The role played by mucosal secretion of 5-HT in evoking the peristaltic reflex has been a conundrum. This role seems to be critical: EC cells secrete 5-HT in response to pressure or mucosal distension (Feldberg and Toh, 1953
; Bülbring and Crema, 1959b
; Grider et al., 1994
; Foxx-Orenstein et al., 1995
). This 5-HT stimulates the intrinsic sensory neurons that initiate the peristaltic (Kirchgessner et al., 1992
; Grider et al., 1994
; Foxx-Orenstein et al., 1995
) or secretory reflexes (Cooke and Reddix, 1994
). The mechanism of mucosal 5-HT inactivation, however, has not been known. If 5-HT is the transmitter at an EC cell-sensory nerve junction, then mucosal 5-HT will have to be removed rapidly to terminate responses to it and prevent receptor desensitization. Indeed, an inactivating mechanism is a criterion used in identifying neurotransmitters (Burnstock, 1983
). The present investigation was thus carried out to identify the putative mechanism for the inactivation of mucosal 5-HT. We tested the hypothesis that the same plasmalemmal 5-HT transporter that is expressed by serotonergic neurons and accounts for the inactivation of 5-HT at synapses in the nervous system is expressed in the mucosa. Because the mucosa contains no serotonergic nerves (Costa et al., 1982
; Furness and Costa, 1982
), the cells that express this transporter would have had to be either nonserotonergic neurons or non-neuronal cells. Our data suggest that uptake of 5-HT in the mucosa plays a necessary role in the peristaltic reflex by preventing the desensitization of 5-HT receptors. Mucosal uptake of 5-HT occurs because crypt epithelial cells synthesize and express a functional 5-HT transporter in their plasma membrane.
Evidence that 5-HT inactivation is important in the peristaltic reflex was derived from in vitro
studies of the reflex-activated propulsion of artificial fecal pellets in the guinea pig distal colon. The peristaltic reflex was evoked reproducibly in this preparation for long periods of time. Because the in vitro
reflex was blocked by tetrodotoxin and inhibited by hexamethonium, it was nerve-dependent. The reflex was also dependent on intact serotonergic mechanisms, because propulsive activity was abolished by desensitization of 5-HT receptors. The effectiveness of tropisetron, a 5-HT3
dual antagonist, and 5-HTP-DP, a 5-HT1P
antagonist, in inhibiting propulsion suggested that both 5-HT3
(Kadowaki et al., 1993
) and 5-HT1P
receptors participate in the reflex. The observation that 5-HTP-DP was more effective when applied to the mucosal rather than the serosal surface of the colon suggests that 5-HTP-DP acts on the mucosa. 5-HT1P
receptors have been located in the mucosal nerve plexus that underlies the intestinal epithelium (Wade et al., 1994
), and 5-HT1P
receptors have been implicated in the activation of the intrinsic sensory neurons in the submucosal plexus that initiate the peristaltic reflex (Kirchgessner et al., 1992
; Foxx-Orenstein et al., 1995
). A site of action on mucosal sensory nerves thus would account for the observed effectiveness of 5-HTP-DP. These observations supported the use of the guinea pig distal colon to test the prediction that blocking the uptake of 5-HT would interfere with the peristaltic reflex.
The actions of fluoxetine and zimelidine, selective inhibitors of 5-HT uptake, supported the hypothesis that the 5-HT transporter plays a role in the peristaltic reflex. A low concentration of fluoxetine potentiated the propulsion of fecal pellets; nevertheless, propulsion was inhibited by fluoxetine at concentrations above 0.1 μM. These observations could be explained if a moderate degree of inhibition of 5-HT uptake by fluoxetine enhances the action of endogenous 5-HT to accelerate propulsion but more complete inhibition of uptake causes 5-HT receptors to desensitize, inhibiting the reflex. The similar action of zimelidine supports the idea that 5-HT accumulation accounts for reflex inhibition; however, nonspecific antagonism of a 5-HT receptor subtype by fluoxetine was not excluded rigorously. Fluoxetine did not affect cholinergic transmission or nerve-driven muscle contraction and thus evidently did not interfere nonspecifically with reflex-driven motility. Physiological experiments alone do not identify which of the potential sites of action of 5-HT are affected by drugs. Because 5-HT is a neurotransmitter of myenteric neurons, as well as a product of EC cells, interference with serotonergic transmission could antagonize the peristaltic reflex at sites in the mucosa or in the myenteric plexus. Although the peristaltic reflex depends on serotonergic transmission, receptor desensitization, 5-HTP-DP, tropisetron, and fluoxetine/zimelidine could have antagonized the reflex in the guinea pig distal colon by blocking the effect of 5-HT on either mucosal sensory nerves or myenteric neurons.
A functional 5-HT transporter was indeed found to be expressed in the mucosa. This transporter was localized to crypt epithelial cells by radioautography; thus, these cells took up 3
H-5-HT by a Na+
-dependent mechanism that was inhibited by fluoxetine. Fluoxetine was shown previously to block mucosal uptake of radiolabeled 5-HT in the rat, although that study assumed that 5-HT was taken up by EC cells (Ternaux et al., 1981
). Most of the EC cells, identified immunocytochemically in the current study by their content of endogenous 5-HT, did not become labeled by 3
H-5-HT. Conceivably, crypt cells might have been mistaken for EC cells in the previous investigation of the rat mucosa. It seems likely that the uptake of 5-HT by crypt epithelial cells enables the mucosal epithelium to act as a 5-HT sink. The uptake of 5-HT by crypt epithelial cells does not prevent the diffusion of 5-HT secreted by EC cells into the lumen of the bowel (Ahlman et al., 1981a
) or from absorption into blood vessels (Toh, 1954
); nevertheless, the presence of the epithelial sink probably causes the concentration of 5-HT in the lamina propria to fall more rapidly, after its secretion by EC cells, than it would have in the absence of the crypt epithelial 5-HT transporter. An apparent barrier, which impedes the mucosal to serosal passage of 3
H-5-HT, has been reported previously (Gershon and Tamir, 1981
; Cooke et al., 1983
). This barrier could be accounted for by the existence of a mucosal 5-HT sink.
Northern analysis indicated that mRNA hybridizing with a full-length cDNA probe encoding the rat brain 5-HT transporter is present in the intestinal mucosa of both guinea pigs and rats, confirming previous data from rat bowel (Hoffman et al., 1991
). This mucosal mRNA was about the same size (3.7 kb) as hybridizing mRNA extracted from rat brainstem and encoded a molecule that was virtually identical in sequence to the rat brain 5-HT transporter (Blakely et al., 1991
). mRNA encoding the 5-HT transporter was also found by Northern analysis to be more abundant in the rat intestinal mucosa than in the rat brainstem. Less hybridizing mRNA was detected in the guinea pig mucosa, probably because the sequence of the guinea pig 5-HT transporter is not identical to that of the rat. In situ
hybridization revealed that cells that contain mRNA encoding the 5-HT transporter were located in exactly the same region of the crypt epithelium as the cells found by radioautography to take up 3
H-5-HT. These cells were most abundant in the basolateral region of intestinal crypts and declined in number along the crypt-to-villus axis. This distribution corresponds to the proliferative zone of epithelial precursor cells (Gordon et al., 1992
). These crypt epithelial cells are known to be secretory (Welsh et al., 1982
) and intestinal Cl−
secretion is stimulated by 5-HT (Cooke and Reddix, 1994
). 5-HT also stimulates the proliferation of crypt epithelial cells (Tutton, 1974
). The 5-HT transporter thus may play a role in epithelial actions of 5-HT.
5-HT transporter-immunoreactive cells were observed to be present in the same regions of the intestinal crypts as the cells that contain mRNA encoding the transporter and take up 3H-5-HT. The transporter protein thus is expressed by crypt epithelial cells and probably accounts for their uptake of 3H-5-HT. The location of 5-HT transporter-immunoreactive cells, however, was not absolutely identical to that of cells revealed by in situ hybridization. Some villus cells contained 5-HT transporter immunoreactivity but not the corresponding mRNA. This difference suggests that the 5-HT transporter continues to be present in a subset of epithelial cells after they ascend beyond the crypt-villus junction and biosynthesis of the transporter ceases. 5-HT transporter immunoreactivity was strikingly marginal in individual cells, consistent with a concentration of the transporter in the plasma membrane. Few of the 5-HT-containing EC cells expressed 5-HT transporter immunoreactivity, accounting for the failure of these cells to become labeled by 3H-5-HT. The expression of the 5-HT transporter in the epithelium, therefore, differs from its expression in the ENS and CNS, where the cells that secrete 5-HT are the cells that also express the 5-HT transporter. Epithelial expression of the transporter is thus a capture and not a recapture mechanism.
Our observations are consistent with the idea that the peristaltic reflex is evoked by the release of 5-HT from mucosal EC cells to stimulate intrinsic sensory nerves. The current observations represent the first report of an inactivating mechanism for 5-HT in the mucosa and thus provide support for the hypothesis that the mucosal action of 5-HT is important in neurally mediated reflexes in the gut.