An Endogenous Tachykinergic NK2/NK3 Receptor Cascade System Controlling the Release of Serotonin from Colonic Mucosa.

5-Hydroxytryptamine (5-HT) released from colonic mucosal enterochromaffin (EC) cells is a major signaling molecule, which participates in the pathophysiological regulation of colonic functions in gut disorder including irritable bowel syndrome (IBS), but the endogenous modulator system for the 5-HT release is not yet well elucidated. Our in vitro studies in guinea-pig colon have indicated that the cascade pathway of neuronal tachykinergic NK3 receptors and NK2 receptors on peptide YY (PYY)-containing endocrine L cells represents an endogenous modulator system for 5-HT release from EC cells and that melatonin, endogenous tachykinins and PYY play important roles in modulation of the release of 5-HT from EC cells via the endogenous NK2/NK3 receptor cascade system. This review aims at examining the potential role of the endogenous tachykinergic NK2/NK3 receptor cascade system controlling the release of 5-HT from EC cells, with special attention being paid to the pathophysiology of gut disorders including IBS.

INTRODUCTION

In the gastrointestinal (GI) tract, 5-hydroxytryptamine (serotonin, 5-HT) is a major signaling molecule, which mediates GI functions (motility and secretion), emesis, nausea and pain [1, 2]. Most of colonic 5-HT is synthesized and released from mucosal enterochromaffin (EC) cells; therefore, alterations in the release of 5-HT from EC cells affect both physiological and pathophysiological colonic functions [2].

Irritable bowel syndrome (IBS) is a GI chronic disorder characterized by abdominal discomfort or pain associated with altered bowel habits. Abnormal levels of chromogranin A (a marker for all endocrine cells) in the colon of IBS patients have been demonstrated [3], thus indicating a role of the colonic endocrine cells (namely, 5-HT-containg EC cells or peptide YY (PYY)-containing L cells), in the regulation of colonic function in IBS patients [4-6]. Indeed, a recent human study [7] suggests that the increased 5-HT release from colonic EC cells contributes to the development of abdominal pain in IBS patients. Therefore, an improved understanding of endogenous modulator system for 5-HT release from EC cells may provide novel insights into the etiology and pharmacotherapy of functional bowel disorders such as IBS.

Our in vitro studies have demonstrated that isolated guinea-pig colon is a helpful in vitro-preparation for studying the mechanism modulating the release of 5-HT from EC cells [8-10]. Experimental data from the in vitro-preparations indicate that melatonin, endogenous tachykinins (TKs; neuropeptides such as substance P and neurokinin A) and PYY play important roles in the modulation of the release of 5-HT from EC cells via a unique endogenous tachykinergic NK2/NK3 receptor cascade system [8-11]. This review focuses on the evidence establishing a potential role of the endogenous NK2/NK3 receptors cascade system controlling the release of 5-HT from EC cells, with special attention being paid to the pathophysiology of gut disorders including IBS.

TACHYKININ RECEPTORS AND 5-HT RELEASE FROM COLONIC MUCOSA

A selective tachykinin NK2 receptor agonist βAla-NKA-(4-10) (0.01-1 μM) evoked a long-lasting 5-HT release from isolated guinea-pig colon in a concentration-dependent manner [8-11]. The 5-HT-releasing action of βAla-NKA-(4-10) was unaffected by tetrodotoxin (TTX), and was also seen in myenteric plexus-free mucosal preparations, thus suggesting that the NK2 agonist-evoked 5-HT release is mediated by activation of NK2 receptors located on non-neuronal cells in the mucosal layer [8]. This agrees with a previous report which showed tachykinin NK2 receptor expression on the surfaces of enterocytes of guinea-pig colon [12]. The L-type calcium channel blocker nicardipine or the syntaxin inhibitor botulinum toxin type C also reduced the long-lasting 5-HT release evoked by βAla-NKA-(4-10) [9], thus suggesting that the NK2 receptor-triggered 5-HT release is mediated via syntaxin-related exocytosis mechanisms. However, an involvement of tachykinin NK1 receptors in the evoked 5-HT release is unlikely because a selective tachykinin NK1 receptor agonist, [Sar9, met (O2)11]-substance P (100 nM) failed to affect basal 5-HT release [9].

A selective tachykinin NK3 receptor agonist senktide also evoked a transient 5-HT release from the isolated guinea-pig colon implicating tachykinin NK3 receptors, in addition to NK2 receptors in modulating 5-HT release from colonic mucosa [8]. As summarized in Fig. , the enhancing effect of senktide on 5-HT release was prevented by both TTX and the NK2 receptor-selective antagonist SR48968, thus suggesting that senktide stimulates the release of neuronal TKs, which in turn enhances 5-HT release via mucosal NK2 receptors. This agrees with previous findings in guinea-pig colon where NK3 receptor activation releases TKs and 5-HT [13, 14]. The 5-HT-releasing action of senktide was also inhibited by hexamethonium, and was not seen in myenteric plexus-free mucosal preparations, thus suggesting that myenteric cholinergic interneurons also participate in the mechanism for NK3 receptor-triggered 5-HT release [8]. Thus, these findings support the view that the cascade of neuronal NK3 receptors and mucosal NK2 receptors represents an endogenous modulator system for 5-HT release from EC cells.

These results are also in line with previous findings (1) in human colon where NK3 receptors present predominantly on myenteric and submucosal plexus neurons, and NK3 receptors come into a play only in pathological conditions such as rescue responses or inflammation [15], and (2) in healthy volunteers where the stimulation of NK2 receptors appears to induce IBS-like symptoms [16]. Moreover, it has been shown that the 5-HT release from mucosal biopsy specimens of IBS patients is increased by 10-fold over controls, and the increased 5-HT release contributes to development of abdominal pain in IBS patients [7]. Therefore, the view that the endogenous tachykininergic NK2/NK3 receptor cascade system can regulate the release of 5-HT from colonic EC cells, has an important implication for the potential role of endogenous TKs in the generation of the symptoms associated with IBS; it can be speculated that endogenous TKs acting through the NK2/NK3 receptor cascade system facilitate the release of 5-HT which in turn affects the colonic function of IBS patients through the activation of 5-HT receptor subtypes.

A sensory nerve-derived neuropeptide, calcitonin gene-related peptide (CGRP, 1-100 nM) has been also shown to produce a TTX-sensitive but atropine-resistant 5-HT release via stimulation of myenteric plexus neurons in the isolated segments of guinea-pig colon [8]. The enhancing effect of CGRP on 5-HT release was also prevented by both the NK2 receptor-selective antagonist SR48968 and the NK3 receptor-selective antagonist SR142801, but not by the NK1 receptor-selective antagonist L703606 [8]. Thus, these findings support the view that CGRP stimulates the release of neuronal TKs, which in turn enhances 5-HT release via the endogenous NK2/NK3 receptor cascade system. Thus, it appears that CGRP-sensitive TKs-releasing neurons play a role in activating the endogenous NK2/NK3 receptor cascade system. This conjecture is also supported by a previous report which showed that CGRP can stimulate intramural tachykinergic neurons in the guinea-pig colon [17]. Furthermore, the evidence for the elevation of the levels of endogenous CGRP and TKs in human diseased colonic mucosa has also been demonstrated [18, 19]; irritants, immunological and inflammatory mediators can release TKs and CGRP within the intestinal wall where these peptides may facilitate the release of 5-HT via the endogenous NK2/NK3 receptor cascade system. Thus, these findings suggest a possible role of the endogenous NK2/NK3 receptor cascade system controlling the release of 5-HT from EC cells in pathophysiological conditions that are associated with an upregulation of CGRP or TKs release.

NK2 RECEPTOR-TRIGGERED 5-HT RELEASE IS MEDIATED BY ENDOGENOUS PYY

The 36 amino-acid long peptide, peptide YY (PYY) is known as a major peptide hormone, which regulates colonic functions (motility and secretion) and appetite by acting via different neuropeptide Y receptor subtypes [20, 21]. In the colon, PYY is mainly synthesized and released into the blood from mucosal L cells. The blood PYY levels have been shown to increase rapidly in response to food ingestion, and the postprandial blood PYY levels remain elevated for several hours [21]. Abnormal levels of PYY in large intestinal endocrine cells have also been reported in patients with IBS [6, 22], indicating a possible role of PYY in the generation of the symptoms associated with IBS.

The NK2 receptor agonist, βAla-NKA-(4-10) (10-100 nM) also evoked a long-lasting and TTX-resistant PYY release from isolated guinea-pig colonic mucosa [11], thus suggesting that activation of NK2 receptors on the mucosal layer induces a long-lasting PYY release from L cells. As shown in Fig. , pretreatment with the neuropeptide Y1 receptor-selective antagonist BIBO3304 attenuated the βAla-NKA-(4-10)-evoked 5-HT release [11]. These findings support the hypothesis that activation of NK2 receptors on PYY-containing L cells provokes the release of PYY, which in turn activates mainly Y1 receptors located on EC cells to induce 5-HT release (Fig. ). This hypothesis is further corroborated by findings that exogenously applied PYY (10 nM) evoked a long-lasting and BIBO3304-sensitive 5-HT release with a time course similar to that found for the NK2 agonist-evoked 5-HT release [11]. Overall, the above observations indicate that endogenously released PYY play a fundamental role in the NK2 receptor-triggered 5-HT release from EC cells; PYY-containing L cells appears to control 5-HT release from EC cells (Fig. ). Given that food intake is a major and physiologically significant factor stimulating the release of PYY from mucosal L cells [21] and both PYY and 5-HT are well-known mediators of nausea and vomiting [23], it is hypothesized that the cascade of NK2 receptors on PYY-containing L cells and Y1 receptors on EC cells (Fig. ) might play a role in the generation of postprandial symptoms (nausea and vomiting) in patients with IBS; in fact, a postprandial increase in plasma 5-HT levels have been shown to be related to the generation of postprandial symptoms in diarrhea-predominant IBS [24, 25]. Therefore, the NK2 receptor-triggered PYY release from colonic mucosa may be expected as a pharmacological target for postprandial symptoms in functional bowel disorders including IBS. Activation of tachykinin NK3 receptors on enteric cholinergic neurons has also been shown to provoke the release of PYY from colonic mucosa [26]. Thus, to elucidate whether the endogenous tachykinergic NK2/NK3 receptor cascade system participates in the mechanism for food intake-induced PYY release, is an interesting topic for future study.

INHIBITION OF NK2 RECEPTOR-TRIGGERED 5-HT RELEASE BY MELATONIN

The 5-HT-derived neurohormone, melatonin (MT) is mainly synthesized in the pineal gland as well as in 5-HT-rich EC cells of the intestinal mucosa [27]. It has been shown that exogenous MT modulates colonic motility and pain in IBS patients [28, 29]; therefore, MT is considered as a possible therapeutic agent for IBS [29, 30]. In the isolated guinea-pig colonic mucosa, MT has been shown to inhibit the NK2 receptor-triggered 5-HT release by acting through MT3 melatonin receptors located on the mucosal layer, thus suggesting a possible role of MT as a negative-regulator for the NK2 receptor-triggered 5-HT release from EC cells [10] (Fig. ). It is not yet known whether or not endogenous MT participates in the pathogenesis of IBS, but oral MT has been shown to reduce abdominal pain and the sensation of bowel urgency in IBS patients [29]. It is therefore to be expected that if low MT concentrations in the colonic mucosa facilitate 5-HT release from EC cells, the enhanced 5-HT release leads to abdominal pain in IBS patients. In fact, it has been shown that IBS patients have lower MT levels compared with healthy controls [31]. Thus, to elucidate whether the endogenous NK2/NK3 receptor cascade system participates in the modulatory effect of MT on colonic motility or pain, is an interesting topic for future study.

CONCLUDING REMARKS

The present review shows that the cascade pathway of NK3 receptors on cholinergic interneurons and NK2 receptors on PYY-containing endocrine L cells represents an endogenous modulator system for 5-HT release from colonic EC cells, and that melatonin, endogenous TKs and PYY play important roles in the modulation of the release of 5-HT from EC cells via the endogenous NK2/NK3 receptor cascade system (Fig. ). Given that TKs, 5-HT-containing EC cells, PYY-containing L cells and melatonin play important roles in the pathophysiological regulation of colonic functions in the diseased gut including IBS, the potential role of the endogenous NK2/NK3 receptor cascade system controlling the release of 5-HT from EC cells, will undoubtedly be a focus of future investigations.