in/. j. biochem. cdl bid. vol. 28. no. 7, pp. 721 73x. 19%€¦ · ip3 acts on specific receptors...

Pergamon In/. J. Biochem. Cdl Bid. Vol. 28. No. 7, pp. 721 73X. 19%

Copyright cj 1996 Elsevier Science Ltd PII: 31357-2725(96)00017-9 Printed in Great Britain. All rights reserved

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REVIEW

Tachykinins: receptor to effector AAMIR M. KHAWAJA, DUNCAN F. ROGERS* Thoracic Medicine, National Heart and Lung institute (Imperial College), Dovehouse Street. London SW3 6LY, U.K.

Tachykinins belong to an evolutionarily conserved family of peptide neurotransmitters. The mammalian tacbykinlns include substance P, neurokinin A and neurokinln B, which exert their effects by hiding to speclllc receptors. These tacbyklnin receptors are divided into three types, designated NK,, NK, and NK,, respectively. Tachykinin receptors have heeu cloned and contaln seven segments spanning the cell membrane, indicating tbeir inclusion in the G-protein-linked receptor family. Tbe continued development of selective agonists and antagonists for each receptor has helped elucidate roles for these mediators, ranging from effects in the central nervous system to the perpetuation of the inflammatory response in the periphery. Various selective ligands have shown both inter- and intraspecies differences in bimling potencies, indicatbrg dlstlnct binding sites in the tacbyklnin receptor. The interaction of tacbyktin with its receptor activates G,, which in turn activates phospholipase C to break down phospbatidyl inositol bispbospbate info inositol trispbospbate (IP,) and dlacylglycerol (DAG). IP3 acts on specific receptors in the sarcoplasmic reticulum to release intracellular stores of Ca’ + , while DAG acts via protein klnase C to open L-type calcium channels in the plasma membrane. The rise in intracellular [Ca*+] induces the tissue response. With an array of actions as diverse as that seen with tacbyklnins, there is scope for numerous therapeutic possibilities. With the development of potent, selective non-peptide antagonists, there could he potential benefits in the treatment of a variety of clinical conditions, including chronic pain, Parkinson’s disease, Alzheimer’s disease, depression, rheumatoid arthritis, irritable bowel syndrome and asthma. Copyright 19 1996 Elsevier Science Ltd

Keywords: Substance P Neurokinin Tacbykinin antagonist Tachykinin receptor G-protein

int. J. Biochem. Cell Biol. (1996) 28, 721-738

INTRODUCTION

Tachykinins are small peptide neurotransmitters that share a common amino acid sequence at their carboxy terminal (Table 1). They are actively involved in the physiology of both lower and advanced lifeforms. The three main mammalian tachykinins are substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), which exert their actions via interaction with a distinct set of receptors. The mammalian tachykinins may contribute to the pathophysiology of a number of human diseases. Their

*To whom all correspondence should be addressed. Received 13 September 1995; accepted 22 January 1996.

implication in disease has prompted the pharmaceutical industry to develop increasingly more potent and selective tachykinin receptor antagonists in attempts to produce therapeutic agents for possible treatment of a diverse range of clinical disorders, including emesis, headache, rheumatoid arthritis, irritable bowel syndrome, cystitis, allergic rhinitis, chronic coughing and sneezing, and asthma.

Interaction of a G-protein-coupled receptor with endogenous ligand is fundamental to cellular signalling and is the basis for the activity of living organisms, from slime mould to mammal. The process following ligand-receptor interaction is governed by complex networks of chemical and molecular pathways, ultimately leading to a physiological response. This system

721

722 Aamir M. Khawaja and Duncan F. Rogers

Table I. Amino acid sequences of the natural mammalian and non-mammalian tachykinins

Mummalian SP NKA NKB Neuropeptide K

Neuropeptide 7

H-Arg-Pro-Lys~Pro~Gln~Gln-Phe-Phe-Gly--Leu-Met-NH, H-His-Lys-Thr-Asp%-Phe-Val-Gly-LeeMet-NH, H-AspMet-His-AspPhe-Phe-Val-Gly-Leu-MNH* H~AspAla-AspSer-Ser-Ile-Glu~Lys~Gln-Val-Ala-Leu-Leu-Lys-Ala-Leu-Tyr-Gly-His-Gly- Gin-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp- H-As~Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-As~Ser-Ph~Val-Gly-Le~ Met-NH2

Non-mammalian Kassinin H-As~Val-Pro-Lys-Ser-As~Gln-Phe-Val-Gly-Leu-Met-NH* Eledoisin Pyr-PreSer-Lys-AspAla-Phe--Ile-Gly-LewMet-NH* Physalaemin Pyr-Ala-Asp-Pr+Asn-Lys-Phe-Tyr-Gly-Leu-NH2

Bold amino acids indicate common sequence at carboxy terminal.

of producing cascading intermediaries (‘second moschata), physalaemin (from the south ameri- messengers’) converts release of a minute can frog Physalaemus bigilonigerus) and sub- amount of neurotransmitter substance to an sequently six other similar non-mammalian- amplified, quantifiable signal to alter tissue derived peptides, suggested that these molecules function. In this review, we will discuss these belonged to a structurally and pharmacologi- steps for the tachykinins. To limit the size of the cally related family (see Erspamer, 1981). In the article, examples of the physiological and 197Os, following the determination of its amino pathophysiological role of tachykinin receptors acid sequence (Chang et al., 1971), it became in the periphery are biased to the airways. apparent that SP, although already known to Information on the peripheral role of share biological activities with the non- tachykinins in other organ systems, as well as mammalian peptides, also shared a common detailed accounts of tachykinin receptors and amino acid sequence (see Table 1). Sub- tachykinin receptor antagonists, can be found in sequently, this group of bioactive molecules was three recent reviews (Maggi et al., 1993; Otsuka collectively termed “tachykinins” (Erspamer and Yoshioka, 1993; Regoli et al., 1994). and Anastasi, 1966).

TACHYKININS

Historical perspective

The first member of the tachykinin family to be discovered was substance P, an endogenous peptide present in both mammalian and non-mammalian species. In 193 1, von Euler and Gaddum discovered that “preparation P”, obtained from horse intestine and brain by alcoholic extraction, caused peripheral vasodi- latation and intestinal contraction in the atropinised rabbit. This extract, renamed substance P (Gaddum and Schild, 1935), was purified and its pharmacological properties were determined. SP exhibited peripheral activity, including lowering of blood pressure and stimulation of gut motility in laboratory animals (Gernandt, 1942), and increased blood flow in the human forearm (Pernow, 1963). SP also exhibited central effects, most notably respiratory stimulation (von Euler and Pernow, 1956).

In 1962, the discovery of the peptide sequence of eledoisin, a biologically active substance found in the mediterranean octopus (Eledone

The name tachykinin resulted from the pharmacological similarity of these peptides to bradykinin, an endogenous inflammatory mediator, which elicited a distinctive slow (brady) contraction of guinea-pig ileum. The tachykinins, while having generally the same pharmacological profile, induced an immediate contraction of the same tissue (tachy, fast). The term “kinin” was originally coined by Schachter and Thain in 1954, when describing a polypeptide wasp venom that shared the “slow-acting” characteristics of bradykinin. “Kinin” was further classified by a committee in charge of recommendations for kinin nomenclature as “a general name indicating a hypotensive polypeptide which contracts most isolated smooth muscle, but relaxes rat duodenum.... In addition, the name may be applied to any polypeptide which is structurally related to bradykinin” (Webster, 1970). However, the tachykinins are not of similar structure to the nonapeptide bradykinin, nor do they relax rat duodenum. Therefore, as such, they are not really kinins. The name, however, remains.

Fifty years elapsed after the discovery of SP before two further mammalian tachykinins were

Tachykinins: receptor to effector -23

described, reported simultaneously by different groups, and which became known as NKA and NKB (Kimura et al., 1983; Maggio et al., 1983; Nawa et al., 1983). The discovery of two newer tachykinins, neuropeptide K (Tatemoto et al., 1985) and neuropeptide y (Kage et al., 1988) brought knowledge of mammalian tachykinins to its current state. Little is known about the physiological roles of neuropeptides K and y, and this review will focus on the three established mammalian tachykinins (SP, NKA and NKB).

Synthesis

SP, NKA and NKB, like all members of the tachykinin family, possess a common carboxy terminal sequence (Table 1). They are derived from two precursor genes, named preprotachykinin A (PPTA), which encodes SP, NKA, neuropeptide K and neuropeptide y, and preprotachykinin B (PPTB), which encodes NKB (Nawa et al., 1983; Kotani et al., 1986; Bonner et al., 1987). Alternative splicing of the PPTA gene gives rise to three distinct mRNAs: a-PPT produces SP; &PPT produces SP, NKA and neuropeptide K; and y-PPT produces SP, NKA, and neuropeptide y (Nawa et al., 1984; Krause et al., 1987; MacDonald et al., 1989). The expression of the individual mRNAs (a-, P-, or ;I-PPT) is species- and tissue-dependent. For example, in bovine central (nervous) tissue, r-PPT is predominantly expressed over B-PPT; the converse is true for peripheral tissue. However, in rat very little a-PPT is expressed at all, although P-PPT and y-PPT are found wherever neurokinins are expressed (Nakanishi, 1987; Carter and Krause, 1990).

Once synthesised, the tachykinins are stored in large granular vesicles in nerve endings (Floor et ul., 1982) awaiting an appropriate stimulus.

Distribution

The tissue distribution of tachykinins has been studied over many years. However, the common carboxy terminal sequence of tachykinins may give rise to cross-reactivity between different tachykinins. Consequently, earlier studies, which used antisera putatively selective for SP, need to be viewed with caution because other tachykinins may have also been detected (Maggio and Mantyh, 1989). Studies with more selective immunoassays (Hua et al., 1985; Lundberg and Saria, 1987; Tateishi et al., 1990; Dey et al., 1990) have shown that SP-like immunoreactivity is widely distributed through-

out the mammalian body, being localised primarily in neurones within the CNS and in peripheral tissue, although there is some localisation in non-neuronal cells including endothelial cells (Loesch et al., 1993; Stones et al., 1995) and inflammatory cells (Metwali et al., 1994). NKA-like immunoreactivity is almost exclusively co-localised with SP, although it is present in smaller amounts. NKB-like immunoreactivity is almost solely confined to the CNS, although a few groups have found NKB-like reactivity in some peripheral tissue. including rat gut (Tateishi et al., 1990).

Metabolism

The magnitude and duration of action of tachykinins is regulated by degradation by two major enzymes, namely angiotensin converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) (Skidgel et cd., 1984; Borson. 1991). The two SP cleavage sites for ACE are between amino acids PheX and Gly’ and between Gly’ and Leu” (see Table 1 ), whereas NEP cleaves SP between amino acids Gin’ and Phe’ and between Gly’ and Leu’“. Although other proteolytic enzymes are active in degrading tachykinins and have been purified, including “substance P degrading enzyme” (SPDE) (Lee et al.. 1981) dipeptidylaminopeptidase (DPAP) (Wang et ul., 199 1) and postproline endopeptidase (PPEP) (Blumberg et al., 1980) the tissue location of the two membrane-bound enzymes, NEP and ACE. is ideal for producing efficient hydrolysis. In the CNS, ACE has been found in human cerebrospinal fluid (Lantz et al., 1991). In the periphery, ACE has been shown to degrade SP in vascular (Rouissi et uf., 1990a) and urinogenital (Rouissi et al.. 1990b) smooth muscle preparations. In the airways, ACE is located principally in vascular endothelial cells, which is consistent with the observation that captopril, an ACE inhibitor, potentiates bronchoconstriction caused by intravenously admin- istered SP (Shore et ~1.. 1988) but not by inhaled SP (Lotvall et al.. 1990). NEP immunoreactivity in the CNS, using autoradiographic techniques. has been shown to closely mirror the immunoreactivity of SP (Matsas et al., 1986). The presence of NEP in the CNS has also been demonstrated by the use of NEP inhibitors, including thiorphan and phosphoramidon (Matsas et al.. 1985). In the presence of phosphoramidon. an increase in SP activity in rat striatal slices is ob- served (Mauborgne et al., 1987). Peripherally,


NEP appears to be involved in tachykinin degradation in various organs, including gastrointestinal and other smooth muscle (see Roques et al., 1993). In airways, in contrast to ACE, NEP is localised predominantly to epithelium and submucosal glands of a number of animal species including humans (Borson, 1991; John- son et al., 1985). Furthermore, NEP inhibitors potentiate the secretory response to SP in vitro in ferret and human airways (Rogers et al., 1989; Webber, 1989) as well as in the nasal passages (Petersson et al., 1989). The potentiat- ing effect of NEP can also be demonstrated in vivo, for example in regulation of the magnitude of cigarette smoke-induced plasma exudation in guinea pig airways (Lei et al., 1995) a response mediated via activation of sensory nerves and release of tachykinins.

TACHYKININ RECEPTORS

At present, three tachykinin receptor types are recognised in mammals. Now designated NK,, NK2, and NKI, they were initially characterised by their preferential affinities for the endogenous tachykinin ligands with potency orders of SP > NKA > NKB at the NK, receptor, NKA > NKB > SP at the NK2 receptor, and NKB > NKA > SP at the NK, receptor (for review see Maggi et al., 1993). The characterisation was not ideal as each of the ligands bound to each of the receptors, albeit with different affinities. However, the classification has been, to an extent, confirmed with the development of novel selective ligands, both agonists and antagonists. Certain selective competitive antagonists bind differentially to the NK, receptor subtype of different species (Patacchini et al., 1991). The same has also been shown for the NK, receptor (Beresford et al., 1991; Longmore et al., 1994). Thus, there may be subtypes of tachykinin receptor and the classification system for these receptors may need to be readdressed.

Structure In 1987, Masu et al. cloned the bovine

stomach NK, receptor and expressed it in Xenopus oocytes. They found that it conformed to a previously discovered “super-family” of receptors. Later, the amino acid sequences of the NK, and NK, receptors were also established and found to belong to the same family (Hershey and Krause, 1990; Shigemoto et al., 1990). This group of receptors contains seven

hydrophobic, transmembrane segments and includes the B-adrenergic receptor and the cholinergic (muscarinic) M, and Mz receptors. It is now established that this receptor family is linked to a GTP-binding protein (G-protein), which is an early step in intracellular signal transduction. In fact, G-protein-linked receptors have been demonstrated as being the signal transduction mechanisms for over a hundred receptor types, and studies showing that guanine nucleotides inhibit tachykinin receptor binding confirm that tachykinin receptors are also of this type (Cascieri and Liang, 1983; Lee et al., 1983; Luber-Narod et al., 1990). All share a similar overall receptor structure comprising an extracellular amino terminal and an intracellular carboxy terminal bridged by seven domains spanning the cell membrane (Strader et al., 1994) (Fig. 1). For the human tachykinin receptor, there is approx. 40% sequence identity between NK,, NK, and NK, receptors (Gerard et al., 1993). The greatest identity, around 75%, is found in the cytoplasmic sequences, with approx. 70% identity in the transmembrane domains. Sequence homology amongst species is, in general, greatest for the NK, receptor, with the rat and human receptor being around 90% homologous. However, homology for the NK2 and NK, receptors is still high, being approx. 85 and 88%, respectively.

Distribution The distribution of tachykinin receptors has

been elucidated using both traditional pharmacology, in the form of radiolabelled ligands, and molecular biology, by targetting receptor mRNA.

The development of radioligands has pro- vided an invaluable tool for investigating not only receptor densities in different tissues (Fig. 2) but also the binding characteristics of novel tachykinin antagonists. [1251]-Bolton Hunter substance P binds to NK, receptors, and [‘251]-iodohistidyl NKA or [4,5-3H-Leu]-NKA binds to NK, receptors. However, because they are based upon the natural agonist, each of these ligands lacks selectivity and will bind to other tachykinin receptor types present in the preparation. A more selective agonist radioligand for the NK, receptor is [“‘I]-Bolton Hunter [Sar’,Met(O,)“]-SP (Lew et al., 1990), and for the NK3 receptor, [3H]-senktide (Guard et al., 1988). Development of selective agonist radioligands for the NK, receptor has so far been unsuccessful. The principal advantage in

Tachykinins: receptor to effector 725

L call r-nedxsne

Intracellular side U

Fig. 1. Putative secondary structure of the rat NK, receptor. Filled circles indicate residues that are conserved between the three tachykinin receptors (NK,, NK,, NKJ. E,-E, indicate the extracellular loops. C,-CI indicate the cytoplasmic loops, TM,TMv,, denote the seven transmembrane segments,

and = indicates the site of disulphide linkage (adapted after Nakanishi, 1993).

using agonist radioligands is that they bind to the same or similar receptor sites as the natural ligand. In recent years, however, radiolabelled antagonists possessing greater affinity for the individual receptor types have been developed (Fig. 2). These include rII+CP 96,345 (Zhang et al., 1995) [‘2sI]-CP 96,345 (Cascieri et al., 1992) [“‘II-L 703,606 (Francis et al., 1994) and [3H]-FK 888 (Miyayasu et al., 1993) for the NK, receptor, and [3H]-SR 48968 (Emonds-Alt et al., 1993) for the NK, receptor. While antagonist radioligands are of use in autoradiographic localisation of binding sites, their value for radioligand binding studies is questionable, because some antagonists bind to sites on the receptor distinct from their endogenous ligand (Gether et al., 1993a).

With the use of radioligands and targetting of receptor mRNAs (Tsuchida et al., 1990; Hershey et al., 1991) the distribution of tachykinin receptors has been extensively mapped. NK, receptors are found both in central nervous tissue and in peripheral organs, including rat striatum, spinal cord, gastrointes-

tinal tract and urinary bladder (Beaujouan et al., 1986; Mantyh et al., 1988), and in ferret airway epithelium and submucosal glands (Meini et al., 1993). NK, receptors are found mainly in peripheral organs, including rat gastrointestinal tract and ferret airway smooth muscle (Tsuchida et al., 1990, Meini et al., 1993). NK, binding sites are confined mainly to the CNS, primarily the cerebral cortex tind the dorsal horn of the spinal cord (Dam et al., 1990) although detectable levels are also found in several rat peripheral tissues, incltiing the urinary bladder and intestine (Tsuchida et al., 1990).

Intracellular efSector.5 The intracellular effecters mediating

tachykinin-induced physiological responses are reasonably well described (Fig. 3). In general in the G-protein-linked receptor class, the G-protein in its “resting state” is linked to one of the intracellular loops of the receptor. The G-protein itself comprises three distinct subunits, termed M, fl and y, which are associated together

at rest and are bound to a guanosine diphosphate (GDP) molecule. The GDP-bound heterotrimeric state is a stable entity. After ligand-receptor interaction, conformational changes within the receptor cause the G-protein to exchange GDP for GTP (guanosine triphosphate). This activates the G-protein, causing dissociation of the c( subunit, which binds to and activates the intracellular effector. The CI subunit hydrolyses GTP to GDP, effectively switching itself off, and reassociates with the /I and y subunits.

The three tachykinin receptor types are coupled to the pertussis-toxin-insensitive G,/G,, family, which is linked to phosphoinositide metabolism. The intracellular effector is phospholipase C (PLC). Stimulation of tachykinin receptors initiates phosphoinositol breakdown in a number of tissues, including rat salivary gland, rat brain and guinea pig tracheal smooth muscle (Hanley et al., 1980; Mantyh et al., 1984; Hunter et al., 1985; Grandordy et al., 1988) leading to the production of inositol 1,4,5- trisphosphate (IP,) and diacylglycerol (DAG). IP, releases calcium from intracellular stores by

acting on specific receptors on the sarcoplasmic reticulum. DAG is believed to increase intracellular calcium by opening voltage-gated Ca2 + channels via protein kinase C (Gallacher et al., 1990) (see Fig. 2).

Although PLC is considered to be the principle intracellular effector for tachykinin receptors, in certain cells, tachykinin receptor activation can also be coupled to adenylyl cyclase activation (presumably via G,) in cultured neuroblastoma cells and in dog thyroid gland (Narumi and Maki, 1978; Yamashita et al., 1983) or to adenylyl cyclase inhibition (probably via Gi) in rat salivary gland (Laniyonu et al., 1988). These results indicate that, depending on the cell type, tachykinins are able to elicit their effects by activating multiple effecters via different G-proteins.

Physiological roles A vast amount of research has gone into

demonstrating physiological roles for the tachykinins. The use of endogenous ligands as pharmacological tools, followed by synthetic selective agonists and, more recently, selective

Fig. 2. Autoradiographic mapping of tachykinin receptors in ferret trachea. (A) and (B) Dark-field photomicrographs showing the respective distibution of binding sites for [“‘I]-Bolton Hunter substance P (non-selective NK, radioligand) and [‘HI-CP 96,345 (selective NK, radioligand). (C) and (D) Bright-field photomicrographs of adjacent sections stained with 1% cresyl fast violet to show structures labelled in (A) and (B). For both ligands, binding sites are localised over the epithelium (Ep) and submucosal glands (G) but not over the cartilage (Ca) or smooth muscle (Sm). For each ligand, labelling is greater over the

epithelium than over the gland. These micrographs were kindly supplied by Dr Judith Mak.

Tachykinins: receptor to effector

Fig. 3. Schematic diagram showing the intracellular effector system for tachykinins. Ligand (natural tachykinin or synthetic agonist) interacts with a tachykinin receptor (NK,, NK? or NK& causing exchange of guanosine diphosphate (GDP) with guanosine triphosphate (GTP) on the GTP-binding G-protein. The a subunit of the G-protein dissociates from the /r and 1: subunits and subsequently activates the intracellular enzyme, phospholipase C (PLC). PLC hydrolyses phosphatidyl inositol bisphosphate (PIP,) into inositol trisphosphate (IP,) and diacylgycerol (DAG). IP, acts on specific receptors to release intracellular stores of calcium from the sarcoplasmic reticulum. DAG acts via protein kinase C (PKC) to open L-type calcium channels in the plasma membrane. This increase in [Ca”+], eventually leads to

the tissue response.

antagonists has enabled collection of a great deal of functional information on tachykinin effects. NK,, NK, and NK3 receptors are widely distributed both centrally and peripherally and, therefore, the administration of tachykinin receptor agonists can produce an array of effects. The most useful studies, therefore, are those in which a physiological function is altered by selective antagonists.

In the CNS, SP is the principal tachykinin neurotransmitter, with the NK, receptor domi- nating. NKA is co-localized with SP in most areas, but is present in comparatively minute quantities, with little or no NK, receptor expression (Tsuchida et al., 1990). The NK, receptor is found in certain areas, including the cerebral cortex, nucleus tractus solitarii and the dorsal root of the spinal cord. Consequently, the majority of the tachykinin effects relate to the action of SP. In the CNS, tachykinins have a neuromodulatory role by causing neuronal membrane depolarization, thought to be via a reduction in inwardly rectifying K+

currents (Minota et al., 1981; Stanfield et al., 1985). SP-containing nerves innervate dopamin- ergic neurones in the substantia nigra (Mahalik, 1988) cholinergic neurones in basal forebrain nuclei (Beach et al., 1987) noradrenergic neurones in the locus coereleus (Guyenet and Aghajanian, 1977) and seretoninergic (containing 5-hydroxy-tryptamine, 5-HT) nerves in rat raphe nuclei (Ljungdahl et al., 1978). Adminis- tration of SP in these areas induces release of neurotransmitters (Reid et al., 1990; Nakajima et al., 1985; Guyenet and Aghajanian, 1977). As neurotransmitter dysfunction in the CNS is believed to be the basis for many neurological diseases (for example Parkinson’s disease, Alzheimer’s disease and anxiety), these studies indicate that tachykinins may play a role in neurodegenerative aetiology.

Substance P has an important role in the central control of respiration (von Euler and Pernow, 1954) being released in the nucleus tractus solitarii (NTS) following cardiovascular and respiratory sensory impulses (Srinivasan


et al., 1991; Morilak et al., 1988). Adminis- tration of SP to the NTS causes an increase in breathing frequency and tidal volume (Chen et al., 1990) and bradycardia (Nagashima et al., 1989). In the substantia gelatinosa and brain- stem, release of tachykinins from central sensory endings may represent an important sensory neurotransmitter function for these peptides.

In peripheral tissue, tachykinins can have both sensory and motor functions. Tachykinins and their receptors are widely distributed. The main source of tachykinins in the peripheral nervous system is a population of C-fibres (sensory nerves), which are sensitive to activation, and also degeneration, by capsaicin, the pungent extract of certain fruits of the Capsicum family (classically, hot red chilli peppers) (Holzer, 1991). When stimulated, these fibres release a number of neuropeptide transmitters, including SP, NKA, and calcitonin gene-related peptide (CGRP), which initiate functional responses. These nerves, therefore, subserve “sensory-efferent” or “local effector” functions (Maggi and Meli, 1987; Holzer, 1988). As these “sensory neuropeptides” are pro-inflammatory, the collective response is termed “neurogenic inflammation.” In the airways, for example, sensory-efferent neurones are widely distributed (Lundberg and Saria, 1987). By use of tachykinin receptor antagonists, sensory efferent nerves have been found, particularly in rodent species, to be involved in numerous airway functions including plasma exudation induced by cigarette smoke (Delay-Goyet and Lundberg, 1991; Hirayama et al., 1993) or antigen challenge (Bertrand et al., 1993), bronchoconstriction induced by sodium metabisulphite (Sakamoto et al., 1994) or electrical stimulation of the vagus nerves (Hirayama et al., 1993), as well as neurogenic mucus secretion (Ramnarine et al., 1994;

Khawaja et al., 1995). It is of great interest to use tachykinin receptor antagonists to determine whether tachykinins mediate similar responses in human airways.

Synthetic receptor agonists and antagonists Table 2 details a number of selective synthetic

agonists and antagonists for each tachykinin receptor type and Fig. 4 gives the structure of a selection of the newer antagonists. They have helped elucidate the physiological roles of the individual receptors, as well as offering potential drug therapy for certain diseases. Success in the development of selective agonists was initially achieved by altering the amino acid sequence of the parent peptide, particularly at the common carboxy terminal (Regoli et al., 1988). The discovery of these potent, selective tachykinin receptor agonists furthered the selectivity of receptor monosystems, in which novel antagonists could be tested on the independent receptors (Regoli et al., 1987) (Table 2).

The search for tachykinin antagonists began in the early 1970s with the so-called “first generation” drugs. These were peptide analogues based upon the amino acid sequence of SP, which was the only mammalian tachykinin known at the time. Initially, these drugs simply had D-amino acids substituted for L-amino acids on the SP backbone. The first tachykinin antagonist developed was [D-Pro*,D-Trp’,qSP (Leander et al., 1981), and although inhibition of SP-induced responses in numerous preparations was demonstrated (Holmdahl et al., 1981; Bjijrkroth et al., 1982) the drug suffered from neurotoxic side effects (Post and Paulsson, 1985). Other drawbacks of the first generation antagonists included poor selectivity (Yachnis et al., 1984) and potent mast cell degranulation (Fewtrell et al., 1982), which led to the search for newer, more selective compounds. The originators of [D-Pro*,D-Trp’.qSP extended

Table 2. Agonists and antagonists at tachykinin receptors

NK NKz NK, Amino acid residues Natural agonist potency Synthetic, selective agonists

Synthetic antagonists

Monoreceptor test system

407 SP>NKA>NKB [Sa?, Met(O#‘]SP [SarqSP sulfone

FK888 CP 99,994*

CGP 49823* RF’ 67580*

GR 203040* Dog carotid artery

398 NKA>NKB>SP

[BAla’] NKA, - ,0 We’ll NKA, 10

L 659,877 MEN 10,627 SR 48968*

Rabbit pulmonary artery

465 NKB>NKA>SP

[MePhe’] NKB Senktide

R487 GR 138,676 SR 142801*

Rat portal vein

*Non-peptide.


,‘I \-I,

% N -42

\-/ 0

# J

Fig. 4. Structures of some commonly used NK, and NK2 receptor antagonists (peptide on the left and non-peptide on the right).

cl cl

t-5 / \ ’ I SC loA w _ Kl 0

\ /

their search for tachykinin antagonists by developing four new and less toxic compounds (Folkers et al., 1984). In the following years, “second generation” antagonists were developed. These were more potent and selective, taking into account the recent classification of the neurokinin receptor types. The discovery of a number of highly selective peptide antagonists at the NK, and NK, receptors (see Table 2) vastly increased knowledge of tachykinin function. A drawback of these antagonists was their peptide structure, which made them susceptible to enzymatic degradation, limiting their in viuo potency and, therefore, their clinical potential. An exception to this may be the polycyclic peptide NK, antagonist, MEN 10,627, which is selective, potent and overcomes many of the problems associated with use of peptides as drugs (Maggi et al., 1994). The “third generation” antagonists were non- peptide compounds, the first of which was the Pfizer compound, CP-96,345 (Snider et al., 199 1) (Fig. 4). This drug, while being potent and

selective for the NK, receptor subtype, was found subsequently to exhibit non-selective activities including binding to L-type calcium channels (Schmidt et al., 1992) and demonstration of non-selective effects on neurotransmission and of local anaesthetic activity (Wang et al., 1994). Newer NK, and NK, selective drugs in this group have now been synthesised with demonstrably fewer non-selective actions (Fig. 4) for example the NK, antagonists CP 99,994 (Desai et al., 1992; McLean et al., 1993) and GR 203040 (Beattie et al., 1995) and the NK, antagonist SR 48968 (Advenier et al., 1992; Emonds-Alt et al., 1992). Most recently, antagonists have also been developed for the NK, receptor, an important step in determining the pharmacology of this elusive receptor (Emonds-Alt et al., 1994; Stables er ~1.. 1994).

Binding sites

Once the primary amino acid sequence of a receptor is determined, the secondary structure can be envisaged (Fig. 1). The tertiary


“three-dimensional” (3-D) structure of a protein is determined from the amino acid sequence, with the aid of molecular chaperones (Hart1 et al., 1994). However, the 3-D structure cannot be readily visualised from the amino acid sequence: human biotechnology is not yet sufficiently advanced to be able to correctly fold a protein. Most research in this area has been in the generation of homology models using primary sequence comparisons and secondary structure predictions, invariably based upon a bacteriorhodopsin template (for example, see Hibert et al., 1991). These models serve as 3-D hypotheses that can be tested experimentally, the importance of which is apparent when, in using chimeric molecular biology techniques, the general vicinity of the ligand binding site can be located. Point mutations and ligand changes corresponding to the amino acid changes are then required to describe the details of the interactions for agonists and antagonists (Fig. 5). Pharmaceutical research in the area of G protein-coupled receptors has focused upon homology models to help in the search for compounds that might eventually become therapeutic agents. Currently, there appears to be little effort in the use of 3-D models to “dock” potential compounds, perhaps due to the low resolution of the majority of these models.

For the tachykinin receptors, numerous studies have been undertaken to discover agonist, antagonist and G-protein binding sites. For the natural agonists (SP, NKA and NKB), evidence suggests that binding sites are not distinct, but overlap and may involve different residues that are close when visualised in 3-D (Strader et al., 1994). The presence of conserved groups of amino acids on the amino terminal (residues 23, 24 and 25) and first intracellular loop (residues 96 and 108) of the three receptor subtypes are thought to interact with the common carboxy terminals of the tachykinins, responsible for their common biological activity (Watling and Krause, 1993). A group of residues on the third extracellular loop is also required for high affinity binding of the endogenous peptides (Fong et al., 1992a). Using site-directed mutagenesis, SP binding has been shown to be affected by substitution of the first transmembrane domain and the amino terminal of the NK, receptor (Gether et al., 1993~). In contrast, NKB interaction with the NK, receptor appears to be towards the carboxy terminal, within the seventh transmembrane domain and the third extracellular loop (Fong

et al., 1992a; Gether et al., 1993~). In addition. substitution of valine with glutamate in residue 97 (first extracellular loop) of the NK, receptor confers on it a higher affinity for NKB. Furthermore, the NK, receptor has a glutamate residue at this position, indicating an interaction point for NKB in the first extracellular loop (Fong et al., 1992a). The binding site for the NK2 receptor appears to be after the fifth transmembrane segment, as NK2 substitution at this segment increases the affinity of the NK, receptor for NKA (Gerard et al., 1993). Binding sites for the intracellular effector system also appear to be different for each receptor. G-protein interaction sites are in the third cytoplasmic loop for NK, receptors and the carboxy terminal loop for the NK, receptor (Blount and Krause, 1993). However, it should be remembered that it is difficult to interpret results of a mutation unless careful comparative studies are done between a variety of mutants and a number of different agonists and antagonists. The effects of a mutation might be due to a direct interaction with the ligand, leading to large changes in potency (approx. l&20-fold). Conversely, the effects of the mutation might be indirect, in that it produces a subtle conformational change in the receptor, which alters the affinity for one ligand compared to another (Fong et al., 1992b).

Binding sites for the antagonists overlap in a way similar to that of binding sites for the natural agonists. The majority of research has been on the NK, receptor. The non-peptide NK, receptor antagonist, CP 96,345, has been shown to interact with the extracellular portion of the transmembrane segments V and VI, specifically residues 183-196 (tmV) and 271-276 (tmV1) (Gether et al., 1993a,b), a glutamine residue (165) in the fourth transmembrane domain (Fong et al., 1994), and histidine (265) in the sixth transmembrane segment (Zoffmann et al., 1993). The species-related binding differences seen for CP 96,345 and other receptor antagonists have been attributed to the non- conserved residues between rat and human NK, receptor. It appears that substituting the serine residue 290 and leucine at 116 in rat, with isoleucine and valine (present in humans), respectively, reverses the difference in binding profiles of CP 96,345 and RP 67580 seen in rat and human (Fong et al., 1992b; Sachais et al., 1993; Jensen et al., 1994). The interaction site for RP 67580 is believed to be in the seventh transmembrane domain, between residues 262

Tachykinins: receptor to effector

Fig. 5. Tachykinin receptor binding model. The human NK, receptor is shown in its three-dimensional conformation based upon the bacteriorhodopsin “footprint”. Side views (left panels) place the extracellular loops at the top; top views (right panels) are from the extracellular side of the receptor. (A) Substance P in its proposed binding site; (B) the NK,-receptor antagonist, CP 96,345, in its binding site. The antagonist binding conformation is derived from pharmacophore mapping and is a reasonable interpretation. The SP conformation and its docking mode is hypothetical but emphasises the difference in size between the natural ligand and the synthetic antagonist. Residues involved only in agonist binding are in yellow, those invoved in antagonist binding are in red, while the green residues are involved in signal transduction. These images were kindly produced for us by Dr Dennis Underwood (Merck Research

Laboratories, West Point, Pennsylvania. U.S.A.).


and 270 (Gether et al., 1994), as is the site for FK888 (both compounds are selective NK, receptor antagonists). A further residue (serine 169) appears to be crucial for RP 67580 binding (Fong et al., 1994). These binding-site studies have also been carried out on newer, non-peptide NK, receptor antagonists, including L- 703,606 and L-161,664 (Huang et al., 1994; Cascieri et al., 1995), perhaps indicating the future direction for designing novel drugs.

Clinical implications

The discovery of a novel endogenous mediator of a physiological response invariably initiates investigation of its possible role in the pathophysiology of human disease. The mammalian tachykinins have been implicated in numerous physiological as well as pathophysiological mechanisms, and are important targets for therapy of a number of disease states. In the central nervous system, tachykinins modulate the release of neurotransmitters from dopamin- ergic, cholinergic and noradrenergic nerves (Maggi et al., 1993). This may have important implications in the pathophysiology of neurodegenerative diseases in which these neurotransmitters are abnormal, most notably Parkinson’s disease, Alzheimer’s disease, depression and anxiety. Tachykinins also appear to play a role in the transmission of pain (McCarson and Krause, 1994; Lembeck et al., 1981; Laneuville et al., 1988), and so a potential, therapeutic antinociceptive agent could be a possibility.

Peripherally, the tachykinins are localized in C-fibres. Although the primary function of C-fibres is still thought to be sensory, the release of sensory neuropeptides from peripheral terminals represents an additional “motor” function only brought into play under conditions of injury or infection. Consequently, these C-fibre neurones have been implicated in the pathophysiology of a number of inflammatory disorders, including rheumatoid arthritis, irritable bowel syndrome, cystitis and allergic rhinitis (Maggi et al., 1993). In the lower airways, sensory-efferent nerves and tachykinins can be readily shown to be involved in respiratory physiology of a number of non-human animal species, whereas in humans it has proved to be diffcicult to find a role for the tachykinins. However, it is possible that the involvement of sensory-efferent nerves and tachykinins increases in inflammatory conditions of the airways, including asthma and chronic bronchitis. For example, during an

exacerbation of asthma, plasma concentrations of SP-like immunoreactivity are double those in healthy controls (Cardell et al., 1994). SP-like immunoreactivity is also increased in the sputum of patients with asthma or chronic bronchitis compared with healthy controls (Tomaki et al., 1993). These data are consistent with the finding of a reduced airway content of SP-like immunoreactivity in the trachea of asthmatic patients (Lilly et al., 1995), which the authors took to indicate augmented SP release followed by degradation, but may be due also to passage of SP into the blood stream and sputum. The latter three observations indicate that asthma and chronic bronchitis are associated with increased sensory-efferent nerve activity, decreased degradation of released tachykinins, or both. In the airways of asthmatic patients, the number and length of SP-immunoreactive nerves have been found to be increased above that in non-asthmatics (Ollerenshaw et al., 1991), although this has not been confirmed (Howarth et al., 1995). Further- more, steady-state mRNA levels for tachykinin NK, receptors, but not NK, receptors, have been shown to be increased in the asthmatic lung (Adcock et al., 1993). In contrast, NK, rather than NK, receptor mRNA has been shown to be increased in membranous bronchi of asthmatics compared with non-smoking controls (Bai et al., 1995). However, any increase in tachykinin mRNA is not necessarily associated with an increased receptor number (Goldie, 1990). It remains to be determined whether increases in nerve density and receptor number would enhance tachykinin effects in the airways and whether these in turn would contribute to pathogenesis of bronchial disease. One indication that they might is that the dual NKJNK, receptor antagonist FK224 inhibits bradykinin-induced bronchoconstriction and cough in asthmatic subjects, responses which are considered to be due to activation of sensory-efferent nerves and release of tachykinins (Ichinose et al., 1992), and reduces sputum production and cough in patients with chronic bronchitis (Ichinose et al., 1993). In contrast, the selective NK, receptor antagonist CP-99,994 does not inhibit hypertonic saline- induced bronchoconstriction or cough in mild asthmatic patients (Fahy et al., 1995). This may indicate that the NK, receptor is more relevant in asthma. It will be interesting to see the results of similar studies using NK, receptor antagonists. However, it should be noted that the


aforementioned studies each investigated lim- ited and artificially induced aspects of asthma. Longer term studies of the effectiveness of tachykinin receptor antagonists in “natural” asthma are undoubtedly required to fully determine the potential of these drugs as therapeutic agents in asthma. Interestingly, sodium cromoglycate, a drug used in the treatment of asthma for over 25 years, has been shown to have tachykinin receptor antagonistic properties (Page, 1994). Long-term trials of reduction of asthma symptoms are required to determine the role of tachykinins in pathogenesis of these diseases and of the therapeutic potential of tachykinin receptor antagonists. It will also be interesting to see whether tachykinin antagonists can differentiate between the rela- tive importance of sensory-efferent nerves and tachykinins in chronic non-productive cough, sneezing and asthma (Karlsson, 1993).

CONCLUSIONS

It is now 64 years since the discovery of SP (von Euler and Gaddum, 1931). During this time, four other mammalian tachykinins have been discovered (NKA, NKB, NPJJ and NPK), three tachykinin receptor types have been cloned (NK,, NK, and NK,), and numerous peptide and non-peptide tachykinin receptor antagonists have been developed. However, various anomalies in these studies (including inter- an intraspecies binding differences) indicate that understanding of the receptor subtypes, and perhaps effector systems, is incomplete. This has important implications in terms of disease therapy, as the diversity of tachykinin effects is immense, ranging from effects in the central nervous system to gastrointestinal effects to inflammatory effects. In order to better charac- terise physiological roles for the tachykinins we need first to understand their pharmacology then develop highly selective ligands to target the appropriate receptor type. With greater understanding of ligand-receptor interactions, the appropriate tissue then be targetted. However, despite the new technologies under- lying rational drug design, serendipity will always have a role to play in drug discovery. It will be fascinating to follow tachykinin research over the next decade to see just how far tachykinins are implicated in human disease.

Acknowledgements-We thank Ciba Geigy (Basel, Switzer- land) for funding A. M. K., and A. Menarini Pharmaceuti-

cals (Florence, Italy), the Cystic Fibrosis Research Trust (U.K.), the Fujisawa Pharmaceutical Co. Ltd. (Osaka, Japan), and Pfizer Central Research (Sandwich, U.K.) for supporting a number of the studies referred to in the text.

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