Regulation of serotonin release from the intestinal mucosa

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REGULATION OF SEROTONIN RELEASE FROM THE INTESTINAL MUCOSA KURT RACKt~ and HARALD SCHWORER*

Department of Pharmacology, Universityof Mainz and *Division of Gastroenterology, Department of Medicine, Universityof GOttingen, Germany Received in final form 18 May 1990

SUMMARY

In the mammalian intestine serotonin (5-hydroxytryptamine, 5-HT) is present in high concentrations in the enterochromaffin cells. The release of 5-HT from the intestinal mucosa is regulated by a complex pattern of neuronal and humoral inputs to the enterochromaffin cells. The enterochromaffin cells appear to be endowed with different inhibitory (az-adrenoceptors, GABAA- and GABAB-receptors, histamine H3-receptors, receptors for vasoactive intestinal polypeptide and somatostatin) as well as stimulatory receptors (fl-adrenoceptors, muscarine and nicotine receptors). The physiological significance of this complex system of receptors is suggested by experiments which demonstrate that the respective intrinsic neurotransmitters (catecholamines, acetylcholine, GABA and vasoactive intestinal polypeptide) released within the gut are involved in the regulation of the release of 5-HT from the enterochromaffin cells. KEYWORDS:enterochromaffin cells, serotonin release, acetylcholine, catecholamines, GABA. ABBREVIATIONS: DMPP, 1,1-dimethyl-4-phenylpiperazinium; ECs, enterochrornaffin cells; GABA, y-aminobutyric acid; 5-HT, 5-hydroxytryptamine, serotonin; 5-HIAA, 5hydroxyindoleacetic acid; TTX, tetrodotoxin; VIP, vasoactive intestinal polypeptide.

INTRODUCTION

5-Hydroxytryptamine (5-HT, serotonin) is present in high concentrations in the gastrointestinal tract and here predominantly in the enterochromaffin cells (ECs) [1]. ECs are dispersed all over the gastrointestinal mucosa and can release 5-HT into the lumen as well as into the portal circulation. In addition, there is evidence, that also serotonergic neurones exist in the intestinal tract [see 2, 3]. However, the amounts of 5-HT present in enteric neurones appear as traces in comparison to the amounts of 5-HT present in ECs. Thus, the concentrations of 5-HT in and the amounts of 5-HT released from intestinal preparations with intact mucosa exceeded about 100-fold the respective values observed in mucosa-free muscle preparations in which, however, the serotonergic neurones are present [4, see also 2, 3, 5]. In rats and mice substantial amounts of 5-HT are also present in mast cells 1043-6618/91/010013-13/S03.00/0

© 1991 The Italian PharmacologicalSociety

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[1]. Therefore, in these species the origin of 5-HT released in the intestine may be more heterogenous than in other mammals. The aim of the present review is to summarize recent results, particularly those obtained in pharmacological studies, which allow the description of a very complex pattern of mechanisms involved in the regulation of 5-HT release from the intestinal mucosa, i.e. from the ECs. The most detailed picture can be drawn for the regulatory mechanisms in the guinea-pig intestine (Fig. 1). However, as far as observations are available from other species it appears that similar regulatory mechanisms may be of importance in all mammals (Table I).

Endogenous

Bz-,igond// \

~

~

" ~ . ~ , " / / Endogenous

Fig. 1. Schematicsynopsis of the complex mechanisms described for the regulation of 5-HT release from enterochromaffin ceils of the guinea-pig small intestine. ~, release; --', stimulation; ~,inhibition. AC, adenylate cyclase; ACh, acetylcholine; Bz, benzodiazepine receptor; GA, GB, GABAA- and GABAB-receptors; a2 and/31, a2- and/31-adrenoceptors; N, M, M1 and M2, nicotine and muscarine receptors; HT3, 5-HT3-receptor; CP, cisplatin; Op, opioid receptor; VIP, receptor for vasoactive intestinal polypeptide; X and XR, inhibitory transmitters and their receptors (partially identified as VIP). For further details and explanations see text. GENERAL CHARACTERIZATION OF 5-HT RELEASE General mechanisms of the synthesis, storage and release of 5-HT in peripheral tissues, including the intestine, have recently been reviewed [6]. However, some aspects concerning the ECs should be discussed in more detail. 5-HT which can be synthesized within the ECs [7, 8] is stored predominantly in large, electron dense secretory granules [9-1 3]. Most of the granules appear to be located near the base

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Table I Outline of the different pharmacological receptor systems involved in the regulation of 5-HT release from enterochromaffin cells Receptor

Effect on 5-HT release

Direct effects on enterochromaffin cells* Nicotine Stimulation Stimulation Muscarine fl-Adrenoceptors a2-Adrenoceptors GABAA GABAB Benzodiazepine Vasoactive intestinal polypeptide (VIP) Somatostatint Histamine-H3 Indirect, neuronally mediated effects Nicotine Muscarine (M~) GABAA Opioidt

Species

Stimulation Inhibition Inhibition Inhibition Inhibition Inhibition

GP, rabbit, pig GP, rabbit, cat, dog, pig GP, rabbit, rat, cat GP GP GP GP GP

Inhibition Inhibition

Rabbit, human~ Pig

Stimulation Inhibition Stimulation Stimulation

Rabbit, dog, GP, cat GP, rabbit GP GP, dog

*Localization of the effects based essentially on experiments which excluded a neuronal mediation, a local paracrine mediation cannot be excluded at present. For details and references see text. GP, guinea-pig. tGood evidence for a direct or indirect mechanism lacking. ~Suggested by the effectiveness in carcinoid syndrome. of the ECs [12] suggesting that 5-HT release across the basolateral membrane (finally appearing in the portal circulation) may be more important. Like the release of catecholamines from chromaffin cells of the adrenal medulla [see 14], the secretion of 5-HT from ECs may also occur via exocytosis. In support of this assumption is the observation that the release of 5-HT, like any exocytotic release, is largely calcium-dependent. Removal of extracellular calcium largely reduced the release of 5-HT from rabbit [15], guinea-pig [4] and pig [16] intestinal preparations, whereas calcium ionophores promoted the release of 5-HT [15]. Experiments which showed that calcium channel blockers can inhibit the release of 5-HT suggest that an influx of calcium into the ECs is a crucial event in the stimulus secretion coupling of the release of 5-HT [15]. Moreover, differential effects of the dihydropyridine nifedipine (blocks selectively L-type calcium channels [see 17]) and of w-conotoxin-GIVA (blocks N-type and some L-type calcium channels [see 17]) on the spontaneous and high potassium evoked release of 5-HT suggest that multiple calcium channels may be involved in the release of 5-HT from ECs [16]. Although direct electrophysiological recordings have not yet been carried out on ECs, experiments in which the release of 5-HT was studied in the presence of

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tetrodotoxin (TTX)--known to block voltage-dependent sodium channels [18]support the idea that fast action potentials carried by TTX-sensitive sodium channels may not be of particular importance. TTX appears to block all the neuronal inputs to the ECs [4, 19-24], but it does not prevent the secretion of 5-HT from ECs nor its modulation by various facilitatory and inhibitory stimuli (see also below). A possible role of voltage-dependent calcium channels on ECs is suggested by the observation that depolarizing concentrations of potassium enhanced the release of 5-HT from the porcine small intestine, and that this was effectively blocked by the calcium channel blockers gadolinium and nifedipine [16].

METABOLISM OF RELEASED 5-HT

5-HT released from the ECs is rapidly metabolized to 5-HIAA already within the intestinal wall. The ratio 5-HIAA/5-HT in the interstitial fluid of the gastric wall determined in vivo [25] or in the portal effluent of the guinea-pig small intestine perfused in vitro varied between about 0.9 and 1.3 (4, 19-24, 26] and was even considerably higher (about 5) in media of incubated porcine intestinal segments [16]. In contrast, the ratio 5-HIAA/5-HT in the tissue was between 0.01 and 0.02 [4, 25] indicating that the acid metabolite is rapidly washed out of the intestine. 5-HIAA formed within the intestine appears to derive almost exclusively from released 5-HT which had again been sequestered by a high affinity uptake mechanism. This is suggested by the effects of imipramine which reduced the portal outflow of 5-HIAA, an effect accompanied by a corresponding increase in the outflow of 5-HT [4]. A high affinity uptake for 5-HT into mucosal cells (presumably ECs) has been observed in intestinal preparations of rats [8] and rabbits [27, 28], but it is also associated with aminergic neurones in the intestine [29].

CHOLINERGIC MODULATION Acetylcholine has been well established as a neurotransmitter in the intestinal tract [see 2, 30] and a cholinergic modulation of 5-HT release from the ECs has been well documented. Both nicotine as well as muscarine receptors appear to be present on ECs and the activation of either of them can mediate a stimulatory effect on the release of 5-HT. However, the contribution of either nicotine or muscarine receptors to the cholinergic modulation of 5-HT release appears to vary between different species and the different parts of the intestinal tract (see below). Acetylcholine has been shown to increase the release of 5-HT into the portal circulation of the dog [31, 32] and into the gastric interstitial fluid in rabbits [25, 33]. The stimulatory effect of acetylcholine in the dog intestine was blocked by atropine [31, 32], indicating the involvement of muscarine receptors, whereas the effect of acetylcholine in the rabbit stomach was only antagonized by hexamethonium, suggesting a dominant role of nicotine receptors. However, muscarine receptor agonists, but not nicotine receptor agonists, stimulated the

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release of 5-HT from the in vitro incubated rabbit duodenum, jejunum and ileum [15, 34, 35]. 5-HT release from the vascularly perfused guinea-pig small intestine into the portal circulation was stimulated by nicotine as well as muscarine receptor agonists and these effects could be localized on the ECs, or at least very near to the ECs, since an indirect, neuronal mediation could be excluded [19, 21]. Moreover, the stimulatory effect of muscarine receptor agonists in these experiments was only seen when the neuronal input to the ECs was blocked by TTX. The facilitation of 5-HT release induced by muscarine receptor activation was about 2-3 times greater than that caused by nicotine receptor activation. In the absence of TTX, muscarine receptor agonists inhibited the release of 5-HT [19, 21]. Therefore, it was concluded that activation of neuronally located muscarine receptors can induce the release of neurotransmitters which are inhibitory to the ECs (see Fig. 1 ). It could be excluded that 7-aminobutyric acid (GABA), which is an important, predominantly inhibitory transmitter in the intestine (see 'GABAergic modulation'), is involved in the above described, neuronally mediated muscarinic inhibition of 5-HT release [23]. However, there is evidence that vasoactive intestinal polypeptide (VIP) is involved in this indirect muscarinic inhibition (see 'Peptidergic modulation'). Experiments with subtype selective muscarine receptor agonlsts and antagonists suggest that Ml-receptors are involved in the indirect, neuronally mediated inhibition of 5-HT release, whereas the muscarine receptors located on ECs had neither typical characteristics of MI-, M 2- n o r M3-receptors [21 ]. Recent experiments on in vitro incubated porcine small intestinal preparations show that in this species 5-HT release is also stimulated by nicotine and muscarine receptor agonists, but differentially to the observations in guinea-pigs, the release of 5-HT induced by the nicotine receptor agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP, in the presence of TTX) was about 3-4 times greater (increase by about 130%) than that caused by the muscarine receptor agonist oxotremorine [16, and unpublished observations]. Only a few comments can be given about mechanisms possibly involved in the cholinergic facilitation of 5-HT release from the ECs. Since both nicotinic and muscarinic stimulatory effects were observed in the presence of TTX [19, 21, 35], an involvement of fast, sodium-dependent, 'neuronal-type' action potentials can be excluded. The muscarinic facilitation of 5-HT release from the ECs was, however, inhibited by several inorganic calcium channel blockers [15], suggesting that muscarine receptor activation may enhance calcium influx through specific, possibly receptor linked channels.

ADRENERGIC MODULATION AND THE ROLE OF cAMP

The intestinal mucosa is innervated by adrenergic nerve fibres which are often in close proximity to the ECs [36]. First evidence for an adrenergic modulation of 5-HT release has been presented by Burks and Long [37] in the dog vascularly perfused intestine. They showed that catecholamines and periarterial nerve stimulation increased the release of 5-HT into the portal circulation. Pettersson et al. [38] observed a decrease in mucosal 5-HT fluorescence in the rat duodenum

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after exposure to different catecholamines, an effect blocked by propranolol, suggesting that fl-adrenoceptor activation caused the release of 5-HT from the ECs. Activation of fl-adrenoceptors increased also the release of 5-HT from the guinea-pig [20] and rabbit [39] intestine. Since the stimulatory effects of isoprenaline were also observed after blockade of the neuronal input (by TTX), the fl-adrenoceptors may be localized directly at the ECs [20, 39]. Finally, these fladrenoceptors may belong to the ill-subtype as the selective fll-adrenoceptor antagonist CGP 20712A blocked the effect of isoprenaline (Schw6rer, Kilbinger and Rack6, unpublished). Interestingly, in cats propranolol partially blocked the stimulatory effect of vagal nerve stimulation on 5-HT release, suggesting that an enhanced release of noradrenaline (shown to be present in abdominal vagal fibres, [40]), may be involved in this response [41]. fl-Adrenoceptors are often linked to the adenylate cyclase [see 42]. Elevation of the intracellular cAMP levels, either by forskolin which can directly stimulate the adenylate cyclase or by the phosphodiesterase inhibitor A H 21-132, also enhanced the release of 5-HT from the guinea-pig intestine [20]. An increase in 5-HT release by forskolin and a phosphodiesterase inhibitor (3-isobutyl-l-methylxanthine) was also observed in the rabbit duodenum [15]. That an increase in intracellular cAMP facilitates 5-HT release from the ECs is finally suggested by stimulatory effects of 8-bromo-cAMP, a stable analogue of cAMP [15]. Furthermore, the observation that a marked potentiation of the effects of isoprenaline and AH 21-132 occurred, when both drugs were simultaneously present in low concentrations [20], indicates that the stimulatory effect of fl-adrenoceptors on ECs involves an activation of adenylate cyclase. Functional studies using 5-HT receptor antagonists suggest that an enhanced 5-HT secretion is involved in the cholera toxin induced fluid accumulation and diarrhoea [43]. Cholera toxin has been shown to decrease 5-HT levels in the ECs of the feline jejunum, suggesting a stimulatory effect on 5-HT release [44]. Since cholera toxin is known to cause a long lasting activation of adenylate cyclase [45, 46], these observations agree well with the above proposed role of adenylate cyclase in the regulation of 5-HT release from the ECs. ECs are also endowed with a-adrenoceptors which mediate an inhibition of 5-HT release. The a2-adrenoceptor agonist clonidine inhibited the release of 5-HT from the guinea-pig intestine [20]. This effect was prevented by the a 2adrenoceptor antagonist tolazoline, but not by the al-adrenoceptor antagonist prazosin [20], demonstrating that a2-adrenoceptors mediated this effect. These aadrenoceptors, like the fl-adrenoceptors, appear to be located directly on the ECs (or at least very near to the ECs), since the inhibitory effect of clonidine was also observed in the presence of TTX [20]. a2-Adrenoceptors are often linked negatively to the adenylate cyclase [see 42] and %-adrenoceptor agonists inhibited the cholera toxin induced intestinal fluid accumulation [47]. Therefore, it may be speculated that in ECs, as in many other systems, a2-adrenoceptors may inhibit the formation of cAMP and thereby reduce 5-HT release, although direct biochemical evidence in support of this idea is lacking. Since an enhanced release of 5-HT may be involved in the pathogenesis of diarrhoea, as shown for the diarrhoea caused by cholera toxin [43] and carcinoid syndrome [48], an inhibitory effect on 5-HT release may contribute to the antidiarrhoeal properties of a2-adrenoceptor agonists [49].

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PEPTIDERGIC MODULATION In neurones as well as in paracrine cells of the gut, numerous peptides are present [see 2, 9, 30], which are thought to be of great significance for the regulation of many intestinal functions. Nevertheless, there is little information about a putative peptidergic modulation of 5-HT release. In the rabbit duodenum somatostatin inhibited the release of 5-HT evoked either by muscarine receptor activation or by enhanced intracellular cAMP [15]. Inhibitory effects on ECs may also be responsible for the therapeutic effects of somatostatin and particularly of the long-acting somatostatin analogue octreotide in patients with carcinoid syndrome [see 50, 51 ]. The release of 5-HT from the guinea-pig intestine is also inhibited by VIP which acts in concentrations as low as 1 p~ [22]. Evidence that endogenous, neuronally released VIP might be involved in the modulation of 5-HT release was obtained in experiments in which the effects of an antibody directed against VIP (G-143, [52]) were studied. This antibody did not affect the spontaneous outflow of 5-HT from the guinea-pig intestine, but abolished the indirect, neuronally mediated inhibition of 5-HT release caused by muscarine receptor agonists [53]. Furthermore, there is some evidence that endogenous opioids also participate in the regulation of 5-HT release from the ECs. In the presence of the opioid receptor antagonist naloxone the stimulatory effect of enhanced intraluminal pressure on 5-HT release from the guinea-pig small intestine (see 'Physicochemical stimuli') was converted into an inhibition [26]. In agreement to these observations, Burks and Long [54] observed a stimulation of 5-HT release from the perfused dog intestine by morphine and related compounds. Although a direct stimulatory action of opioids on the ECs cannot be excluded at present, the general inhibitory properties of opioids on transmitter release suggest that the facilitation of 5-HT release by opioids may be the result of the suppression of inhibitory transmitters [26].

GABAERGIC MODULATION There is good evidence that GABA is a neurotransmitter in the intestinal tract [see 55, 56], but in addition there is some evidence that a non-neuronal, paracrine GABAergic system may also be of functional significance in the gastrointestinal tract [57, 58]. In the guinea-pig intestine, both direct inhibitory and indirect stimulatory GABAergic effects appear to be involved in the regulation of 5-HT release from the ECs (see Fig. 1) [23, 24, 59]. GABAA-receptor activation can induce the release of neuronal acetylcholine [60, 61] which then can stimulate the release of 5-HT from the ECs (see above). Thus, the GABAa-receptor agonist muscimol had concentration- and time-dependently a biphasic effect on the release of 5-HT. At a low concentration (1 ktM) muscimol caused a transient increase in 5-HT release which was followed by an inhibitory response. In the presence of TTX or hyoscine (scopolamine) the stimulatory effect of muscimol was prevented and converted into an inhibitory response. This shows that the GABAergic stimulation of 5-HT release was mediated by the neuronal release of acetylcholine and that in addition, inhibitory GABA A-receptors may be present on the ECs (or

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at least very near to the ECs). At a higher concentration (10ktM) the direct inhibitory effect of muscimol was dominant. Experiments with the GABA Areceptor antagonist bicuculline suggest that both the indirect GABAergic facilitation and the direct GABAergic inhibition of 5-HT release may be activated by endogenous GABA which appears to derive, at least in part, from a nonneuronal pool. This latter conclusion is supported by the observation that bicuculline affected the release of 5-HT also in the presence of TTX, i.e. after blockade of propagated neuronal activity. Therefore, these experiments support the above-mentioned concept of a non-neuronal GABAergic system in the intestine. In addition, the ECs may also be endowed with inhibitory GABAB-receptors, since baclofen reduced the release of 5-HT in a stereospecific manner, also in the presence of TTX [23]. Generally, GABAA-receptors are regulated by benzodiazepine receptors [see 62, 63]. Experiments in which the effects of the benzodiazepine receptor agonist midazolam were studied showed that both the above described indirect, neuronally mediated GABAergic facilitation and the direct GABAergic inhibition of 5-HT release are augmented by benzodiazepine receptors [24, 59]. Most interestingly, the benzodiazepine receptor antagonist flumazenil (Ro 15-1788) affected the release of 5-HT in a manner opposite to the effects caused by midazolam [24, 59]. Since flumazenil has been characterized as a benzodiazepine receptor antagonist without inverse agonisfic properties [for references see 24], it was concluded that functionally significant amounts of endogenous ligands for the benzodiazepine receptor may be present in the guinea-pig intestine.

HISTAMINERGIC MODULATION Histamine is also distributed all over the intestinal tract, particularly present in mast cells. In addition, there is evidence that, at least in the gastric mucosa, histamine is also stored in EC-like cells [64]. Histamine has been shown to inhibit the release of 5-HT from the porcine intestine. This effect was almost completely blocked by the H3-receptor antagonist thioperamide, whereas cimetidine (H 2receptor antagonist) only slightly attenuated the effect of histamine. Mepyramine (Hi-receptor antagonist) did not affect the inhibitory action of histamine [65]. The involvement of H3-receptors is further suggested by the observation that the selective H3-receptor agonist (R)a-methylhistamine was about 10 times more potent than histamine [16]. Finally, the inhibitory effect of (R)a-methylhistamine was also observed in the presence of TTX, suggesting that the H3-receptors may be located directly at (or very near to) the ECs [16].

EFFECTS OF CISPLATIN AND ROLE OF 5-HT3-RECEPTORS Cisplatin is a cytotoxic agent which is used in antineoplastic chemotherapy. However, the application of cisplatin is associated with severe emesis which often limited its therapeutic use. Recently, it was observed that 5-HT3-receptor antagonists can prevent the cisplatin-induced emesis, suggesting that the release of 5-HT might be involved [see 66, 67]. Since cisplatin altered the mucosal levels of

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5-HT and 5-HIAA [68] and vagotomy prevented the cisplatin-induced vomiting in ferrets [69], an effect of cisplatin on the release of 5-HT from the ECs appeared to be possible. Indeed, cisplatin stimulated the release of 5-HT from the guinea-pig intestine, an effect prevented by TTX [70]. Hexamethonium and hyoscine (scopolamine) also inhibited the zisplatin evoked 5-HT release. Most surprisingly, the 5-HT3-receptor antagonist ondansetron (GR 38032F), in picomolar concentrations, also prevented the stimulatory effect of cisplatin [70]. Further experiments with additional 5-HT3-receptor antagonists (ICS 205-930 and MDL 72222) confirm the role of 5-HT3-receptors for the cisplatin-induced release of 5-HT from the intestinal mucosa (Schw6rer, Rack6, Kilbinger, unpublished). These observations all together suggest that the release of 5-HT from the ECs is regulated by a 'positive feedback loop' which involves cholinergic interneurones. Excitatory 5-HT3-receptors appear to enhance the ganglionic activation of these cholinergic neurones. Cisplatin, by a so far unknown mechanism, appears to augment this 'positive feedback loop', possibly by sensitizing excitatory 5-HT3-receptors at preganglionic parasympathetic nerve endings (see Fig. 1 ). PHYSICOCHEMICAL STIMULI

Lowering the intraluminal pH can stimulate the release of 5-HT from the ECs [71-73]. Intraluminal instillation of hypertonic glucose or sucrose solutions can also enhance the release of 5-HT [74, 75]. Increasing the intraluminal pressure has also been shown to stimulate the release of 5-HT from the ECs into the lumen as well as into the portal circulation [22, 26, 76]. This facilitation of 5-HT release by the enhanced intraluminal pressure appears to be mediated indirectly via the release of stimulatory neurotransmitters, since in the presence of TTX, elevation of the intraluminal pressure decreased the release of 5-HT into the portal circulation [22].

CONCLUDING REMARKS

The studies summarized in the present review show that ECs are endowed with numerous, functionally important receptor systems. This very differentiated receptive system appears to enable the ECs to integrate a very complex pattern of neuronal and humoral modulatory inputs. Although the present review did not deal with functional aspects of 5-HT in the intestine, it should be emphasized that the general distribution of ECs all over the intestinal tract together with the ability of these cells to respond to the various demands underlines the important role that ECs play in the regulation of intestinal functions. ACKNOWLEDGEMENTS

The authors' own work was supported by the Deutsche Forschungsgemeinschaft and the Forschungsrat Rauchen und Gesundheit. We would like to thank Prof. H. Kilbinger for his support and Ms B. Hering for the drawing of the scheme.

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