Stroking human jejunal mucosa induces 5-HT release and Clminus secretion via afferent neurons and 5-HT4 receptors

John M. Kellum, Francisco C. Albuquerque, Michael C. Stoner, and R. Paul Harris

Department of Surgery, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0161


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

5-Hydroxytryptamine (5-HT) release and neural reflex pathways activated in response to mucosal stroking were investigated in muscle-stripped human jejunum mounted in modified Ussing chambers. The mucosa was stroked with a brush at 1/s for 1-10 s. Mucosal stroking resulted in a significant increase in the concentration of 5-HT in the mucosal bath within 5 min. It also was associated with a reproducible positive change (Delta ) in short-circuit current (Isc), which was abolished by inhibitors of chloride secretion. Capsaicin and hexamethonium significantly inhibited the Delta Isc but not the release of 5-HT. The Delta Isc was inhibited by TTX but not by atropine. It was also inhibited by the 5-HT3,4 receptor antagonist tropisetron (10 µM) and the 5-HT4,3 receptor antagonist SDZ-205-557 (10 µM) but not by preferential antagonists of 5-HT1P, 5-HT2A, or 5-HT3 receptors. These results suggest that mucosal stroking induces release of mucosal 5-HT, which activates a 5-HT4 receptor on enteric sensory neurons, evoking a neuronal reflex that stimulates chloride secretion.

serotonin; serotonin receptors; enteric neurons; intestinal secretion; short-circuit current; chloride; capsaicin; 5-hydroxytryptamine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

SINCE THE DISCOVERY that 60-90% of mammalian serotonin [5-hydroxytryptamine (5-HT)] stores are found in the gastrointestinal tract, the physiological role of 5-HT in alimentary function has been the subject of intense research (5). 5-HT has been found in enteric neuronal terminals where it functions as a neurotransmitter, but much larger amounts are synthesized and stored in the enterochromaffin (EC) cells in the crypts (8).

Frieling et al. (4) speculated that reflex circuits in the enteric nervous system mediate complex integrative functions that control and coordinate contractility of the longitudinal and circular muscle layers and regulate transport function in the epithelium. They noted that previous studies involving distending the intestinal wall, stroking the mucosa, irritating the mucosa chemically, or inducing inflammation of the viscera had reported activation of mechanosensitive and "silent" visceral afferent neural fibers that both initiate a stereotypic propulsive response and stimulate chloride secretion. Mucosal enteric sensory fibers often lack the selectivity assigned to them, responding to more than one stimulus modality, and are more appropriately considered polymodal (16).

Sidhu and Cooke (17) recently reported that stroking of guinea pig colonic mucosal sheets, mounted in modified Ussing chambers, induced a change (Delta ) in short-circuit current (Isc) mediated via a neuromucosal reflex involving both muscarinic and serotoninergic 5-HT1P receptors. This model provides an opportunity to examine a potential physiological role of endogenous intestinal 5-HT and to define reflex circuits in the enteric nervous system. The goal of the present study was to examine whether the effect of mucosal stroking on Isc was associated with release of serotonin. We postulated that release would occur and induce a Delta Isc via an enteric sensory neural pathway involving a 5-HT4 receptor.


    EXPERIMENTAL METHODS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental protocol. Segments of jejunum were obtained from patients having gastric bypass surgery for obesity. The experimental protocol was approved by the Committee for the Conduct of Human Research at the Medical College of Virginia/Virginia Commonwealth University. All patients gave informed consent for use of discarded tissue in these experiments. The excised jejunum was washed in iced Krebs and opened longitudinally along the mesenteric border. The muscularis propria was excised by sharp scissor dissection, leaving an intact mucosal sheet. Adjacent sections of the mucosal sheet were mounted in four parallel Ussing chambers (cross-sectional area of 2.1 cm2) for each experiment and subjected to short-circuit conditions by voltage clamp (World Precision Instruments, Sarasota, FL). Tissue was transported on ice and mounted within 15 ± 5 min after removal from the patient. The tissue was short circuited for the duration of the experiments with the exception of brief interruptions for reading the potential difference. Histological examination of this preparation has consistently demonstrated the absence of neural ganglia (11). Both sides of the tissue were bathed in Krebs-Ringer buffer (final concentration of 115 mM NaCl, 25 mM NaHCO3, 24 mM K2HPO3, 0.4 mM KH2PO4, 1.2 mM CaCl2 · 2H2O, and 1.2 M MgCl2 · 6H2O) containing glucose (10 mM) on the serosal and the mucosal sides. The tissue was gassed (95% O2-5% CO2) and maintained at 37°C by a jacketed water bath.

The tissue equilibrated for 30 min, after which it was stroked at one stroke per second 1-10 times with a fine 2-mm paintbrush mounted through a side port opposite the mucosal side (see Fig. 1). The first stroke was in a left-to-right direction, and the subsequent stroke was in the opposite direction. The Isc was measured throughout the experiment and before and after stroking. Stroking was done in the presence and absence of 5-HT receptor agonists and antagonists as well as neural, cholinergic-receptor and chloride-channel blockers. Test agents were added to both sides of the chamber 15 min before stroking. The maximum change in Isc after the strokes was recorded (this usually occurred within the first 2 min). Separate chambers were used for each experiment, since Bunce et al. (1) has shown that repeated washing of the same tissue decreases the amplitude of subsequent 5-HT-induced responses.



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Fig. 1.   A: time course for 5-hydroxytryptamine (5-HT) release, as measured by HPLC, in response to 5 mucosal strokes delivered at 1 stroke/s. Values are means ± SE of 5-HT concentrations in the buffer bathing the mucosal surface. B: comparison of 5-HT release from unstroked (control) and stroked human jejunal mucosa. Release was from the mucosal surface and is expressed as mean ± SE of total 5-HT release in the first 5 min after stroking. * P < 0.05, Student's t-test for paired data.

The following concentrations (based on the referenced source) of antagonists or neurotoxins were used: 1 µM TTX (n = 15) (10), 10 µM capsaicin (n = 6) (8), 1 µM atropine (n = 9) (4), 100 µM hexamethonium (n = 10) (4, 11, 17, 18), 10 µM 5-hydroxytryptophan dipeptide (5-HTP-DP; n = 8) (Michael D. Gershon, personal communication), 0.1 µM ketanserin (n = 8) (6, 18), 1 µM ondansetron (n = 10) (6), 10 µM tropisetron (n = 7) (6, 11), 10 µM SDZ-205-557 (n = 8) (6), the serosal loop diuretic 100 µM bumetanide (n = 6) (12), and the chloride channel blocker 100 µM anthracene-9-carboxylic acid (A-9C; n = 8) (13). The serosal bath was washed before the second stroke.

5-HT release. 5-HT was measured using HPLC with electrochemical detection. Before and at 5-min intervals after mucosal stroking, samples were taken from the Ussing chamber buffer solutions, which bathed the serosal and mucosal surfaces and which contained 1 mM pargyline and ascorbate to prevent oxidation of 5-HT. Because inhibitory 5-HT4 receptors have been reported on EC cells, 5-HT concentrations were also measured in the presence of 10 µM SDZ-205-557 both before and at 5-min intervals after stroking. Samples were centrifuged at 1,000 g for 20 min and filtered (0.2-µm Acro LC13 filter, Gelman Sciences, Ann Arbor, MI) to remove exfoliated cells that could artificially increase assayed 5-HT levels; samples were then stored at 4°C in the dark. The chromatography system consisted of a Waters 501 solvent delivery system (Water, Milford, MA), which was equipped with pulse damperers, WISP model 712 intelligent sample processor, making 5-µl sample injections, and a Novapak C18 column (3.9 × 150 mm) with a guard column containing a µ-Bondapak C18 GuardPak. The electrochemical detector was a Waters Model 460 with a glassy carbon electrode operating at 0.65 V relative to the reference electrode. Mobile phase was 0.1 mM EDTA with 0.075 mM 1-octanesulfonic acid, sodium salt for ion pairing. Chromatograph analysis was performed with an integrating Waters 740 data module. The system can routinely detect 5-HT at concentrations of 3 nM.

Chemicals. 5-HT creatinine sulfate, TTX, capsaicin, atropine, hexamethonium bromide, bumetanide, and A-9C were purchased from Sigma Chemical (St. Louis, MO). Ketanserin tartate, tropisetron and SDZ-205-557 were obtained from Research Biochemicals International (Natick, MA), and N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP) was purchased from The New York Research Foundation for Mental Hygiene (New York, NY). Ondansetron was obtained from Glaxo Pharmaceutical (London, UK).

Statistical analysis. Values are expressed as means ± SE. Statistical comparisons were made using one-way ANOVA or Student's t-test for paired data, using the statistical package Statistical Analysis System (SAS Institute, Cary, NC).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

Stroking human jejunal mucosa, mounted in modified Ussing chambers, caused a significant increase in the release of serotonin from the mucosal but not from the serosal surface. Release was evident as early as 5 min after stroking, and the concentration in the mucosal bathing solution continued to rise over 30 min (Fig. 1, A and B). Pretreatment with the 5-HT4 antagonist SDZ-205-557 had no significant effect on either baseline 5-HT levels [0.9 ± 0.2 vs. 1.3 ± 0.4 ng/ml (controls)] or the rise in 5-HT levels 15 min after stroking [0.3 ± 0.1 vs. 0.4 ± 0.1 ng/ml (controls); n = 6].

Stroking induced an increase in Isc within 2 min, which was statistically significant (P < 0.01, Student's t-test, paired data, n = 7) in comparison with unstroked tissue (Fig. 2A). Stroking a second time, after Isc was allowed to return to baseline levels, which normally took from 10-15 min, induced a change in Isc that was not statistically significantly different compared with that following the initial stroking (Fig. 2B). For this reason, in the remainder of this study, comparisons of the response to the initial stroking (control) were made with that to the repeat stroking, which was done in the presence of various antagonists and neurotoxins. Because baseline Isc and Delta Isc were highly variable from one subject to another, each tissue served as its own control. The variation in baseline and the reactivity are most likely related to differences in stripping technique and intrinsic differences in tissue reactivity from individual to individual. Tissue with high conductivity (suggesting tissue anoxia) at any time during the experiment was excluded from this study.



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Fig. 2.   A: comparison of positive change in short-circuit current (Delta Isc), expressed as percent change from baseline Isc, in unstroked and stroked jejunal mucosa at 5 min. * P < 0.05, Student's t-test for paired data. B: comparison of Delta Isc in successive strokes separated by a 20-min recovery period. There was no significant difference in the means by Student's t-test for paired data.

When stroking was increased from 1 to 10 strokes (1, 2, 5, 10 strokes), which was done in separate chambers to avoid desensitization, the Isc rose in a stroke-dependent manner (Fig. 3). Maximum Delta Isc was observed within the initial 5 min.


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Fig. 3.   Delta Isc in separate chambered mucosal sheets subjected to variable numbers of stokes. Differences in the mean values among the treatment groups are greater than would be expected by chance. P < 0.05 using one-way ANOVA (Bonferroni's all pairwise multiple-comparison procedures).

Although neither TTX nor capsaicin significantly reduced baseline Isc, bumetanide (P < 0.01), A-9C (P < 0.05), and hexamethomium (P < 0.05) all significantly inhibited baseline Delta Isc. A-9C (100 µM, n = 8), a chloride channel blocker, and bumetanide (100 µM, n = 6), a loop diuretic and basolateral sodium-potassium-chloride transport blocker, virtually abolished the Delta Isc in response to stroking (Table 1). The neural sodium channel blocker, TTX (1 µM, n = 15), significantly reduced the Delta Isc induced by stroking. The nicotinic receptor antagonist, hexamethonium (100 µM, n = 10), but not the muscarinic cholinergic receptor antagonist, atropine, reduced the Delta Isc in response to stroking. Capsaicin (10 µM, n = 5) caused significant inhibition of the Delta Isc in response to stroking (Table 1). Capsaicin had no effect on the release of 5-HT in response to stroking. Release was 5.9 ± 2.6 and 5.9 ± 1.8 ng · cm-2 · 5 min-1 in the absence and presence of capsaicin (P = 0.84, n = 5), respectively.

                              
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Table 1.   Effect of drugs on mucosal stroking responses in human jejunum

The following 5-HT receptor antagonists did not have any significant effect on the change in Isc induced by stroking: the 5-HT1P antagonist 5-HTP-DP, the 5-HT2A antagonist ketanserin, and the 5-HT3 antagonist ondansetron (see Table 2). It should be noted that, whereas Table 1 demonstrates the baseline, maximum absolute, and change in Isc in response to stroking in the absence and presence of antagonists, Table 2 illustrates only the Delta Isc response to stroking. The 5-HT3,4 receptor antagonist tropisetron and the 5-HT4,3 receptor antagonist SDZ-205-557 were associated with inhibition, which was significant (P < 0.05, Student's t-test, n = 9) at 10 µM (Table 2). The inhibition was concentration dependent for SDZ-205-557 (Fig. 4).

                              
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Table 2.   Effect of 5-HT receptor antagonists on mucosal stroking responses in human jejunum



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Fig. 4.   Percent change in Isc in relation to baseline Isc in the presence of preferential antagonists at 5-HT1P [5-hydroxytryptophan dipeptide (5-HTP-DP)], 5-HT2 (ketanserin), 5-HT3 (ondansetron), and 5-HT4 (SDZ-205-557) receptor antagonists. Only SDZ-205-557 was associated with a concentration-dependent inhibition of stroke-induced Delta Isc.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that stroking human jejunal mucosa induces both 5-HT release and a rise in Isc. Thus the human intestine responds to mucosal stroking in the same way as described for guinea pig colon by Sidhu and Cooke (17). The 5-HT release noted in the present study is most likely from EC cells, since our model does not contain neural ganglia and is devoid of serotoninergic neurons.

The transport response in human jejunum is similar to that of guinea pig colon in several respects. The abolition of the stroke-induced change in Isc by both a chloride channel blocker, A-9C, and a loop dieuretic and basolateral sodium-potassium-chloride transport blocker, bumetanide, demonstrates that chloride ion secretion in the human jejunum, like that in the guinea pig colon, is responsible for the stroke-induced change in Isc. The response was also sensitive to TTX, as Sidhu and Cooke (17) found in the guinea pig.

These findings in the human jejunum differ from those in guinea pig colon in that the Isc response to stroking in the human jejunum is mediated via the 5-HT4 receptor and a capsaicin-sensitive afferent neural pathway, whereas in the guinea pig colon it is mediated via a 5-HT1P receptor. Also, in contrast to the findings in guinea pig colon, the response in human jejunum was inhibited by a nicotinic but not by a muscarinic, cholinergic receptor antagonist, whereas in the guinea pig colon the reflex was abolished by atropine. In the present study, the addition of hexamethonium significantly depressed baseline Isc, as well as maximum Isc, in response to stroking, casting some doubt on the role of nicotinic receptors in the mediation of the stroking response in human jejunum. The disparity may reflect species or anatomic (colon vs. jejunum) differences or they may reflect differences in the stripping techniques. It should be noted that chambered human jejunal mucosa also differs from that described by Sidhu and Cooke (17) in that the human jejunum is stripped of both myenteric and submucosal neural ganglia, whereas the guinea pig preparation retained the submucosal plexus.

Previous in vitro studies in animal models have confirmed that 5-HT induces intestinal chloride ion secretion. Zimmerman and Binder (19) demonstrated this phenomenon in muscle-stripped sheets of rat distal colon mucosa mounted in Ussing chambers by measuring isotopic sodium and chloride flux. By demonstrating that furosemide and chloride-free solutions abolish the Delta Isc in a similar preparation of guinea pig ileum, Cooke and Carey (2) demonstrated that this response to exogenous 5-HT is caused by chloride ion secretion. Kellum et al. (11) reported isotopic flux studies, demonstrating that significant chloride ion secretion occurs in human jejunal mucosal sheets in response to exogenous 5-HT; the Delta Isc was accounted for by electrogenic chloride ion secretion. Thus human jejunum appears to respond to 5-HT in vitro in a fashion similar to the responses of guinea pig ileum and rat distal colon. However, stripped human jejunum behaves more like rat distal colon than guinea pig ileum or colon in its chloride secretory response to exogenous 5-HT in that the response is insensitive to atropine and has a largely TTX-insensitive component (11).

In this study, tropisetron at 10 µM and the preferential 5-HT4,3 receptor antagonist SDZ-205-557 (10) significantly antagonized the stroke-induced rise in chloride ion secretion. Tropisetron (<1 µM) is a highly preferential 5-HT3,4 antagonist with a high affinity for the 5-HT3 receptor (14, 15). The lack of inhibition by ondansetron makes the participation of the 5-HT3 receptor unlikely. In contrast, at >= 1.0 µM, tropisetron acts also as an antagonist at the 5-HT4 receptor (15). The combination of a lack of inhibition by ondansetron and the significant inhibition of the Delta Isc in response to stroking by 10 µM tropisetron makes it highly likely that the 5-HT receptor involved in this reflex is a 5-HT4 receptor.

The mediatory role of 5-HT4 receptors is also supported by the findings with SDZ-205-557, which has been recently characterized as a preferential 5-HT4,3 receptor antagonist with very weak affinity for the 5-HT3 receptor (10). The estimated pA2 from the present study was 7.8 for the antagonist compared with the reported pKi value of 6.5 (2) for the 5-HT3 and 7.5 for the 5-HT4 receptors, respectively (2).

Grider and Jin (9) recently reported that capsaicin applied directly to the mucosa inhibits the peristaltic reflex in response to mucosal stroking, presumably by blocking a mucosal enteric sensory neuron. The present study found that 10 µM capsaicin significantly inhibited the Delta Isc in response to stroking yet did not affect the same response to exogenous 5-HT. Whereas nicotinic, but not muscarinic, blockade inhibited the response to stroking, hexamethonium had a generally depressant effect on both baseline and stroke-induced Delta Isc. Two possible circuits can explain these findings. The more likely is that the mechanical deformation caused by stroking induces 5-HT release from EC cells that activates 5-HT4 receptors on enteric sensory neurons. These sensory neurons activate a neural-mucosal reflex resulting in chloride secretion. The other possibility is that stroking excites a capsaicin-sensitive enteric sensory neuron that either directly or through an interneuron induces release of 5-HT from EC cells that directly stimulates 5-HT4 receptors or crypt enterocytes to induce chloride secretion. The latter circuit is less likely because of the finding that the same concentration of capsaicin had no effect on 5-HT release from the mucosal surface. The hypothesis that stroking induces release of 5-HT from EC cells by mechanical deformation is also supported by the fact that the release in the present study is not significantly affected by pretreatment with SDZ-205-557, despite the fact that Gebauer et al. (7) reported evidence for the presence of a 5-HT4 receptor on the EC cell inhibitory to the release of serotonin.

This mechanism is analagous to that reported by Foxx-Orenstein et al. (3) for the initiation of the peristaltic reflex by mucosal stroking in human jejunum. These authors found that stroking induced concomitant release of 5-HT from mucosal stores and calcitonin gene-related peptide (CGRP) from sensory neurons. The participation of 5-HT receptor subtypes was species dependent in that the release of CGRP and the initiation of the peristaltic reflex were inhibited in the guinea pig colon by a combination of 5-HT3 and 5-HT4 or 5-HT1P receptor antagonists, whereas these responses were inhibited only by the 5-HT4 receptor antagonist in human bowel. Thus it appears that enteric neurons activated by mucosal stroking through 5-HT release from EC cells is a common pathway in both species; however, the subtype of 5-HT receptor mediating the response is different in human jejunum and guinea pig colon. Both the peristaltic motor response (3) and the secretion of chloride (present study) induced by mucosal stroking in human jejunum are mediated by the 5-HT4 receptor.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. M. Kellum, Dept. of Surgery, MCV/VCU, PO Box 980161, Richmond, VA 23298-0161 (E-mail: jkellum{at}hsc.vcu.edu).

Received 17 February 1998; accepted in final form 7 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

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5.   Gaginella, T. Serotonin in the intestinal tract: a synopsis. In: Serotonin and Gastrointestinal Function, edited by T. S. Gaginella, and J. J. Galligan. Boca Raton, FL: CRC, 1995, p. 1-9.

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Am J Physiol Gastroint Liver Physiol 277(3):G515-G520
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