRP release and increased mechanical responses in a concentration-dependent fashion. Unlike mechanical stimulation, SCFAs did not stimulate the release of BDNF. The response to mechanical stimulation of the mucosa was greatly enhanced in the presence of a low concentration of acetate and was accompanied by corresponding increases in 5-HT and CGRP release. CGRP release was correlated with 5-HT release and with the mechanical components of the response.

METHODS

Measurements of the peristaltic reflex in a compartmented flat-sheet preparation of the rat colon. All procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Virginia Commonwealth University. The peristaltic reflex was measured in a three-compartment, flat-sheet preparation of the middle to distal colon from the rat as described in detail previously (12). This region was chosen so that the peristaltic reflex and sensory pathway activated by SCFAs could be compared with those described for the peristaltic reflex induced by mechanical stimulation of the colonic mucosa (12, 13, 15, 16). A 5-cm segment of the colon was opened, pinned mucosal side up in a tissue bath, and separated into three compartments by vertical partitions sealed with vacuum grease. Each compartment contained 2 ml of a Krebs-bicarbonate medium maintained at pH 7.4 and composed of (in mM) 118 NaCl, 4.8 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, 25 NaH2CO3, and 11 glucose. For assays of CGRP and BDNF, the medium also contained 0.1% BSA, 10 µM amastatin, and 1 µM phosphoramidon. For the assay of 5-HT, the medium also contained 10 µM pargyline.

The peristaltic reflex was initiated by stroking the mucosa with a fine brush (2–8 strokes at a rate of 1 stroke/s) or the addition of a SCFA to the central compartment. The SCFAs (0.5–100 mM final concentrations) tested were the sodium salts of acetate, propionate, and butyrate adjusted to neutral pH. In some experiments, the stroking stimulus was repeated in the presence of a low concentration of acetate (1 mM). Ascending contraction of the circular muscle was measured in the orad peripheral compartment and descending relaxation was measured in the caudad peripheral compartment using force-displacement transducers attached to the muscle layers, as previously described (12). For measurements of CGRP, BDNF, and 5-HT, either mucosal strokes were applied to the central compartment five times each during a 15-min period or a SCFA (5 mM) was applied for a 15-min period. In some experiments, two to eight strokes were applied in the presence of 1 mM acetate. Media from the central and both peripheral compartments were collected and frozen for a subsequent radioimmunoassay of CGRP or ELISA of 5-HT and BDNF. Experiments were repeated in the presence of the preferential 5-HT3 receptor antagonist 1-methyl-N-[8-methyl-8-azabicyclo(3.2.1)-oct-3-yl]-1H-indazole-3-carboxamide maleate (LY-278584; 10 µM), the preferential 5-HT4 antagonist 1-[(2-methylsulfonylamino)ethyl]-4-piperidinyl-methyl-1-methyl-1H-indole-3-carboxylate maleate salt (GR-113808A; 10 µM), and the preferential CGRP receptor antagonist hCGRP8–37 (10 µM). These concentrations have been shown to be effective in previous studies (12, 13, 15) using this preparation of the rat colon and to be selective for these receptors.

Measurements of the velocity of propulsion in an isolated whole segment of the guinea pig colon. The velocity of propulsion was measured in an isolated whole segment of the guinea pig colon using artificial clay pellets that mimicked native fecal pellets in size and shape, as previously described (19). These experiments were done in the guinea pig colon because the guinea pig pellet propulsion model is well established and the isolated whole rat colon does not rapidly propel fecal pellets. In a previous study, we (13) have shown that the underlying peristaltic reflex in the guinea pig and rat are similar in that both are initiated by the sequential release of 5-HT and CGRP. Krebs-bicarbonate medium, similar to that used for measurements of the peristaltic reflex, was continuously perfused at a rate of 0.25 ml/min via a polyethylene-10 catheter introduced via the caudad end of the segment. Basal velocity was measured by inserting a pellet into the orad end of the segment and calculating the time it took for it to traverse a fixed distance. At 5-min intervals, a second pellet and then a third pellet were inserted, and the basal velocity was determined from the mean of the three values. Acetate (1 mM) was added to the perfusate, and the velocity of propulsion was measured again in a similar fashion. The velocity of propulsion was calculated in millimeters per second.

Measurements of CGRP, BDNF, and 5-HT. CGRP was measured by a radioimmunoassay as described previously using antibody RIK 6006 (13). The limit of detection of the assay was 2.6 fmol/ml, and the IC50 was 34.7 ± 6.2 fmol/ml. The antibody reacts fully with CGRP but does not cross react with amylin, calcitonin, substance P, neurokinin A, somatostatin, BDNF, VIP, [Met]-enkephalin, 5-HT, LY-278584, or GR-113808A.

BDNF was measured by ELISA as described previously (16). All samples were first acidified to pH <3.0 with 1 N HCl for 15 min and then readjusted to neutral pH before the assay was performed. The limit of detection of the assay was 0.2 fmol/ml, and the range of the assay was 0.2–557.0 fmol/ml. The antibody reacts fully with BDNF but not with nerve growth factor, neurotrophin-3, neurotrophin-4, CGRP, VIP, substance P, or 5-HT.

5-HT was chemically derivatized to N-acetyl-5-HT and measured by ELISA. The limit of detection of the assay was 0.5 µM, and the IC50 was 4.2 ± 0.5 µM. N-acetyl-5-HT antibody reacts fully with N-acetyl-5-HT but not with 5-HT, 5-hydroxy-3-indole acetic acid, 5-hydroxytryptophol, melatonin, substance P, VIP, CGRP, BDNF, LY-278584, or GR-113808A.

The amounts of 5-HT, BDNF, or CGRP released into each compartment were determined from the volume of medium (2 ml) and concentration in each compartment and was expressed as picomoles of 5-HT or femtomoles of CGRP or BDNF per 100 mg wet tissue weight per minute. The wet tissue weight of each compartment was determined at the end of the experiment.

Materials. CGRP, CGRP antiserum RIK 6006, and 125I-labeled CGRP (125I-CGRP) were purchased from Bachem-Peninsula (Torrance, CA). The 5-HT ELISA kit was purchased from ICN (Costa Mesa, CA), and the BDNF ELISA kit was purchased from Promega (Madison, WI). LY-278584 was purchased from Research Biochemicals (Natick, MA). Sodium salts of acetate, butyrate, and propionate, amastatin, phosphoramidon, pargyline, and all other chemicals and reagents were purchase from Sigma Chemicals (St. Louis, MO). GR-113808A was a gift from Drs. G. Kilpatrick and B. Bain (GlaxoSmithKline Research and Development, Middlesex, UK).

RESULTS

Activation of the peristaltic reflex by SCFAs. The addition of Na-acetate, Na-propionate, or Na-butyrate in the range of 0.5–100 mM at neutral pH 7.2 to the central compartment of the three-compartment preparation of the rat colon caused a concentration-dependent contraction of the circular muscle in the orad compartment (ascending contraction) and relaxation in the caudad compartment (descending relaxation) (Fig. 1). Maximal responses were not significantly different, and EC50s ranged from 1.5 to 5.5 mM.


Figure 1
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  		Fig. 1. Concentration-dependent stimulation of ascending contraction and descending relaxation by short-chain fatty acids (SCFAs). SCFAs were added to the central compartment of a three-compartment flat-sheet preparation of the rat colon. Ascending contraction and descending relaxation of the circular muscle were measured in the orad and caudad peripheral compartments, respectively. Acetate, butyrate, and propionate were added as sodium salts buffered to pH 7.2. Each concentration-response curve was obtained from a separate colonic preparation. Four separate preparations were used for each agent. Values are means ± SE of 4 experiments with each agent.

 
Release of 5-HT, BDNF, and CGRP by SCFAs. In previous studies (13, 15, 16), we showed that mechanical stimulation of the mucosa in the central compartment elicited 5-HT, BDNF, and CGRP release in proportion to the intensity of stimulation. To determine whether chemical stimulation using SCFAs elicited a similar pattern of release, 5-HT, BDNF, and CGRP were measured in the central, caudad, and orad compartments in the basal state and after the addition of 5 mM of acetate, propionate, or butyrate to the central compartment.

Basal release of 5-HT into the central, orad, and caudad compartments was similar (means ± SE, 6.8 ± 1.1 pmol·100 mg–1·min–1). The addition of 5 mM acetate, propionate, or butyrate to the central compartment induced an 8- to 10-fold increase in 5-HT release into the central compartment but not into the caudad or orad compartments (Fig. 2). The amounts of 5-HT released by 5 mM acetate, propionate, and butyrate were not significantly different (75.6 ± 9.7, 59.5 ± 8.1, and 64.0 ± 9.4 pmol·100 mg–1·min–1, respectively, corresponding to 10.9 ± 2.0-, 7.8 ± 2.0-, and 8.4 ± 3.2-fold increases above the basal level). The amounts of 5-HT in the orad and caudad compartments did not differ significantly from basal levels (range: 7.2 ± 2.1–9.6 ± 1.4 pmol·100 mg–1·min–1).


Figure 2
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  		Fig. 2. Selective release of serotonin (5-HT) and CGRP at the site of stimulation. Krebs-bicarbonate medium was added to each of the three compartments of a flat-sheet preparation of the rat colon for 15 min, and media were collected for measurements of basal 5-HT and CGRP release. Fresh medium was then added to all three compartments; the medium in the central compartment contained (in addition) acetate, propionate, or butyrate to a final concentration of 5 mM. The media in each compartment were collected after 15 min for measurements of 5-HT and CGRP release. 5-HT release (8- to 11-fold above basal) and CGRP release (2- to 3-fold above basal) occurred in the central compartment (solid bars) but not orad and caudad compartments (hatched bars) upon exposure to each SCFA separately. Basal 5-HT release was 6.8 ± 1.1 pmol·100 mg–1·min–1; basal CGRP release was 4.8 ± 0.5 fmol·100 mg–1·min–1. Values are means ± SE of 4–6 experiments.

 
Basal release of CGRP into the three compartments was also similar (mean ± SE, 4.8 ± 0.5 fmol·100 mg–1·min–1) but were 1,000-fold lower than that of 5-HT. The addition of 5 mM of acetate, propionate, or butyrate caused a two- to threefold increase in CGRP release above basal levels in the central compartment only (Fig. 2). The amounts of CGRP released by acetate, propionate, and butyrate were not significantly different (14.6 ± 2.1, 17.8 ± 4.6, and 15.6 ± 3.0 fmol·100 mg–1·min–1, respectively, corresponding to 2.0 ± 0.3-, 2.8 ± 0.8-, and 2.4 ± 0.4-fold increases above the basal level). The amounts of CGRP in the orad and caudad compartments did not differ significantly from basal levels (range: 5.0 ± 1.3–6.5 ± 0.7 fmol·100 mg–1·min–1).

In contrast to 5-HT and CGRP, the addition of the three SCFAs to the central compartment did not elicit a significant release of BDNF into any compartment (basal BDNF release: 0.9 ± 0.2 fmol·100 mg–1·min–1; BDNF release in the presence of 5 mM SCFA range: 0.7 ± 0.3–0.9 ± 0.3 fmol·100 mg–1·min–1).

Dependence of CGRP release on 5-HT release and activation of 5-HT4 receptors. Previous studies (13, 15) have shown that CGRP release induced by mechanical stimulation of the mucosa was dependent on 5-HT release and was mediated by the activation of 5-HT4 receptors. The effect of mucosal stimulation on CGRP release was inhibited by the addition of a selective 5-HT4 receptor antagonist and mimicked by the addition of a selective 5-HT4 receptor agonist to the central compartment.

Release of CGRP into the central compartment was measured after the addition of 5 mM acetate, propionate, or butyrate in the absence or presence of the selective 5-HT4 receptor antagonist GR-113808A (10 µM) or the 5-HT3 receptor antagonist LY-278584 (10 µM). The 5-HT4 receptor antagonist inhibited CGRP release induced by acetate, propionate, or butyrate by 58 ± 6%, 70 ± 8%, and 74 ± 8%, respectively (P < 0.01, n = 4; Fig. 3). The 5-HT3 receptor antagonist had no effect on CGRP release (Fig. 3).


Figure 3
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  		Fig. 3. Inhibition of SCFA-induced CGRP release by 5-HT4 receptor antagonist. CGRP release in response to the addition of 5 mM acetate, propionate, or butyrate to the central compartment of a three-compartment preparation of the rat colon was determined as described in Fig. 2. Each agent was added separately first alone and then in combination with either 10 µM of the 5-HT3 receptor antagonist LY-278584 or 10 µM of the 5-HT4 receptor antagonist GR-113808A. Values are means ± SE of 4–6 experiments with each SCFA. **P < 0.01, significantly different from control.

 
Dependence of the peristaltic reflex on sequential release of 5-HT and CGRP and activation of 5-HT4 receptors. Previous studies (13, 15) have shown that ascending contraction and descending relaxation induced by mechanical stimulation of the mucosa were dependent on release of CGRP, which, in turn, was dependent on the release of 5-HT and activation of 5-HT4 receptors. Ascending contraction and descending relaxation were therefore measured in the orad and caudad compartments, respectively, in response to the addition of 5 mM acetate, propionate, or butyrate in the absence or presence of the 5-HT4 receptor antagonist GR-113808A (10 µM), the 5-HT3 receptor antagonist LY-278584 (10 µM), or the CGRP receptor antagonist hCGRP8–37 (10 µM).

The addition of the 5-HT4 receptor antagonist to the central compartment inhibited ascending contraction in response to acetate, propionate, or butyrate by 76 ± 12%, 63 ± 8%, and 69 ± 9%, respectively, and descending relaxation by 67 ± 8%, 69 ± 5%, and 78 ± 6%, respectively (Fig. 4). The extent of inhibition of ascending contraction and descending relaxation by GR-113808A was similar for all three SCFAs. The addition of the 5-HT3 receptor antagonist LY-278584 had no effect on ascending contraction or descending relaxation (Fig. 4).


Figure 4
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  		Fig. 4. Inhibition of SCFA-induced ascending contraction and descending relaxation by 5-HT4 receptor antagonist. Ascending contraction (A) and descending relaxation (B) in response to the addition of 5 mM acetate, propionate, or butyrate to the central compartment of a three-compartment preparation of the rat colon were measured in the orad and caudad compartments, respectively, as described in Fig. 1. Each agent was added separately first alone and then in combination with either 10 µM of the 5-HT3 receptor antagonist LY-278584 or 10 µM of the 5-HT4 receptor antagonist GR-113808A. Values are means ± SE of 4–6 experiments with each SCFA. **P < 0.01, significantly different from control.

 
The addition of the CGRP receptor antagonist hCGRP8–37 (10 µM) to the central compartment inhibited ascending contraction in response to acetate, propionate, or butyrate by 64 ± 9%, 67 ± 4%, and 56 ± 9%, respectively, and descending relaxation by 67 ± 7%, 55 ± 6%, and 61 ± 4%, respectively (Fig. 5). The extent of inhibition of ascending contraction and descending relaxation by hCGRP8–37 was similar for all three SCFAs.


Figure 5
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  		Fig. 5. Inhibition of SCFA-induced ascending contraction and descending relaxation by CGRP receptor antagonist. Ascending contraction (A) and descending relaxation (B) in response to the addition of 5 mM acetate, propionate, or butyrate to the central compartment of a three-compartment preparation of the rat colon were measured in the orad and caudad compartments, respectively, as described in Fig. 1. Each agent was added separately first alone and then in combination with the CGRP receptor antagonist hCGRP8–37 (10 µM). Values are means ± SE of 4 experiments with each SCFA. **P < 0.01, significantly different from control.

 
Effect of combined mechanical and chemical stimulation of the mucosa on 5-HT and CGRP release and the peristaltic reflex. The patterns of 5-HT and CGRP release and effects of 5-HT receptor antagonists on the peristaltic reflex induced by mechanical and chemical stimulation of the mucosa suggested that both modalities of stimulation activated the same pathways. This notion was investigated further in experiments involving combined chemical and mechanical stimulation of the mucosa. Mechanical mucosal stimuli (2–8 strokes) were applied to the central compartment first alone and again in the presence of 1 mM acetate. 5-HT and CGRP release were measured in the central compartment, whereas ascending contraction and descending relaxation were measured in the orad and caudad compartments, respectively.

The stimulus-response curves for both 5-HT and CGRP release as well as those for ascending contraction and descending relaxation showed significant shifts to the left in the presence of 1 mM acetate (Figs. 6 and 7). The increase in 5-HT release at various levels of stimulation was more than additive (4.4- to 13.9-fold above the basal level), whereas the increase in CGRP release appeared to be additive (Fig. 6, A and B). The increase in ascending contraction or descending relaxation followed more closely the pattern of increase in CGRP release (Fig. 7, A and B).


Figure 6
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  		Fig. 6. Augmentation of 5-HT and CGRP release by combined mechanical (stroking) and chemical (SCFA) stimulation of colonic mucosa. 5-HT release (A) and CGRP release (B) were measured as described in Fig. 2. Mechanical stimulation (2–8 strokes) or chemical stimulation (1 mM acetate) was applied separately to the mucosa in the central compartment of a three-compartment preparation of the rat colon. The experiments were then repeated, during which time mechanical stimulation was applied in the presence of 1 mM acetate. Media in the central compartment were collected after each stimulus and assayed for 5-HT and CGRP. Values are means ± SE of 6 experiments. **P < 0.01 and *P < 0.05, significantly different from mechanical stimulation alone.

 

Figure 7
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  		Fig. 7. Augmentation of ascending contraction and descending relaxation by combined mechanical (stroking) and chemical (SCFA) stimulation of the colonic mucosa. Ascending contraction (A) and descending relaxation (B) were measured in the orad and caudad compartments, respectively, as described in Fig. 1. Mechanical stimulation (2–8 strokes) or chemical stimulation (1 mM acetate) was applied separately to the mucosa in the central compartment of a three-compartment preparation of the rat colon. The experiments were then repeated, during which time mechanical stimulation was applied in the presence of 1 mM acetate. Values are means ± SE of 6 experiments. **P < 0.01 and *P < 0.05, significantly different from mechanical stimulation alone.

 
Correlation of CGRP release and the muscle response. Ascending contraction or descending relaxation elicited by mucosal stroking alone or in combination with 1 mM acetate was closely correlated with the amount of CGRP released. Data obtained by various levels of mechanical stimulation of the mucosa or combined mechanical and chemical stimulation were described by the same curve (Fig. 8).


Figure 8
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  		Fig. 8. Correlation of CGRP release and mechanical response to mucosal stimulation. Results on CGRP release and mechanical responses to mucosal stimulation alone and in the presence of 1 mM acetate were plotted to display the dependence of ascending contraction and descending relaxation on the amount of CGRP released. The concentration-response curves indicated that ~50% of maximal contraction (1.2 g) or maximal relaxation (0.8 g) was elicited by an ~150% increase in CGRP release.

 
Stimulation of propulsive activity in the isolated guinea pig colon by an intraluminal perfusion with acetate. Propulsive activity was measured in isolated whole segments of the guinea pig colon using artificial pellets inserted at the orad end. The velocity of propulsion was measured as the average time taken for three successive pellets inserted at 5-min intervals into the orad end to traverse a fixed distance. During the measurement of basal velocity, the segment was perfused with Krebs-bicarbonate medium via a polyethylene-10 catheter inserted at the caudad end. The addition of 1 mM acetate to the perfusate increased propulsive velocity by 94.1 ± 12.5% (P < 0.01), from a basal value of 1.1 ± 0.1 mm/s to a measured value of 2.1 ± 0.3 mm/s.

DISCUSSION

This study provides strong evidence that SCFAs in the physiological range stimulate 5-HT release, which activates 5-HT4 receptors located on intrinsic CGRP-containing sensory neurons and triggers a characteristic colonic peristaltic reflex consisting of contraction orad (ascending contraction) and relaxation caudad (descending relaxation) to the site of stimulation. This is the first study to demonstrate the effects of SCFAs on both limbs of the peristaltic reflex. Previous studies have focused exclusively on the contractile effects of SCFAs. The main steps that lead to the activation of the peristaltic reflex by SCFAs were identical to those initiated by mechanical stroking of the mucosa (12, 13, 15); however, unlike the pathway activated by mechanical stroking, the pathway activated by SCFAs did not appear to lead to release of the neurotrophin BDNF, which has been previously found to enhance the peristaltic reflex induced by mechanical stimulation (16). Neither mechanical stroking of the mucosa nor application of SCFAs involved muscle stretch or distension; thus, the effect is due to the SCFA itself. Furthermore, the present study demonstrated for the first time that the effects of mechanical stroking and chemical stimulation were synergistic and correlated with the amount of CGRP release. Both CGRP release and the mechanical components of the reflex were dependent on the activation of 5-HT4 receptors.

The effects observed in the present study are unlikely due to pH alone because we used the SCFAs at neutral pH where they are sodium salts so as to eliminate any effects directly attributable to pH. The potential role of pH has been studied in several settings, and it has been generally found that SCFAs are effective at neutral pH. In a direct measurement of an intestinal afterhyperpolarization (AH) neuron response in the guinea pig, it was found that mucosal application of acetate at neutral pH was effective (2). In a study (2) in which SCFAs were found to inhibit motility, neutral SCFAs were found to be ineffective compared with acidified SCFAs. In contrast, when SCFAs were reported to stimulate motility and transit, they were found to be more effective at neutral pH than at acidic pH (6, 22, 33). Similarly, a recent study (11) found that while SCFAs stimulate motility at neutral pH and acidic pH, the effect was not attributable to pH because perfusion of acidic solutions alone did not affect the rat colon. Thus, while the pH may play a role in more proximal regions where the pH tends to be acidic, it seems unlikely that it plays a major role in the distal colon, where pH is higher (30, 37, 38).

Acetate, propionate, and butyrate applied in the range of 0.5–100 mM to the colonic mucosa elicited concentration-dependent ascending contraction and descending relaxation with an EC50 of ~5 mM. At this concentration, each SCFA induced 5-HT release (8- to 11-fold above the basal level) and CGRP release (2- to 3-fold above the basal level). This concentration is within the physiological range of single SCFAs reported for the distal colon (1, 4, 37). Considering that the total concentration of all SCFAs can be anywhere from 20 to 100 mM, the total physiological release of 5-HT and CGRP could be even greater depending on the nature of the diet, transit times, and luminal pH (1, 4, 37, 38).

Depending on the type of measurement, approach, species, and concentration, both excitatory and inhibitory effects of SCFA have been reported. Inhibitory effects were noted mainly at higher nonphysiological concentrations (100–500 mM) (5–7, 20, 36), whereas lower physiological concentrations (<100 mM) were excitatory or had no effect (2, 11, 20, 21, 28, 29, 33, 39). In a study by Cherbut et al. (7) where colonic spike bursts and transit were measured, an intraluminal infusion of SCFA resulted in a decrease in spike bursts that mediate the "to-and-fro" movement of luminal contents, whereas those that mediate propulsive motility were increased, resulting in increased rates of transit. This is consistent with the enhancement of the peristaltic reflex identified in the present study. Also consistent with the findings of the present study are numerous studies demonstrating an increase in motility, contractility, transit rate, or pellet output induced by SCFAs in the dog (21, 28), guinea pig (20), and rat (2, 11, 29, 33, 39). Also in agreement with the present study, the majority of these studies found the excitatory effect of SCFAs in the lower range of physiological concentrations (<100 mM).

In general, the excitatory effects of SCFAs have been thought to be dependent on enteric neural reflexes that lead to activation of enteric cholinergic motor neurons (11, 21, 24, 29, 33), although direct effects on smooth muscle cells (28) and enteric neurons (2, 17, 18) as well as effects mediated by local release of prostaglandin have also been demonstrated (29). There is also general agreement that the SCFA-induced reflex is dependent on the presence of an intact mucosa (2, 11, 21, 24, 29, 39). This is consistent with the results of the present study demonstrating that the initial step in the response to SCFAs is release of 5-HT from mucosal sources. The subsequent release of CGRP and initiation of the peristaltic reflex are dependent on 5-HT since they are inhibited strongly by 5-HT4 but not 5-HT3 receptor antagonists.

Two recent studies (11, 29) in the rat colon have also demonstrated a key role of 5-HT in mediating the response to SCFAs, although the conclusions differ somewhat from the present study. Fukumoto et al. (11), using an in vivo preparation, demonstrated increased pellet propulsion and colonic transit in response to SCFAs. They postulate that 5-HT activates 5-HT3 receptors on vagal afferents to initiate a vagovagal reflex that ultimately activates cholinergic motor neurons in the myenteric plexus. This is based on the demonstration of SCFA-induced 5-HT release and inhibition of the reflex by a 5-HT3 receptor antagonist, perivagal capsaicin, or vagotomy. This study (11) differs from the present study in several important ways that may explain the differences in conclusions. First, a combination of SCFAs was administered to a final concentration of 50–200 mM rather than administration of individual SCFAs. Second, the pH of the solution was acidic rather than neutral, although the authors demonstrated that acidic solutions of similar pH but without SCFAs had no effect on transit or motility. Third, the motility and transit experiments were done in vivo, where components extrinsic to the enteric nervous system are intact and can modify the enteric pathways. Fourth, the SCFA mixture was administered through a catheter in the cecum and placed so as to empty into the proximal colon. Thus, the site of activation in this study could be the proximal colon, where vagal activity and 5-HT receptors could differ from the distal colon, which was used in our study. Finally, these authors did not test a 5-HT4 receptor antagonist, so the role of 5-HT4 receptors cannot be completely excluded. In contrast with the study of Fukumoto et al. but in agreement with our study, Mitsui et al. (29) found that circular muscle strips from the distal colon responded to low concentrations (10 µM–10 mM) of butyrate and propionate, but not acetate, with a phasic and tonic contraction. The former was neurally mediated, dependent on the presence of the mucosa, and was inhibited by a 5-HT4 antagonist. Unlike our study, this reflex response was not inhibited by a CGRP antagonist, although this might be the result of the low concentration (1 µM) of antagonist used in this study. Mitsui et al. concluded that SCFAs caused 5-HT release, which activated a 5-HT4 receptor on an intrinsic primary afferent neuron. Neither of these studies examined the effect of intraluminal SCFAs on the descending inhibitory component of the peristaltic reflex. Considering these two studies and the present study, it is possible that SCFAs activate a vagovagal, 5-HT3-mediated pathway in the proximal colon and an intrinsic 5-HT4/CGRP pathway in the distal colon and that enhanced colonic transit is the result of both pathways.

The mechanism by which SCFAs cause 5-HT release is not known; however, recently discovered SCFA receptors offer an intriguing possibility. Two orphan G protein-coupled receptors (GPRs), GPR41 and GPR43, have been recently cloned and demonstrated to be receptors for SCFAs (3, 32). A comparison of the characteristics of these SCFA receptors and the results of the present study suggest that one of these receptors, GPR43, may mediate the release of 5-HT induced by SCFAs in the colon. GPR43 was activated by the anion of the neutral sodium salt of the SCFAs and was not dependent on acidic pH. Second, the EC50 for the interaction of various SCFAs with GRP43 was on the order of 0.5–1.5 mM, similar to the EC50s determined in the present study. Finally, acetate, butyrate, and propionate were essentially equipotent at the GPR43 receptor, consistent with the findings in the present study showing that all three SCFAs were equipotent at stimulating the release of 5-HT, CGRP, and the peristaltic reflex. A similar conclusion about the role of GRP43 was reached by John et al. (20), based on the equivalent potency of SCFAs in inducing peristalsis in the guinea pig ileum. This notion is also supported by the recent study bu Karaki et al. (23) demonstrating that GPR43 is expressed by mucosal cells of the rat distal ileum and colon. GRP43 was expressed at low levels by all enterocytes, but the main site of expression was on mucosal PYY-containing enteroendocrine cells and on 5-HT-containing mucosal mast cells of the lamina propria (23). There was no evidence of GPR43 expression by enteric neurons, smooth muscle cells, or, surprisingly, 5-HT-containing enteroendocrine cells (23). This suggests that it is the mucosal mast cell that responds to SCFAs to release 5-HT and activate the local peristaltic reflex, whereas the 5-HT-containing enteroendocrine cell is likely the source of 5-HT released by mucosal mechanical stimulation. This dual source of 5-HT may also explain why BDNF is not released in conjunction with SCFA stimulation, like it is with mechanical stimulation. BDNF and 5-HT are costored in enteroendocrine cells (14) and thus likely coreleased by mechanical stimulation; however, since the mucosal mast cell is the site of SCFA-induced 5-HT release, it may not be accompanied by BDNF release, consistent with the findings in the present study.

In vivo, it is likely that the passage of intraluminal contents exposes the colonic mucosa to both mechanical stimulation as the pellet passes over the mucosa and chemical stimulation by products of digestion, such as SCFAs, in the fecal material. In the present study, we tried to mimic this by combining mechanical (2–8 mucosal strokes) and chemical (1 mM acetate) stimulation of the mucosa. The presence of acetate caused significant increases in 5-HT and CGRP release and corresponding increases in the magnitudes of ascending contraction and descending relaxation above that induced by mechanical stimulation alone. The enhanced mechanical responses were closely correlated with the amount of CGRP release. This augmentation of mucosal mechanical stimulation in the presence of SCFA is consistent with the notion that the initiating event for each involves a different source of 5-HT. Mechanical stimulation likely leads to release of 5-HT from mucosal enteroendocrine cells, whereas stimulation by SCFAs likely leads to release from mucosal mast cells via activation of GPR43. The net result is an augmentation of 5-HT release similar to that measured in the present study.

The significant increase in 5-HT and CGRP release and the corresponding increase in the magnitude of the reflex in the presence of a low concentration of acetate suggest that small variations in the intraluminal concentrations of SCFAs could potentiate the peristaltic response to mechanical stimulation of the mucosa engendered by the passage of digesta through the colon and lead to more effective propulsion of intraluminal contents. Direct measurements of propulsive activity in isolated whole segments of the guinea pig colon using artificial fecal pellets showed that perfusion of the lumen with a low concentration of acetate (1 mM) doubled the velocity of propulsion. The passage of pellets under these conditions provides a mechanical mucosal stimulus comparable with experimental stroking of the mucosa that is enhanced by the presence of acetate. Thus, the enhancement of the underlying peristaltic reflex results in enhanced propulsion similar to that observed in vivo.

In summary, the present study demonstrates that the presence of SCFA in the distal colon stimulates the peristaltic reflex similar to that induced by mechanical stimulation of the mucosa. The mechanism is similar in that the major pathway in both involve the release of 5-HT, activation of 5-HT4 receptors, release of CGRP, and activation of enteric reflex pathways, leading to stimulation of both ascending contraction and descending relaxation. The pathways seem to differ slightly in that BDNF is released by mechanical stimulation and enhances the reflex, whereas BDNF does not seem to be released as part of the response to SCFAs. The present study also demonstrates an interaction when multiple stimuli are present such that the response to mechanical stimulation of the mucosa is enhanced in the presence of low concentrations of SCFAs. This latter effect is evident in the enhancement of colonic propulsion in the presence of similar concentrations of SCFAs. Thus, colonic propulsion in the in vivo setting is likely to be mediated by the combined effects of mechanical and chemical stimulation of the mucosa by luminal contents.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34153.

FOOTNOTES


Address for reprint requests and other correspondence: J. R. Grider, Dept. of Physiology, Virginia Commonwealth Univ., Box 980551, Richmond, VA 23298 (e-mail: jgrider{at}vcu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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