I. Introduction

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Seventy years ago, von Euler and Gaddum described an unidentified substance present in alcoholic extracts of equine brain and intestine that in the rabbit displayed a potent stimulant action on the jejunum and a hypotensive action that was distinct from all compounds then known to stimulate the gut and that was referred to as "P" on the tracings and the protocols.

Using semipurified preparations, numerous biological studies of its activity were carried out, but many efforts have been made to isolate the active substance. After some unsuccessful attempts on horse intestine, substance P (SP) was isolated in a pure form from bovine hypothalamus, and 40 years later, its structure was established by Chang and Leeman (1970). SP was then isolated in a pure form and sequenced also from horse intestine (Studer et al., 1973). SP was one of the most extensively studied active substances during the half-century since its discovery, and for many years, it was believed to be the only mammalian tachykinin considered to be a neuropeptide. This belief was firmly put to rest only in 1983 with the discovery of neurokinin A (NKA) and neurokinin B (NKB) (Kangawa et al., 1983; Kimura et al., 1983) that differ from SP in their pharmacological activity, both peripheral and central, and in their preference for different tachykinin receptor subtypes.

The story of the identification of SP was very similar to that leading to the discovery of nonmammalian tachykinins. In 1947, while investigating the occurrence of biogenic amines, especially serotonin in the posterior salivary glands of a Mediterranean octopod, Eledone moschata, an unidentified substance was found that again lowered blood pressure in rabbits and dogs, stimulated isolated preparations of intestinal smooth muscle, and caused profuse salivation in dogs and rats (Erspamer, 1949). The structure of this substance, first called moschatin and then eledoisin, was established in 1962 (Anastasi and Erspamer, 1962; Erspamer and Falconieri Erspamer, 1962). In the same year, it was found that extracts of the skin of the South American leptodactylid frog Physalaemus biligonigerus (formerly fuscumaculatus) also displayed eledoisin-like activity. Also the elucidation of the structure of physalaemin (Anastasi et al., 1964; Erspamer et al., 1964) recalled that of eledoisin and similarly was followed in rapid succession by the identification of a number of other related peptides in the skin, in the brain and gut of amphibians, and in brain and gut of submammalian species (from birds to agnata).

These peptides, all called tachykinins, represent the largest known peptide family, including members occurring in different animal species from low invertebrates to mammals. The possible occurrence of authentic tachykinins in invertebrates was confirmed by the isolation of two tachykinins from the salivary glands of a mosquito (Champagne and Ribeiro, 1994) and by the occurrence in nervous structures of the insect Locusta migratoria of four related peptides (the locustatachykinins) having structure homology with the vertebrate tachykinins (Schoofs et al., 1990a,b).

The identification of the locustatachykinins was soon followed by the isolation of similar peptides in other insects and in crabs, echinoid worms, and molluscs. It will be shown that locustatachykinin-like peptides, the number of which is destined to grow, have full citizenship right in the tachykinin peptide family, which with more than 40 members represents one of the largest, if not the largest, family in the peptide world.

The purpose of this review is, first, to keep the reader up to date on the different occurrences, species distributions, and localizations of the numerous members of the tachykinin peptide family. Second, because the identification and study of nonmammalian tachykinins has contributed conspicuously to the explosive progress of knowledge in the field of mammalian active tachykinins, we put in evidence the extensive pharmacological studies on the nonmammalian tachykinins (TKs) (eledoisin, physalaemin, and kassinin) whose availability preceded by years that of the corresponding mammalian peptides.

	II. Occurrence and Species Distribution of Tachykinin-Like Peptides

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At present among the numerous families of neuropeptides, which are evolutionarily the oldest neurotransmitters, perhaps even older than acetylcholine and catecholamines, four tachykinin-like peptides seem to occupy a very important position.

a. Substance P-like immunoreactivity (SP-LI) was localized in the primitive nervous system of Hydra (Taban and Cathieni, 1979; Grimmelikhuijzen et al., 1981; Pierobon et al., 1989), in the cerebral ganglion of the locust (Benedeczky et al., 1982), in the central nervous system of the cockroach Periplaneta americana (Verhaert and De Loof, 1985), in the retina and eyestalk neurones of the lobster Palinurus interruptus (Mancillas et al., 1981), in the eye stalk of the fiddler crab Uca pugilator (Fingerman et al., 1985), in the somatogastric system of the crab Cancer borealis and the lobster P. interruptus and Homarus americanus (Goldberg et al., 1988), in tissues of the earthworm Lumbricus terrestris (Aros et al., 1980; Kaloustian and Edmands, 1986), in the adult nervous system of the fly Sarcophaga bullata (Sivasubramanian, 1990), in the cricket Teleogryllus communis (Lembeck et al., 1985), in the brain and central ganglia of the bowfly Calliphora vomitoria and of Drosophila (Lundquist et al., 1994), in the brain, corpora cardiaca, and corpora allata of the insect Leucophaea madeirae (El-Salhy et al., 1983), in the central nervous system of the mollusc Limulus polyphemus (Mancillas and Selverstone, 1985), and in the nervous system of several parasitic trematode worms (Bush and Gupta, 1988) including Schistosoma mansoni (Gustafsson, 1987), Diphyllobathrium dendriticum (Gustafsson et al., 1986), Fasciola hepatica (Magee et al., 1989), and Diclidophora merlangi (Maule et al., 1989).

In a large number of invertebrate phyla from coelenterates to molluscs, in addition to the usual SP-like tachykinin, an NKA-like peptide has also been found. However, it is highly improbable that authentic SP or authentic NKA is present in invertebrates. First, because retention time of the invertebrate tachykinins in elution from reverse-phase HPLC columns never coincided with retention time of the mammalian tachykinins; and second, because authentic SP and/or NKA was never found, even in lower submammalian species (amphibian, fish, and agnata). It has also been shown that radioimmunoassay and immunohistochemical techniques are often insufficient to distinguish between structurally related peptides because of frequent lack of selectivity of the pertinent antisera. Callitachykinin II, for example, was recognized not only by an antiserum to the locustatachykinin (in both peptides the C-terminal residue is Arg-NH₂) but also by an antiserum to the amphibian kassinin having, like all other classical tachykinins, the C-terminal residue Met-NH₂ (Lundquist et al., 1994). Moreover, preincubation of locustatachykinin antibody with SP and preincubation of SP antibody with locustatachykinin blocked subsequent immunolabeling of the somatogastric nervous system in C. borealis, indicating that a member of the locustatachykinins is likely to be the source of the previously identified SP in the nervous system (Blitz et al., 1995).

b. Schoofs et al. (1990a,b) first described the occurrence in insects, more precisely in extracts of brain, corpora cardiaca-corpora allata, and suboesophageal ganglion of Locusta migratoria of five peptides, the locustatachykinins, which exhibited sequence homologies (up to 45%) with the vertebrate tachykinins, especially with amphibian and fish tachykinins. The locustatachykinins were completely inactive in all bioassay preparations used for the vertebrate tachykinins but showed a myotropic action in the insect intestine, eliciting a potent contraction of the cockroach hindgut (Winther et al., 1998).

The prediction of Schoofs' group in their first paper (Scoofs et al., 1990b) that "the peptides discovered in this study may be just the first in a whole series of substances from arthropod species to be identified as tachykinin family peptides" was correct even beyond any expectation. Up to the present, as many as 20 locustatachykinin-like peptides were isolated not only from various other arthropods, but also from an echinoid worm and from molluscs (Nassel, 1999). Table 1, reporting the present, probably provisional situation, shows that invertebrate tachykinin-like peptides are linear peptides with 8 to 15 amino acid residues and that, with the exception of the Leucophaea tachykinin-related peptide LemTRP10, they have at their C terminus an amidated Arg residue instead of the amidated Met residue, which is peculiar without exception to all classical tachykinins, including the invertebrate tachykinins (eledoisin and sialokinin I and II). Lem TRP1 is present also in two elongated forms with 17 (LemTRP2) and 19 (LemTRP3) amino acid residues, respectively (Winther et al., 1999).

In the light of appearance on the screen of the locustatachykinins and of the fact that locustatachykinin antisera may cross-react with SP, it is probable that in several and perhaps in most cases, the SP-LI described in a variety of invertebrates must be ascribed to locustatachykinin-like peptides. Thus, the locustatachykinin-like peptides of invertebrates must be considered authentic tachykinins, being either the primitive representatives of the tachykinin peptide family from which the vertebrate tachykinins have evolved by simple substitution of the C-terminal residue Arg-NH₂ with Met-NH₂ or an evolutionary adaptation for the invertebrates of a common ancestral tachykinin prototype already possessing the C-terminal Met-NH₂ residue.

c. Authentic tachykinins with the classical C-terminal pentapeptide sequence Phe-(Tyr/Ile)-Gly-Leu-Met-NH₂ occur in non-neuronal, epithelial cells of the posterior salivary glands of the Mediterranean octopods E. moschata and Eledone aldovrandi (eledoisin, up to 100 nmol/g wet tissue) (Erspamer and Falconieri Erspamer, 1962) and in the salivary glands of the mosquito Aedes aegypti (sialokinins I and II) (Champagne and Ribeiro, 1994): eledoisin, pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH₂; sialokinin I, Asn-Thr-Gly-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH₂; sialokinin II, Asp-Thr-Gly-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH₂.

These peptides display the full spectrum of activity of the mammalian tachykinins and bind to the same receptors. It is remarkable that eledoisin occurs only in Eledone but not in the strictly related Octopus vulgaris. Yet, the salivary glands of both Eledone and Octopus contain large amounts of biogenic amines: serotonin (up to 2 µmol/g), octopamine, tyramine, and histamine.

B. Prevertebrate Tachykinin-Like Peptides

1. Amphioxus lanceolatus. Radioimmunoassay combined with HPLC suggests the occurrence of small amount of SP-LI in brain and spinal cord of this prevertebrate species (Lembeck et al., 1985).

2. Tunicata (Protocordata). Using immunohistochemical techniques only, the presence of an antigen related to substance P has been demonstrated in the neuronal ganglion (Fritsch et al., 1979), gill epithelium (Fritsch et al., 1980), and alimentary tract (Fritsch et al., 1982) of the ascidian Ciona intestinalis. More recently, the occurrence of tachykinins in C. intestinalis tissues was re-examined by O'Neil et al. (1987) using specific antisera for the C terminus (C) of SP and the N terminus (N) of mammalian SP and NKA, completed with immunohistochemistry and reverse-phase HPLC of the tissue extracts. It was found that only C-SP-LI (not N-SP-LI) occurs both in cells of the ganglia and in peripheral neurons, together with but separately from N-NKA-LI. Only C-SP-LI was found in endocrine cells of the pharynx. However, we conclude that already at the prevertebrate stage of chordate evolution, the tachykinin family is represented by at least two distinct members that are provided by separate cell populations, none of which was identical with either mammalian SP or mammalian NKA.

The formidable enlargement of the tachykinin peptide family is consequent to systematic studies conducted on the one side on the amphibian skin and on the other side on the brain and intestines of submammalian vertebrates, mainly in their cold-blooded classes: reptiles, amphibians, fish, and agnata. The story of the group of the amphibian skin peptides began in 1964 with isolation and structure elucidation of physalaemin (Erspamer et al., 1964), followed by the systematic screening of peptide contents in the skin of as many as 600 amphibian species from all over the world, which resulted in the discovery and isolation of numerous neuropeptides, belonging to a dozen distinct families, among which is that of the tachykinins (with 21 members).

The fruitful search for tachykinin peptides in brain and gut of submammalian vertebrates started in 1986 with the isolation and structure elucidation of scyliorhinins I and II from dogfish intestine (Conlon et al., 1986a). At the end of 1998, a list of as many as 24 novel tachykinins was available, 12 from brain and 12 from gut. Skin tachykinins and brain/gut tachykinins will be discussed separately.

1. Amphibian Skin Tachykinins. Table 2 summarizes the present situation. The great majority of amphibian skin peptides have the classical C-terminal pentapeptide sequence: Phe-X-Gly-Leu-Met-NH₂. However, important exceptions are represented by: 1) some tachykinins from the skin of the Australian frog Agalychnis callidryas, namely AC-AR1, -AR2, and -AR3 with the C-terminal pentapeptide sequence Phe-Tyr-Pro-Gly-Met-NH₂ and AC-AR4 with sequence Phe-Tyr-Pro-Val-Met-NH₂; and 2) hylambatin from the skin of the South-African frog Hylambates maculatus with the C-terminal pentapeptide sequence Phe-Tyr-Gly-Met-Met-NH₂. It is evident that in the C-terminal pentapeptide only the Phe residue at position 5 from the C terminus and Met-NH₂ are immutable.

2. Brain and Gut Tachykinins. Table 3 summarizes the present situation. All of the tabulated tachykinins, with the exception of ranatachynin D, show the classical C-terminal pentapeptide Phe-X-Gly-Leu-Met-NH₂. Of considerable interest is the fact that in goldfish, cod, and trout NKA-like peptides, the usual acidic Asp residue at position 7 from the C terminus, crucial for receptor NK2/NK3 selectivity, is replaced by the neutral Asn residue.

View this table: [in this window] [in a new window]   TABLE 4 Amino acid sequence of submammalian vertebrate -neuropeptides

D. Mammalian Tachykinins

1. Mammalian Tachykinins and Their Biosynthesis. Until now, only three tachykinins have been isolated and sequenced from mammalian tissues: SP, NKA (neuromedin L, neurokinin, and substance k), and NKB (neurokinin and neuromedin k). NKA is present also in two elongated forms, neuropeptide K and neuropeptide- (Table 5).

	III. Localization of Tachykinin-Like Peptides

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We have previously shown that tachykinins constitute one of the largest families of peptides in the world whose members are present in all animal species, from lower invertebrates to mammals. Tachykinins possess a widespread distribution in the central and peripheral nervous system that is undoubtedly the major source of these peptides. However, tachykinins have also, like numerous other peptides and like all biogenic amines, a limited and species-dependent, but not negligible, distribution in non-neuronal structures represented by the irregular and sparse localizations in which they display known and unknown functions. In the first localization (neuronal cells), the active compounds act as neurotransmitters/neuromodulators, in the second (non neuronal cells) as autocrine, paracrine, or endocrine regulators.

A. Non-Neuronal Localization

1. Amphibian Skin. The most complex and rich localization of non-neuronal tachykinins is certainly the amphibian skin, that may be considered a huge factory and storehouse of a variety of tachykinins. However, an important characteristic of the skin peptides is their extreme irregularity in occurrence and richness: some amphibian species are extremely rich in active peptides, others, even closely related species, are lacking of active peptides. These findings contribute to the incomprehension of the physiological significance of the skin tachykinins and of that of the skin biogenic amines, especially indolealkylamines that are similarly present, sometimes in enormous amounts in the skin. The occurrence in the skin of a such variety of neuropeptides and amines may perhaps be explained by the common embryogenic origin of the integument and the nervous system from the primitive ectoderm.

2. Invertebrate Salivary Glands. The significance of eledoisin, as with that of the large amounts of biogenic amines, occurring in the posterior salivary glands of E. moschata but not in the glands of any related Octopus species is again completely obscure. On the contrary, the sialokinins occurring in the salivary glands of A. aegypti may be interpreted as an evolutionary selection of the peptides as vasodilators, in connection with the habit of feeding in blood of this insect (Champagne and Ribeiro, 1994).

3. Normal Mammalian Tissues. All of the non-neuronal localizations of the tachykinins concern mainly SP, and the occurrence of the peptide was established only by immunohistochemistry, using selective SP-antisera. SP occurring (together with serotonin) in populations of argentaffin/chromaffin and argyrophil/acidophil (with no serotonin) cells of the mammalian and probably also of lower vertebrate (ascidian and fish) intestine may act either as paracrine hormone or as a true hormone after release into the blood stream. It is possible that SP found in mammalian blood originates prevalently from the SP-containing cells of the intestinal mucosa. More than 60 years ago, Vialli and Erspamer (1933) described the so-called "acidophil basigranular cells" of the dog and cat large intestine, and it would be worthwhile checking to see if they are, like the argyrophil cells of the human large intestine, SP-producing cells. The acidophil cells are, in fact, neither argentaffin nor chromaffin, that is they do not contain serotonin. Little is known as to whether and under which conditions SP is released from the endocrine cells of the gastrointestinal mucosa. For example, release of SP from the enterochromaffin cells of the rat caecum mucosa seems to be inhibited by serotonin and calcium-free medium (Simon et al., 1992). Moreover, that SP may be released from the gut endocrine cells into the blood stream is strongly suggested by the evidence that legation of all intestinal blood vessels and evisceration in the cat significantly lowered SP plasma levels (Gamse et al., 1978) and by the fact that portal venous blood contains about 4 times more SP than peripheral blood (Pernow, 1983).

Data on the distribution and localization of neuronal tachykinins in the CNS and periphery have been obtained by a combination of HPLC with radioimmunoassay and/or by immunohistochemistry (Hokfelt et al., 1975, 1977; Pernow, 1983; Maggio, 1985).

Regional specific antisera directed against the C-terminal region of the tachykinins have been generally used, a condition that is unfortunate for discrimination between the various tachykinins (Maggio, 1988). The low specificity of several antisera and the different tissue extraction methods (Lindefors et al., 1985; Brodin et al., 1986) may explain some differences encountered in the literature on the localization of the three mammalian tachykinins.

Central nervous system.  Distribution of the tachykinins in the CNS has been extensively studied only in the rat (Otsuka and Yoshioka, 1993). Data on other species are scanty. As expected, SP is generally cosynthesized, colocalized, and cosecreted with NKA. The values of immunoreactive SP in various areas of the rat brain are: olfactory tubercle, 300 pmol/g of wet tissue; amygdala, 383 pmol/g of wet tissue; nucleus caudatus, 247 pmol/g of wet tissue; globus pallidus, 332 pmol/g of wet tissue; septum, 116 to 405 pmol/g of wet tissue; hypothalamus, 208 to 626 pmol/g of wet tissue; habenula, 377 pmol/g of wet tissue; posterior pituitary, 489 pmol/g of wet tissue; thalamic nucleus, 25 pmol/g of wet tissue; globus pallidus, 332 pmol/g of wet tissue; substantia nigra, 1725 to 2580 pmol/g of wet tissue; periaqueoductal central gray, 590 to 994 pmol/g of wet tissue; locus caeruleus, 332 pmol/g of wet tissue; nuclei parabranchiales, 546 pmol/g of wet tissue; medulla oblungata, 95 to 436 pmol/g of wet tissue; dorsal horn of the spinal cord, 1070 pmol/g of wet tissue; and ventral horn, 134 pmol/g of wet tissue (Kanazawa and Jessell, 1976; Douglas et al., 1982). The concentration of SP may reach values as high as 2 to 3 µg/g of wet tissue.

In rats, both the density and the distribution of SP-containing neurons is changing significantly during the period just before birth, the days after birth, and into the adult period. SP-staminal cells and fibers reached maximum levels between postnatal days 5 and 15. Then density generally decreased (Inagaki et al., 1982; Sakanaka et al., 1982). The distribution of NKA is less known and in the rat brain, seems to be similar to that of SP, with clearly different locations, however, in several regions. As revealed by immunohistochemistry, NKB-containing perikarya were detected in the main and accessory olfactory bulb, some cortical regions, the olfactory tubercule, the n. accumbens, the septum, the neostriatum, several hypothalamic nuclei, the superior colliculus, the substantia nigra, the medullary reticular formation, and the external caudate nucleus (Kanazawa et al., 1984; Merchenthaler et al., 1992). NKB is also located in the spinal cord, predominantly in the dorsal horn, while it is present in negligible amounts in dorsal root ganglia and dorsal roots (Ogawa et al., 1985).

In the cat brain, kassinin-LI (NKA-LI) has a widespread distribution with the highest concentration present in substantia nigra, hypothalamus, and caudate n.; moderate levels in thalamus, brain stem, and spinal cord; and low levels in the cortex and cerebellum. Distribution of NKA-LI paralleled that of SP, although the ratio between the two peptides varied throughout the different areas (Hunter et al., 1985).

In human brain, the areas most rich in immunoreactive SP were: amygdala, 25 to 340 pmol/g of wet tissue; nucleus caudatus, 113 to 370 pmol/g of wet tissue; putamen, 81 to 380 pmol/g of wet tissue; globus pallidus, 518 to 1800 pmol/g of wet tissue; hypothalamus, 125 to 135 pmol/g of wet tissue; substantia nigra, 1264 to 4720 pmol/g of wet tissue; and locus caeruleus, 199 pmol/g of wet tissue (Gale et al., 1978; Emson et al., 1980; Cooper et al., 1981).

Data on the occurrence of SP and SP-like peptides in the frog and fish brain are presented by Inagaki et al. (1981).

Gut.  The main sources of the neuronal tachykinins in the gut are: a) the intrinsic enteric neurons of the myenteric plexus, b) the intrinsic enteric neurons of the submucosal plexus, and c) the extrinsic primary afferent fibers. The most quantitatively important source of tachykinins in the gut is the enteric nervous system, which has its cells in the wall of the intestine and supplies all gastrointestinal effector systems. The mammalian gastrointestinal tract contains both SP and NKA and various extended forms of these tachykinins.

Neurons that contain only SP (alone or with other nontachykinin transmitters) are considered to be intrinsic sensory neurons (Holzer and Holzer-Petsche, 1997a, 1997b). In addition to these neurons, extrinsic efferent nerve fibers also display a small, but distinct contribution to the SP/NKA immunoreactivity in the gut. These fibers originate from dorsal root ganglia and reach the periphery via sympathetic or parasympathetic nerves, passing through prevertebral ganglia. The extrinsic efferent nerves project predominantly to the vessels in the intestinal wall but they also supply the lamina propria of the gastrointestinal mucosa. There are considerable species-dependent quantitative differences in the location of the tachykinins in the various gut segments, in the concentration of the peptides and in the density of SP/NKA-containing fibers.

In most species, the highest concentrations of tachykinins in the gut are found in pylorus, gastric fundus, duodenum, and jejunum (Pearse and Polak, 1975; Lazarus et al., 1980; Hunter et al., 1985; Gates et al., 1989). In the guinea pig small intestine, the bulk of SP and NKA, which are stored in the same synaptic vesicles, is associated with the myenteric plexus in longitudinal muscle.

Concerning NKB, the peptide is generally considered to be absent from human, porcine, guinea pig, and rat intestine, which is consistent with the absence of PPT-B expression in the enteric nervous system of the rat but is in contrast with other results, showing that human and rat intestine contains minute amounts of NKB (Holzer and Holzer-Petsche, 1997a) and even more so with data showing that highly specific antiserum to NK3 receptors detected them in nervous myenteric and submucosal neurons (Grady et al., 1996). SP- but not NKA- and NKB-immunoreactivity is present also in the gall bladder and bile duct and in the pancreas, both around blood vessels and in the acini and the islets (Otsuka and Yoshioka, 1993).

Respiratory tract.  RIA and immunohistochemistry have demonstrated the presence of SP and NKA in the respiratory tract of various mammalian fibers. In the trachea and bronchi, SP-immunoreactive fibers have been found in the smooth muscle layer and around local ganglion cells. In the bronchial tree, most of the SP-positive fibers are of vagal origin; but in the lung, the fibers are both of vagal and thoracic spinal origin. (Nilsson et al., 1977; Lundberg et al., 1983; Saria et al., 1985; Manzini et al., 1989).

Blood vessels.  Data on the occurrence of SP-containing fibers are rather scanty and old. SP-like immunoreactivity has been observed in fibers of the adventitia and media in various blood vessels, such as feline cerebral arteries (Liu Chen et al., 1986), guinea pig intestinal vasculature (Furness et al., 1982), and rat portal vein (Barja and Mathison, 1982). The majority of SP-containing perivascular fibers are of sensory, capsaicin-sensitive origin.

Urinary system.  The distribution of SP and NKA has been extensively studied in renal pelvis and ureter and especially in the urinary bladder of several species (Sharkey et al., 1983; Gibbins et al., 1985; Maggi et al., 1987). Capsaicin treatment results in an almost complete disappearance of the tachykinin-immunoreactive fibers, suggesting that the major sources of tachykinins in the urinary bladder are sensory fibers (Maggi and Meli, 1988; Maggi et al., 1988).

Skin.  In human digital skin, SP- and NKA-immunoreactivity is present in free nerve endings in dermal papillae and epidermis (Dalsgaard et al., 1985; Bjorklund et al., 1986). SP-like immunoreactive fibers are also found in the skin of the rat and cat (Hokfelt et al., 1977). Treatment with capsaicin in rats caused a 70% depletion of SP-like immunoreactivity in various skin areas, suggesting that SP is present mainly in primary afferent C-fibers (Holzer, 1991).

Immune system.  Tachykinin-containing primary capsaicin-sensitive afferent nerves are present in lymphoid organs, such as thymus, spleen, lymph nodes, and lymphoid aggregates in the lung and nasal mucosa. Their distribution is prevalently perivascular, but some fibers penetrate within the follicles. Both SP and NKA have been detected in rat thymus, spleen, and lymph nodes by radioimmunoassay. In addition to NKA, neuropeptide K and an eledoisin-like peptide also occur in the guinea pig thymus (Geppetti et al., 1987). However, non-neuronal sources of tachykinins are also present in the immune system. Using an anti-NKA antiserum, positive immunostaining was observed in staminal cells throughout the thymic parenchyma of the rat with predominance in the medullary area (Ericsson et al., 1990).

Because endothelial cells express SP-LI (Linnik and Moskowitz, 1989; Ralevic et al., 1990), the vascular endothelium of lymphoid organs may be the source of non-neuronal tachykinins at this level. Moreover, there is evidence that certain immune cells such as eosinophils and macrophages synthesize and release SP (for review, cf. Maggi, 1997).

Blood.  Quantitative data on the concentration of immunoreactive SP in blood plasma are very variable with a wide range of values obtained by the different authors, indicating that nonspecific factors are probably interfering with the assay of immunoreactive SP. This is particularly true when unextracted plasma was used. As previously stated, the major part of circulating SP evidently originates from the intestine (Pernow, 1983). Values were as follows: man 70 to 300 and 50 to 620 fmol/ml; dog, 40 to 50 fmol/ml; and calf, 165 and 18 fmol/ml for unextracted and extracted plasma, respectively.

	IV. Relationships between Structure/Activity Receptor Selectivity

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Tachykinins, yet defined as peptides having the characteristic C-terminal pentapeptide Phe5-Xaa4-Gly-Leu-Met-NH₂, are identified as "aromatic tachykinins" when Xaa is an aromatic amino acid residue (Phe or Tyr) and "aliphatic tachykinins" when Xaa is an aliphatic amino acid residue (Val or Ile). All natural tachykinins are amidated at their C terminus, and this function is crucial for biological activity. Deamidated peptides are virtually inactive (Erspamer, 1994).

Structure/activity relationship studies established that the C-terminal pentapeptide was essential but not sufficient for the biological activity of the tachykinins. In fact, the C-terminal pentapeptide of eledoisin and physalaemin (Bernardi et al., 1964; Regoli et al., 1994b) like that of all other examined tachykinins was virtually inactive. The minimum chain length required for activity was six residues. These studies also recognized the Phe residue at position 5 from the C terminus and the amidation at the C terminus to be crucial for biological activity, both occurring in all vertebrate and invertebrate tachykinins, as well as the presence of the C-terminal Arg-NH₂ in the locustatachykinin-like peptides. The biological activity of the tachykinins depends on their interaction with three G protein-coupled receptorsNK1, NK2, and NK3which share considerable structural homology, reflecting their common mechanism of action.

Receptors are small proteins of 350 to 500 amino acid residues, belonging to the family of rhodopsin-like membrane structures. The tachykinin receptor displaying higher affinity for SP was termed NK1, the receptor showing higher affinity for NKA was termed NK2, and the receptor showing higher affinity for NKB was termed NK3. It should be emphasized that, up to date, all naturally occurring tachykinins may act as agonists on all three receptor types, although sometimes with considerably different affinities (Regoli et al., 1987, 1994a; Maggi et al., 1993).

Parallel bioassay on a number of isolated and in situ test systems using the natural tachykinins and selective synthetic analogs, radioligand binding studies, and the use of antagonists with increasing potency and selectivity have led to the conclusion that all of the three main tachykinin receptors are heterogeneous entities, with NK1, NK2, and NK3 subtypes (Maggi et al., 1993; Quartara and Maggi, 1997, 1998). The main second messenger system coupled to activate the three known receptor subtypes is the stimulation of phospholipase C, leading to phosphoinositol breakdown and elevation of intracellular calcium (Guard and Watson, 1991). At high tachykinin concentrations, an adenylate cyclase stimulation and cAMP formation may also come into play (Nakajima et al., 1992).

The extracellular loops of these G-protein coupled receptors probably have the specific function of selecting a ligand, whereas the interaction of the ligand with transmembrane domains is responsible for receptor activation. Tachykinin peptides, therefore, presumably contain a sequence that interacts with the extracellular loops of the receptor and a sequence that interacts with transmembrane domains. Recent findings conclusively allowed clarifying the crucial importance for and the influence on receptor selectivity and activity of some key amino acids in the tachykinin sequence (Severini et al., 2000).

The Pro residue is a well represented residue in the natural tachykinins. It is nearly always located in the N-terminal moiety of the peptide sequence and has a clear-cut preference for positions 8 and 10 from the C terminus. In the majority of natural NK1 receptor-preferring tachykinins, a Pro residue is present at position 8, adjacent to the crucial neutral or basic residue occupying position 7. Proline in this position could modify the conformation of the C-terminal sequence of the tachykinin peptides and helps to increase their affinity and selectivity for the NK1 receptor. Cascieri et al. (1992) have suggested that all tachykinins containing Pro at position 8 from the C terminus, for example SP, have greatly reduced affinity for NK2 and NK3 receptors, and they have attributed this behavior to the preferred conformation of the Pro-containing peptides for the NK1 receptor and unfavorable for NK2 and NK3 receptors.

	V. Tachykinin-Like Peptides: Pharmacological Actions

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The TKs display a number of potent pharmacological actions in the periphery and in the central nervous system. In the present chapter, analysis is limited essentially to the pharmacological actions of the nonmammalian TKs (eledoisin, physalaemin, and even kassinin) available in pure form several years before the structures of SP, NKA, and NKB were elucidated. As a consequence, the pharmacology of the TKs is based largely on the study of amphibian physalaemin, kassinin, and on molluscan eledoisin.

Whereas results obtained with eledoisin and kassinin, multireceptor agonists, do not exactly mimic results obtained with either NKA or NKB, results obtained with physalaemin, a selective NK1 agonist, are perfectly superimposable, with negligible quantitative differences, on those later obtained with SP, the mammalian selective NK1 receptor agonist.

A. Cardiovascular System

1. Systemic Arterial Blood Pressure Tachykinins administered to the anesthetized dog by the parenteral route are the most potent among all known hypotensive agents. On the dog blood pressure, physalaemin was 2 to 2.5 times less potent than SP, 10 to 20 times more potent than kassinin, and 3 to 4 times more potent than eledoisin (Erspamer, 1981). When administered by rapid intravenous injection, physalaemin was 200 to 1000 times more potent than bradykinin, and 600 to 2000 times more potent than histamine (Bertaccini et al., 1965). Physalaemin was very effective in antagonizing the pressor effects of noradrenaline and angiotensin II given at doses 100 and 10 times higher, respectively.

2. Regional Circulation. Coronary bed.  Physalaemin and, to a considerably lesser extent eledoisin and SP (Losay et al., 1977) displayed a very potent vasodilator action on the dog coronary vascular bed not only when given by intracoronary administration, but also when given by intravenous infusion. A transient, 50% increase in coronary flow was obtained by rapid intracoronary injection with 0.1 pmol/kg and a 100% increase with 1 pmol/kg of physalaemin. Eledoisin was 200 times less active and nitroglycerin, 10,000 times less active.