tachykinins in the gut. part i. expression, release and ... · and nka interact with other enteric...

Pha-01. Ther. Vol. 73, No. 3, pp. 173-217, 1997 Copyright0 1997 Elsevier Sciencelnc.

ISSN 0163s7258197 $32.00 c

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Associate Editor: 1. Wessh

Tachykinins in the Gut. Part I. Expression, Release and Motor Function

Peter Holrer* ad Ulrike Holzer-Pets&e DEPARTMENTOFEXPERIMENTALANDCLINICALPHARMACOLOGY, UNlVERSlTYOFGRAZ,UNIVERSITiiTSPLATZ4.A-8010GRAZ,AUSTRlA

ABSTRACT. The preprotachykinm-A gene-derived peptides substance P and neurokinin (NK) A are expressed in distinct neural pathways of the mammalian gut. When released from intrinsic enteric or extrinsic primary afferent neurons, tachykinms have the potential to influence both nerve and muscle by way of interaction with three different types of tachykinin receptor, termed NK,, NK, and NK, receptors. Most prominent among the effects of tachykinins is their excitatory action on gastrointestinal motor activity, which is seen in virtually all regions and layers of the mammalian gut. This action depends not only on a direct activation of the muscle through NK, and/or NK, receptors, but also on stimulation of excitatory enteric motor pathways through NK, and/or NK, receptors. In addition, tachykinins can inhibit motor activity by stimulating either inhibitory neuronal pathways or interrupting excitatory relays. A synopsis of the available data indicates that endogenous substance P and NKA interact with other enteric transmitters in the physiological control of gastrointestinal motor activity. Derangement of the regulatory roles of tachykinins may be a factor in the gastrointestinal dysmotility associated with infection, inflammation, stress and pain. In a therapeutic perspective, it would seem conceivable, therefore, that tachykinin agonists and antagonists are adjuncts to the treatment of motor disorders that involve pathological disturbances of the gastrointestinal tachykinin system. PHARMACOL. THER. 73(3):173-217, 1997. @ 1997 Elsevier Science Inc.

KEY WORDS. Substance P. neurokinin A. enteric nervous system, intestinal motility, peristalsis, intestinal motor disorders.

CONTENTS

1. INTRODUCTION. . . . . . . . . . . . . . .174 2. OCCURRENCE OFTACHYKININSIN

THEENTERICNERVEPLEXUSESAND MUSCLELAYERS. . . . . . . . . . . . . . .174 2.1. OVERVIEW. . . . . . . . . . . . . . .174 2.2. IDENTITYANDMOLECULAR

BIOLOGYOFTACHYKININSIN THEMAMMALIANGUT . . . . . . . .175

2.3. REGIONALANDSPECIESDIFFERENCES 1NTHETACHYKININDISTRIBUTION INTHEGUT..............176

2.4. TACHYKININS IN THEENTERIC NERVEPLEXUSESAND MUSCLELAYERS. . . . . . . . . . . .176 2.4.1. INTRINSICENTERIC

NEURONS. . . . . . . . . ...176 2.4.2. EXTRINSIC PRIMARY

AFFERENTNEURONS. . . . . .178 2.5. TACHYKININSINTHE

BILIARYTRACT . . . . . . . . . . . .179 3. RELEASEOFTACH~KININSIN

THEGUT . . . . . . . . . . . . . . . ...179 3.1. OVERVIEW. . . . . . . . . . . . . . .179 3.2. RELEASEOFTACHYKININSINWTRO.~~~ 3.3. RELEASEOFTACHM(ININSINVIVO. .180

4. TACHYKININPHARMACOLOGY. . . . . . .181 4.1. OVERVIEW...............~~~ 4.2. TACHYKININRECEPTORAGONISTS. .181 4.3. TACHYKININRECEPTOR

ANTAGONISTS. . . . . . . . . . . . .181

4.4. MOLECULARTACHYKININRECEPTOR PHARMACOLOGY . . . . . . . . . . .183

4.5. TRANSDUCTIONMECHANISMS. . . .183

*Corresponding author.

4.6. FACTORSDETERMININGTHE ACTIVITYOFTACHYKININSAT THEIRRECEPTORS. . . . . . . . . . .184 4.6.1. DEGRADINGENZYMES. . . . .184 4.6.2. RECEPTORDESENSITIZATION

ANDINTERNALIZATION. . . .184 5. TACHYKININSANDGASTROINTESTINAL

MOTILITY . . . . . . . . . . . . . . . . . .184 5.1. OVERVIEW...............~~~ 5.2. EXCITATORYMOTOREFFECTSOF

TACHYKININSINTHEGUT . . . . . .185 5.2.1. ESOPHAGUS . . . . . . . . . .185 5.2.2. STOMACH . . . . . . . . . . .186 5.2.3. 8MALLINTBSTINE . . . . . . .187 5.2.4. LARGEINTESTINE . . . . . . .190

5.3. EXCITATORYMOTOREFFECTS OFTACHYKININSINTHE BILIARYTRACT . . . . . . . . . . . .192

5.4. INHIBITORYM~T~REFFECTSOF TACHYKININSINTHEGUT . . . . . .192 5.4.1.

5.4.2.

5.4.3.

5.4.4.

INHIBITORYEFFECTSDUETO MECHANISMSTHATHAVENOT BEENFULLYIDENTIFIED. . . .192 ~NHIBITORYEFFECTSDUETO PREJUNCTIONALINHIBITION OFTRANSMITTERRELEASE . .193 ~NHIBITORYEFFBCTSDLJETO STIMULATION OFINHIBITORY ENTERICNEURONS. . . . . . .193 INHIBITORYEFFECTSDUETO STIMULATION OF SYMPATHETICNEURONS. . . .193

174 P. Holzer and U. Holzer-Petsche

5.5. PHYSIOLOGICAL ROLES OF 5.6. TACHYKININS IN GASTROINTESTINAL MOTOR ACTIVITY . . . . . . . . . . .194 5.5.1. ROLES OF TACHYXININS

RELEASED FROM ENTERIC NEURONS. . . . . . . . . . . . 194

5.5.2. ROLES OF TACHYXININS

PATHOPHYSIOLOGICAL IMPLICATIONS OF TACHYKININS IN GASTROINTESTINAL MOTILITY 5.6.1. PATHOLOGICAL CHANGES

IN TACHYKININ-MEDIATED MOTOR CONTROL . . . . .

5.6.2. THERAPEUTIC PROSPECTS RELEASED FROM EXTRINSIC ACKNOWLEDGEMENTS ........... AFFERENT NERVE FIBERS ... 199 REFERENCES ................

. .201

. .201

. .202

. .203

. .203

ABBREVIATIONS. ACE, angiotensin-converting enzyme; ACh, acetylcholine; CCK, cholecystokinin; CGRP, calcitonin gene-related peptide; ChAT, choline acetyltransferase; EFS, electrical field stimulation; EJP, excitatory junction potential; EPSP, excitatory postsynaptic potential; GABA, y-aminobutyric acid; 5HT, 5hydroxytryptamine; 5HTR, 5HT receptor; LES, lower esophageal sphincter; LT, leukotriene; MP, myenteric plexus; NA, noradrenaline; NANC, nonadrenergic noncholinergic; NEP, neutral endopeptidase; NK, neurokinin; NO, nitric oxide; NPy, neuropeptide y; NPK, neuropeptide K; PPT, preprotachykinin; SMP, submucosal plexus; SNS, sympathetic nerve stimulation; SP, substance P; TTX, tetrodotoxin; VIP, vasoactive intestinal polypeptide; VNS, vagal nerve stimulation.

1. INTRODUCTION

Ever since substance P (SP) was discovered to occur in the intestine and to contract gastrointestinal smooth muscle

(von Euler and Gaddum, 193 l), the gut has been a model system in which to study the presence, actions, physiological and pathophysiological implications of SP and related

peptides. SP is the most widely known representative of a family of small biologically active peptides, the tachykinins

(Erspamer, 1981), which consists of a large number of nonmammalian and mammalian members. The subject of this

review is the group of mammalian tachykinins that, besides SP, comprises neurokinin (NK) A and NKB as the princi-

pal members (Table 1). Being considered of potential im-

portance for the understanding and therapy of human disease, the tachykinins have become one of the best studied groups of neuropeptides, with detailed information being

available on the molecular biology of peptide (Nakanishi, 1987) and receptor (Nakanishi, 1991) expression. Con- comitantly, exceptional progress has been made in tachyki-

nin pharmacology, culminating in the discovery and development of selective agonists and selective nonpeptide

antagonists for all three types of tachykinin receptor,

termed NKr, NK, and NK, receptors (Guard and Watson,

1991; Maggi et al., 1993~; Regoli et al., 1994; Maggi, 1995).

Within the gastrointestinal tract, tachykinins are important messenger molecules of enteric neurons that are intrinsic to

the gut and that control various aspects of digestive activity. Regulation of motor activity by tachykinins is an area that has been studied most intensively ever since SP was discov-

ered to occur in the gut (Barth6 and Holzer, 1985; Maggi et al., 1993~; Otsuka and Yoshioka, 1993; Dockray, 1994; Holzer- Petsche, 1995; Shuttleworth and Keef, 1995), and it is with

this area of gastrointestinal pharmacology that the present review is concerned. By integrating the wealth of information

that has become available through the use of molecular bio-

logical, immunocytochemical, physiological and pharmacological techniques, it is sought to define the physiological

and pathophysiological implications of tachykinins in gut motility in a comprehensive manner and to point out therapeutic perspectives for gastrointestinal motility disorders.

2. OCCURRENCE OF TACHYKININS IN THE ENTERIC NERVE PLEXUSES AND MUSCLE LAYERS 2.1. Overview

The mammalian gut contains both SP and NKA, and various N-terminal-extended forms of these tachykinins, which

all are derived from alternative splicing of the primary tran-

TABLE 1. Amino acid sequence and tachykinin receptor preference of mammalian and some nonmammalian tachykinins

Tachykinin Amino acid sequence

Tachykinin

receptor preference

SP NKA NPK

WY)

NKB Physalaem Eledoisin Kassanin

Iin

H-Arg-Pro-Lys-Pro-G1n-Gln-Phe-Phe-Gly-Leu-Met.NH2 H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met.NHr

H-Asp-Ala-Asp-Ser-Ser-Ile-Glu-Lys-Gln-Val-Ala-Leu-Leu- Lys-Ala-Leu-Tyr-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-

His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met.NHr H-Asp-Ala-Gly-His-GIy-Gln-Ile-Ser-His-Lys-Arg-

His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met.NHr H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met.NHr

pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met.NHr pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Be-Gly-Leu-Met.NHr

H-Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met.NH~

NKi > NK, > NK, NKr > NK3 > NKi NK, > NK, >> NK,

NK2 > NK, >> NK3

NK3 >> NK, = NK, NKi > NK, > NK, NK, >> NK, = NK, NK, > NK, >> NK,

Data from Regoli et al. (1994) and Ho&r-Petsche (1995).

Tachykinins and Gut Motor Functions 175

scripts of the preprotachykinin (PPT)-A gene. In contrast,

there is little evidence that the PPT-B gene with its major

product NKB is expressed within the digestive system to

any substantial degree. In terms of cellular sources of tachykinins in the enteric nerve plexuses and muscle layers, two

principal systems have been identified: intrinsic enteric neurons and extrinsic primary afferent nerve fibers. The quantitatively most important source of tachykinins is the

enteric nervous system, which has its cell bodies in the my-

enteric (MP) and submucosal (submucous; SMP) plexuses and supplies all gastrointestinal effector systems (Fig. 1). The SP-containing enteric neurons in the guinea-pig intes-

tine have been mapped to such a precision that their pro-

jections within and between the nerve plexuses and to the

muscular and other effector systems are well understood.

2.2. identity and Molecular Biology

of Tachykinins in the Mammalian Cjut

The tachykinins are a family of biologically active peptides whose common structural feature is the C-terminal amino acid sequence Phe-X-Gly-Leu-Met-NH2, where X is an aro-

matic (Phe or Tyr) or hydrophobic (Val or Ile) residue (Ta-

ble 1). The mammalian tachykinins that have been identi-

fied to date are SP, NKA, NKB and the N-terminally extended forms of NKA, neuropeptide K (NPK) and neu-

ropeptide y (NPy). The tachykinins are phylogenetically very old peptides, and there is a large and still growing list

of nonmammalian tachykinins that are not dealt with here in detail. It should not go unnoticed, though, that the pro-

totypes of nonmammalian tachykinins, which include physalaemin, eledoisin and kassinin (Table l), have played

DRG c

MG -1

ORAL ABORAL

FIGURE 1. Schematic diagram of known projections of SP- immunoreactive neurons within the enteric plexuses and to the muscle layers of the guinea-pig intestine. The possibility that extrinsic afferent neurons send axons into the MP is based on functional rather than morphological evidence. Where known, the co-existence with other neuropeptides or neuronal markers is indicated. BV, blood vessel; CB, calbindin; CM, circular muscle; CR, calretinin; DRG, dorsal root ganglion; DYN, dynorphin; ENK, enkephalin; LM, longitudinal muscle; MG, mesenteric prevertebral ganglion; MM, muscularis mucosae; MU, mucosa; NF, neurofilament protein.

an important role in the discovery of mammalian tachyki-

nins and in the development of mammalian tachykinin re-

ceptor pharmacology (Maggi et al., 1993~; Otsuka and

Yoshioka, 1993; Regoli et al., 1994). As other regulatory peptides, the mammalian tachyki-

nins are derived from larger precursor peptides, the PPTs, which are encoded by two different PPT genes (Nakanishi, 1987). One, known as PPT-A or PPT-I, contains the se-

quences encoding both SP and NKA, whereas the other,

PPT-B or PPT-II, encodes NKB. The primary RNA tran- script of PM-A is alternatively spliced to produce four different forms of PPT-A mRNA (Nakanishi, 1987; Carter

and Krause, 1990; Harmar et al., 1990), termed a-PPT (or (-w-PM-A), p-PPT (or P-PPT-A), y-PPT (or -y-PM--A) and

6-PPT (or E-PM-A). SP can be produced by translation of

all four PPT-A mRNAs, while sequences coding for NKA are found in P-PPT-A and y-PPT-A only. NPK and NP-y are

encoded by P-PPT-A and r-PPT-A, respectively, whereas 6-PPT-A gives rise to a 22 amino acid C-terminal peptide unique to this form of PPT (Harmar et al., 1990). The PPT-

B gene is transcribed to two slightly different forms of PPT- B mRNA, both of which encode NKB (Kotani et al., 1986).

The abundance of SP and NKA in the gut predicts the

presence and expression of the PPT-A gene in this organ.

In the enteric nervous system of the rat gastrointestinal

tract, it is y-PPT-A that accounts for as much as 80-90% of the tachykinin-encoding mRNA, while P-PPT-A com-

prises about lo-20% and (Y-PPT-A less than 1% of the to-

tal SP/NKA-encoding mRNA (Stemini et al., 1989). It recently has been shown that 6-PPT-A is also expressed in

the rat small and large intestine, but is less abundant than y-PPT-A and P-PPT-A (Khan and Collins, 1994). The prevalence of PPT-A mRNAs encoding both SP and NKA

implies that in most, if not all, enteric neurons, SP coexists

with NKA. This conjecture is supported by radioimmuno-

logical and immunohistochemical measurements of SP and NKA levels in the human, porcine, feline, canine, guinea-

pig and rat gut (Deacon et al., 1987; Too et al., 1989;

Christofi et al., 1990; McDonald et al., 1990; Takeda et al.,

1990; HellstrBm et al., 1991; Schmidt et al., 1991; Shuttle- worth et al., 1991; Maggi et al., 199213; Moussaoui et al.,

1992). Although the molar ratios of SP/NKA sometimes deviate considerably from l:l, it has been shown that SP and NKA are colocalized in, and coreleased from, the same synaptic vesicles of the guinea-pig, porcine and canine en-

teric nervous system (Deacon et al., 1987; Christofi et al., 1990; McDonald et aI., 1990; Schmidt et al., 1991; Broad et al., 1992). NPK p ro uced d by translation of /3-PPT-A mRNA has been found to occur in the guinea-pig and feline intestine, and has been suggested to act as a precursor, which during packaging into storage vesicles for axonal transport, is converted to NKA (Deacon et al., 1987; Hell- strnm et al., 1991). This inference is consistent with the finding that, unlike SP and NKA, NPK is apparently not released from enteric neurons (Christofi et al., 1990; Hell- striim et al., 1991), although NPK and NPK-( l-24) are lib- erated from carcinoid tumor cells (Ahlman et al., 1988;


Conlon et al., 1988). The expression of NPy is species-specific inasmuch as this product of y-PPT-A is present in the

rabbit, but not guinea-pig, intestine (Kage et al., 1988). NKB is absent from the human, porcine, guinea-pig and rat

gut (Deacon et al., 1987; Too et al., 1989; Christofi et al., 1990; McDonald et al., 1990; Takeda et al., 1990; Schmidt et al., 1991; Shuttleworth et al., 1991; Maggi et al., 1992b; Moussaoui et al., 1992), which is consistent with the absence of PPT-B expres-

sion in the enteric nervous system of the rat intestine (Sternini, 1991). One laboratory, though, holds that the rat (Tateishi et al., 1990) and human (Kishimoto et al., 1991) gut contains minute amounts of NKB. These discrepancies are not under-

stood, but may be the result of either species differences in the activity of the PPT-B gene, which is expressed in the bovine (Kotani et al., 1986), but not rat, (Sternini, 1991) intestine, or differences in the post-translational processing of PPT-B.

2.3. Regional and Species Differences

in the Tachykinin Distribution in the (jut

Radioimmunological studies have provided a wealth of quantitative information on the distribution of tachykinins in the gastrointestinal tract and have shown that there are marked variations in the tachykinin levels between different regions and layers of the gut and between different species. The regional distribution of tachykinins in the human, rabbit, rat and guinea-pig gut is similar inasmuch as the tissue concentrations of SP and NKA are low in the esophagus, intermediate in the stomach and high in the intestine, the

highest levels being usually measured in the small intestine (Holzer et al., 1980a, 1981,1982; Brodin et al., 1983; Ferri et al.,

1987; Wattchow et al., 1987; Tateishi et al., 1990; Kishimoto et al., 1991; McGregor and Conlon, 1991). In contrast, the distribution of SP and NKA in the porcine and feline gut shows little variation and is fairly even between the esophago- gastric region and the rectum (Holzer et al., 1982; Brodin et al., 1983; McGregor et al., 1984; Schmidt et al., 1991). Species differences also exist with regard to the concentrations of SP

in the different layers of the gastrointestinal wall. The levels of SP and NKA in the external muscle layer of the guinea- pig, rat, rabbit, ferret and feline gut are considerably higher than in the mucosa/submucosa layer (Holzer et al., 1981, 1982; Leander et al., 1982; Brodin et al., 1983; Deacon et al.,

1987; Greenwood et al., 1990), whereas the mucosa/submucosa layer of the human and equine intestine contains similar or even higher concentrations of SP than the external

muscle layer (Brodin et al., 1983; Llewellyn-Smith et al., 1984; Ferri et al., 1987; Burns and Cummings, 1993).

2.4. Tmhykinins in the Enteric

Nerve Plexuses and Muscle Layers 2.4.1. Intrinsic enteric neurons.

2.4.1.1. Regional dishibution of tachykininergic enteric tmmms .

The muscle layers (longitudinal muscle, circular muscle, muscularis mucosae) of the gut receive, in general, a dense supply by tachykinin-containing nerve fibers, most of which originate from intrinsic enteric neurons. In all mam-

malian species that have been examined, SP and NKA are

present in somata of the MPs and SMPs and in varicose nerve fibers that emanate from these networks of enteric neurons. Although there are regional and species differences in the density of the tachykininergic innervation of the gut (Leander et al., 1982; Brodin et al., 1983; Keast et al.,

1985, 1987; Wattchow et al., 1987; Burns and Cummings, 1993), the general distribution of SP/NKA-containing somata and axons is very similar in most species. The projections of these neurons have been identified with neuroanatomical lesion and tracing techniques, and their chemical coding (that is, the characteristic combination of coexist- ing transmitters, neuropeptides and other neuronal markers) has been revealed by immunocytochemistry. If all

observations are taken together, it would appear that tachykinin-positive nerve fibers connect the ganglia within the

MPs and SMPs, link the two plexuses with each other and issue projections to the longitudinal muscle, circular muscle and muscularis mucosae. A second, although quantitatively

less important, source of tachykinins in the gastrointestinal muscle layers is extrinsic primary afferent nerve fibers.

In the esophagus of opossum, guinea-pig, rabbit, pig and humans, SP-positive neurons have been localized to both the MPs and SMPs (Leander et al., 1982; Aggestrup et al., 1986; Keast et al., 1987; Wattchow et al., 1987; Christensen et al., 1989; Singaram et al., 1991), whereas in the feline esophagus, SP-positive neurons are present in the MP only (Leander et al., 1982). These intrinsic neurons issue SP-immunoreactive fibers that run in the plexuses themselves and innervate the

longitudinal muscle, circular muscle and muscularis mucosae,

but not the striated muscle layers of the esophagus. The lower esophageal sphincter (LES) of humans and pig is also supplied by SP-expressing nerve fibers (Aggestrup et al., 1986).

The innervation of the mammalian stomach differs from that of other regions of the gut inasmuch as the MP seems to be the only source of enteric neurons supplying all layers of the gastric wall, as the SMP is largely absent. Accord- ingly, myenteric neurons expressing SP have been found to

innervate the longitudinal muscle, circular muscle and muscularis mucosae of the murine, rat, guinea-pig and canine stomach (Minagawa et al., 1984; Ekblad et al., 1985; Mawe et al., 1989; Furness et al., 1991; Schemann et al.,

1995). Further analysis has confirmed that all SP-containing axons in the gastric circular muscle of the dog originate

from the MP because they disappear after myectomy (removal of the MP), but are left unaltered by extrinsic denervation of the stomach (Furness et al., 1991). As many as 32% of all myenteric neurons in the guinea-pig stomach contain SP along with choline acetyltransferase (ChAT) (Schemann et al., 1995). On the basis of their chemical coding, these myenteric SP neurons can be subdivided into at least three populations, the major subgroup expressing enkephalin-like immunoreactivity (Schemann et al., 1995).

The muscle layers of the small and large intestine are innervated by SP/NKA-containing axons that originate either from the MP or SMP of enteric neurons. This has been demonstrated in a large variety of mammalian species in-

Tachykinins and Gut Motor Functions

eluding humans (Costa et al., 1980, 1981; Jessen et al., 1980;

Schultzberg et al., 1980; Leander et al., 1982; Brodin et al., 1983; Llewellyn-Smith et al., 1984; Keast et al., 1985; Ek- blad et al., 1985, 1987; Aggestrup et al., 1986; Wattchow et al., 1987, 1988; Furness et al., 1990; Heinicke and Kieman, 1990; Singaram et al., 1991; Sang and Young, 1996).

2.4.1.2. Chemicd coding of tachykininergic enteric neurons. The

tachykinin-expressing enteric neurons in the guinea-pig intestine, which have been studied in most detail, can be di- vided into many categories, depending on their projection

and chemical coding (Fig. 1). Almost all myenteric and submucosal SP neurons in the guinea-pig intestine co-express ChAT and can be further subgrouped by their content of calbindin/calretinin, neurofilament protein triplet and dynorphin/enkephalin-like immunoreactivity, whereas vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) synthase do not coexist with tachykinins in the same

enteric neurons of the guinea-pig, murine and canine gut

(Llewellyn-Smith et al., 1988; Brookes et al., 1991a,b, 1992; Steele et al., 1991; Costa et al., 1992; Fumess et al., 1992; Berezin et al., 1994; Schemann et al., 1995; Sang and

Young, 1996). Co-expression of enkephalin-like immunoreactivity and SP has also been found in enteric neurons of the human and feline gut (Domoto et al., 1984; Watt- chow et al., 1988). Tachykininergic enteric neurons in the murine small and large intestine co-express calretinin, y-aminobutyric acid (GABA) or 5-hydroxytryptamine

(5-HT) to various degrees and show some distinct differences from the chemical coding of SP neurons in the guinea-pig gut (Sang and Young, 1996). The physiological

roles of neurons depend on their chemical coding, and by synopsis of their chemical codes, morphological character-

istics and projections, it has been possible to deduce the functional identity of tachykininergic enteric neurons as sensory neurons, interneurons and motoneurons (Furness

and Costa, 1987; Steele et al., 1991; Brookes et al., 1991a,b, 1992; Furness et al., 1992; Sang and Young, 1996).

2.4.1.3. Projections of cuchykininqic enteric mmns. SP-containing structures are found in all three network layers of the MP within the guinea-pig small intestine: the primary

plexus, which is composed of the myenteric ganglia and in-

ternodal strands; the secondary plexus, which is made of narrow circumferential strands of fibers running either in or on the surface of the circular muscle layer; and the tertiary plexus (longitudinal muscle plexus), with its branching fibers lying between the internodal strands and being closely

apposed to the surface of the longitudinal muscle layer (Costa et al., 1980, 1981; Brookes et al., 1992).

Neuroanatomical tracing studies (Fumess and Costa, 1987) have led to the identification of at least seven different projections of myenteric SP neurons in the guinea-pig small intestine (Fig. 1).

( 1) Some myenteric neurons containing SP issue ascending projections typical of interneurons (Costa et al., 1981; Brookes et al., 1991b).

(2)

(3)

(4)

(5)

(6)

(7)

177

The longitudinal muscle of the guinea-pig small intes-

tine is innervated by as many as one-quarter of all myenteric neurons, about 50% of which contain SP (Brookes et al., 1991b, 1992). Fibers of SP/NKA-containing myenteric neurons also supply the longitudinal muscle of the guinea-pig colon and the corresponding

tenia of the cecum and colon (Shuttleworth et al., 1991; Fumess et al., 1992; Messenger, 1993). A major target of myenteric SP neurons in the guinea- pig gut is the circular muscle layer (Costa et al., 1981;

Messenger and Fumess, 1990; Brookes et al., 1991a; Steele et al., 1991; Messenger, 1993), in which as many as 50% of all neurons may contain SP (Llewellyn- Smith et al., 1988). A part of the myenteric SP neurons projecting to the circular muscle co-expresses ChAT

and issues ascending projections of short length. The other part of myenteric neurons that supply the circular muscle has oral projections of intermediate length (Costa et al., 1981; Brookes et al., 1991a). In the face of

all their characteristics, the short- and long-projection neurons to the circular muscle are considered to be ex-

citatory cholinergic motor neurons (Costa et al., 1981; Llewellyn-Smith et al., 1988; Brookes et al., 1991a), whose varicose fibers may come as close as 15-20 nm to

the circular muscle (Llewellyn-Smith et al., 1988). Some myenteric SP neurons interconnect the MP and SMP (Messenger, 1993). Other myenteric SP neurons that co-express enkephalin- and dynorphin-like immunoreactivity function as

excitatory motor neurons to the muscularis mucosae (Steele and Costa, 1990; Messenger, 1993). Still another group of myenteric SP neurons sends ax-

ons into the mucosa, being intrinsic sensory neurons (Song et al., 1991).

There is little evidence that SP-containing neurons of the SMP in the guinea-pig gut project to the muscle layers, their major targets of innervation being the mucosa (Costa et al., 1981; Song et al., 1992) and submucosal blood vessels (Vanner and Surprenant, 199 1) .

The projections of enteric SP neurons in the rat, canine

and porcine intestine show some distinct differences to those seen in the guinea-pig gut. Thus, myenteric SP neurons in the rat small intestine issue descending projections,

whereas those in the colon are ascending (Ekblad et al., 1987, 1988). To the contrary, SP-expressing neurons in the SMP of the rat small intestine give off both orally and

anally directed fibers (Ekblad et al., 1987). In the canine small intestine, both ascending and descending projections

emanate from myenteric SP neurons, which supply the circular muscle layer and contribute to the interconnection of the MPs and SMPs (Daniel et al., 1987). SP-positive neu-

rons in the SMP also give off both orally and anally directed fibers in the canine small intestine, whereas in the colon, only ascending projections have been visualized (Furness et al., 1990). The innervation of the circular muscle likewise differs between the small and large intestine:


while all SP in the circular muscle of the canine colon is derived from myenteric neurons, it is only the outer part of the circular muscle in the small intestine that is exclusively innervated by myenteric SP neurons, whereas the inner

part is supplied by both myenteric and submucosal SP neurons (Furness et al., 1990). It is noteworthy in this context that SP does not contribute to the innervation of the inter-

stitial cells of Cajal in the dog gut (Berezin et al., 1990), although SP binding sites are concentrated in the inner zone

of the circular muscle layer where the interstitial cells are located (Mantyh et al., 1988). The porcine small intestine

contains many SP neurons in the MP, which issue orally directed projections, and in the inner SMP (Meissner plexus), whereas SP neurons are comparatively rare in the outer SMP,

also called Schabadasch plexus (Timmermans et al., 1990).

2.4.1.4. Morphology and ultrastructure of tachykininergic enteric neurons. Morphologically, SP neurons in the MP of the por-

cine small intestine belong to the group of uniaxonal Type I neurons that have multiple short dendrites (Scheuermann

et al., 1991), and in this respect, are comparable with myenteric SP neurons of the guinea-pig gut, most of which can

be categorized as Dogiel Type I neurons (Brookes et al., 1991a, 1992; Song et al., 1991). The fibers of SP-containing

enteric neurons are varicose axons, with SP being found in the varicose (vesiculate) as well as intervaricose (nonvesic- ulate) portions of nerve processes (Llewellyn-Smith et al.,

1984, 1988, 1989). SP has been localized both in large granular vesicles of myenteric neurons (Probert et al., 1983; Feh& et al., 1984; Llewellyn-Smith et al., 1984, 1988, 1989;

Berezin et al., 1990) and in the cytoplasm outside the synaptic vesicles, a localization that is true both for the vari-

cosities of nerve fibers and the nerve cell bodies (Llewellyn- Smith et al., 1984, 1989). Although SP-positive varicosities

make extensive contacts with other nerve processes or somata in the human and guinea-pig small intestine, only a

very small proportion of the contacts show morphologically identifiable synaptic specializations (Llewellyn-Smith et al., 1984, 1989). Studies in the cat and rat small intestine have confirmed that SP-like immunoreactivity is associated with myenteric synapses and that SP-positive processes take both pre- and postsynaptic positions (Feh& and Wenger,

1981; Feh& et al., 1984).

2.4.2. Extrinsic primary afferent neurons. Since extrinsic denervation fails to alter the tachykininergic innervation of the MP and the circular muscle layer of the guinea- pig, rat, canine and porcine gut (Costa et al., 1980, 1981; Malmfors et al., 1981; Ekblad et al., 1987, 1988; Llewellyn- Smith et al., 1988; Furness et al., 1990, 1991), it is obvious that intrinsic enteric neurons are the major source of SP in the external muscle layer of the gut. This inference is supported by the finding that chemical ablation of extrinsic afferent neurons by the neurotoxin capsaicin fails to alter the tissue content of SP and NKA in the rat gastrointestinal tract (Holzer et al., 1980a; Geppetti et al., 1991; McGregor and Conlon, 1991). However, there is immunohistochemi-

cal evidence that extrinsic neurons do make a small, but appreciable, contribution to the tachykininergic innervation of the gastrointestinal muscle layers (Fig. 1). Most of these extrinsic SP/NKA-containing nerve fibers in the gut are

primary afferent neurons that have their cell bodies in dorsal root ganglia and reach the gut via sympathetic (splanch- nit, colonic and hypogastric) and sacral parasympathetic

(pelvic) nerves while passing through prevertebral ganglia and forming collateral synapses with sympathetic ganglion

cells (Lindh et al., 1983, 1988, 1989; Matthews and Cuello, 1984; Sharkey et al., 1984; Ekblad et al., 1987, 1988; Su et

al., 1987; Green and Dockray, 1988). The extrinsic tachykinin-expressing neurons differ from

intrinsic enteric neurons with regard to their chemical coding (Costa et al., 1986) and sensitivity to capsaicin (Barth6 and Holzer, 1985; Holzer, 1991). Thus, spinal afferents disappear from the wall of the gastrointestinal tract when the

gut is surgically severed from its extrinsic nerve supply or the experimental animals are treated with a neurotoxic

dose of capsaicin (Furness et al., 1982; Hayashi et al.,

1982a,b; Minagawa et al., 1984; Papka et al., 1984; Gibbins

et al., 1987; Su et al., 1987; Green and Dockray, 1988). Their chemical coding in the rat, guinea-pig and canine di-

gestive tract is very characteristic, inasmuch as SP, NKA and calcitonin gene-related peptide (CGRP) are co-expressed in extrinsic afferents, but not in enteric neurons (Gibbins et al., 1987; Lee et al., 1985; Uddman et al., 1986; Su et al.,

1987; Green and Dockray, 1988; Kirchgessner et al., 1988; Lindh et al., 1988; Sternini et al., 1992). In addition to spinal afferents, the gut is also supplied by vagal afferents that

have their cell bodies in the nodose and jugular ganglia, but there is little evidence that vagal afferent nerve fibers contrib-

ute significantly to the gastrointestinal SP content (Hayashi et al., 198213; Lindh et al., 1983; Green and Dockray, 1988).

In the guinea-pig small intestine, extrinsic SP-containing axons of dorsal root ganglion origin project primarily to submucosal blood vessels, but make also some contribution

to the SP-positive fiber network in the SMP (Costa et al., 1980, 1981; Furness et al., 1982). The pylorus of the guinea- pig is innervated by extrinsic afferent nerve fibers that contain SP and CGRP and that, to the most extent, represent spinal afferents reaching the pylorus via the celiac ganglion, whereas vagal afferent nerve fibers are of minor importance (Lindh et al., 1983, 1989). In the rat stomach, extrinsic af-

ferent nerve fibers expressing SP and CGRP supply not only the SMP and submucosal blood vessels, but also the MP and the longitudinal and circular muscle layers (Mina- gawa et al., 1984; Green and Dockray, 1988). While vagal afferent neurons expressing SP do not innervate the rat stomach to any significant extent (Green and Dockray, 1988), it seems as if the distal esophagus and gastric corpus of the cat is innervated by vagal SP-containing fibers that project into the MP (Hayashi et al., 1982b), whereas the extrinsic supply of SP fibers in the MP of the gastric antrum and duodenum is derived from spinal afferents running through the splanchnic nerves and the celiac ganglion (Ha- yashi et al., 1982a,b).


2.5. Tachykinins in the Biliary Tract

As in the gut, the tachykinins present in the hepatobiliary

system are derived from two principal sources: intrinsic neurons and extrinsic primary afferent nerve fibers. In the

gallbladder of humans, pigs, dogs, cats and guinea-pigs, SP has been localized to cell bodies of both ganglionated nerve

plexuses that are analogous to the intrinsic nerve plexuses in the gut (Cai et al., 1983; Keast et al., 1985; Dahlstrand et al., 1988; Goehler et al., 1988; Mawe and Gershon, 1989; Sternini et al., 1992; Talmage et al., 1992; Sand et al., 1993, 1994; De Giorgio et al., 1995). The fibers of these intrinsic

SP neurons supply all layers and regions of the gallbladder and innervate the bile duct and sphincter of Oddi. The chemical coding of the intrinsic SP neurons that do not

contain CGRP differs from that of extrinsic afferent nerve fibers that characteristically co-express CGRP and tachyki-

nins and are most abundant around blood vessels of the gallbladder (Goehler et al., 1988; Maggi et al., 1989~; Mawe and Gershon, 1989; Sternini et al., 1992; De Giorgio et al.,

1995; Goehler and Sternini, 1996).

3. RELEASE OF TACHYKININS IN THE GUT

3.1. Overview

Depolarizing stimuli cause release of SP and NKA from in-

trinsic enteric and extrinsic primary afferent neurons, the two major neuronal sources of tachykinins in the gut. The calcium dependency of the release process points to an exo-

cytotic mechanism, which is an important criterion for es- tablishing tachykinins as gastrointestinal neurotransmitter

substances. Apart from direct immunochemical evidence for a stimulus-evoked release of SP and NKA, there is a wealth of indirect evidence that tachykinins are released in response to a variety of stimuli, as revealed by pharmacolog-

ical blockade of the actions of endogenous tachykinins. These findings are dealt with when the physiological and pathophysiological implications of tachykinins in the different gastrointestinal effector systems are discussed.

3.2. Release of Tachykinins in vitro

There is a multitude of radioimmunological data to show that nerve stimulation causes release of tachykinins, partic-

ularly SP, within the gastrointestinal tract in vitro (Table 2). Experiments with the MP-longitudinal muscle layer of the guinea-pig small intestine have demonstrated that nerve depolarization, either by raising the extracellular K+ concentration or by electrical field stimulation (EFS), releases SP-like immunoreactivity, the extent of which depends on the K+ concentration or frequency of stimulation (Baron et al., 1983; Holzer, 1984). Since the stimulus- evoked release of SP from the gut is prevented by removal of extracellular Ca*+ (Baron et al., 1983; Angel et al., 1984; Holzer, 1984; Kwok and McIntosh, 1990; Broad et al., 1992), it would appear that SP is released by a calcium- dependent exocytotic process. Indirect evidence indicates that the influx of Caz+ supporting the release process takes

place via o-conotoxin-sensitive N-type calcium channels

(Katsoulis et al., 1993; Maggi et al., 1994b).

As is expected for a gastrointestinal messenger molecule, the liberation of tachykinins is under the facilitatory and inhibitory influence of a number of intestinal neurotrans-

mitters and hormones. Among the substances that enhance the spontaneous release of SP and NKA from intestinal

neurons are acetylcholine (ACh) acting via nicotinic receptors, the ganglionic stimulant dimethylphenylpiper- azinium, cholecystokinin (CCK) octapeptide, bombesin,

neurotensin and leukotriene (LT) D, (Table 2). The stimulus- evoked release of SP and NKA is enhanced by naloxone, but inhibited by a methionine-enkephalin analogue (Holzer, 1984), noradrenaline (NA), ACh acting via muscarinic re-

ceptors (Schmidt et al., 1992), adenosine acting via Al receptors and 5HT acting via 5eHTlA receptors (5-HT,,Rs)

(Table 2). In a physiological context, it is important to note that mechanical stimuli that are known to regulate di-

gestive activity, such as distension of the gut wall, increase spontaneous tachykinin release (Table 2), which is under the control of excitatory nicotinic acetylcholine receptors

and inhibitory a-adrenoceptors (Donnerer et al., 1984a,b; Grider, 1989a,b; Jensen and Holmgren, 1992; Schmidt et al., 1992).

Although it is sometimes difficult to ascertain whether the released tachykinins originate from intrinsic enteric or

extrinsic afferent neurons, it nevertheless is possible to dif-

ferentiate between these two sources by making use of capsaicin, which first selectively stimulates and then defunc- tionalizes extrinsic primary afferent, but not intrinsic

enteric, neurons (Barth6 and Holzer, 1985; Holzer, 1991). In the case of the MP-longitudinal muscle layer of the

guinea-pig small intestine, it is safe to assume that the bulk of SP is released from intrinsic enteric neurons because acute exposure to capsaicin fails to cause any appreciable

release of SP (Holzer, 1984) and because some 90% of the SP present in this layer is contained in neurons that are insensitive to the neurotoxic action of capsaicin (Murphy et

al., 1982). The situation is different when peptide levels are mea-

sured in the outflow of the vascularly perfused small intestine of the guinea-pig, in which it is possible to demonstrate

tachykinin release from both intrinsic and extrinsic neurons. Exposure of this preparation to intravascular capsaicin causes considerable release of SP, which is absent in intesti-

nal segments taken from animals pretreated with a neurotoxic dose of capsaicin, whereas SP release elicited by distension or exposure to a ganglionic stimulant is left unaffected by capsaicin pretreatment and hence, reflects peptide release from enteric neurons (Donnerer et al.,

1984a). The K+- evoked release of SP-like immunoreactivity from the vascularly perfused rat stomach and duodenum is mimicked by capsaicin (Kwok and McIntosh, 1990;

Fujimiya and Kwok, 1995), which suggests that a significant portion of the peptide originates from the perivascular plexus of extrinsic afferent nerve fibers. Extrinsic afferent neurons are evidently the source of tachykinins that capsai-


tin releases from isolated slices of the stomach or gallbladder (Renzi et al., 1988, 1991; Maggi et al., 1989c), since the

ability of capsaicin to liberate SP or NKA is lost in tissues obtained from capsaicin-pretreated animals.

Little is known as to whether or not, and under which

conditions, tachykinins are released from endocrine cells of the gastrointestinal mucosa (Simon et al., 1992). In addition, SP may be released from eosinophils and other tachy-

kinin-expressing immune cells that accumulate or reside within the gastrointestinal wall (Weinstock and Blum, 1990), but it is not known whether these sources are of physiological significance under nonpathological circum-

stances.

3.3. Release of Tachykinins in vivo

The findings of a stimulus-evoked, calcium-dependent

release of tachykinins in vitro are complemented by observations showing that SF’ and NKA are released from the gastrointestinal tract in oivo (Table 3). Vagal nerve stimulation (VNS) in anesthetized cats releases SP into the lumen of the stomach and upper small intestine and increases the

plasma levels of SP in the portal vein (Table 3). Feeding or administration of bombesin increases the concentrations of

SP in the jejunal lumen and peripheral blood of the dog (Table 3), whereas the SP level in the human plasma does not change after ingestion of a meal (Parker et al., 1995). Release of NKA into the bloodstream of the cat is further-

more evoked by application of 0.10 M HCl to the serosal surface of the jejunum, while intraluminal application of 0.15 M HCl causes release of SP and NKA into the rat duodenum. Similarly, acidification seems to increase the release of SP-like immunoreactivity into the lumen of the hu-

man stomach (Table 3). In the feline colon, it is electrical stimulation of the pelvic nerves, mechanical stimulation of

the anus or distension of the rectum that have been found to increase the plasma levels of SP and NKA (Table 3).

The release of SP caused by serosal acidification and VNS is not altered by atropine or hexamethonium (Gronstad et al., 1987; Brunsson et al., 1990). Conversely, the SP release

evoked by ingestion of a meal or administration of bombesin is reduced by atropine and propranolol (Jaffe et al., 1982; Ferrara et al., 1987). The source of SP and NKA has not

been determined in these experiments, but the SP appear-

TABLE 2. Release of tachykinins in the gut in pritro

Peptide Tissue released Stimulus to evoke release Inhibition of evoked release Reference

Guinea-pig gastric corpus SP Capsaicin slices

Rat gastric corpus slices SP High [K+] NKA Capsaicin

Guinea-pig gallbladder slices SP Capsaicin Human jejunum segments SP, NKA Stretch Guinea-pig small intestine SP High [K+]

MP-longitudinal muscle layer EFS ACh Bombesin, CCK or neurotensin LTD, Naloxone (in opiate-tolerant gut)

Guinea-pig small intestine SP, NKA High [K+] MP varicosities Rat ileum segments SP EFS Rat colon segments SP, NKA Stretch Rabbit colon segments SP EFS

Capsaicin Canine colon SP EFS or bombesin

muscularis mucosae Vascularly perfused stomach SP Balloon distension

of rainbow trout Vascularly perfused rat stomach SP High [K+] or capsaicin Vascularly perfused rat SP Capsaicin

duodenum Vascularly perfused guinea-pig SP Pressure distension or

small intestine ganglionic stimulant CCK Capsaicin

Vascularly perfused porcine SP, NKA Atropine ileum ACh or periarterial nerve

stimulation1 Vascularly perfused canine ileum SP EFS

Renzi et al., 1988

Geppetti et al., 1991 Renzi et al., 1991 Maggi et al., 1989a,c Grider, 198913

Cal+ removal or enkephalin Holzer, 1984 Ca*+ removal or TTX Baron et al., 1983, Holzer, 1984 Hexamethonium or TTX Holzer, 1984 TTX Holzer, 1984 TTX Bloomquist and Kream, 1987 TTX Want and Tsou, 1989 CaZ+ removal, adenosine A, Broad et al., 1992, 1993

agonists or 5-HTm agonists TTX

Ca*+ removal or TTX

Belai et nl., 1987 Grider, 1989a Snape et al., 1989 Mayer et al., 1990a Angel et al., 1984

Ca*+ removal

TTX, hexamethonium or NA

Hexamethonium or TTX Capsaicin pretreatment

Hexamethonium

Jensen and Holmgren, 1992

Kwok and McIntosh, 1990 Fujimiya and Kwok, 1995

Donnerer et al., 1984a,b Donnerer et al., 1984b Donnerer et al., 1984a

Schmidt et nl., 1992 Schmidt et al., 1992

Manaka et al., 1989

1These stimuli were effective in releasing SP and NKA only in the presence of atropine and phentolamine.


TABLE 3. Release of tachykinins in the gut in vivo

Tissue Peptide released Stimulus to evoke release Reference

Lumen of human stomach Lumen of guinea-pig stomach Lumen of rat stomach Lumen of feline stomach

Lumen of rat duodenum SP, NKA Lumen of feline duodenum and jejunum SP Lumen of canine jejunum SP Portal vein plasma of cat SP Peripheral vein plasma of dog SP

Mesenteric vein plasma of cat Colonic vein plasma of cat

NKA SP, NKA

SP SP SP SP

Luminal acid Intravenous capsaicin Intragastric capsaicin VNS Intravenous ACh or adrenaline Luminal ACh Luminal acid VNS Ingestion of a meal VNS Ingestion of a meal Bombesin Acid applied to the jejunal serosa Pelvic nerve stimulation Rectal distension Mechanical stimulation of the anus

Mueller et al., 1991 Renzi et al., 1988 Hayashi et al., 1996 Uvnss-Wallensten, 1978 Uvnas-Wallensten, 1978 Uvn2s-Wallensten, 1978 Smedfors et al., 1994 Grizinstad et al., 1985b, 1987 Ferrara et al., 1987 Granstad et al., 1985a Jaffe et al., 1982; Ferrara et al., 1987 Jaffe er al., 1982; Ferrara et al., 1987 Brunsson et al., 1990 HellstrGm et al., 1991 Hellstram et al., 1991 HellstrGm et al., 1991

ing in the gastric lumen of the rat and guinea-pig after in-

tragastric or i.v. administration of capsaicin (Renzi et al., 1988; Hayashi et al., 1996) is likely to originate from extrinsic afferent nerve fibers.

4. TACHYKININ PHARMACOLOGY 4.1. Overview

This section is designed to give a brief introduction to con- temporary tachykinin pharmacology and thus, to provide a

background against which the actions of tachykinins in the

various effector systems of the gut can be appreciated. Three receptors for tachykinins (NK,, NK,, NK3) have

been cloned and characterized to have seven transmem-

brane spanning segments, to be coupled to G-proteins and to be linked to the phosphoinositide signaling pathway.

The structure of the tachykinin receptors exhibits species differences, and there may be further receptor subtypes

whose existence has not been proven yet by molecular pharmacological methods. Although NK, receptors are

considered to be SP-preferring, NK2 receptors NKA-preferring and NK3 receptors NKB-preferring receptors, SP, NKA

and NKB are full agonists at all three tachykinin receptors. These receptors, however, can be differentiated by use of

receptor-selective synthetic agonists and antagonists. The activity of tachykinins at their receptors is regulated by

membrane-bound proteases and by ligand-initiated receptor down-regulation (desensitization), which is likely due to internalization of stimulated receptors.

4.2. Tachykinin Receptor Agonists

Three distinct types of tachykinin receptors have been identified by functional and binding studies: NK1, NK2 and NK, receptors (Guard and Watson, 1991; Maggi et al.,

1993~; Regoli et al., 1994; Maggi, 1995). Pharmacologi-

cally, NK1, NKz and NK3 receptors are defined on the basis of different agonist and antagonist affinities. The mammalian tachykinins SP, NKA and NKB, whose C-terminal

hexapeptide sequence seems to be the minimal requirement for recognition, show little selectivity towards the different

tachykinin receptors (Table 1) and are capable of acting as full agonists at all receptor types (Guard and Watson, 1991; Maggi et al., 1993~; Regoli et al., 1994; Maggi, 1995). How-

ever, there are synthetic tachykinin analogues that display considerable receptor selectivity, and Table 4 lists some of the receptor-selective agonists that are commonly in use for

agonist characterization of tachykinin receptors. A range of

receptor-selective radioligands for the biochemical and au-

toradiographic study of tachykinin receptor types is also available (Mussap et al., 1993). It should not go unnoticed here that not all biological actions of tachykinins are solely

encoded by their C-terminal sequence and that the N-terminal sequence may be relevant for certain activities of these peptides (Maggi et al., 1993~; Regoli et al., 1994;

Maggi, 1995).

4.3. Tachykinin Receptor Antagonists

The occurrence of three tachykinin receptor types has been

confirmed by the development of competitive and selective receptor antagonists, some of which point to the existence

of further tachykinin receptor subtypes (Regoli et al., 1994;

Maggi, 1995). The available tachykinin antagonists can be roughly grouped into three categories, the first group of which was discovered when certain L-amino acids in the

sequence of SP were replaced by D-amino acids. This first class of competitive antagonists is exemplified by [D- Pro2,D-Trp7,9]-SP and [D-Argl,D-Trp7J,Leull]-SP (spant- ide) and is characterized by low activity, restricted selectivity for different tachykinin receptor types and nonspecific effects, including antagonism of bombesin, release of histamine, local anesthetic and neurotoxic effects (Maggi et al., 1993c).

Further modification of the tachykinin amino acid sequence gave way to competitive tachykinin antagonists characterized by improved potency, selectivity towards NK, or NK2 receptors, and lack of nonspecific effects. Of the an-


tagonists listed in Table 4, GR-82,334, FK-888, R-396, GR-

94800, MEN-10376 and MEN-lo,627 fall into this second group of peptidic receptor-selective tachykinin antagonists. GR-82,334 is a conformationally restrained analogue of physalaemin, and in this way, has become a metabolically

stable antagonist at NK1 receptors (Hagan et al., 1991), while FK-888 is a dipeptide-derived compound that is a highly selective NK, receptor antagonist (Fujii et al., 1992). Most of the peptidic NK2 receptor antagonists are linear

peptides, R-396 being a hexapeptide (Dion et al., 1990),

GR-94,800 a heptapeptide (McElroy et al., 1992) and MEN-lo,376 a heptapeptide derived from substitution of

NKA-(4-10) (Maggi et al., 1991). In contrast, MEN-lo,627

is a polycyclic hexapeptide derivative that turned out to be one of the most potent and selective NK, antagonists

(Maggi et al., 1994a).

TABLE 4. Tachykinin receptor-selective agonists and antagonists

A new era in tachykinin receptor pharmacology began with the discovery and subsequent development of highly

active nonpeptide tachykinin antagonists for all three tachykinin receptor types. Most of the compounds that at present are available are selective for the NK1 receptor, a few examples being given in Table 4. The first nonpeptide antagonist at NK, receptors was the quinuclidine com-

pound CP-96,345 (S nl ‘d er et al., 1991). Although being a

potent, selective and competitive antagonist in binding

studies, CP-96,345 has serious nonspecific effects including high affinity to L-type calcium and other membrane chan-

nels (Guard et al., 1993; Tamura et al., 1993; Maggi, 1995). While the tachykinin antagonistic activity resides in the

(-) isomer of the molecule, the nonspecific effects are shared by the ( -) and (+ ) isomers of CP-96,345. The (+ ) isomer, therefore, allows to differentiate between the non-

Selective

Tachvkinin Selective agonists antagonists

receptor Ligand Reference Ligand

NK1 SP methyl ester

[Sarg]-SP sulphone

[Pro9]-SP

[pGlue,L-Pro9]-SP-(6-11) (septide)’

NKz [PAlaB]-NKA-(4-10) [NleulO]-NKA-(4-10)

NK, Succinyl-[Aspe,N-MePhes]-SP-(6-11) (senktide)

[MePhe7]-NKB

Guard and Watson, 1991 GR-82,334

Drapeau et al., 1987

Lavielle et al., 1988

Laufer et al., 1986

Rover0 et al., 1989 Drapeau et al., 1987

Wormser et al., 1986

Drapeau et al., 1987

FK-888

(X-96,345

CP-99,994

RP-67,580

SR-140,333

R-396 GR-94,800

MEN-lo,376

MEN-lo,627 SR-48,968

SR-142,801

PD-161,182

%I Reference

7.59LM 7.17CM

8.81LM 7.53CM

8.11LM 8.17CM

N.A.

7.37LM

9.65LM

N.A. 8.85CM

6.44CM

N.A. 7.83CM

9.27LM

N.A.

Hagan et al., 1991; Maggi et al., 1994e; Maggi, 1995

Fujii et al., 1992; Maggi et al., 1994e, Maggi, 1995

Snider et al., 1991; Maggi et al., 1994e; Maggi 1995

McLean et al., 1993

Garret et al., 1991; Maggi, 1995

Emonds-Alt et al., 1993a

Dion et al., 1990 McElroy et al., 1992;

Maggi et al., 1994e Maggi et al., 1991,

1994e Maggi et al., 1994a Emonds-Alt et al.,

1992; Maggi et al., 1994e

Emonds-Alt et al., 1994; Patacchini et al., 1995

Boden et al., 1996

‘Note that septide is not necessarily a selective NK, receptor agonist, but may preferentially label another receptor tentatively called “septide-sensitive” receptor (Glowinski, 1995). The pKa (equilibrium dissociation constant) values were evaluated by the use of receptor-selective NK,, NK, and NK, receptor agonists and their contractile effects on the guinea-pig ileum longitudinal (LM) or circular (CM) muscle. N.A., quantitative estimates of affinity in the guinea-pig ileum are not available.

Antagonist code explanations: GR-82,334, pGLu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-D.Pro(spiro-y-lactam)Leu-Trp.NHZ; FK-888, N*-[(4R)-4-hydroxy- l-( l-mechyl-1H-indol-3-yl)carbonyl-prolyl]-N~me~hyl~N-phenylme~hyl~3~(2~naph~hyl)~ I a aninamide; CP-96,345, (-)-(2S,3S)-cis-2-(diphenylmethyl). N-[(2-methoxy-phenyl)-methyl]-l-azabicyclo[2.2.2]octan-3-amine; CP-99,994, (+)-(2S,3S)-3-(2-methoxybenzylamino)-Z-phenylpiperidine; RP-67,580, (3aR,7aR)-7,7-diphenyl-2-[l-imino-2-methoxy~phenyl)~e~hyl]perhydroisoindol~4-one; SR-140,333, (S)1-(2-3-[3-(3,4-dichlorophenyl)-l-(3-isopropoxyphe~ nyl-acetyl)piperidin-3-yl]-ethyl]-4-phenyl-l-azoniabicyclo[2.2.2]-octane chloride; R-396, Ac-Leu-Asp-Gin-Trp-Phe-Gly.NH2; GR-94,800, PhCO-Ala-Ala- D.Trp-Phe-D.Pro-Pro-Nle.NHZ; MEN-20,376, H-Asp-Tyr-D.Trp-Val-D.Trp-D.Trp-Lys.NH*; MEN-10,627, cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2P- 5p); SR48,968, (-)-N-methyl-N[4-acetyl amino-4-phenyl-piperidino-2-(3,4-dichlorophenyl)bu~l]~be~amide; SR-142,801, (3)-(N)-(l-[3-( l-benzoyl-3-(3,4-

dichlorophenyl)piperidin-3-yl)propyl]-4-phenylpiperidin~4~yl~~N-me~hylace~amide; PD-161,182, [S-(R*,S*]-(2-[2,3-difluorophenyl]-l-methyl~l~[(7~ure~ idoheptyl)carbamoyl]ethyl)carbamic acid 2 methyl-l-phenylpropyl ester.


specific and tachykinin-antagonistic activities of the mole-

cule. A similar precaution is in place when other nonpep-

tide antagonists are being used, although nonspecific effects are generally less prominent than with (X-96,345.

A follower of CP-96,345, which reportedly overcomes most drawbacks of the parent compound, is (Y-99,994 (McLean et al., 1993). Two other nonpeptide NK, receptor

antagonists that were developed soon after the discovery of CP-96,345 are the perhydroisoindole compound RP-67,580

(Garret et al., 1991) and SR-140,333 (Emonds-Alt et al., 1993a). Unlike SR-140,333, CP-96,345, CP-99,994 and

RP-67,580 exhibit marked species differences in their activity. CP-96,345 and CP-99,994 possess much greater affinity

to, e.g., the human and guinea-pig NK, receptor than to

the receptor in the rat and mouse, whereas the reverse is true for RP-67,580. This species difference in antagonist affinity results from small variations in the receptor domains

to which the nonpeptide antagonists bind (Maggi et al.,

1993c, Regoli et al., 1994; Maggi, 1995). Nonpeptide antagonists of NK, and NK, receptors also

have become increasingly available (Table 4). SR-48,968

(Emonds-Alt et al., 1992) is a NK2 antagonist that has been in use for several years, while SR-142,801 and PD-161,182

are the first, much-awaited prototypes of potent and selective NK7 antagonists (Emonds-Ah et al., 1994; Boden et al., 1996).

A number of pharmacological studies suggests that there may be additional types of tachykinin receptors or that the hitherto identified NK,, NK2 and NKj receptors may be

subdivided into further subtypes (Maggi et al., 1992a, 1993~; Regoli et al., 1994; Glowinski, 1995; Maggi, 1995; Portbury et al., 1996). These claims are based on differences

in agonist/antagonist potency or antibody reactivity, which may reflect tissue and/or species variants (homologues) of

tachykinin receptors, post-transcriptional or post-translational receptor variations, disparities in the ligand recognition epitopes or differences in the second messenger cou-

pling domains. The pharmacological heterogeneity of NK, receptors is exemplified by the “septide-sensitive” receptor, which is particularly sensitive to septide, a synthetic SP an-

alogue originally designed to be a selective NK, receptor agonist (Petitet et al., 1992; Maggi et al., 199313). This “septide-sensitive” receptor differs from typical NKl receptors with regard to both agonist and antagonist affinities, but has not been identified and characterized by molecular

pharmacological techniques yet (Glowinski, 1995). It is important to note in this context, that the NK, receptor can exist in two isoforms, which vary in the length of their in-

tracellular carboxyl terminal tail (Fong et al., 1992) and which in the CNS, are expressed in a region-specific manner (Mantyh et al., 1996).

4.4. Molecular Tachykinin Receptor Phmmacoi.ogy

The pharmacological and biochemical evidence for the existence of three distinct types of tachykinin receptor has been verified by the cloning and molecular pharmacologi-

cal characterization of NK,, NK, and NKj receptors in the

rat (Nakanishi, 1991). S b q u se uent cloning of tachykinin

receptors in other mammalian species, including those of humans, has confirmed this heterogeneity of tachykinin receptors and, in addition, has shown the existence of small species differences in the amino acid sequence and structure of the receptors (Regoli et al., 1994; Maggi, 1995).

These species differences are responsible for the species-related potency differences of nonpeptidg NK1 receptor antagonists

(Maggi et al., 1992a, 1993~; Regoli et al., 1994; Maggi, 1995). Analysis of the binding epitopes for agonists and an-

tagonists has come up with what has been called a “pharmacological heresy” by Leslie L. Iversen. Although nonpeptide antagonists may behave competitively in binding and

functional assays, they do not bind to the same epitopes of the receptor protein to which the agonists bind. As a consequence, the antagonists do not displace the agonists from

their binding sites, and the apparently competitive interaction with the agonists seems to result from a conforma-

tional change in the structure of the receptor, which re- stricts the agonist’s access to their binding epitopes (Maggi,

1995). In addition, the binding domains for various agonists may differ in one and the same receptor, and the ago-

nist binding epitopes may be distinct from the domains re-

sponsible for signal transduction (Maggi, 1995).

4.5. Transduction Mechanisms

The cloned tachykinin NK1, NK2 and NK3 receptors be-

long to the superfamily of G-protein-coupled receptors with seven transmembrane spanning domains, an extracellular

N-terminus, and an intracellular C-terminal segment. The G-proteins that mediate the effects of tachykinin receptor

stimulation in the gut have not been fully characterized yet, but seem to vary in different gastrointestinal effector systems (Sun et al., 1993; Sohn et al., 1995). In terms of second mes-

senger systems, it appears as if the phospholipase C/phosphoinositide signaling pathway is the major transduction system that is operated by all three types of tachykinin re-

ceptors (Guard and Watson, 1991). Coupling to other second messenger systems, such as adenylate cyclase or phospholipase Al, seems to be of relevance in certain cells that

have been transfected with tachykinin receptor cDNAs, but is unlikely to play a major role under physiological conditions. The phosphoinositide signaling pathway is also utilized in the gut in which activation of NK,, NK2 and NKI

receptors causes rapid appearance of inositol phosphates, a response that is independent of membrane depolarization,

calcium influx or intracellular calcium mobilization (Holzer and Lippe, 1985; Guard et al., 1988; Hall and Morton, 1991; Petitet et al., 1993). In contrast, there is little evidence that tachykinins enhance the formation of cyclic AMP and cyclic GMP in the digestive tract and salivary glands (Walling et al., 1977; Lee et al., 1983; Watson, 1984;

Palmer et al., 1987; Kowal et al., 1989; Hellstriim et al., 1994). The membrane-effector pathways that are regulated by phosphoinositide turnover and that bring about the bio-


logical actions of tachykinins are dealt with in detail when the implications of tachykinins in the various gastrointestinal effector systems are discussed.

4.6. Factors Determining the Activity of Tachykinins at their Receptors 4.6.1. Degrading enzymes. The activity of tachykinins at

their receptors in certain tissues is cut short by peptide- cleaving enzymes. While there is unequivocal evidence

that tachykinin-degrading enzymes are also present in the gastrointestinal tract (Watson, 1983; Bunnett et al., 198513; Hall et al., 1990), there is less unanimity as to whether or not these enzymes are major determinants of the biological

activity of tachykinins. Of the pertinent peptidases present

in the digestive system, the best characterized is neutral endopeptidase (NEP, enkephalinase, endopeptidase 24.11, EC 3.4.24.11). NEP is a cell membrane-bound enzyme that

has been localized to gastrointestinal mucosa and muscle (Bunnett et al., 1985a, 1988, 1993; Nau et al., 1985, 1986;

Sterchi et al., 1988). When the enzyme that can cleave both SP and NKA is inhibited by phosphoramidon or

thiorphan (Nau et al., 1986; Bunnett et al., 1988), the capsaicin-induced release of SP in the guinea-pig isolated gall-

bladder is augmented (Maggi et al., 1989a). Furthermore, inhibition of NEP increases the availability of exogenous

SP for binding to tachykinin receptors in the rat ileum (Iwamoto et al., 1988) and enhances the peptide’s potency

to contract the guinea-pig gallbladder and the rat and ferret small intestine (Djokic et al., 1989; Maggi et al., 1989a).

The ability of SP, but not NKA, to cause anion secretion in the rat colonic mucosa (Cox et al., 1993) and the contractile activity of SP, but not NKA or NKB, in the circular

muscle of the human esophagus and LES (Huber et al., 1993a,b) are likewise enhanced by phosphoramidon, whereas inhibition of NEP is without effect on the ability of

SP to contract the rat stomach in viva (Holzer-Petsche, 1991) and the longitudinal muscle of the guinea-pig colon in vitro (Briejer et al., 1993).

Two other enzymes that are present in the gut and that may degrade tachykinins are angiotensin-converting enzyme (ACE, peptidyl dipeptidase A, kininase II, EC 3.4.15.1) and an aminopeptidase. The action of ACE to

cleave SP in the rat stomach is inhibited by captopril (Or- loff et al., 1986), but ACE does not seem to be of relevance in determining the availability of tachykinins at their re-

ceptors since captopril fails to alter the contractile activity of SP in the rat and ferret small intestine (Djokic et al., 1989). A membrane-bound aminopeptidase has been isolated from the longitudinal muscle layer of the guinea-pig small intestine. This enzyme, which is inhibited by bestatin and amastatin, is able to hydrolyze NKA, but not SP (Nau et al., 1986; Shimamura et al., 1991), but there is no information as to whether or not it may influence the biological activity of tachykinins in the gut.

Many studies have failed to show that combined inhibition of NEP, ACE and aminopeptidase alters the peak biological activity of tachykinins in the human colon and in

the guinea-pig small and large intestine (Hall et al., 1990; Maggi et al., 1990a, 1994e; Giuliani et al., 1991). The effect of NKB to cause hyperpolarization and relaxation of the circular muscle of the guinea-pig colon, though, is substan-

tially enhanced by combined exposure to thiorphan, captopril and amastatin (Maggi et al., 1994f), as is the activity of

SP to cause salivary secretion (Cascieri et al., 1984; Rouissi

et al., 1993). In the guinea-pig tenia ceci, the peak contractile effect of tachykinins is left unaltered by phosphoramidon, captopril and bestatin, whereas the offset of the re-

sponses is markedly prolonged (Hall et al., 1990).

4.6.2. Receptor desensitization and internalization.

While peptidases do not seem to be important determinants of the relative biological activity of tachykinins in

the gut, it has long been known that the actions of tachykinins, especially those of SP and NK1 receptor agonists, un- dergo rapid desensitization. This phenomenon, which is

fairly receptor-specific, pertains to SP effects such as depolarization of myenteric neurons (Johnson et al., 1981; Sche-

mann and Kayser, 1991), contraction of gastrointestinal smooth muscle (Barth6 and Holzer, 1985), anion secretion

in the intestinal mucosa (Kachur et al., 1982; Perdue et al., 1987; Yarrow et al., 1991; Parsons et al., 1992), salivary secretion (Pernow, 1983; Sugiya and Putney, 1988; Soltoff et al., 1989; Yoshimura and Nezu, 1991) and exocrine secretion from the pancreas (SjGdin et al., 1994). SP desensitiza-

tion has been explained in many ways, and it seems now very plausible that tachykinin receptor desensitization is re-

lated to a rapid “down-regulation” of the receptors (Sugiya and Putney, 1988; Sjiidin et al., 1994). Although not examined

specifically in the gut yet, it would appear that agonist- evoked activation of NK1 receptors is followed by rapid en- docytosis of the stimulated receptors, this process of receptor internalization resulting in a depletion of high affinity-state

NK1 receptors from the cell surface (Bowden et al., 1994).

5. TACHYKININS AND GASTROINTESTINAL MOTILITY 5.1. Overuiew

Tachykinins enhance motor activity in virtually all regions

and layers of the mammalian gut. In many instances, this action depends not only on a direct activation of the muscle, but also on stimulation of enteric motor neurons that excite the muscle by release of ACh. Although ridden by species differences, the distribution of motility-controlling tachykinin receptors is such that, typically, NK, receptors are located on smooth muscle cells and NKj receptors on enteric neurons, while NK, receptors reside on both muscle and nerve. Besides their prominent excitatory action, tachykinins can also exert inhibitory influences on motor activity by stimulating either inhibitory neuronal pathways or interrupting excitatory relays (Fig. 2). There is abundant evidence that endogenous SP and NKA participate in the neural stimulation of motility and do so in synergism with


ACh, with which they are coreleased from enteric neurons.

This synergistic action, which seems to be particularly relevant for the enteric control of peristaltic motor activity,

needs to be borne in mind when the implications of tachykinins in gastrointestinal motor regulation are analyzed and

considered as a potential target for therapeutic intervention.

5.2. Excitatory Motor Effects of Tachykinins in the C&t

The ability of SP to stimulate intestinal motor activity was one of the actions that led to the discovery of this peptide. Given appropriate experimental conditions, tachykinins contract gastrointestinal smooth muscle in virtually all

mammals investigated. The excitatory motor effects of tachykinins pertain to all regions of the digestive system

from the esophagus down to the rectum and to all muscle

layers, including the longitudinal muscle, the circular muscle and the muscularis mucosae. What is different between species, regions and muscle layers, though, is the type of ex-

citatory motor effect observed (e.g., phasic and/or tonic contractions), the types of tachykinin receptor involved, and the effector systems and mechanisms participating in

the motor responses to specific tachykinin receptor activation. In many instances, tachykinins influence gastrointes-

tinal motor activity not only by a direct effect on the muscle, but also by an action on other motility-regulating systems. The latter action is in keeping with the ability of

tachykinins to excite enteric neurons and to alter the re-

lease of many gastrointestinal neurotransmitters and hormones, an activity that is reviewed in the companion arti-

cle (Holzer and Holzer-Petsche, 1997). The information on the excitatory motor actions of SP and related peptides in the gut is so broad and diverse that in a review like this,

only work aimed at defining the type of tachykinin receptors, their location and specific transduction mechanisms

DRG @-I TWCGRP

ORAL ABORAL

LM

SM

FIGURE2. Schematic diagram of tachykinin neurons and tachykinin receptors in enteric motor pathways of the guinea- pig gut. Neuronal somata are depicted by circles, muscle cells by diamonds. Receptors are set in italics. +, excitation; -, inhibition; CM, circular muscle; DRG, dorsal root ganglion; LM, longitudinal muscle; M, muscarinic acetylcholine receptor; N, nicotinic acetylcholine receptor; S, enteric sensory neuron; TK, tachykinin.

can be discussed. Other aspects of the regulatory role of ta-

chykinins in gastrointestinal motility have been reviewed

elsewhere (Pemow, 1983; Barth6 and Holzer, 1985; Otsuka

and Yoshioka, 1993; Holzer-Petsche, 1995; Shuttleworth and Keef, 1995).

5.2.1. Esophagus

5.2.1.1. Tachykinin actions, tachykinin receptors and mediator systems. Tachykinins have been found to contract the lon-

gitudinal muscle, circular muscle and muscularis mucosae of the mammalian esophagus, to increase the LES pressure, and to facilitate the swallowing of fluid after application of

SP to the guinea-pig pharynx (Jin et al., 1994). Isolated preparations of the circular muscle of the human esoph-

ageal body and LES are contracted by SP, NKA and NKB,

the rank order of potency being NKA > NKB > SP (Huber et al., 1993a,b). Since only NK,, but not NK, and NK3, re-

ceptor-selective agonists mimic the response to NKA and the effect of NKA is inhibited by SR-48,968, but not CP- 96,345, it follows that the motor action of tachykinins is

mediated by NK2 receptors on the circular muscle because the NKA-evoked contraction is left unaltered by tetrodotoxin (TTX) and atropine (Huber et al., 1993a,b). The SP-

induced contraction of the muscularis mucosae of the opossum, feline, canine and guinea-pig esophagus likewise re-

mains unchanged by TTX and atropine (Christensen and Percy, 1984; Kamikawa and Shimo, 1984).

While the motor response of the rat esophagus muscu-

laris mucosae to NKA is brought about by NK2 receptors (Croci et al., 1994a), it may be mostly NK, receptors that

mediate the tachykinin effects on the muscularis mucosae of the opossum esophagus, given that only NK1 agonists behave as full agonists in this species, although SP, NKA,

NKB, as well as NK,, NK, and NK,, receptor-selective agonists all contract the muscle and exhibit cross-desensitization (Daniel et al., 1989). Small amounts of binding sites for

SP, presumably of the NK, type, are also present in the feline LES (Rothstein et al., 1991) in which SP enhances

spike activity and causes contraction (Reynolds et al., 1984;

Sohn et al., 1995). In the ferret LES, though, SP causes only a brief contraction, which is followed by a prolonged relaxation. This relaxant response is mediated by NK1 receptors

as it is prevented by CP-96,345 (Blackshaw et al., 1994). The canine LES is contracted by both SP and NKA, whereas NKB is inactive (Sandler et al., 1991). While the contraction in response to NKA is slow in onset, long-last- ing and due to a direct action on the muscle, the response to SP is quick, phasic and blocked by TTX and atropine

(Sandler et $., 1991). It would appear, therefore, that NK, receptors on the muscle of the canine LES are matched by NK, receptors on intramural nerves, a situation which is

similar to that in the rat esophagus in which SP binding sites have been localized to neurons of the SMP (Wieder-

mann et al., 1987).

5.2.1.2. Transduction mechanisms. The transduction mechanisms that underlie the tachykinin-evoked contraction


have been addressed in the guinea-pig and feline esophagus

only. The contractile action of SP on the guinea-pig esophagus muscularis mucosae, which is blocked by Ca*+ removal, but remains unaffected by verapamil (Kamikawa and Shimo, 1984), seems to arise primarily from mobiliza-

tion of intracellular Cal+. The contraction that SP induces in the circular muscle of the feline esophagus (Leander et

al., 1982) is mediated by the G,,GiI type of G-proteins, phosphatidylcholine-specific phospholipase, influx of ex-

tracellular Ca2+ and a protein kinase C-dependent pathway

(Sohn et al., 1995). Importantly, the transduction mecha-

nisms utilized by SP in the LES of the cat are remarkably distinct from those operated in the esophageal body (Sohn et al., 1995). Thus, the motor response of the feline LES to

SP depends on the G,G,, type of G-proteins, a phosphati- dylinositol-specific phospholipase C, inositol trisphos-

phate-mediated release of intracellular Ca*+ and a calmodulin-dependent pathway (Sohn et al., 1995).

5.2.2. Stomach. 5.2.2.1. Tuchykinin actions, tuchykinin receptors and

mediator systems.

5.2.2.1.1. Rat. Tachykinins influence the motor activity

of the rat stomach and pylorus in vitro and in uiuo. Intraperi- toneal or intraarterial injection of SP and other tachykinins

influences gastric emptying and gastrointestinal transit of a

liquid meal in a complex manner, and the prevailing effect (acceleration or inhibition) depends on the concomitant activation of parasympathetic and sympathetic reflexes

(Mange1 and Koegel, 1984; Holzer, 1985; Silkoff et al., 1988; Holzer-Petsche, 1991; Chang et al., 1992; Valdovinos et al., 1993). Close arterial administration of tachykinins

contracts the rat stomach, the order of potency (NKA > SP > SP methyl ester) being indicative of the prevalence of

NK, receptors in this region of the gut (Holzer-Petsche et

al., 1987; Holzer-Petsche, 1991). The contractile effect of

NKA on isolated circular muscle strips of the rat gastric corpus seems to be due to a direct action on the muscle,

while the effect of SP is inhibited by TTX and atropine (Holzer-Petsche et al., 1987). The neural component in the motor response to SP is consistent with the immunohistochemically visualized presence of NK1 receptors on neu-

ronal structures within the MP of the rat stomach (Sternini et al., 1995). The SP-induced excitatory motor response in the circular muscle of the rat antrum and pylorus in vitro

also involves cholinergic neurons and depends in part on the release of 5-HT acting via 5-HT2Rs (Lidberg et al., 1985). The latter finding is reminiscent of the ability of SP to release 5-HT from the stomach of the rainbow trout (Holmgren et al., 1985).

The situation in the longitudinal and circular muscle of the rat gastric fundus is different inasmuch as the motor effects of tachykinins in this region are not affected by TTX and atropine and hence, appear to be purely myogenic responses (Mussap and Burcher, 1993; Smits and Lefebvre, 1994). The rank order of potency (NKA > NKB > SP) and the inability of RP-67,580 to prevent tachykinins from con-

tracting the fundus points to the predominance of NK2 re-

ceptors, which are susceptible to antagonism by MEN- 10,376 and other NK2 antagonists (Mussap and Burcher, 1993; Smits and Lefebvre, 1994). Functional NK, receptors appear to be present on the longitudinal muscle (Smits and

Lefebvre, 1994), but seem to be absent from the circular muscle (Mussap and Burcher, 1993). This functional situation is not quite matched by the autoradiographic distribution of tachykinin receptors in the rat gastric fundus in

which distinct NK1 binding sites are present on the circular

muscle, although NK, receptors prevail in this muscle layer and are the only tachykinin receptors found in the muscu-

laris mucosae. The musularis mucosae may, in addition, contain some NK3 binding sites, whereas the longitudinal muscle layer lacks appreciable binding sites for any tachyki-

nin receptor ligand (Burcher et al., 1986, 1993; Mussap and Burcher, 1993 ) .

5.2.2.1.2. Guinea-pig. The actions of tachykinins in cir-

cular muscle strips of the guinea-pig stomach are complex and involve both excitatory and inhibitory motor responses

(Jin et al., 1993). The relaxant response to NK, agonists is converted to contraction after treatment of the strips with TTX, whereas a TTX/atropine-insensitive contraction is

the only response seen after selective NK2 receptor activa-

tion. The presence of excitatory NK1 and NK2 receptors on the muscle is confirmed by experiments with dissociated

circular muscle cells from the guinea-pig stomach (Jin et al., 1993). Conversely, senktide is very weak in contracting dissociated muscle cells, which is in keeping with the absence

of any NK, receptors mediating contraction of intact muscle strips (Jin et al., 1993).

5.2.2.1.3. Cat. The feline pylorus expresses high numbers of SP binding sites compared with the adjacent regions of

the antrum and duodenum (Rothstein et al., 1991), but the pharmacological identity of these receptors is not known.

Close arterial administration of SP elicits atropine-sensitive contractions of the cat’s stomach and pylorus (Edin et al.,

1980; Lidberg et al., 1983; Barber and Burks, 1987). Analy sis of the motor actions of SP in vitro shows that the circular muscle of the feline corpus, antrum and inferior portion of the pylorus is effectively contracted by SP, whereas the su-

perior part of the pylorus and the longitudinal muscle of the stomach are poorly reactive to the peptide (Merlo and Co- hen, 1989). The SP-evoked contraction of the gastric circular muscle is reduced by TTX and atropine, whereas the motor response of the inferior pylorus is insensitive to atropine, although being blunted by TTX (Merlo and Cohen, 1989).

5.2.2.1.4. Dog. Systemic administration of SP, NKA and NKB to the dog induces phasic contractions of the stomach and stimulates gastroduodenal motility, these effects being inhibited by atropine (Kuwahara and Yanaihara, 1987; Ito et al., 1993; Shibata et al., 1994). Analysis of the actions of tachykinins in strips of longitudinal and circular muscle from the canine antrum shows that SP, NKA and NKB


stimulate both the frequency and amplitude of spontaneous contractions, whereas no effect on tone is observed (Mayer

et al., 1986; Koelbel et al., 1988a). The chronotropic action of tachykinins in either muscle layer appears to result from stimulation of NK1 receptors that are located directly on the muscle cells (Mayer et al., 1986; Koelbel et al., 1988a). The inotropic effect of tachykinins on the circular muscle and that of NKA on the longitudinal muscle are also myo- genie responses that are supposedly brought about by NK,

receptors, whereas the inotropic effect of SP and NKB on the longitudinal muscle is reduced by TTX and atropine

(Mayer et al., 1986; Koelbel et al., 1988a). The presence of tachykinin receptors on neurons is corroborated by the

finding that SP, NKA and NKB are all able to enhance the release of [3H]-ACh from enteric neurons in the canine an-

trum, a response that is inhibited by TTX and enkephalin (Koelbel et al., 1988a,b). Pharmacological dissection of the tachykinin receptors with the use of receptor-selective ago-

nists indicates that in the circular muscle layer of the canine antrum, pylorus and duodenum in ho, NK1 receptors are distributed to both muscle and neurons, whereas NK,

receptors are restricted to the muscle of the antrum and pylorus and NK3 receptors are exclusively expressed on neu-

rons within the antrum and duodenum (Allescher et al.,

1989).

5.2.2.2. Transduction mechanisms. The transduction mechanisms underlying the contractile effect of SP have been

examined with dissociated smooth muscle cells from the toad stomach. The SP-induced contraction is associated with depolarization of the muscle cell membrane and generation of action potentials (Sims et al., 1986). Analysis of the membrane events indicates that SP inhibits a K+ out-

ward current, which decreases the membrane conductance

and causes depolarization (Sims et al., 1986), and increases a high-threshold, slowly inactivating Cal+ current (Clapp

et al., 1989).

5.2.3. Small intestine. 5.2.3.1. Tachykinin actions, tachykinin receptors and

mediator systems. 5.2.3.1.1. Guinea-pig. The longitudinal muscle of the

guinea-pig ileum has long been considered to be a NK, monoreceptor bioassay tissue if tachykinins are tested in the presence of indomethacin and an-opine (Regoli et al., 1984; Holzer-Petsche et al., 1985; Croci et al., 1994a). Pharmaco-

logical and biochemical studies indicate that all tachykinin receptors present on dispersed longitudinal muscle are of the NK, type (Souquet et al., 1987), but this inference is based on the activity of SP, NKA and NKB only and needs to be corroborated by the use of receptor-selective ligands. Notwithstanding the predominance of NK, receptors, there is pharmacological evidence that the longitudinal muscle also contains a few NK2 receptors (Nguyenle et al., 1996).

NKI receptor binding to the longitudinal muscle is easily demonstrated in tissue homogenates or muscle cell suspen- sions (Souquet et al., 1987; Tousignant et al., 1991), but is

less prominent in tissue autoradiograms (Burcher et al.,

1986). Unexpectedly, immunocytochemistry has failed to

show the presence of NK, receptors on the longitudinal muscle of the guinea-pig intestine, which suggests that the receptors on this muscle are a subtype of NK, receptors (Portbury et al., 1996). This contention is supported by pharmacological and ligand binding studies that point to the existence of ‘atypical’ tachykinin receptors in the

guinea-pig gut. One of these receptors is the “septide-sensitive” tachykinin receptor (Petitet et al., 1992; Burcher and

Stamatakos, 1994), which is thought to be a subtype of the

NKI receptor, although its precise identity relative to NK, receptors remains to be established (Glowinski, 1995). An- other receptor that seems to be present on both muscle and nerve is labelled with scyliorhinin II and appears to be an entity separate from typical NK1, NK2 or NK, receptors

(Mussap and Burcher, 1994). The longitudinal muscle layer with attached MP con-

tains another population of tachykinin receptors that are located on enteric neurons and whose pharmacological properties are distinct from the NK1 receptors on the mus-

cle (Holzer and Lembeck, 1980; Nemeth et al., 1983; Fos-

braey et al., 1984; Chahl, 1985a; Laufer et al., 1985; Feath- erstone et al., 1986; Kilbinger et al., 1986). These receptors

have now been characterized as NK, receptors that are stimulated by senktide and other NK, receptor-selective ag-

onists (Guard and Watson, 1987; Guard et al., 1990; Ramirez et al., 1994) and blocked by SR-142,801 (Croci et al., 1995; Emonds-Alt et al., 1994). As reviewed in the companion article (Holzer and Holzer-Petsche, 1997), acti-

vation of NKI receptors causes release of ACh from myenteric neurons, which is accompanied by a TTX-sensitive

contraction of the longitudinal muscle (Holzer and Lem- beck, 1980; Laufer et al., 1985; Guard and Watson, 1987). Atropine also inhibits the contraction due to NK3 receptor

activation, but is less effective than TTX (Laufer et al.,

1985; Guard and Watson, 1987), because the contractile response to NK, agonists is mediated by release of both ACh and endogenous tachykinins stimulating NK, receptors on the muscle (Guard and Watson, 1987; Corsi et al.,

1994; Croci et al., 1995). Binding studies have confirmed

the presence of NKI receptors in the MP/longitudinal muscle layer of the guinea-pig small intestine (Burcher et al.,

1986; Guard et al., 1990). Furthermore, the MP contains NK, receptors (Burcher et al., 1986; Portbury et al., 1996), which when stimulated give rise to ACh release (Guard and Watson, 1991).

Like the longitudinal muscle, the circular muscle of the guinea-pig small intestine is potently stimulated by tachykinins. In full thickness segments of the small intestine, it is primarily phasic contractions, superimposed on a tonic response, that are induced by SP, NKA and NKB. The responses to SP and NKB are inhibited by TTX and atropine, whereas the effect of NKA is myogenic in origin (Holzer et al., 1980b; Costa et al., 198513; Barth6 and Pethii, 1992; Su- zuki et al., 1994b). Further analysis involving tachykinin receptor-selective agonists has shown that all three tachyki-


nin receptors contribute to the contractile effects of tachykinins in circular muscle strip preparations (Maggi et al., 1990a). The motor response to NK, receptor activation

is partially reduced by TTX, but left unaffected by atropine, which suggests that NK, receptors are present on both non-

cholinergic neurons and circular muscle cells (Maggi et al.,

1990a). Immunocytochemistry has confirmed that NK, re-

ceptors are present on myenteric and submucosal neurons of the guinea-pig gut, whereas in the circular muscle layer, it is only interstitial cells that express immunoreactive NK1

receptors (Portbury et al., 1996). This observation has been taken to suggest that NK1 receptor-mediated excitation of

the circular muscle may be indirect through activation via interstitial cells (Portbury et al., 1996).

NK, receptors seem to be exclusively present on muscle

cells, since contractions evoked by NK2 agonists are not af-

fected by TTX, atropine or the N-type calcium channel blocker o-conotoxin (Maggi et al., 1990a, 1994d). Interest-

ingly, the sensitivity of the circular muscle to NK, receptor stimulation seems to be counteracted by endogenous pros-

tanoids that relax the muscle (Maggi et al., 1994d). NK, receptors, on the other hand, are restricted to en-

teric neurons, as the contraction induced by senktide is abolished by TTX and reduced by atropine and o-conotoxin (Maggi et al., 1990a, 1993a). Since removal of the

MP attenuates, but does not prevent, contractions due to NK3 stimulation, it is thought that NKI receptors are present on somata and axons of myenteric motor neurons

(Maggi et al., 1994d). These motor neurons utilize both ACh and endogenous tachykinins as their neuroeffector

transmitters, since combined administration of atropine and a NK, antagonist is necessary to significantly blunt the

motor response to senktide (Maggi et al., 1994d). Abolition of the contractile effect of senktide discloses a relaxant response to NK, activation, which arises from stimulation of inhibitory motor pathways to the circular muscle (Maggi et

al., 1993a, 1994d). The distribution of NK1 and NK, receptors to the circular

muscle layer and of NK1 and NK3 receptors to myenteric

pathways supplying the muscle seems to be uniform from the duodenum down to the ileum (Maggi et al., 1990a, 1994e; Zagorodnyuk et al., 1995) an is in overall accordance with d

the autoradiographic localization of binding sites for SP to

the circular muscle and MP, whereas NKA binding sites occur on the muscle only (Burcher et al., 1986; Burcher and Bomstein, 1988). Unresolved issues of tachykinin receptor pharmacology in the circular muscle of the guinea-pig small intestine relate to the functional implications of NK, receptors on interstitial cells, the apparent absence of typical NK1 receptors from circular muscle cells (Portbury et al., 1996), the identity of the “septide-sensitive” tachykinin receptors (Maggi et al., 199313, 1994e) and the possible intraspecies heterogeneity of NK2 receptors (Maggi et al., 1994e).

As is expected from their prominent action on the circu-

lar muscle, tachykinins influence peristaltic motor activity in isolated segments of the guinea-pig small intestine in a receptor-selective manner (Holzer et al., 1995). NK2 and

particularly NKj agonists facilitate intestinal peristalsis, whereas SP first stimulates and then inhibits peristaltic motility. The facilitatory effect of SP is prevented by atropine (Barth6 et al., 1982b) and involves stimulation of NK, receptors (Holzer et al., 1995), while the secondary inhibitory

effect is mimicked by the NK1 agonist SP methyl ester and inhibited by the NK, antagonist CP-99,994 (Holzer et al.,

1995).

5.2.3.1.2. Rat. Experiments with dispersed longitudinal and circular muscle cells indicate that the musculature of the rat small intestine expresses all three tachykinin receptors (Hellstriim et al., 1994). This inference derives from

experiments with selective receptor protection and from the observations that SP, NKA and NKB, as well as NK,,

NK2 and NK3 receptor agonists are all able to potently contract the muscle (Hellstr6m et al., 1994). The presence of

NK1, NK, and NK, receptors on the longitudinal muscle has been confirmed by the ability of tachykinin receptor- selective agonists to contract isolated segments of the rat il-

eum in a TTX-, atropine- and o-conotoxin-insensitive manner (Willis et al., 1993). NK, receptor activation

evokes a predominantly tonic motor response, while NKI receptor activation is followed primarily by phasic contractions, and NK2 receptor agonists cause a mixed phasic/tonic

stimulation of the muscle (Willis et al., 1993). These agonist-dependent motor responses in the in vitro ileum are different from those seen in the in viva duodenum in which

phasic contractions are the predominant response to NK, agonists, whereas stimulation of NK2 receptors gives rise to

a tonic type of contraction (Maggi et al., 1986). Functional in vitro studies with receptor-selective ago-

nists and antagonists show that the circular muscle of the rat small intestine bears both NK1 and NK2 receptors (Maggi and Giuliani, 1995). The NK2 agonist @Alas]- NKA-(4-10) accelerates transit through the rat small intestine (Tramontana et al., 1994), which attributes NK, receptors a particular role in propulsive motility. In the rat

duodenum, this receptor is unusual, as it is comparatively insensitive to NK2 receptor antagonists (Croci et al., 1994a; Rahman et al., 1994), which, however, are active in other

regions of the rat gut (Croci et al., 1994a,b). The NK, receptors have been characterized by ligand binding to tissue homogenates of the rat duodenum and ileum and localized

by autoradiography to the longitudinal and circular muscle layers (Burcher et al., 1986; Bergstram et al., 1987; Emonds- Alt et al., 1993b). NK1 receptors are found in the inner part of the circular muscle layer, in which they may be confined to interstitial cells, and in the enteric nerve plexuses of the rat small intestine (Burcher et al., 1986; Stemini et al., 1995). The presence of NK, receptors has been demonstrated by functional studies only (Laufer et al., 1988; Hell- str6m et al., 1994).

An observation whose significance is not fully understood yet relates to NPy, which is considerably more potent than NKA in contracting the longitudinal muscle of the rat duodenum, in disrupting migrating motor complexes and in


causing irregular spiking activity, whereas SP and SP methyl ester are hardly active (Rahman et al., 1994). The actions of

NPy are reduced by hexamethonium and atropine, but are left unaffected by NKi or NKz antagonists (Rahman et al., 1994), and it remains open whether they are mediated by NK, or other tachykinin receptors that are present on both muscle and nerve.

5.2.3.1.3. Dog. Autoradiography has identified the exist-

ence of binding sites for SP and NKA, but not NKB, in the canine small intestine. While SP and NKA binding sites are present on the muscularis mucosae and external muscle

layers, it is only SP binding sites that are seen in the MP (Mantyh et al., 1988). The binding of SP to the circular

muscle appears to be confined to the inner surface of this muscle layer (Mantyh et al., 1988) and has been further

studied in plasma membrane fractions and characterized as reflecting a NK1 receptor site (Muller et al., 1988). Func-

tional studies indicate that both NKr and NKz receptors are operant in the circular muscle of the canine ileum in vitro and in viva and, when stimulated, cause excitation of the

muscle (Muller et al., 1988; Watson et al., 1994; Daniel et al., 1995). The pharmacology of these receptors requires further study, though, as neither the NK, antagonist CP-

96,345 nor the NKz antagonist SR-48,968 is able to inhibit the contractile effect of SP or a NKr agonist, whereas the motor response to NKA is prevented by a combination of

the two antagonists (Daniel et al., 1995). NKI agonists are inactive in altering motility of the ileum (Daniel et al.,

1995), while in the duodenum, they can induce neurally mediated contractions (Allescher et al., 1989).

5.2.3.1.4. Humans. Functional studies with isolated gut segments and autoradiographic receptor mapping indicate

that both NK, and NKz receptors are expressed in the human small intestine. Binding sites for SP occur on the longitudinal muscle, MP, circular muscle and muscularis mucosae, whereas binding sites for NKA are present on the

muscle, but absent from the plexus (Gates et al., 1988). The

longitudinal muscle of the ileum is contracted by NKA, SP and NKz receptor-selective agonists, whereas NKr agonists

are virtually inactive (Maggi et al., 1989b). This observation and the much higher potency of NKA compared with

that of SP show that it is exclusively NKz receptors that determine the excitatory action of tachykinins on the longitudinal muscle. The situation in the circular muscle is not much different, because also here NKz receptors seem to be the prevailing species of tachykinin receptor, although the existence of functional NK, receptors has also been demon-

strated. The circular muscle contracts in response to SP, NKA, as well as NKr and NKz receptor-selective agonists (Maggi et al., 1990b, 1992b). These motor responses apparently are brought about by a direct action on the muscle,

since they are not affected by atropine (Maggi et al., 1990b). NK, receptors seem to be absent from the human small intestine because NK3 agonists fail to stimulate motor activity (Maggi et al., 1990b) and specific binding sites for NKB are not demonstrable (Gates et al., 1988).

5.2.3.1.5. Other species. Excitatory motor effects of ta-

chykinins on the small intestine have been documented in

many species. NK, type binding sites are present in the cir-

cular, and to a lesser extent in the longitudinal, muscle of the cat’s small intestine and in the ileocecal sphincter of

this species (Rothstein et al., 1989, 1991). The contractile effect of SP on the feline ileum and ileocecal sphincter in-

volves cholinergic neurons, whereas that in the adjacent colon is a myogenic response (Rothstein et al., 1989). NK, binding sites have also been localized to the ileal smooth muscle of sheep (Keefer and Mong, 1990), and it is by NK,

receptors that the tachykinin-induced stimulation of motility in the rabbit jejunum is predominantly brought about

(Holzer-Petsche et al., 1985). In contrast, the tachykinin- induced contraction of the circular muscle in the horse

small intestine relies primarily on activation of NKz and

some NK, receptors (Belloli et al., 1994). NK, receptors mediating contraction of the longitudinal muscle have also been identified in the mouse small intestine (Goldhill et al.,

1995a).

5.2.3.2. Transduction mechanisms. At the level of the muscle cell membrane, SP evokes depolarization, initiates

slow waves and elicits action potentials in the longitudinal and circular muscle of the guinea-pig and porcine small intestine (Bauer and Kuriyama, 1982; Fujisawa and Ito, 1982;

Niel et al., 1983; Wechsung and Houvenaghel, 1992; Zagor- odnyuk et al., 1995). Patch clamp analysis of whole longitu-

dinal muscle cells from the guinea-pig ileum shows that SP induces an inward current that is mainly carried by external

Na+ (Nakazawa et al., 1990). The SP-induced depolarization of the circular muscle in the guinea-pig ileum is followed by a prolonged increase in membrane resistance,

which is thought to arise from a decrease in the K+ conductance (Niel et al., 1983). Depolarization and discharge of action potentials are likewise seen when the circular muscle

is exposed to NK, or NK, receptor agonists (Zagorodnyuk et al., 1995). The spiking activity and associated contrac-

tion are prevented by nifedipine, whereas the depolariza-

tion is attenuated, but not abolished (Maggi et al., 1994e,g; Zagorodnyuk et al., 1995). It is worth noting in this context that the signaling pathway operated by NKz receptors dif-

fers markedly between the circular muscle of the guinea-pig ileum and colon inasmuch as the contraction caused by @Alas]-NKA-(4-10) ’ p is revented by nifedipine in the il-

eum, but left unaltered in the colon (Maggi et al., 1994e). The SP-evoked contraction of the guinea-pig ileum lon-

gitudinal muscle consists of an initial peak contraction that

is soon followed by a submaximal tonic contraction. This course of force development is paralleled by a phasic/tonic course of increased intracellular Ca*+ concentration (Mat-

thijs et al., 1990). A na ly sis of the underlying transduction mechanisms has shown that the initial peak contraction

depends on release of Cal+ from intracellular stores, since it persists after removal of external Ca*+ or K+-induced depolarization of the muscle. Conversely, the delayed tonic motor response relies on influx of Ca*+ as it is prevented by re-


moval of external Ca2+ or blockade of voltage-sensitive

calcium channels (Holzer and Lippe, 1984; Matthijs et al., 1990). The time course of the initial peak contraction is closely paralleled by SP-induced generation of inositol

phosphates, which suggests that the initial mobilization of intracellular Cal+ is mediated by inositol trisphosphate (Holzer and Lippe, 1985). C onsistent with this inference is the observation that the SP-evoked formation of inositol

phosphates is not changed by removal of external Ca*+ or blockade of voltage-sensitive calcium channels (Holzer and

Lippe, 1985; Watson et al., 1990). Protein kinase C does

not seem be of relevance for the initial peak contraction,

but exerts a negative control over the delayed phase of tonic contraction (Holzer and Lippe, 1989; Sasaguri and Watson, 1990).

Stimulation of polyphosphoinositide turnover seems to be the relevant second messenger mechanism for the NK1, NK,, NK3 and “septide-sensitive” tachykinin receptor in

the guinea-pig ileum longitudinal muscle/MP layer (Guard et al., 1988; Barr and Watson, 1993; Petitet et al., 1993). SP-induced generation of inositol phosphates is also seen in

the rat ileum longitudinal muscle (Watson, 1984), whereas SP, NK,, NK2 and NK3 receptor-selective agonists fail to al-

ter cyclic AMP formation (Watson, 1984; HellstrGm et al., 1994). Experiments with dispersed muscle cells from the rat

small intestine show that NK,, NK, and NK, receptors are all coupled to the same transduction mechanism that

causes muscle contraction via elevation of the intracellular Cal+ concentration (HellstrGm et al., 1994). There are some differences, though, between the tachykinin-operated signaling pathways in the longitudinal and circular muscle layers of the rat small intestine. The tachykinin-evoked

contraction of the longitudinal muscle is suppressed by removal of external Ca*+ or exposure to calcium channel

blockers, while the motor response of the circular muscle is

resistant to inhibition by methoxyverapamil (Willis et al.,

1993; Hellstriim et al., 1994).

5.2.4. Large intestine. 5.2.4.1. Tachykinin actions, tachykinin receptors and mediator system. 5.2.4.1.1. Guinea-pig. Experiments with SP, NKA and ta-

chykinin receptor-selective agonists indicate that the longitudinal muscle layer of the guinea-pig cecum (tenia ceci) and colon contains NK1, NK2 and NKI receptors (Hall and Morton, 1991; Shuttleworth et al., 1991; Briejer et al., 1993; Croci et al., 1995). SP, NKA, NK, and NK, agonists act directly on the muscle, as their contractile effects are left unaltered by TTX, hyoscine or atropine (Hall and Morton, 1991; Shuttleworth et al., 1991; Briejer et al., 1993). The use of receptor-selective antagonists has shown that NK, and NKZ receptors are the prevailing species of tachykinin receptor on the muscle (Briejer et al., 1993; Croci et al.,

1995). The motor responses mediated by these two receptors are qualitatively different inasmuch as the SP-evoked contraction is fast and phasic, whereas the contractile effect of NKA is slow in onset and of long duration (Shuttleworth

et al., 1991). Although some NK, receptors may be present on the longitudinal muscle (Hall and Morton, 1991), they do not seem to be of functional importance because the

bulk of excitatory motor response to NKI agonists is neurally mediated and prevented by ?TX (Croci et al., 1995). A combination of atropine and SR-48,968 likewise is able to suppress NK3 agonist-evoked contractions, which dem- onstrates that activation of NK, receptors on enteric neu-

rons causes contraction through release of ACh and endogenous tachykinins acting via NK2 receptors on the muscle

(Croci et al., 1995). Binding sites for SP and NKA are found on the longitu-

dinal and circular muscle of the guinea-pig colon, with additional binding sites for SP being present in the muscularis

mucosae and the MP and SMP (Burcher et al., 1986). The expression of NK1 receptors by myenteric and submucosal

neurons has been confirmed immunocytochemically, whereas immunoreactive NKI receptors seem to be absent from the longitudinal and circular muscle layers (Portbury et al., 1996).

As in the longitudinal muscle, the myogenic contraction of the circular muscle in response to tachykinins is mediated by NK1 and NK2 receptors (Maggi et al., 1994b,e). Interest-

ingly, SP gives rise to a fast phasic contraction, while the

motor response to NKA is slow in onset and sustained, a di- vergence that is related to different transduction mechanisms being coupled to NK, and NK, receptors (Maggi et al.,

1994s). Pharmacological analysis with receptor-selective antagonists suggests that there are two NK1 receptor subtypes in the circular muscle of the guinea-pig ileum and colon and that the NK, receptors in the colon are different

from those in the ileum (Maggi et al., 1994e). The motor activity of the guinea-pig colon circular muscle is controlled

furthermore by NK, receptors, which are exclusively located on enteric neurons and which cause excitation of the mus-

cle via release of ACh (Zagorodnyuk and Maggi, 1995).

5.2.4.1.2. Rat. Cross-desensitization between SF’, NKA, septide and senktide suggests that the motility of the rat coionic circular muscle is under the control of all three tachykinin receptors (Chang et al., 1991). This view is in line with the expression of mRNAs for all three tachykinin re-

ceptors in the rat colon, the predominating forms coding for NK1 and NK2 receptors (Tsuchida et al., 1990). Autora- diographic binding sites for SP and NKA prevail in the circular muscle, while little binding is seen in the longitudinal muscle (Burcher et al., 1986; Geraghty and Burcher, 1992). The MP and SMP display many binding sites for SP (Burcher et al., 1986) that immunohistochemically have been characterized as NK1 receptors (Sternini et al., 1995). The neurons bearing NK, receptors are frequently in close contact with varicose nerve fibers immunoreactive for SP (Sternini et al., 1995). The presence of NK, receptors on muscle and nerve is in keeping with the effects of SP and NKA to cause both myogenic and cholinergically mediated contractions of the rat isolated colon circular muscle (Chang et al., 1991; Scheurer et al., 1994). In piivo spike ac-

Tachvkinins and Gut Motor Functions 191

tivity and motility of the rat colon is stimulated by NKI, but not NK1, agonists (Julia et al., 1994), and the facilitatory ef-

fect of tachykinins on defecation in the conscious rat seems likewise to be a predominantly NK2 receptor-mediated response (Croci et al., 1994b). The tachykinin-induced contraction of the muscularis mucosae of the rat colon is also due to stimulation of NKZ receptors (Astolfi et al., 1993), which is fitting with the autoradiographic observation that only binding sites for NKA are present in this layer of the

colon (Burcher et al., 1986).

5.2.4.1.3. Dog. The longitudinal and circular muscle lay

ers of the colon express mRNAs for all three tachykinin re-

ceptors, whereas in the muscularis mucosae and SMP, NK1 and NKs, but not NKL, receptor mRNAs are found (Khan et al., 1995). Autoradiographically, though, no NKB binding sites have been visualized in the colon, while the vari-

ous muscle layers contain both SP and NKA binding sites, and the only binding sites localized to the enteric nerve

plexuses are SP binding sites (Mantyh et al., 1988). The excitatory effects of SP in the canine colon in viva include

both tonic and phasic contractions, which come about in part by a direct action on the muscle and in part by stimula-

tion of cholinergic neurons (Hou et al., 1989; Keef et al., 1992). The order of potency with which tachykinins (NKA > NKB > SP) contract the circular muscle indicates a predominance of NK2 receptors (Shuttleworth et al., 1993).

This notion is supported by the contractile activity of the NK2 receptor agonist @Alas]-NKA-(4-10) and its suscepti- bility to blockade by SR-48,968 (Basilisco and Phillips, 1994). The stimulant action of SP on the muscularis muco-

sae of the canine proximal colon is not blocked by TTX and hence, likely a myogenic response (Angel et al., 1984).

5.2.4.1.4. Humans. The longitudinal and circular muscle layers of the human isolated colon are very sensitive to

NKA and NK2 receptor agonists, while SP, NK1 and NK3 agonists are comparatively weak in causing a contraction (Giuliani et al., 1991; KGlbel et al., 1994). It appears, there-

fore, as if tachykinins contract the human colon predominantly via stimulation of NK2 receptors. However, the muscle layers of the human colon exhibit not only autoradiographically visible binding sites for NKA, but also

specific binding sites for SP, which are thought to reflect NK, receptors (Gates et al., 1988; Korman et al., 1989;

Mantyh et al., 1994). Add t i ional SP binding sites occur in

the enteric nerve plexuses of the human colon (Gates et al., 1988), which is likely to explain why the stimulant motor

responses to SP, NKA and NKB are partially inhibited by TTX, atropine or hexamethonium (Kiilbel et al., 1994).

5.2.4.1.5. Other species. Tachykinin-induced contractions of the longitudinal muscle in the hamster colon are mediated predominantly by NKZ receptors (Maggi et al., 1992a). Close arterial administration of tachykinins contracts the feline distal colon in ho, an effect that is most potently evoked by NKA and that is reduced by TTX or atropine, but not hexamethonium (Fgndriks and Delbro, 1985; Hell-

striim et al., 1991). The circular muscle of the feline proximal colon in vitro is stimulated by SP, septide and NKA,

but not by senktide, which together with cross-desensiti-

zation experiments, suggests that NK1 and NK,, but not NKI, receptors are operant in the cat’s colon (Chang et al., 1991). Although the motor responses to SP and NKA in

vitro are reduced by atropine (Chang et al., 1991), it appears as if the in viva contractile effect of SP in the proximal colon of the cat is largely independent of nerves (Rothstein et al.,

1989), which is in keeping with the abundance of SP bind-

ing sites on the circular muscle of this gut region (Rothstein

et al., 1991). The longitudinal and circular muscle of the rabbit colon

responds to SP, NKA and NKB with an increase in the amplitude of phasic contractions and with a tonic contraction if higher agonist concentrations are employed (Koelbel et al., 1989). The SP-evoked longitudinal contraction of the

distal colon, which is particularly sensitive to the peptide,

involves a myogenic component that supposedly is mediated by NK1 receptors and a neurogenic component that is

inhibited by hexamethonium and atropine (Koelbel et al., 1989). The SP-evoked stimulation of cholinergic neurons is corroborated by the ability of tachykinins to increase the release of ACh (Koelbel et al., 1989). The effect of SP,

NKA and NKB to contract the circular muscle of the rabbit colon, though, seems to be independent of nerves (Koelbel

et al., 1989). Cholinergic contractions in response to SP are also seen in the pig’s colon (Bayat et al., 1991).

5.2.4.2. Transduction mechanisms. Sucrose gap experiments indicate that SP or NK1 agonists depolarize the tenia ceci and circular muscle of the guinea-pig colon, increase the

membrane conductance and cause action potential activity (Benham and Bolton, 1983; Zagorodnyuk et al., 1994;

Zagorodnyuk and Maggi, 1995). The rise of conductance is

reduced after the tenia has been exposed to media deficient in Na+ or Cl- (Benham and Bolton, 1983). Depolarization, enhancement and prolongation of the slow wave plateau potential and induction of spike activity are also seen in the circular muscle of the canine colon (Huizinga et al., 1984),

and intracellular recordings show that SP and NKA excite myocytes of the canine colon predominantly by activation

of a nonselective cation conductance that leads to membrane depolarization (Lee et al., 1995). Analysis of the membrane events in longitudinal muscle cells from the rabbit colon reveals an additional action of SP on Ca*+-acti-

vated K+ channels (Mayer et al., 1989, 1990~). Low concentrations of SP, which are subthreshold for contraction, activate large conductance K+ channels, an effect that is blocked by nifedipine or Ca*+ removal. Conversely, concentrations of SP that cause contraction lead, after a transient activation, to a prolonged inhibition of K+ channels, which is prevented by Ca*+ removal, but not nifedipine (Mayer et al., 1989, 1990~). In addition, NK, receptors are coupled, via pertussis toxin-sensitive G-proteins, to large conductance Cl- channels that are activated in response to receptor stimulation (Sun et al., 1993).


Investigations in the circular muscle of the guinea-pig colon have disclosed that there are fundamental differences in the transduction mechanisms operated by NK, and NK2 receptors. The depolarization caused by the NK, agonist

[PAlas]-NKA-(4-10) is considerably smaller than that

evoked by [Sarg]SP sulphone and not, like that in response to NK1 receptor stimulation, associated with an increase in

membrane conductance (Zagorodnyuk et al., 1994). Fur- thermore, [PAlas]-NKA-(4-10) fails to evoke action potentials in most instances, while this is an invariable com-

ponent of the excitatory response to [Sarg]SP sulphone. Although the action potentials evoked by both NK1 and NK2 receptor stimulation are prevented by nifedipine, it is only the contraction caused by [Sarg]SP sulphone that is

suppressed by nifedipine, whereas the contractile effect of @Alas]-NKA-(4-10) is hardly reduced (Maggi et al., 19948;

Zagorodnyuk et al., 1994). Removal of external Ca2+, however, depresses depolarization and contraction seen in re-

sponse to both NK, and NK, agonists. It follows that activation of the NK, (Zagorodnyuk et al.,

1994) and “septide-sensitive” (Maggi et al., 1994e) receptor

contracts the circular muscle of the guinea-pig colon via an electromechanical coupling process, whereas the excitatory response to NK, receptor stimulation is preferentially due

to pharmacomechanical coupling (Zagorodnyuk et al., 1994; Zagorodnyuk and Maggi, 1995). This differential sig-

naling is reflected in the fast and slow motor responses caused by SP and NKA, respectively (Maggi et al., 1994s). Similar variations in the transduction mechanisms oper-

ated by different tachykinin receptors have been noted in

the mouse distal colon in which the SP-induced myogenic contraction of the longitudinal muscle is prevented by

nifedipine or Ca*+ removal, whereas the response to kassinin is resistant to these manipulations (Fontaine and Leb- run, 1989). In terms of second messenger mechanisms, it appears that depolarization-independent formation of inosi- to1 phosphates is causally involved in the tachykinin-induced

contraction of the guinea-pig tenia ceci (Hall and Morton, 1991). Subsequent release of Ca*+ from internal stores and

activation of a calmodulin signaling pathway are essential for the ability of SP to contract the internal anal sphincter of the rabbit (Bitar et al., 1990).

5.3. Excitatory Motor Effects

of Tachykinins in the BitiaT Tract

Tachykinins have long been known to contract the gallbladder and sphincter of Oddi in humans, dogs, cats, rabbits and guinea-pigs (Starke et al., 1968; Mate et al., 1986; Shook and Burks, 1986; Feeley et al., 1987; Meldrum et al., 1987; Dahlstrand et al., 1988; Hashimoto et al., 1988; Guo et al., 1989; Maggi et al., 1989a; Yau, 1990). There is broad agreement that the contractile effect of tachykinins on the gallbladder is due to a direct action on the musculature because SP is able to contract isolated muscle cells from the canine gallbladder (Severi et al., 1988) and the tachykinin- evoked contraction of the gallbladder is left unaltered by

TTX and atropine (Mate et al., 1986; Meldrum et al., 1987; Dahlstrand et al., 1988; Yau, 1990). In the guinea-pig gallbladder, though, SP is able to depolarize neurons (Mawe, 1990) and NKA causes release of ACh, which, however, contributes little to the contractile effect of the peptide

(Yau, 1990).

The tachykinin-evoked contraction of the guinea-pig gallbladder is mediated by NK, receptors, a contention sup-

ported by the high potency of NKA compared with that of SP and by the use of tachykinin receptor-selective agonists and antagonists (Shook and Burks, 1986; Maggi et al.,

1989a,c; Yau, 1990; Patacchini and Maggi, 1992; Patac- chini et al., 1994). The observation that an inhibitor of protein kinase C prevents the contractile effect of NKA points

to an involvement of the diacylglycerol second messenger pathway in the action of the peptide (Yau, 1990). The ef-

fect of SP, but not that of NKA, is enhanced by thiorphan and captopril (Maggi et al., 1989a).

5.4. Inhibitory Motor Efiects of Tachykinins in the (jut

5.4.1. Inhibitory effects due to mechanisms that have not been fully identified. Although excitatory motor effects are the prevailing responses that tachykinins elicit in the gut, it is now obvious that these peptides can also inhibit gastrointestinal motor activity. These inhibitory ef-

fects are invariably the result of tachykinins acting on neurons and are brought about either by stimulation of

inhibitory neural pathways to the muscle or by prejunctional interruption of excitatory relays. One of the first re-

ports in this respect came from Fox and Daniel (1986), who noted that close arterial injection of low doses of SP, which

are devoid of contractile activity, inhibit spontaneous and electrically stimulated motor activity in the canine small intestine in uiuo. The tachykinin receptor causing inhibi-

tion of motility has been characterized as being of the NK1 type (Fox et al., 1986; Daniel et al., 1995). Evidence for a neurogenic inhibition of motor activity has also been obtained in the canine colon (Hou et al., 1989) and feline ile-

ocecal junction (Rothstein et al., 1989), in which SP-induced contractions are augmented by ganglionic blockade, and in the guinea-pig ileum (Burcher and Stamatakos, 1994) and

rat distal colon (Scheurer et al., 1994) in which the contractile effects of septide and SP, respectively, are enhanced by -I-TX.

Since the inhibitory motor action of SP in the canine intestine is prevented by atropine and TTX, it is held that the peptide releases ACh to act presynaptically on muscarinic acetylcholine autoreceptors that inhibit excitatory neuromuscular transmission (Fox and Daniel, 1986). This conjecture is compatible with the failure of tachykinins to release VIP, a relaxant transmitter of inhibitory motor neurons (Watson et al., 1994). It is not known yet which mediators are responsible for the delayed inhibition of peristalsis, which in the guinea-pig isolated ileum is caused by SP and the NK1 agonist SP methyl ester (Holzer et al., 1995). It has been noted previously that when the initial stimulant effect of SP on peristalsis is prevented by atro-


pine, inhibition of peristaltic motor activity is the only re-

sponse evoked by the tachykinin (Barth6 et al., 198213).

5.4.2. Inhibitory effects due to prejunctional inhibition of transmitter release. Another mechanism by which tachykinins can interrupt excitatory neurotransmission to gastrointestinal muscle is portrayed by the ability of SP to in-

hibit electrically induced release of ACh from the canine gastric antrum (Mayer et al., 1990b) and the MI? of the guinea-pig small intestine (Kilbinger et al., 1986; Loffler et al., 1994). The tachykinin receptor involved in either effect

has been characterized as being of the NK1 type (Kilbinger et al., 1986; Mayer et al., 1990b; Loffler et al., 1994). Prejunctional inhibitory effects of tachykinins, though, are

not exclusively linked to NK, receptors (Fig. 2). Thus, it

has been shown that after occlusion of any excitatory effect, senktide inhibits electrically evoked cholinergic twitch

contractions in the guinea-pig colon circular muscle (Giuliani and Maggi, 1995). This prejunctional effect of the NK, agonist does not depend to any significant degree on the formation of NO (Giuliani and Maggi, 1995). The observation that atropine-resistant off-contractions of the

human colon in response to EFS are increased after desensitization of the strips to NKA, but not SP, has been taken to

suggest the existence of prejunctional NK2 receptors on noncholinergic enteric neurons (Kolbel et al., 1994).

5.4.3. Inhibitory effects due to stimulation of inhibitory enteric neurons. An important mechanism by which tachykinins can inhibit gastrointestinal motor activity is re-

flected by the ability of these peptides to activate inhibitory motor pathways to the muscle. This conjecture has been substantiated by the immunocytochemical colocalization of NK1 receptors and NO synthase in enteric neurons of the guinea-pig gut, NO being a transmitter of descending interneurons and inhibitory motor neurons (Portbury et al.,

1996). Fittingly, activation of NKi and NK, receptors relaxes the circular muscle of the guinea-pig stomach through

stimulation of inhibitory motor neurons (Jin et al., 1993).

This inhibitory motor effect is associated with a release of VIP and increased formation of NO (Fig. 2). Analysis of the interaction between these relaxant messenger molecules indicates that although NO in part may contribute to the release of VIP, the major role of NO is to mediate the relaxant action of VIP in the myocytes (Jin et al., 1993).

NK2 agonists fail to relax the muscle, but attenuate the VIP release, NO production and relaxation caused by NK, and

NK, receptor agonists (Jin et al., 1993). These data suggest that muscle relaxation arises from stimulation of excitatory NK, and NK, receptors on inhibitory motor neurons that

are negatively controlled by prejunctional inhibitory NK2 receptors (Jin et al., 1993).

NK3 receptor-evoked stimulation of inhibitory motor neurons releasing NO can also be demonstrated in the circular muscle of the guinea-pig small and large intestine and the rat colon (Maggi et al., 1993a, 1994d,f; Scheurer et al., 1994; Giuliani and Maggi, 1995). The contraction of the

guinea-pig ileum circular muscle, which normally is in-

duced by senktide, is converted to relaxation when the con-

tractile effect of the NKs agonist is prevented by o-conotoxin (Maggi et al., 1993a, 1994d). The relaxation is prevented by TTX and an inhibitor of NO synthase (Maggi

et al., 1993a). A similar mechanism depending on the formation of NO as relaxant neurotransmitter operates in the circular muscle of the guinea-pig colon (Maggi et al., 1994f;

Giuliani and Maggi, 1995). The relaxation that NK, receptor agonists elicit under conditions that prevent cholinergic, adrenergic, NKi and NK2 receptor-mediated neu- retransmission is associated with a hyperpolarization of the

circular muscle (Maggi et al., 1994f; Zagorodnyuk and

Maggi, 1995). Since the effect of senktide to cause NO-dependent relaxation of the muscle is more potently antagonized

by SR-142,801 than the NO-independent effect to inhibit

cholinergic twitches, an intraspecies heterogeneity of NK3 receptors in the guinea-pig enteric nervous system has been

proposed (Giuliani and Maggi, 1995). Another line of work has shown that NK, receptors re-

siding on enteric neurons may be responsible for an additional type of inhibitory motor response in the circular

muscle of the guinea-pig colon (Fig. 2). When cholinergic, adrenergic and NK, receptor-mediated neurotransmission is

occluded, NKA causes depolarization of the muscle, which is superimposed by a series of inhibitory junction potentials

(Zagorodnyuk and Maggi, 1995). The inhibitory component of the response to NKA is brought about by NK2 receptors and is abolished by TTX or a combination of

apamin and an inhibitor of NO synthase. These findings in-

dicate that NKz receptors are present on enteric neurons and that activation of these receptors stimulates inhibitory nonadrenergic noncholinergic (NANC) motor neurons to the circular muscle of the guinea-pig colon (Zagorodnyuk and Maggi, 1995).

5.4.4. Inhibitory effects due to stimulation of sympathetic neurons. Tachykinins can inhibit gastrointestinal motil-

ity by stimulating sympathetic neurons supplying the gut. Intravenous injection of tachykinins causes a transient relaxation of the rat duodenum in uiuo, which is followed by phasic and tonic contractions of the muscle (Giuliani et al., 1988). Pharmacological analysis indicates that the inhibi-

tory motor response is mediated by NK, receptors and brought about by stimulation of noradrenergic sympathetic neurons reaching the duodenum via the celiac ganglion (Giuliani et al., 1988). This observation needs to be seen in

context with the ability of SP, NKA and senktide to depolarize postganglionic neurons in the prevertebral sympathetic ganglia (Otsuka and Yoshioka, 1993). While the receptors responsible for this effect in the rat prevertebral

ganglia have not been identified yet, it is NKr and NKs receptors that mediate the depolarizing effect of tachykinins in the guinea-pig celiac and inferior mesenteric ganglion (Zhao et al., 1993). If the tachykinin-evoked depolarization is strong enough to trigger action potentials, it is likely that


propagated activity in postganglionic sympathetic nerve fibers reaches the gut and causes shutdown of motility.

5.5. Physiological Roles of Tachykinins in Cjastrointestind Motor Activity 5.5.1. Roles of tachykinins released from enteric neurons.

5.5.1.1. Involvement oftachykinins in motor res@mses to ekctic$ OT chemical nerve stimulation. The pioneering observation of

Paton and Zar* that the atropine/hyoscine-resistant contractions, which in the guinea-pig ileum longitudinal muscle can

be evoked by trains of electrical pulses, are attenuated by SP

desensitization, has since been reproduced in many laborato- ries (Franc0 et al., 1979; Barthi, et al., 1982d; Gintzler and

Scalisi, 1983; Mat&k and Bauer, 1986; Jin et al., 1989). The implication of endogenous SP or NKA has been proved by the use of tachykinin antagonists (BjGrkroth, 1983; Gintzler and Hyde, 1983; Taylor and Kilpatrick, 1992). It follows

that tachykinins are released from enteric neurons and contribute to the electrically evoked contraction, although this

contribution is revealed only when the prevailing cholinergic motor effect is prevented. A role of endogenously re-

leased tachykinins in the excitation of the longitudinal and circular muscle of the guinea-pig small and large intestine is

corroborated by the ability of SP desensitization and tachykinin antagonists to inhibit noncholinergic excitatory junction potentials (EJPs) (Bauer and Kuriyama, 1982; Niel

et al., 1983; Taylor and Bywater, 1986; Crist et al., 1991; Zagorodnyuk et al., 1993, 1995).

Electrically evoked contractions due to tachykinin release have been noted in many regions of the mammalian gut, and Table 5 presents a summary of the data obtained in

a broad range of species. In addition, tachykinins partici-

pate in a variety of neurogenic contractions that are induced by chemical stimulation of excitatory enteric motor

neurons. As is listed in Table 5, SF’ desensitization or tachy-

kinin antagonists attenuate the contractions caused by ganglionic stimulants, GABA* receptor agonists, bombesin, CCK and CCKA receptor agonists, pituitary adenylate cyclase-activating peptide, VIP, neurotensin, LTD,, histamine H, receptor agonists or 5-HTR agonists. The finding that the noncholinergic contractions evoked by 5-HT,R agonists are blunted by NK1 receptor desensitization, while

those evoked by 5-HT,R agonists are inhibited by NKj receptor desensitization (Ramirez et al., 1994), suggests that 5-HT& are located on tachykininergic enteric motor neurons, while 5-HT4Rs reside on tachykininergic enteric sensory neurons or intemeurons. Other examples of tachykininergic motor responses include the withdrawal contractions of gut segments made tolerant to opiates, adenosine or cloni-

dine (Table 5). It is not clear whether tachykinins play a role in the neu-

ral regulation of biliary motility. The electrically induced contraction of the guinea-pig gallbladder is blocked by atro-

*Paton, W. D. M. and Zar, M. A. (1966) Evidence for tranmsission of nerve effects by substance P in guinea pig longitudinal muscle strip. In: Abstracts of the IIIrd International Pharmacology Congress, Sao Paula, p. 9.

pine, but left unaffected by tachykinin antagonists (Mel- drum et al., 1987; Maggi et al., 1989a,c). In contrast, the contraction of the feline gallbladder and sphincter of Oddi,

which is evoked by VNS, is reduced by tachykinin receptor antagonism (Dahlstrand et al., 1988). Further studies are needed to investigate this issue.

5.5.1.2. Complementary and cooperative motor actions ofendo-

genous tachykinins. Although it has been obvious for some time that tachykinins contribute to a diversity of excitatory

motor responses in the mammalian gut, it is only recently

that the tachykinin receptors stimulated by endogenous tachykinins are being identified. In agreement with the predominance of NK, receptors on the longitudinal muscle of the

guinea-pig ileum, it has been found that electrically evoked noncholinergic contractions are blocked by NK,, but not NK2, receptor antagonists (Taylor and Kilpatrick, 1992). The same is true for the noncholinergic nerve-mediated

contractions induced by CCKA receptor agonists or NKI receptor agonists (Dal Forno et al., 1992; Corsi et al., 1994; Croci et al., 1995). Conversely, it is in keeping with the

more prominent role of NK2 receptors in the longitudinal muscle of the guinea-pig colon that the noncholinergic mo-

tor responses to electrical nerve stimulation in this gut region are mediated by both NK1 and NKZ receptors (Kojima and Shimo, 1995), while those elicited by stimulation of

neural NK3 receptors are predominantly brought about by muscular NK2 receptors (Croci et al., 1995). NK2 receptors are also prominently involved in the delayed contractile response to electrical nerve stimulation in the longitudinal

muscle of the mouse small intestine (Goldhill et al., 1995a) and in the electrically evoked atropine-resistant contraction of the canine colon circular muscle (Shuttleworth et

al., 1993). Other examples of a good match of the receptors

stimulated by exogenous and endogenous tachykinins are the circular muscle of the guinea-pig duodenum (Zagorod-

nyuk et al., 1995) and rat small intestine (Maggi and Giuliani, 1995) in which NK, and NK2 receptors cooperate to bring about electrically evoked NANC contractions.

It is important to note, however, that in many tissues, there is some disparity between the receptors identified with exogenous tachykinin receptor agonists and those activated by endogenously released SP and NKA. For in-

stance, the circular muscle of the human ileum contains functional NK, and NK2 receptors, yet electrically evoked noncholinergic contractions are exclusively mediated by NK2 receptors (Maggi et al., 1992b). It is also primarily NK2 receptors that in the circular muscle of the guinea-pig ileum, are responsible for the tachykinin-mediated contractions caused by EFS or distension of the gut wall (Barth6 et al., 1992; Holzer et al., 1993; Holzer and Maggi, 1994; Su- zuki et al., 1994a), which contrasts with the involvement of both NK1 and NK2 receptors in the muscle responses to exogenous tachykinins (Maggi et al., 1990a, 1994e). Al- though a small contribution of. NK, receptors has been seen in another study (Maggi et al., 1994c), it seems as if NK1 and NK2 receptors are differentially distributed in the


circular muscle such that endogenously released tachyki-

nins, unlike exogenously applied peptides, reach predomi-

nantly one type of receptor only. It need be realized in this

context that within the musculature of the guinea-pig, rat and canine small intestine, SP/NKr receptors may be confined to the interstitial cells at the inner surface of the circular muscle layer, but probably are absent from the circular

muscle cells themselves (Burcher et al., 1986; Mantyh et al., 1988; Stemini et al., 1995; Portbury et al., 1996), although

the presence of a second receptor isoform (Fong et al., 1992; Mantyh et al., 1996) cannot be neglected.

The apparent differences that in the guinea-pig colon circular muscle exist between the receptors identified with exogenous tachykinin receptor agonists and those activated by endogenously released SP and NK have been explained in a different way. As in the small intestine, the circular muscle layer in the guinea-pig colon contains both NK1

and NK, receptors, yet the noncholinergic EJPs and contractions evoked by brief trains (1 set) of electrical pulses

are antagonized by NKr, but left unaffected by NK,, recep-

tor antagonists (Zagorodnyuk et al., 1993). When, however, the duration of the electrical pulse trains is extended, the additional recruitment of some NKz receptors by endogenously released tachykinins becomes apparent and the contractions are abolished by a combination of NK, and NKz

receptors only (Maggi et al., 1994b,g). Temporal analysis indicates that NK, antagonists depress the initial and later phases of the NANC contractile response to a similar degree, while the later, but not initial, phase is also reduced by

NKz antagonists (Maggi et al., 1994g). This is consistent with the finding that activation of NK, receptors on the

guinea-pig colon circular muscle gives rise to a fast and phasic contraction, whereas activation of NKz receptors causes a slow and sustained excitation of the muscle due to stimu-

lation of different signaling pathways (Maggi et al., 1994g). It thus becomes increasingly clear that NANC contrac-

tions in the guinea-pig and rat intestine are cooperatively mediated by endogenously released SP and NKA acting preferentially via NK, and NK, receptors, respectively

(Maggi et al., 1994b,c,g; Kojima and Shimo, 1995; Maggi and Giuliani, 1995; Zagorodnyuk et al., 1995). This cooperative and, as shown in the circular muscle of the guinea-pig

colon, complementary way of transmitting neurogenic motor responses pertains to both the excitatory and inhibitory motor actions of tachykinins (Jin et al., 1993; Maggi et al., 1994b,g; Zagorodnyuk et al., 1995).

5.5.1.3. Corransmission b acetylch&e and tachykinins. It has long been thought that cholinergic and tachykininergic neurons represent two different populations of excitatory enteric neurons because cholinergic contractions can be evoked by single pulses of electrical current, whereas tachykinin-mediated contractions are seen only when high-frequency electrical stimulation is applied in the presence of an atropine-like drug. This conjecture, however, is at odds with the immunohistochemical finding that ACh and tachykinins are co-expressed in most enteric neurons

(Llewellyn-Smith et al., 1988; Steele et al., 1991; Brookes et al., 1991a,b, 1992; Fumess et al., 1992). This observation

has an important bearing on the question as to whether or

not ACh and tachykinins are co-released and play a

cotransmitter role in the excitatory enteric motor pathways. Reevaluation of the question as to whether or not neurogenic contractions of the gut are comediated by ACh

and tachykinins indeed does support the notion of cotrans-

mission by ACh, NKA and SP (Fig. 2). Experiments with the circular muscle of the guinea-pig colon have revealed

that cholinergic and tachykinin-mediated NANC contractions are evoked at comparable frequencies and intensities of electrical pulses, although cholinergic transmission

seems to precede tachykininergic transmission (Maggi et al., 1994~). ACh and tachykinins also comediate senktide- and

CCK-induced contractions of the guinea-pig ileum and tenia coli (Guard and Watson, 1987; Corsi et al., 1994; Maggi et al., 1994d; Croci et al., 1995).

Another aspect of cholinergic/tachykininergic cotransmission relates to the type of interaction. Although not in-

vestigated in a systematic manner, it is apparent that in certain cases, ACh and tachykinins interact in a synergistic,

rather than an additive, manner. Thus, it has been found that the contractile response of the guinea-pig ileum circular muscle to senktide is inhibited only by a combination of atropine and a NKz receptor antagonist, but not by either antagonist alone (Maggi et al., 1994d). Postjunctional syner-

gism between ACh and tachykinins acting via NK, receptors has also been observed to play a role in the ascending reflex contraction of the guinea-pig ileum circular mus-

cle induced by distension of the gut wall (Holzer et al., 1993). The observation that atropine alone virtually abol-

ishes contractions evoked by submaximal grades of distension and a NK2 antagonist reduces them by some 70% can-

not be explained by an additive model of cotransmission and suggests that blockade of the action of one cotransmitter blunts the activity of the other (Holzer et al., 1993). A similar situation applies to peristaltic motility in the guinea- pig ileum (Holzer and Maggi, 1994), where occlusion of

NKz receptors alone has little effect, but abolishes peristalsis when combined with a threshold concentration of atropine

(Fig. 3). It follows that synergism between ACh and tachykinins is an important element of enteric neuromuscular

cotransmission (Holzer and Maggi, 1994), which needs to be considered before the relative contribution of ACh and tachykinins to the transmission process can be deduced from the ac-

tions of one antagonist alone (Shuttleworth and Keef, 1995).

5.5.1.4. Prejunctional modulation of rachykinin-mediated motor reseonses. A complicating factor in cholinergic/tachykininergic cotransmission is the possibility that the postjunctional synergism between ACh and tachykinins is matched by a prejunctional antagonism of the two cotransmitters. Data to support such a contention have been obtained in the muscularis mucosae of the opossum esophagus in which the early cholinergic contraction due to EFS is inhibited by atropine, whereas the delayed tachykinin-mediated con-


TABLE 5. Pharmacological evidence for physiological roles of tachykinins in gastrointestinal motor activity

Tissue Response under study Inhibition by Reference

Chicken esophagus LM of opossum

esophagus MM of opossum

esophagus Guinea-pig

esophagus LES of cat LES of ferret LES of dog CM of rat gastric

corpus Stomach and pylorus

of cat Stomach of dog

Small intestine of dog CM of human small

intestine Rat stomach/small

intestine CM of rat small

intestine LM of rat small

intestine LM or murine small

intestine Guinea-pig small

intestine

CM of guinea-pig small intestine

LM of guinea-pig small intestine

Contraction due to EFS NC contraction due to EFS

Contraction due to EFS or capsaicin

Swallowing of water

Contraction due to acid1 or bombesin Relaxation due to acid* Contraction due to capsaicinl Contraction due to EFS or capsaicin

Contraction due to VNS or SNS

NANC contraction due to EFS or SNS

Contraction due to MNS NC contraction due to stretch or EFS

Gastrointestinal transit of a liquid meal

NANC contraction due to EFS

NANC contraction due to MNS

NC contraction due to EFS

Atropine-resistant peristalsis

Atropine-compromised peristalsis Hexamethonium-resistant peristalsis NC contraction due to EFS

NC poststimulus excitation due to EFS Late EJP due to EFS NANC EJP due to EFS Ascending NC EJP due to mucosal

stroking or distension NC ascending contraction due to distension

Action potential activity due to distension Nonadrenergic contraction due to MNS NC contraction due to capsaicin NC contraction due to CCK NC contraction due to EFS

Nonadrenergic contraction due to MNS

Contraction due to capsaicin

SP-DESENS, TKR-ANT SP-DESENS

SP-DESENS, TKR-ANT

NK,R-ANT

SP-DESENS, TKR-ANT SP-DESENS, NK,R-ANT TKR-ANT SP-DESENS, TKR-ANT

SP-DESENS, TKR-ANT

SP-DESENS

TKR-ANT TKR-ANT; NK2R-ANT

TKR-ANT

NKIR-/NK*R-PNTs

TKR-ANT

NK,R-ANT, LTD.,-ANT

SP-DESENS, TKR-ANT

NK,R-ANT TKR-ANT TKR-ANT NK,R-/NK2R-ANTS

TKR-ANT TKR-ANT NKIR-/NKzR-ANTS TKR-ANT

TKR-ANT NK,R-/NK*R-ANTS

SP-DESENS SP-DESENS, TKR-ANT NK,R-/NK2R-ANTS SP-DESENS SP-DESENS, TKR-ANT

NKIR-ANT SP-DESENS, TKR-ANT

SP-DESENS, TKR-ANT

NK,R-ANT

Neya et al., 1990 Crist et al., 1986

Domoto et al., 1983; Robotham et al., 1985

Jin et al., 1994

Reynolds et al., 1984, 1986 Blackshaw et al., 1994 Sandler et al., 1993 Holzer-Petsche et al., 1989; Holzer-

Petsche and Moser, 1993 Delbro et al., 1983; Lidberg et al.,

1983 Sakai and Daniel, 1984; Nakazato et

al., 1987 Neya et al., 1989 Grider, 198913; Maggi et al., 1992b

Holzer et al., 1986a

Maggi and Giuliani, 1995

Wali, 1985

Goldhill et al., 1995a

Barth6 et al., 1982a,b; Costa et al., 1985a

Holzer and Maggi, 1994 Barth6 et al., 1989 Costa et al., 198513 Barth6 et al., 1992; Suzuki et al.,

1994a; Zagorodnyuk et al., 1995 Niel et al., 1983 Crist et al., 1991 Zagorodnyuk et nl., 1995 S;Fhlan9nmess, 1988; Smith et

Cost: et al., 1985b; Holzer, 1989 Barth6 et al., 1992; Holzer et al., 1993; Maggi et al., 1994~

Yokoyama and North, 1983 Takaki et al., 1990 Barth6 et al., 1994 Holzer et al., 1980b Paton and Zar, 1966; Franc0 et al.,

1979; Barth6 et al., 1982d; Bjsrkroth, 1983; Gintzler and Hyde, 1983; Gintzler and Scalisi, 1983; Mat&k and Bauer, 1986; Jin et al., 1989

Taylor and Kirkpatrick, 1992 Barth6 et al., 1982~; Grbovie and

RadmanoviC, 1983; Takaki et al. , 1987

Barth6 et al., 1982~; Chahl, 1982; Tsou et al., 1982; BjGrkroth, 1983; Barth& 1988; Maggi et al., 1988

Croci et al., 1994a

(continued)


TABLE 5. Continued

197

Tissue Response under study Inhibition by Reference

LM of guinea pig small intestine

CM of feline small intestine

Ileocecal sphincter of cat

Guinea-pig tenia ceci

NANC ascending contraction due to EFS Ascending excitation due to EFS

Contraction due to colonic distension

CM of guinea-pig colon

Guinea-pig colon LM of guinea-pig

colon

NC off-contraction due to EFS NC contraction due to NK,R agonists NC contraction due to EFS

NC pressure waves due to distension NC contraction due to EFS

CM of rat colon Contraction due to CCK Ascending NC contraction due to stretch

LM of murine colon NC contraction due to neurotensin CM of rabbit colon NC off-contraction due to EFS

LM of rabbit colon Feline colon CM of canine colon MM of canine colon Human colon

Contraction due to capsacin Contraction due to SNS NC contraction due to EFS Contraction due to bombesin or EFS Contraction due to EFS

NC contraction due to CCK

Contraction due to GABA,R agonists NC contraction due to ganglion stimulant NC contraction due to NK,R agonists

NC contraction due to PACAP NC contraction due to VIP Contraction due to H2R agonist Contraction due to 5-HT NC contraction due to 5-HT3R agonist NC contraction due to 5-HT.+R agonist Contraction due to LTD, NC contraction due to neurotensin NC off-contraction due to NO NC contraction due to opioid withdrawal

Contraction due to clonidine withdrawal Contraction due to adenosine withdrawal NANC EJP due to EFS

SP-DESENS, TKR-ANT

NK,R-ANT

SP-DESENS, TKR-ANT SP-DESENS NK,R-DESENS, TKR-ANT NK,R-ANT SP-DESENS, TKR-ANT SP-DESENS, TKR-ANT SP-DESENS SP-DESENS, TKR-ANT NKIR-DESENS NK,R-DESENS SP-DESENS, TKR-ANT SP-DESENS NKI-R-ANT SP-DESENS, TKR-ANT

TKR-ANT TKR-ANT SP-DESENS, TKR-ANT

SP-DESENS SP-DESENS

SP-DESENS

TKR-ANT NK,R-ANT NK,R-/NK,R-ANTS

NK,R-/NK,R-ANTS NKIR-/NK2R-ANTS

TKR-ANT TKR-ANT, SP/NKA

antibody SP-DESENS SP-DESENS, TKR-ANT

TKR-ANT SP-DESENS, TKR-ANT NK,R-ANT SP-DESENS, SP antibody TKR-ANT

Hutchison and Dockray, 1981; Barth6 et al., 1983; Chang et al., 1984; Garzdn et al., 1987; Lucaites et al., 1991 Dal Fomo et al., 1992; Corsi et al.,

1994 Tonini et al., 1987 Franc0 et al., 1979 Guard and Watson, 1987 Corsi et al., 1994; Croci et al., 1995 Katsoulis et al., 1993 Katsoulis et al., 1992 Barker and Ebersole, 1982 Chahl, 1983a; Buchheit et al., 1985 Ramirez et al., 1994 Ramirez et al., 1994 Bloomquist and Kream, 1987 Monier and Kitabgi, 1980 Barth6 and Lefebvre, 1994 Gintzler, 1980; Tsou et al., 1982;

Chahl, 1983b, 1986; Tsou et al., 1985

Chahl, 198513 Chahl, 1990 Bauer and Kuriyama, 1982; Taylor

and Bywater, 1986 Jin et al., 1988 Miolan and Niel, 1988

Rothstein et al., 1990

Leander etal., 1981 Croci et al., 1995 Zagorodnyuk et al., 1993; Maggi et

al., 1994b,g Giuliani et nl., 1993 Kojima and Shimo, 1995

Wiley and Owyang, 1987 Grider and Makhlouf, 1986; Grider,

1989a Fontaine and Lebrun, 1985 Koelbel et al., 1989; Snape et al.,

1989 Mayer et al., 1990a Fandriks et al., 1985 Shuttleworth et al. , 1993 Angel et al., 1984 Larsson et al., 1987

1Acid or capsaicin administered into distal esophagus. ANT, antagonist; CM, circular muscle; DESENS, desensitization; GABAAR, GABA* receptor; H,R,

histamine H, receptor; 5-HT,R, 5-HT, receptor; 5-HT,R, 5-HT, receptor; LM, longitudinal muscle; MM, muscularis mucosae; MNS, mesenteric nerve stim-

ulation; NC, noncholinergic; NK,R, NK, receptor; NK,R, NKZR receptor; NK,R, NK, receptor; PACAP, pituitary adenylate cyclase activating peptide;

TKR, tachykinin receptor.

traction is enhanced (Domoto et al., 1983). This result,

which has been further analyzed, suggests that ACh inhib-

its the release of the cotransm.itter by a prejunctional site of action. There is also circumstantial evidence that prejunc-

tional muscarinic acetylcholine receptors within the MP

inhibit the tachykininergic slow excitatory postsynaptic potential (EPSP) (Morita et al., 1982). Whether prejunctional

SP receptors control the release of ACh has not been ad-

dressed specifically, but it is worth remembering that tachy-

kinins can inhibit the electrically evoked release of ACh

from enteric neurons (Kilbinger et al., 1986; Mayer et al.,

1990b; Liiffler et al., 1994; Giuliani and Maggi, 1995).

In agreement with the prejunctional modulation of SP release from enteric neurons by adenosine, opioid peptides

198

MEN-lo,376 (10 @VI) I l

0.5 z-

&

2 7 B 0 1 5 min

FIGURE 3. Recording of the effect of the NKI receptor antagonist MEN-10,376, administered alone or after addition of atropine, on peristaltic motor activity of the guinea-pig small intestine in vitro. The concentrations of the drugs in the organ bath are given in brackets. Note that MEN-lo,376 alone had little effect, but abolished peristalsis in the presence of a threshold concentration of atropine, which by itself caused only a small inhibition of motility. Data from Holzer and Maggi (1994).

and NA (Table 2), it has been found that tachykinin-medi-

ated neurogenic contractions of the guinea-pig ileum longitudinal muscle are inhibited by adenosine (Barth6 et al., 1985; Christofi et al., 1990), opiate receptor agonists (Mon- ier and Kitabgi, 1981; Barth6 et al., 1982d; Gintzler and

Scalisi, 1983; Leander et al., 1984), olz-adrenoceptor ago-

nists (Barth6 et al., 1983; Galligan, 1993) and sympathetic nerve stimulation (SNS) (Barth6 et al., 1983). Circumstan-

tial evidence indicates that NO (Wiklund et al., 1993), GABA (Cherubini and North, 1984) and somatostatin

(Monier and Kitabgi, 1981) are other factors that inhibit the release of tachykinins from enteric neurons.

5.5.1.5. Enteric refix contructions and intestinal peristalsis.

5.5.1.5.1. Enteric reflex contractions in vitro. There has been a long-standing interest in the potential role of tachykinins in the regulation of enteric motor reflexes and pro-

pulsive motility of the gut (Barth6 and Holzer, 1985). Tak- ing all data together, it is evident that SP and NKA do play a role in the coordination and execution of enteric motor

programs, although analysis of their precise role is progress- ing slowly due to the complexities of chemical coding and cotransmission in the enteric nervous system. Characteris- tic of enteric motor reflex pathways is their polarity, which is evident from the pharynx down to the anus and ensures the unidirectional transport of the chymus. The swallowing

of fluid in the guinea-pig is reduced by intrapharyngeal application of FK-888, which suggests the involvement of tachykinins acting via NK, receptors in the pharyngo-esophageal propulsion (Jin et al., 1994). The relative contribution of tachykinins to excitation of the opossum esophageal smooth muscle is most prominent in the distal part of this gut region (Crist et al., 1986) in which SP and NKA partic-

P. Holzer and U. Holzer-Petsche

ipate in the acid-controlled activity of the LES. Acidifica- tion of the cat’s lower esophagus causes spike activity and contraction of the LES, which is blunted by SF’ desensitization and a tachykinin antagonist (Reynolds et al., 1984).

Conversely, the ferret LES responds to esophageal acidification with relaxation, a response that is also blocked by a NK, receptor antagonist (Blackshaw et al., 1994).

The function of tachykinins in propagated motor activity

has been most thoroughly studied in the small and large in-

testine of the guinea-pig and rat. In the guinea-pig ileum, it has been shown that tachykininergic enteric neurons con-

tribute to the longitudinal muscle contraction that is evoked by electrical stimulation of nerves in the submucosal. layer (Jin et al., 1989). Apart from these submucosal- longitudinal muscle pathways, tachykinins are prominently involved in excitatory ascending pathways, causing excita-

tion and contraction of the longitudinal and circular muscle orally to the site of stimulation. Thus, the ascending

slow wave and spike activity caused by electrical nerve stimulation in the feline small intestine is blocked by SP

tachyphylaxis (Miolan and Niel, 1988). Likewise, the contraction of the guinea-pig ileum longitudinal muscle re-

corded orally to the site of electrical nerve stimulation is in-

hibited by SP desensitization, whereas the descending relaxation remains unaffected (Jin et al., 1988).

Since radial distension of the intestinal wall is believed to be the adequate stimulus for the induction of propagated enteric motor reflexes and peristalsis, considerable efforts have been taken to analyze the role of tachykinins in the

ascending contraction and descending relaxation of intestinal circular muscle in response to stretch or distension of

the gut wall (Fig. 2). These polarized motor reflexes involve distension-sensitive enteric sensory neurons, interneurons,

orally projecting excitatory motor neurons to cause ascend-

ing contraction and aborally projecting inhibitory motor neurons to cause descending relaxation (Furness and Costa, 1987). SP desensitization, tachykinin immunoneutraliza- tion and the use of tachykinin antagonists have shown that the ascending reflex contraction caused by wall distension or stroking of the mucosa (Table 5) involves ACh and tachykinins as excitatory cotransmitters, whereas the descend-

ing relaxation seems to be independent of tachykinins acting via NK, and NK2 receptors (Costa et al., 1985b; Grider

and Makhlouf, 1986; Smith and Furness, 1988; Grider, 1989a,b; Holzer, 1989; Smith et al., 1990; Barth6 et al.,

1992; Holzer et al., 1993; Maggi et al., 1994~). Further analysis with NK1 and NK2 receptor-selective

antagonists indicates that tachykinins serve as neuromuscular transmitters of the ascending reflex contraction, causing excitation of the guinea-pig ileum circular muscle preferentially by activation of NK, receptors (Barth6 et al., 1992; Holzer et al., 1993), although some involvement of NKl receptors is also evident (Maggi et al., 1994~). The stretch- induced ascending contraction, but not descending relaxation, of the human jejunum and rat colon (Grider, 1989a,b) is associated with a release of both SP and NKA, and the involvement of either peptide in the ascending


contraction of the rat colon circular muscle has been proven by the inhibitory effects of specific SP and NKA an-

tisera (Grider, 1989a). It has long been held that tachyki-

nins are primarily responsible for the atropine/hyoscine- resistant contraction seen in response to high degrees of distension or stretch (Costa et al., 1985b; Grider and Ma- khlouf, 1986; Grider, 1989a,b; Holzer, 1989; Smith et al.,

1990). However, tachykinin antagonists do cause some inhibition of the ascending reflex contraction in the absence of atropine (Grider and Makhlouf, 1986; Holzer, 1989; Bar- thb et al., 1992; Holzer et al., 1993), and a comparison of the effects of atropine and a NK2 antagonist in the guinea- pig ileum indicates that ACh and tachykinins synergize in causing excitation of the muscle in response to submaxi-

mally effective grades of distension (Holzer et al., 1993),

which is consistent with the coexistence and presumed core- lease of ACh, SP and NKA from enteric motor neurons.

While there is little doubt that enteric tachykinins sub- serve transmission from excitatory motor neurons to the

muscle, the function of tachykinins in neuroneuronal relays of enteric motor reflexes is less well characterized. The expression of SP in enteric sensory neurons and interneurons and the abundant presence of tachykinin receptors on enteric neurons is consistent with the possibility that tachykinins play a role as transmitters between sensory neurons

and interneurons or interneurons and motor neurons (Fig. 2). Experiments involving desensitization to the NK7 re-

ceptor agonist senktide have shown that tachykinins acting

via NKI receptors participate in the transmission from enteric sensory neurons to both ascending and descending

motor pathways, but apparently do not contribute to transmission from ascending and descending interneurons (Johnson et al., 1996).

5.5.1.5.2. Intestinal peristalsis in ui~ro. The implications

of SP and NKA in excitatory enteric motor reflexes have a bearing on integrated propulsive motility in the gut because

intestinal peristalsis results from sequential activation of the ascending excitatory and descending inhibitory re-

flexes, which by moving aborally, propel the contents of the intestine in the same direction (Fumess and Costa,

1987). A contribution of tachykinins to peristaltic muscle

excitation has been shown directly in the circular muscle of the guinea-pig small intestine in which the noncholinergic EJPs observed during peristalsis are inhibited by SP desensitization or chymottypsin (Yokoyama and North, 1983). However, when judged from the effects of tachykinin antagonists on peristalsis in the guinea-pig small intestine, there is little indication that SP and NKA play any major role in the coordination of peristaltic motor activity (Bar-

thb et al., 1982a,b, 1989; Costa et al., 1985a; Holzer and Maggi, 1994; Holzer et al., 1995). Only when cholinergic neuromuscular transmission is blocked by atropine, has it been possible to demonstrate a significant contribution of endogenous tachykinins to the maintenance of propulsive motility (Barth6 et al., 1982a,b; Costa et al., 1985a). Further analysis has shown that synergism between ACh and tachy-

kinins acting via NK2 receptors (Fig. 3) is of particular rele-

vance when the relative roles of the two cotransmitters are

assessed (Holzer and Maggi, 1994). While in the absence of

atropine NK2 receptor antagonists cause only a minor inhibition of peristalsis, they invariably abolish peristalsis when cholinergic transmission has been compromised by a

threshold concentration of atropine (Holzer and Maggi, 1994).

It seems possible that cotransmission by ACh and tachy-

kinins is not only of relevance for neuromuscular, but also for neuroneuronal transmission within the pathways sub- serving peristaltic motor activity. This notion derives from the finding that the residual peristaltic activity, which is

seen when ganglionic transmission is suppressed by hexamethonium and the effect of endogenous opioid peptides is

prevented by naloxone, is blocked by a tachykinin antagonist with supposed activity at NK? receptors (Barth6 et al.,

1989). The validity of this inference has not been con-

firmed yet by the use of tachykinin receptor-selective antagonists. A role of transmission via NK, and NK, receptors in intestinal peristalsis is consistent with the ability of both NKZ and NK7 receptor-selective agonists to facilitate peri-

staltic motor activity (Holzer et al., 1995). Conversely, NK, receptor agonists inhibit, and NK, antagonists cause some facilitation of, peristaltic motor activity (Holzer et al.,

1995). Although not yet further analyzed, these data at- tribute transmission via NK, receptors an inhibitory role in peristalsis.

5.5.1.5.3. Propulsive motility in ~ipio. The finding that i.p. administration of a tachykinin antagonist inhibits gas-

tric emptying and gastrointestinal transit in the rat supports the notion that tachykinins are involved in the regulation of propulsive motility in viva (Holzer et al., 1986a). This finding is analogous to a study in the guinea-pig colon in

viva in which atropine-resistant pressure waves caused by distension of the colonic wall are inhibited by a combina-

tion of NK, and NK, receptor antagonists (Giuliani et al., 1993).

5.5.2. Roles of tachykinins released from extrinsic

afferent nerve fibers. 5.5.2.1. Local roles of ufferent nerve-derived tachykinins in the

gut wall. Sensory nerve stimulation with capsaicin exerts both excitatory and inhibitory effects on the in vitro and in viwo motor activity of the opossum, guinea-pig, rat, rabbit and canine gut (Barth6 et al., 1982c, 1994; Barth6 and

Holzer, 1985, 1995; Robotham et al., 1985; Holzer-Petsche et al., 1989; Mayer et al., 1990a; Sandler et al., 1993). Simi- lar effects are elicited by antidromic electrical stimulation of intestinal afferents in the mesenteric nerves when sympathetic neurotransmission has been blocked. The ability of mesenteric nerve stimulation to cause contraction in the guinea-pig, rat and canine intestine is prevented by capsai- tin-induced defunctionalization of afferent neurons (Bar- th6 et al., 1982~; Grbovik and RadmanoviC, 1983; Wali, 1985; Takaki et al., 1987, 1990; Neya et al., 1989).


The mechanisms that underlie the motor effects of sensory nerve stimulation in the intestine are multifactorial (Fig. 2). There is ample evidence that tachykinins partici-

pate in the excitatory motor responses of the gut to capsai- tin and mesenteric nerve stimulation, but it is not clear whether they originate from extrinsic afferent nerve fibers or from enteric neurons that are stimulated by neurotransmitter substances released from the stimulated afferent

nerve fibers. SP, nevertheless, is released by capsaicin from the intestinal wall (Donnerer et al., 1984a; Maggi et al.,

1989~; Mayer et al., 1990a), and the contribution of tachykinins to the contractile effect of electrical and capsaicin-

evoked afferent nerve stimulation in the opossum, guinea-

pig, rat, rabbit and canine gut is well documented. Thus, both the cholinergic and particularly the noncholinergic

components of the contractile response to sensory nerve activation are inhibited by SP desensitization or tachykinin antagonists (Barth6 et al., 1982c, 1994; Chahl, 1982; Tsou et al., 1982; Bjiirkroth, 19836; Grbovik and RadmanoviC,

1983; Robotham et al., 1985; Wali, 1985; Takaki et al.,

1987, 1990; Maggi et al., 1988; Barthb, 1988; Holzer-Petsche et al., 1989; Neya et al., 1989; Mayer et al., 1990a; Sandler et

al., 1993). The tachykinin receptors involved in the capsai-

tin-evoked contraction of the guinea-pig ileum longitudinal

muscle are predominantly of the NK1 type (Croci et al.,

1994a), whereas the contraction of the circular muscle involves both NK, and NK2 receptors (Barth6 et al., 1994).

A synopsis of the effects of atropine/hyoscine, tachykinin

antagonists and TTX has led to the proposal that the tachykinins involved in the motor response to capsaicin are primarily released from enteric neurons and activate NK1/NK2 receptors on the intestinal smooth muscle (Barth6 et al., 1994). As enteric tachykinin-releasing neurons are them-

selves not sensitive to capsaicin (Barth6 et al., 1982d; Holzer, 1984, 1991; Barth6 and Holzer, 1985, 1995), it is

held that excitation of enteric neurons is the consequence of transmitter release from afferent nerve fibers (Barth6 et

al., 1994). The identity of these transmitters is uncertain, although tachykinins have long been suspected to play a

role, given that activation of NK, receptors releases ACh and tachykinins from enteric neurons. Since, however, the NK3 receptor antagonist SR-142,801 fails to block the contractile motor response of the guinea-pig ileum to capsaicin (Patacchini et al., 1995), it would appear that the afferent nerve-derived transmitters that stimulate enteric neurons are nontachykinin in nature and remain to be identified.

The tachykinin-mediated contractions that vagal or splanchnic nerve stimulation elicits in the canine and feline stomach, pylorus and small intestine are believed to re-

sult from antidromic activation of extrinsic afferent neurons, which through peripheral transmitter release, stimulate cholinergic enteric motor neurons (Delbro et al., 1983; Lid- berg et al., 1983; Nakazato et al., 1987; Neya et al., 1989). The same mechanism may explain the tachykinin-mediated contraction of the feline colon in response to lumbar SNS (Ftindriks et al., 1985). Again, the source of the tachykinins involved in these responses is uncertain, and has

been suggested to be either extrinsic afferent nerve fibers (Delbro et al., 1983; Lidberg et al., 1983; Fgndriks et al., 1985) or intrinsic enteric neurons (Neya et al., 1989).

Afferent nerve-derived tachykinins may also have a bearing on biliary motility. Capsaicin releases SP from the

guinea-pig gallbladder (Table 2) and causes contraction of the bladder, an effect that is mediated by tachykinins as it is

blocked by a receptor-nonselective tachykinin antagonist (Maggi et al., 1989a,c). CGRP, which is coreleased with SP, relaxes the gallbladder, an effect that is enhanced by blockade of tachykinin receptors (Maggi et al., 1989~). This ob-

servation shows that SP and CGRP have antagonistic ef-

fects on gallbladder motility, an inference that is supported by the ability of CGRP to inhibit the bladder contraction seen in response to SP (Hashimoto et al., 1988).

5.5.2.2. Participation of tuchykinins in short-loop rejlexes of

prevewbral sympathetic ganglia. Another mechanism by which

tachykinins released from afferent nerve fibers may control gastrointestinal motility relates to their ability to depolarize

sympathetic ganglion cells in the prevertebral ganglia (Ot- suka and Yoshioka, 1993). Spinal afferent nerve fibers con-

taining SP send axon collaterals into the ganglia to make synaptic contacts with postganglionic sympathetic neurons

(Kondo and Yui, 1981; Matthews and Cuello, 1984; Green and Dockray, 1988; Lindh et al., 1988). There is evidence that the noncholinergic slow EPSP in the celiac and infe-

rior mesenteric ganglion of the guinea-pig is mediated, at least in part, by SP and NKA (Jiang et al., 1982; Konishi et al., 1983; Saria et al., 1987) acting via NK, receptors (Zhao et al., 1993). Distension of the guinea-pig colon (Parkman

et al., 1993) or electrical stimulation of the lumbar colonic nerve (Stapelfeldt and Szurszewski, 1989) releases SP in the inferior mesenteric ganglion, and the slow EPSP, which in certain somata is triggered by colonic distension, is attenuated by capsaicin-induced ablation of primary afferent neu-

rons (Kreulen and Peters, 1986) or SP tachyphylaxis (Peters

and Kreulen, 1986). These observations suggest that primary

afferent neurons from the gut, which send tachykinin-releasing axon collaterals into prevertebral sympathetic ganglia, participate in short-loop reflexes that control gastrointestinal effector systems via stimulation of sympathetic output.

5.5.2.3. Participation of tachykinins in vagal and spinal reflexes.

Apart from short-loop reflexes relayed in the prevertebral sympathetic ganglia, it need also be considered that vagal and spinal reflexes involving tachykininergic afferents may participate in the autonomic and central nervous control of intestinal motility. While there is ample evidence that tachykinins play a role in a variety of vagal and spinal reflexes (Otsuka and Yoshioka, 1993), the implication of such tachykinin-mediated reflexes in the regulation of intestinal motor activity is still little studied. Distension of the feline colon causes contraction of the ileocecal sphincter, which is mediated by a spinal sympathetic reflex. A participation of tachykinins is deduced from inhibition of the reflex by SP tachyphylaxis (Rothstein et al., 1990), but the site at which tachykinins participate in the reflex has not been de-


termined. However, there is evidence that activation of

NK, receptors in the rat CNS contributes to the reflex inhibition of gastrointestinal motility caused by visceral and somatic pain (Julia et al., 1994; Holzer-Petsche and Ror- dorf-Nikoli’c, 1995).

5.6. Pathophysiological lmplicatiuns of Tachykinins in Cjastrointestinul Motility 5.6.1. Pathological changes in tachykinin-mediated motor control. 5.6.1.1. Motor disturbances caused by intestinal anaphylaxis and

inflammation. Despite the wealth of informatjon regarding the actions of tachykinins on gut motility and their impli-

cations in gastrointestinal motor control, there is comparatively little information as to whether the operationality of the tachykinin system is altered in gastrointestinal disease and may contribute to disease-related motor disturbances. There is mounting evidence, though, that some of the motor changes associated with intestinal anaphylaxis, infec-

tion, inflammation and stress are related to functional alterations of intrinsic enteric or extrinsic primary afferent

neurons releasing tachykinins. Capsaicin-sensitive afferent

neurons are nociceptive neurons that are responsive to tissue irritation and injury, and it is tempting to assume that tachykinins released from their peripheral terminals con-

tribute directly to motor dysfunction, while tachykinins released from their central endings participate in reflex disturbances of motor activity. It is pertinent in this respect to recall that many gastrointestinal disorders go along with changes in tachykinin and/or tachykinin receptor expression, changes that are reviewed in detail in the companion article (Holzer and Holzer-Petsche, 1997).

Tachykinins seem to be involved in the allergen-induced disruption of myoelectrical activity in the upper small in-

testine of ovalbumin-sensitized rats, since the allergen- evoked motor disturbance is attenuated by CP-96,345 or pretreatment of the rats with a neurotoxic dose of capsaicin

(Fargeas et al., 1993). Perivagal capsaicin pretreatment and a NK, antagonist likewise prevent the shutdown of myoelectrical activity, which in the rat cecocolonic region, is caused by drug-induced degranulation of mast cells, which strengthens the concept that capsaicin-sensitive vagal affer-

ent nerve fibers and tachykinins acting via NK, receptors play a crucial role in allergy/anaphylaxis-associated disturbances of gastrointestinal motility (Castex et al., 1994).

The sites at which NK1 antagonists act to prevent anaphylaxis-induced dysmotility are not known; conceivably, they could be NK1 receptors within the enteric nerve plexuses of the rat intestine (Sternini et al., 1995) or at the projection targets of capsaicin-sensitive afferent nerve fibers within the CNS.

Whether inflammation-related changes in biliary motility involve tachykininergic neurons remains to be eluci- dated. SP is released from afferent nerve fibres in the guinea-pig gallbladder (Table 2) and could potentially contribute to spasm of the biliaty tract under conditions of bile stone obstruction, biliary infection or cholecystitis. On the

other hand, the neutrophil-derived oxidant monochloram-

ine has been found to reduce the CCK- and NKA-evoked

contraction of the guinea-pig gallbladder, which suggests that certain inflammatory mediators may depress biliary tract motility (Moummi et al., 1991).

An implication of tachykinins in some of the motor changes associated with infection is illustrated by the observation that defecation induced by Salmonella enteritidis en-

dotoxin is partially reduced by the NKZ receptor antagonist

SR-48,968 (Croci et al., 199413). Since infection with Trichinella spiralis causes a marked down-regulation of the

SP-degrading enzyme, NEP, in the inflamed rat jejunum (Hwang et al., 1993), it appears conceivable that the intes-

tinal hypermotility seen in Trichinella infection arises from enhanced availability of endogenously released tachykinins at their receptors.

Inflammatory bowel disease is a multifactorial entity that

perturbs all gut functions, and there is good reason to assume that the pertaining changes in motility involve tachykinins, given that tachykinin and tachykinin receptor ex-

pression can be profoundly altered in this disorder (Holzer and Holzer-Petsche, 1997). Potential roles of tachykinins

in the disease process are envisaged from the interactions

that in the normal gut, exist between tachykinins and inflammatory mediators, such as prostaglandins and LTs. It, hence, may be postulated that imbalances in these interactions during inflammation give rise to significant changes in motor activity. LTD, has been found to increase the re-

lease of SP from enteric neurons (Bloomquist and Kream, 1987; Goldhill et al., 1995a), whereas tachykinins, in turn, are able to stimulate the formation of both prostaglandins

and leukotrienes (Kuwahara and Cooke, 1990; Mayer et al., 1990b; Rangachari et al., 1990; Takeuchi et al., 1991; Yau et al., 1991; Parsons et al., 1992; Parrish et al., 1994).

This interaction of inflammatory factors with tachykininergic neurons may be a contributory factor in the dysmotility associated with intestinal inflammation. Such a

scenario is further reflected by the observation that colitis induced by trinitrobenzene sulphonic acid, acetic acid, mitomycin C or T. spiralis infection compromises the contractility of the rat distal colonic longitudinal muscle in response to SP and ACh (Grossi et al., 1993). An imbalance

in the possible interactions between tachykinins and inflammatory mediators may also be deduced from the finding that after induction of colitis with trinitrobenzene sul-

phonic acid, the responsiveness of the rabbit colonic muscularis mucosae to SP is normal, while that to leukotrienes and prostaglandins is diminished (Percy et al., 1993). Con- versely, ricin-induced ileitis in the rabbit is associated with increased LTD, formation and up-regulation of electrically evoked noncholinergic contractions, which presumably are mediated by tachykinins, as they are inhibited by SP desensitization (Goldhill et al., 1995b). Circumstantial evidence suggests that alterations in rat colonic motility caused by platelet-activating factor, to some extent, may also be related to changes in the tachykinin motor control system (Deshpande et al., 1994).

202

5.6.1.2. Motor disturbances caused by stress and pain. Prom- inent among the pathological factors that have a significant impact on gut motility are stress and pain. Restraint stress- induced defecation in the rat is inhibited by the NK1 antag

onist RP-67,580, which has been interpreted to reflect a role of enteric SP in stress-induced colonic hypermotility,

because pretreatment with a neurotoxic dose of capsaicin is

without effect (Ikeda et al., 1995). The NK2 antagonist SR- 48,968 does not affect defecation in response to stress (Ikeda et al., 1995), but reduces the defecation caused by

the aZ-adrenoceptor antagonist idazoxan (Croci et al., 199413). Although not understood, this discrepancy indicates that different tachykinin receptors, at possibly different sites within the defecation reflex pathways, are activated by stress and idazoxan. Postoperative ileus is another

example of stress-related shutdown of gastrointestinal mo-

tility, caused by anesthesia and intra-abdominal surgery. Apart from disturbances of local motor control systems,

postoperative ileus also involves a spinal reflex composed of capsaicin-sensitive afferents and sympathetic efferents (Holzer et ai., 198613). .Interestingly enough, preoperative adminis-

tration of a tachykinin antagonist has been found to reduce postoperative ileus in the rat and to hasten the return of myoelectrical activity in the small intestine (Espat et al.,

1995). As stress- and pain-related disturbances of gut motor ac-

tivity involve the CNS, a multiplicity of sites at which tachykinins contribute to the motor changes can be envis-

aged. Apart from its direct action on intestinal muscle and enteric nerves, SP may influence motility by its ability to

sensitize (Holzer-Petsche and Rordorf-NikoliC, 1995) or stimulate (Lew and Longhurst, 1986; Barber and Burks, 1987; Cervero and Sharkey, 1988) primary afferent nerve fibers. This presumably indirect action on extrinsic afferents seems to be mediated by NK1 receptors (Holzer- Petsche and Rordorf-Nikolik, 1’995) and is likely to facili-

tate intestinointestinai motor reflexes relayed in the prevertebral ganglia or CNS. Stimulation of NK, receptors in the rat CNS contributes to the reflex inhibition of colonic

motility in response to painful rectal distension (Julia et al., 1994) and to the reflex relaxation of the stomach in response to capsaicin-induced stimulation of afferent nerve fibers in the peritoneal cavity or skin (Holzer-Petsche and

Rordorf-NikoU, 1995). NK, receptors do not seem to con-

-tribute to the intestinal manifestations of visceral pain, but play a role in somatic responses to painful rectal distension (Julia et al., 1994).

In context with the involvement of central NKI receptors in pain-related shutdown of gastrointestinal motility, it is worth noting that NK1 receptor antagonists are effective anti-emetic drugs that prevent vomiting caused by a variety of stimtili (Bountra%et al., 1993; Tattersall et al., 1993; Wat- son et al., 1995). This anti-emetic action of NK1 antagonists, which has been studied in ferrets and dogs, is most likely due to interruption of the emetic reflex within the CNS (Bountra et al., 1993; Tattersall et al., 1993; Watson et al., 1995).

P. Holzer and U. Holzer-Persche

5.6.1.3. Dysmotility associated with diabetes and Hirschsprung’s disease. The alterations of the gastrointestinal tissue levels of SP in experimental diabetes (Holzer and Holzer-Petsche,

1997) are accompanied by variable changes in the motor actions of SP in the rat small intestine. One report holds

that the efficacy of NKA, but not SP or NKB, is enhanced

after the induction of diabetes with streptozotocin, while the potency of NKA is left unchanged (Mathison and Davison, 1988), whereas in other studies, a decrease in the

efficacy of SP (Liu et al., 1988) or an increase in the potency without alteration of the efficacy of SP has been seen (Pinna et al., 1995). Although the electrically evoked release of SP from the isolated ileum remains unaltered in streptozotocin-diabetic rats (Belai et al., 1987), it would seem that certain components of the motor actions of tach-

ykinins are altered in diabetic rats, but it is not clear whether these alterations are causally related to, or are an

epiphenomenon of, disordered gastrointestinal motor activity. There is also uncertainty as to whether aganglionic coionic segments taken from patients with Hirschsprung’s dis-

ease are normally sensitive to SP (Larsson et al., 1987) or lack responsiveness to the peptide (Tomita et al., 1994). The absence of propulsive motor activity in Hirschsprung’s disease is thought to derive from the complete lack of inhibitory enteric motor neurons in the aganglionic seg-

ments, which results in defective muscle relaxation, con- striction and complete halt of peristalsis (Johanson et al., 1991; Larsson, 1994).

5.6;2. Therapeutic prospects. The inference that tachykinins are involved in a number of motor disturbances

raises the possibility that tachykinin receptor-selective antagonists could therapeutically be exploited for the correc- tion of disordered gut motility. This approach may prove successful if the tachykinin receptors involved in the dysmotility syndromes can be identified and if tachykinin receptor ligands can be developed that improve the disor-

dered motor function, but spare other tachykinin-mediated responses. It will equally be important to appreciate that tachykinins are messengers within a multifactorial motor control system and that manipulation of the tachykinin sys-

tem alone may not be therapeutically sufficient. Notwith- standing these limitations, it is challenging to pursue the ,further development of tachykinin antagonists, because

rhese drugs may be used to selectively counteract the up- regulation of tachykinins/tachykinin receptors seen in certain gastrointestinal disorders.

From the available information, it would seem that tachykinin antagonists could be useful in the therapeutic man- agement of motor disturbances associated with intestinal anaphylaxis, intestinal infection, inflammatory bowel disease, stress- and pain-related dysmotility, including the irri- table bowel syndrome, diabetes and postoperative ileus. Some of the tachykinin receptor-selective antagonists al- ready possess an interesting spectrum of activity. NK2 receptor activation in the rat, guinea-pig and human intestine increases motility and if stimulation is exaggerated,


may cause muscle spasms. The NK2 antagonist SR-48,968 is

spasmolytic in the rat small intestine inasmuch as it pre-

vents the increase in small intestinal transit caused by a NK2 agonist (Tramontana et al., 1994), but lacks any con- stipating activity (Croci et al., 199413). Besides the use of

antagonists, it may also be considered that tachykinin- selective agonists or drugs that activate tachykininergic neurons may be beneficial in states of hypomotility due to a deficiency of the tachykinin system. This line of progress is illustrated by a mot&n agonist that is developed as a prokinetic drug to facilitate postprandial gastric motility and whose prokinetic activity in the dog’s stomach depends on

tachykinins, as it is attenuated by the NK, receptor antagonist FK-888 (Shiba et al., 1995).

Acknowkdgemem-The authors’ work was supported by the Austrian Sci- ence Foundation (grants P4641-MED, P5552-MED, P7845-MED, P7858- MED, P9473-MED and P-11834-MED), the Jubilee Foundation of the Austrian National Bank (grants 4207 and 4905) and the Franz Lanyar Foundation of the Graz University School of Medicine.

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