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Vol. 54, Issue 2, 285-322, June 2002
Consiglio Nazionale delle Ricerche-Institute of Neurobiology and Molecular Medicine, Rome, Italy (C.S.); Department of Human Physiology and Pharmacology "Vittorio Erspamer", University "La Sapienza", Rome, Italy (G.I., G.F.-E., V.E.); and Department of Pharmaceutical Sciences, Università degli Studi, Ferrara, Italy (S.S.)
Abstract
I. Introduction
II. Occurrence and Species Distribution of Tachykinin-Like Peptides
A. Invertebrate Tachykinin-Like Peptides
B. Prevertebrate Tachykinin-Like Peptides
1. Amphioxus lanceolatus.
2. Tunicata (Protocordata).
C. Submammalian Vertebrate Tachykinins
1. Amphibian Skin Tachykinins.
2. Brain and Gut Tachykinins.
D. Mammalian Tachykinins
1. Mammalian Tachykinins and Their Biosynthesis.
III. Localization of Tachykinin-Like Peptides
A. Non-Neuronal Localization
1. Amphibian Skin.
2. Invertebrate Salivary Glands.
3. Normal Mammalian Tissues.
B. Neuronal Localization
IV. Relationships between Structure/Activity Receptor Selectivity
A. Residue Occupying Position 7 from the C Terminus
B. Residue Occupying Position 4 from the C Terminus
C. Residue Occupying Position 6 from the C Terminus
D. Amino Acid Substitutions in the C-Terminal Tripeptide
E. Pro Residue in the N-Terminal Sequence
V. Tachykinin-Like Peptides: Pharmacological Actions
A. Cardiovascular System
1. Systemic Arterial Blood Pressure
2. Regional Circulation.
B. Gastrointestinal Tract
1. Motility.
a. In Vitro Experiments.
b. In Vivo Experiments.
2. Secretions.
C. Airways System
D. Urogenital Tract
E. Immune System
F. Central Nervous System
Action on discrete selected brain areas.
G. Pain
H. Neurogenic Inflammation
I. Miscellaneous Pharmacological Actions
1. Lachrymal Secretion.
2. Histamine Release.
VI. Tachykinins in Human Diseases and Therapeutics
A. Tachykinin Receptor Agonists
B. Tachykinin Receptor Antagonists
VII. General Conclusions
References
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The tachykinin peptide family certainly represents one of the largest peptide families described in the animal organism. So far, more than 40 tachykinins have been isolated from invertebrate (insects, worms, and molluscs), protochordate, and vertebrate (skin, gastrointestinal tract, peripheral and central nervous system) tissues. Substance P (SP), first identified by bioassay as early as 1931 but sequenced only in 1971, several years after the elucidation of the structure of eledoisin from molluscan tissues and of physalaemin from amphibian skin, may be considered as a prototype of the tachykinins. Hitherto, as many as 19 tachykinins have been isolated from amphibian integument, and eight additional peptides have been isolated from amphibian gut and brain. Counterparts of skin tachykinins in mammalian tissues are SP, neurokinin A, and neurokinin B. Three main receptor subtypes for the tachykinins have been identified (NK1, NK2, and NK3), but their number is probably destined to increase. It is obvious that the peripheral and central effects of the tachykinins may substantially vary depending on the activation of different receptor subtypes. Matters are further complicated by the frequent capacity of the single tachykinins to bind, although with different affinity, to more receptors. It has been recognized that tachykinins have a variety of effects in physiological and pathological conditions, and there is evidence suggesting intrinsic neuroprotective and neurodegenerative properties of these neuropeptides. This review provides an update on the current body of knowledge regarding tachykinin occurrence and distribution in the animal kingdom, from the lowest invertebrates to man, and the physiological and pharmacological actions of tachykinins outlining the pregnant importance of this large peptide family.
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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.
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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. Invertebrate Tachykinin-Like Peptides
It is possible separate the tachykinin-like peptides in invertebrates into three groups: a) tachykinins identified by RIA and/or immunohistochemistry, occasionally accompanied by HPLC separation, but not isolated or sequenced; b) tachykinin-related peptides of the locustatachykinin type, isolated and sequenced, which have a C-terminal Arg-NH2 residue instead of the usual Met-NH2 residue present in all of the classical vertebrate tachykinins; and c) authentic tachykinins having structure and biological activity identical with those of the vertebrate tachykinins.
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).
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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 C. Submammalian Vertebrate Tachykinins
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 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 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.
).), 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.), 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).). 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.
TABLE 2
Amino acid sequence of natural amphibian skin tachykinins
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.
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-neuropeptides, as shown in Table
4. From the above sequences, it is
evident that none of the submammalian -neuropeptides is identical
with the corresponding mammalian peptide. Substantial differences
in the amino acid composition may be seen not only in the flanking
sequence but in all examined fish, even in the NKA-like C-terminal
decapeptides.
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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).
TABLE 5
Amino acid sequence of mammalian tachykinins
; Lazarus et al., 1980), using an
antiserum specifically recognizing the N-terminal region of
physalaemin, was able to detect a physalaemin-LI in a number of tissues
of three mammalian species (guinea pig, mouse, and rat) with peaks in
guinea pig and mouse gastric fundus, pylorus, and duodenum (up to 18 pmol/g lyophilized tissue). Moreover, physalaemin antiserum caused a
clear-cut immunostaining in a population of cells of the Brunner's
gland of the guinea pig duodenum, and several other examples of the
expression of physalaemin-, eledoisin-, and kassinin-LI may be found in
carcinoids (see Section III.A.4.).
Mammalian tachykinins are derived from two preprotachykinin genes: the
PPT-A gene, which encodes the sequences of SP, NKA, and neuropeptide K
and neuropeptide-, and the PPT-B gene, which encodes the sequence of
NKB (Nawa et al., 1983; Kotani et al., 1986; Bonner et al., 1987;
Krause et al., 1987).
The precursor RNA from PPT-A is alternatively processed to yield
three different mRNAs (Nawa et al., 1984). The three precursor proteins
from which the mRNA codes are designated 
-, 
-, and -PPT;
-PPT, which generates SP; -PPT, which generates SP, NKA, and
neuropeptide K; and -PPT, which generates SP, NKA, and
neuropeptide-. The biological significance of the alternative
splicing of PPT-A is unknown. The relative proportion of -, -,
and -PPT mRNAs is markedly species dependent. For example, -PPT
is the predominant form expressed in human basal ganglia (Bannon et
al., 1992), whereas -PPT prevails in the bovine brain (Nawa et al.,
1984).
-PPT mRNA is abundant in the brain, whereas - and -PPT mRNAs
are found mainly in peripheral tissues (Nakanishi, 1987). PPT-B mRNA is
found in the brain (hypothalamus) and intestine (Kotani et al., 1986).
Tachykinins are liberated from their precursors by the action of
specific processing proteases. Typical cleavage points are Lys-Arg,
Arg-Arg, and Arg-Lys doublets and the cleavage is carried out by six
groups of proteolytic enzymes called convertases (Chretien et al.,
1989; Steiner et al., 1992; Marcinkiewicz et al., 1993). COOH-terminal
amidation after cleavage is generated from the precursor sequence,
Gly-Leu-Met-Gly-Lys-Arg, in which Gly acts as the amide donor.
As with all known neurotransmitters, neuronal tachykinins are also
released from the nerve ending after a calcium-dependent mechanism in
response to application of physiological and nonphysiological stimuli
(electrical stimulation, potassium, or capsaicin depolarization) (Maggi
et al., 1993). Concerning release, two points are firmly established.
First, like that of biogenic amines, which are considered "rapid
transmitters" and which under certain conditions may be released massively, release of neuropeptides, considered "slow" transmitters or modulators, is probably discrete and long lasting. Second, at the
nerve terminals, especially in brain and in the autonomic nervous
system, a release of a single transmitter is improbable, and, at any
rate, must represent an exception. The concept of co-release of
different peptides, amines, amino acids, and purines is now generally
accepted after the immunohistochemical demonstration of the costorage
in the granular material of single neurones of more active substances
(Hokfelt et al., 1986).
Once released, the tachykinins may be attacked, cleaved, and
inactivated by a number of proteolytic enzymes, which, however, act
with considerably different intensity on the different tachykinins. The
most vulnerable peptide seems to be SP, whereas peptides having at
their N-terminal the pGlu residue seem much more resistant to enzyme
attack. In the proteolytic degradation of SP, three enzymes seem to
display a predominant role: dipeptidyl-amino peptidase, postproline
endopeptidase, and cathepsin D (Regoli et al., 1994a).
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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).
). Among the amines, serotonin is
always present in argentaffin/chromaffin carcinoids, originating from
the malignant growth of the enterochromaffin cells. They are
prevailingly present in the midgut and appendix. Serotonin, however, is
lacking in argyrophil nonchromaffin carcinoids, which may be present in
the foregut, hindgut, lung (mostly bronchial carcinoids) and various
other organs (Creutzfeldt and Stockmann, 1987). Argyrophil carcinoids
may originate in the colon and probably in other sites as well, from
the population of argyrophil, serotonin-lacking cells described by
Sokolski and Lechago (1984). Peptides (bradykinin, enteroglucagon, and
especially tachykinins) may occur both in chromaffin (together with
serotonin) and in argyrophil nonchromaffin carcinoids (Wilander et al.,
1977, 1979).
Because in normal endocrine cells of the gut and other organs
only SP has been detected by immunochemistry, as expected, the tachykinin most frequently identified not only in the primary carcinoid
tumor but also in its metastases was SP, together with its fragment
SP(5-11), both in normal and oxidized form (Gamse et al., 1981; Conlon
et al., 1985; Roth et al., 1985; Theodorsson-Norheim et al., 1985;
Bishop et al., 1989). However, NKA, together with its fragments
NKA(4-10) and NKA(5-10) and its extended form neuropeptide K, may
also frequently be present in carcinoids (Roth et al., 1985;
Theodorsson-Norheim et al., 1985; Conlon et al., 1986b; Bishop et al.,
1989); and, more rarely, an eledoisin-like immunoreactivity (eledoisin-LI) was observed together with a neurokinin B-LI
(Theodorsson-Norheim et al., 1985) and an oxidized physalaemin-LI
(Conlon et al., 1985).
SP-LI was also found, together with NKA-LI, in bronchial carcinoids
(Creutzfeldt and Stockmann, 1987; Bishop et al., 1989), in ovarian
carcinoids (Skrabanek et al., 1980; Strodel et al., 1984), and in a
medullary carcinoma of the thyroid (Skrabanek et al., 1979).
Tachykinins (NKA-, neuropeptide K-, and eledoisin-like peptides) were
produced also by carcinoid tumors in culture (Norheim et al., 1987) and
both 5-HT- and SP-LI were found in cytoplasmatic granules isolated from
an intestinal argentaffin carcinoid, supporting the view that in this
case SP is costored with 5-HT in the granules of the enterochromaffin
cells (Alumets et al., 1977).
Pheocromocytomas. Two pheocromocytomas showed SP-LI and
SP sulfoxide-LI (Gamse et al., 1981), and another pheocromocytoma showed NKB-LI (Conlon et al., 1985). After subcellular fractionation, SP-LI and catecholamines were enriched in the chromaffin granular fraction, making it unlikely that SP-LI originates from nerve terminals
(Gamse et al., 1981).
Lung carcinoma. Only a few data on the content of tachykinins
in these carcinoid tumors are available: up to 2 ng/g fresh tissue of
SP-like peptides, 1.2 ng/g being represented by authentic SP (Gamse et
al., 1981). This content is surprisingly low, compared with the content
of serotonin (from 1 µg to 2.5 mg per g fresh tissue) in argentaffin
carcinoids (Stacey, 1966). Moreover, Lazarus et al. (1983) have
presented evidence that a human small cell carcinoma may contain a
tachykinin peptide (1-1.6 ng/g) that has structural and biological
activity similar to that of the amphibian physalaemin.
Because carcinoid tumors and their metastases are authentic
endocrine glands, releasing into the blood stream biogenic amines and
tachykinins, levels of these compounds may increase in plasma and even
in urine. TK-LI was found in 75% of the 65 carcinoid patients examined
(Norheim et al., 1984). The major component in plasma eluted in
ion-exchange chromatography was in a different position from that of
the usual tachykinins. Similar results were obtained by Conlon et al.
(1986b) in three of four carcinoid patients, with NKA-LI up to 1 nmol/ml plasma and SP-LI up to 345 fmol/ml. Moreover, in urine samples
of 79% of the 48 carcinoid patients examined in another study, the
TK-LI material was 8 times more elevated than in healthy subjects. The
immunoreactive material was heterogeneous, with some components
coeluting with oxidized NKA and neuropeptide K (Bergstrom et al.,
1995). In the urine of patients with argentaffin carcinoids, the
concentration of 5-hydroxyindolacetic acid, the main metabolite of
serotonin, was 100 times more elevated than in healthy subjects
(Stacey, 1966).
All of these findings on carcinoid tumors demonstrate that
tumoral epithelial, non-neuronal cells of the mammalian intestine and
other organs are capable of expressing and storing not only SP, as
expected, but several other tachykinins as well, certainly NKA and its
extended form neuropeptide K, but also NKB and kassinin-, eledoisin-,
and physalaemin-like peptides, i.e., peptides occurring in normal
tissues only in submammalian species.
Patients with argentaffin carcinoid tumors and their metastases very
often exhibit a typical syndrome characterized by flushing, diarrhea,
asthma, cyanosis, and right-side valvular disease. Creutzfeldt and
Stockmann (1987) have considered tachykinins to be coresponsible only
for vasodilation (flushing) and not of the other symptoms. Secretory
diarrhea and enhanced motility, important features of the carcinoid
syndrome, do not seem to be attributable to SP, but instead to NKA and
to eledoisin-like peptides (Makridis et al., 1999).
We can conclude that carcinoid tumors are not pure tachykinin-secreting
tumors and do not contribute, unlike other endocrine tumors
(gastrinomas and vipomas), to the understanding of the physiological
significance and function of the tachykinins.
B. Neuronal Localization
Nervous tissue represents by far the most important localization of the tachykinins in invertebrates and vertebrates. A more or less dense network of tachykininergic fibers, which release their content upon adequate stimulation, permeates all vertebrate tissues close upon a very rich population of different receptors located on the membrane of neuronal and non-neuronal cells. In certain cerebral areas, the concentration of tachykinins may be on the order of nanomoles.
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.
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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 receptors
NK1, 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).
A. Residue Occupying Position 7 from the C Terminus
The amino acid in position seven from the C-terminal of tachykinins seems to address the peptide ligand toward the receptor. SP and tachykinins with a neutral or basic residue in this position have a preference for the NK1 receptor. Neutral residues are generally hydrophilic, and proline in position eight from the C-terminal can increase affinity for the NK1 receptor. Tachykinins with an acidic or a couple of acidic residues in position 7 or 6 and 7 from the C-terminal addressed the peptides toward the NK2 and NK3 receptors. Interestingly, the second extracellular loop has four acidic and four basic residues in the rat NK1 receptor, three acidic and two basic residues in the NK2 receptor, and one acidic and five basic residues in the NK3 receptor.
B. Residue Occupying Position 4 from the C Terminus
In all natural tachykinins, position 4 from the C terminus is occupied either by an aromatic amino acid residue (Phe, Tyr) in the aromatic tachykinins or by an aliphatic, branched amino acid residue (Val, Ile) in the aliphatic tachykinins. The presence of an aromatic residue invariably determines selectivity or increases the selectivity of the peptide for the NK1 receptor. This is true not only when a neutral or basic amino acid residue occupies position 7 from the C terminus but also when an acidic residue occupies position 7. The couple of aromatic residues (Phe-Tyr or Phe-Phe) present in the "message domain" of the tachykinins provide specific binding interactions with transmembrane domains of NK1 receptor.
C. Residue Occupying Position 6 from the C Terminus
The presence of a Pro residue in position 6 from the C terminus causes a profound decay of biological activity. The negative contribution of Pro6 could be related to a distortion in the interaction of the C-terminal sequence of the peptide (Phe-Xaa-Gly-Leu-Met-NH2) with all tachykinin receptors. In the Pseudophryne güntheri tachykinins, a Glu residue occupies position 6 from the C terminus. The couple of acidic residues Asp7-Glu6 present in PG-SP1 and PG-KII could, therefore, be responsible for the marked shift of receptor selectivity toward the NK3 receptor. This shift is quite evident for PG-KII (an aliphatic tachykinin) and much less evident for PG-SP1 (an aromatic tachykinin) in which the Phe-Tyr sequence induces NK1 receptor selectivity.
D. Amino Acid Substitutions in the C-Terminal Tripeptide
To date, six natural peptides have single or double amino acid substitutions in the C-terminal tripeptide Gly-Leu-Met-NH2: Pro (AC-AR2, AC-AR4) or Ala (ranatachykinin D) for Gly; and Val (AC-AR4) or Pro (ranatachykinin D) or Gly (AC-AR2) or Met (hylambatin) for Leu. None of these substitutions affected the peptides' receptor selectivity, only their receptor affinity or potency.
E. Pro Residue in the N-Terminal Sequence
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.
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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.
Intravenously injected in the sheep, eledoisin showed a hypertensive response of slow onset, probably attributable to some arousal of the animals (Ormas et al., 1975).
In cat and in rat, the effect of physalaemin was considerably
less intense, with high variability and tachyphylaxis. Finally, in the
decapitated chicken, physalaemin regularly elicited a biphasic response
consisting of a brief hypotensive phase followed by a more intense and
sustained dose-related pressure increase (Bertaccini et al., 1965). The
rise in pressure observed in sheep and chicken was blocked by
sympatholytic drugs and by pretreatment with reserpine, thus,
indicating a release of cathecolamines from the adrenal medulla and/or
other stores.
Conversely, the hypotensive effect of the tachykinins was not modified
by any of the usual autonomic blocking agents, thus suggesting a direct
effect on the vascular smooth muscle.
Physalaemin, ranakinin, SP, and NKB produced a dose-dependent decrease
in arterial blood pressure in the toad, Bufo marinus. A
selective NK1 antagonist had no effect on the blood pressure fall
elicited by ranakinin and SP, suggesting the existence of an NK1
receptor subtype different from mammalian NK1 receptor (Courtice et
al., 1993).
In the bowfin Amia calva, a teleost fish, the bolus
injection into the bulbus arteriosus of 0.1 to 10 nmol/kg of the bowfin SP resulted in a significant and dose-dependent rise in vascular resistance and blood pressure and a fall in cardiac output without changes in heart rate. Those effects lasted 5 to 10 min (Waugh et al.,
1995b). Similarly, in the teleost fish rainbow trout, both the trout SP
and the trout NKA at intraaortic doses of 1 nmol/kg increased systemic
and celiac vascular resistance leading to hypertension, bradycardia,
and decrease of cardiac output. After in vitro perfusion of the aortic
and celiac mesenteric vascular bed, the peptides dose dependently
increased the vascular resistance. It may be concluded that in teleost
fish, the fish tachykinins are potent vasoconstrictor agents (Kagstrom
et al., 1996).
In the conscious, unanaesthetized dogfish Scyliorhinus
canicula, intravenous injection of either dogfish SP or
scyliorhinin I (up to 5 nmol) produced no change in arterial blood
pressure, pulse amplitude, and heart rate. Injection of greater amounts of the peptides (10-50 nmol) produced a slight increase in blood pressure (Waugh et al., 1993). However, in the unrestrained spiny dogfish Squalus acanthia, the intravenous injection of
scyliorhinin I and NKA caused hypotension, due to a general
vasodilation, with transient increase in mesenteric blood flow and a
prolonged increase in celiac blood flow. The peptides did not increase
heart rate (Kagstrom et al., 1996).
In human volunteers, eledoisin given by rapid intravenous injection
(threshold 15-20 pmol/kg) decreased blood pressure, caused spinal
fluid hypertension, increased the rate of respiration and caused skin
vasodilation, particularly in the head. Rise in blood pressure produced
by 3 to 5 nmol/kg angiotensin or 40 to 60 nmol/kg noradrenaline was
inhibited or reversed by 1 to 2 nmol/kg eledoisin injected 5 s
previously (Sicuteri et al., 1963).
In other experiments, the intravenous infusion of 0.6 nmol/kg/min
eledoisin or 0.2 nmol/kg/min physalaemin produced only a 20 mm Hg
pressure fall that lasted 5 min. Basal levels of pressure returned
despite the continued infusion (De Caro et al., 1966).
SP also decreased blood pressure. A significant difference from the
basal level was found at an infusion rate of 200 pmol/kg/min or higher
(Eklund et al., 1977).
These results were substantially confirmed by Evans et al. (1988), who
found that both SP (3 pmol/kg/min) and NKA (64 pmol/kg/min) did not
change systolic blood pressure, whereas diastolic pressure fell
significantly only after SP infusion. Moreover both peptides increased
heart rate and body temperature, with skin flushing. SP was 6 to 20 times more potent than NKA.
Heart. Electrocardiogram tracings recorded from anesthetized
dogs given an intravenous infusion or a subcutaneous injection of
physalaemin, at doses approximately 1000 higher than the threshold hypotensive dose, produced only moderate electrocardiographic changes
mainly attributable to hypotension (Bertaccini et al., 1965). In a
detailed study, the following percentage changes in a number of
cardiovascular parameters have been observed in dog after intravenous
injection of 4 pmol/kg physalaemin: heart rate, +17.8; mean systemic
arterial pressure, 
22.3; mean pulmonary arterial pressure, +1.8; mean
left atrial pressure, 1; mean right atrial pressure, +0.5; myocardial
contractile force, +17.5; cardiac output, +52; total peripheral
resistance, 65.2; and pulmonary vascular resistance, 35.4 (Nakano
et al., 1968). Similar results were obtained with eledoisin (Nakano,
1964, 1965).
The effect of SP was substantially the same as that observed with
physalaemin. At infusion rates ranging from 3 to 450 pmol/kg/min, SP
invariably induced a dose-dependent increase of cardiac output mostly
due to a larger stroke volume. SP at concentrations up to 50 pmol/ml
had no effect either on the isolated guinea pig auricles or the
perfused rabbit heart, suggesting that SP has no direct effect on the
heart (Burcher et al., 1977).
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.
). Increase in coronary flow and decrease in coronary
vascular resistance also was observed after intravenous infusion of
eledoisin (Beretta Anguissola et al., 1966).
Skeletal muscle. The vessels of the skeletal musculature of
the hindlimbs of dogs were by far the most sensitive to tachykinins of
any vascular bed. Doses of eledoisin as low as 10 fmol injected into
the peronal artery caused an increase in blood flow, both in the intact
and denervated gastrocnemius plantaris muscle. Denervation enhanced the
potency of eledoisin (Bergamaschi and Glasser, 1963, 1964). Physalaemin
was 50 times more potent than eledoisin and 50,000 times more potent
than nitroglycerin (Bergamaschi et al., 1966; Fregnan and Glasser,
1968). In other experiments, close arterial injection of SP caused a
dose-related vasodilation in adipose tissue and skeletal muscle of the
dog only with doses starting from 10 nmol (Pernow and Rosell, 1975).
SP was also a potent vasodilator in humans. Infusion of 0.7 pmol/kg/min
into the brachial artery significantly increased the forearm blood
flow, with increases of oxygen consumption in both cutaneous and muscle
blood. At the infusion rate of 70 pmol/kg/min, there was a bright red
flushing of the skin, particularly in the neck and head, with a
subjective feeling of warmth in the same regions, accompanied by
tachycardia (Eklund et al., 1977). No effect of SP could be seen on
internal carotid blood flow (Samnegard et al., 1978).
Liver. Portal or femoral infusions of SP, eledoisin, and
physalaemin (2-20 pmol/kg/min) increased blood flow in the hepatic artery and vein of the dog. Portal infusions were less effective, thus,
indicating a highly inactivating capacity of the liver. Hepatic
arterial and venous pressures decreased, whereas sinusoid and portal
pressure increased during peptide infusion. As a consequence, hepatic
arterial and outflow resistances decreased. SP was the most potent
peptide, followed by physalaemin (38%) and eledoisin (10%). When
given by close arterial infusion, the peptides also consistently
increased blood flow in the hepatic artery (Melchiorri et al., 1977).
These observations were confirmed by Takaori et al. (1989), who found
that intravenous physalaemin (5 pmol/kg) caused dose-dependent
increases in mesenteric arterial blood flow (70%) and portal venous
blood flow (77%) in the dog.
Lung. Intravenous eledoisin (0.1-1 nmol/kg) did not
increase, and sometimes slightly reduced, pulmonary arterial pressure in the guinea pig; it always increased pressure in the rabbit. In the
isolated, blood-perfused rabbit lung preparation, eledoisin produced a
potent vasoconstriction from threshold doses of 0.01 to 0.1 pmol/kg.
Tachyphylaxis was obvious. Bradykinin was 1000 times less effective
(Hauge et al., 1966). In the dog NKA was much more potent than SP in
decreasing tracheal vascular resistance (Salonen et al., 1988).
Skin. The skin vasculature of the dog was far less sensitive
than the vessels of the musculature. In man, injection of 0.2 nmol
eledoisin into the brachial artery produced digital vascular responses
consisting of an increase in the skin temperature and a consistent
increase in total digital volume, despite a decrease in inflow volume.
Responses seem to indicate a closure of the arteriovenous anastomoses
(De Pasquale and Burch, 1966).
Infusion of SP into the rat femoral artery dose dependently produced
vasodilation (threshold 0.1 pmol/kg/min) that was inhibited by
mepyramine (Lembeck and Holzer, 1979).
Brain. Eledoisin infused intravenously in the dog at 0.01 nmol/kg/min decreased cerebral blood flow (22%), with an increase (+20%) in vascular resistance (Beretta Anguissola et al., 1966). In
human subjects, the intravenous infusion of eledoisin (1-15 pmol/kg/min) influenced neither the cerebral blood flow and vascular resistance nor the cerebral metabolic rate of oxygen and glucose (Bianchi Porro et al., 1965).
Mechanism of vasodilation and hypotension. All of these
findings demonstrate that in some mammalian species, exogenous
tachykinins display a potent dilation of regional musculature
accompanied by the fall of systemic blood pressure, in other mammalian
and nonmammalian species the peptides display inconstant and variable effects: hypotensive/hypertensive or even frank hypertensive responses. Thus, the intervention of endogenous tachykinins in the regulation of
blood pressure and regional circulation is certainly possible but
irregular and unpredictable. At any rate, it is hardly conceivable that
the tachykinins display a significant role in the cardiovascular system
similar to that of noradrenaline, serotonin, angiotensin, prostacyclins, etc. This does not exclude that in man the tachykinins may contribute to the control of vascular tone of the cutaneous vessels
of some areas. So, it has been suggested that tachykinin (SP and NKA)
release is co-involved in the pathogenesis of flushing episodes (not
accompanied by edema!) occurring in the carcinoid disease. To our
knowledge, tachykinin antagonists have never been used in this disease.
The trial could be rewarding from both a pathogenetical and a
therapeutical point of view.
The striking hypotensive effects of the tachykinins observed in some
animal species, as a consequence of intense vasodilation in several
peripheral vascular beds, must be considered a direct effect of the
peptides on the blood vessel wall. However, Regoli et al. (1987),
D'Orléans-Juste et al. (1985, 1986), and Mastrangelo et al.
(1987), in agreement with previous observations (Furchgott, 1983, 1984)
on other vasodilators, found that in isolated strips of arteries and
veins that were maximally contracted by noradrenaline, the relaxing
effect of the tachykinins could be obtained only when the endothelium
was intact. This clearly indicated that the site of action of the
tachykinins (like that of acetylcholine, bradykinin, neurotensin, and
bombesin) was not the smooth muscle cell, as formerly believed, but the
endothelium. Thus, the tachykinins may act to promote the release of
endogenous factors (prostacyclins, endothelium-delivered relaxing
factors, and nitrous oxide) from the endothelium that is able to reduce
the tone of the arterial smooth muscle fibers. However, whereas dog
carotid artery without endothelium is insensitive to all tachykinins,
thus, suggesting an exclusive location of its receptors in the
endothelium, this is not true for the other vascular preparations. The
rabbit pulmonary artery, for example, may be relaxed or contracted by
tachykinins, depending on its basal tone. At high tone levels
(premedication with noradrenaline), relaxation was predominant; whereas
at low tone levels, contraction occurred. Relaxation may be due to the activation of NK1 receptors (SP, physalaemin) located in the
endothelium, and contraction may be caused by activation of NK2
receptors (NKA, kassinin) located on the arterial smooth muscle. In the
rabbit pulmonary artery possessing endothelium, the relaxing potency of
SP was 3 to 4 times higher than that of NKA or kassinin; in the artery
without endothelium, the contracting potency of SP was 30 to 120 times
lower than that of NKA or kassinin (D`Orléans-Juste et al.,
1986). Similarly, in the rat portal vein (with intact endothelium and
not pretreated with noradrenaline), contraction elicited by NKB and
kassinin was brought about by NK3 receptors, presumably located on the
smooth muscle membrane (Mastrangelo et al., 1987).
In the intact animal, response of the vasculature to tachykinins is
complex, depending on the animal species, density in the smooth muscle
cells, and the endothelium of the different receptor types, as well as
the kind of tachykinins administered or released. Whereas in dogs and
rabbits, the NK1-preferring tachykinins (SP, physalaemin) regularly
cause a dose-dependent intense hypotension with no sign of
tachyphylaxis, the same tachykinins in cats, sheeps, rats, and pigeons
may produce moderate hypotension with obvious tachyphylaxis,
hypotension/hypertension, or frank hypertension, thus, indicating a
complex activation of different receptor types, and perhaps a different
availability of relaxing factors in the endothelium (tachyphylaxis).
Capillary permeability. The capillary permeability was
enhanced by intradermal injection of eledoisin and physalaemin in the rat, guinea pig, and man. Eledoisin was 2 to 10 times more potent than
histamine in all animal species and more potent than bradykinin in man
(De Caro, 1963). Intradermal injection of eledoisin at doses exceeding
1 pmol in man caused pain, local edema, and erythema; whereas when
injected intraperitoneally, intramuscolarly, or subcutaneously into
human subjects at doses up to 17 to 50 nmol, the peptide failed to
elicit pain (Kantor et al., 1967). Similarly, intradermal injection of
0.1 to 10 µM SP in man induced flare, wheal, and itching, which was
similar to the response induced by histamine and, like histamine, was
blocked by antihistaminic drugs (Hagermark et al., 1978).
Introduced in the mouse pleural cavity, SP (ED50 = 14 nM) caused a long-lasting recruitment of leukocytes and a small
but evident exudation. These effects were partially inhibited by NK1 receptor antagonists and were mediated by nitric oxide (Frode-Saleh et
al., 1999). Finally, close-arterial infusion of SP, from doses as low
as 0.1-0.5 pmol/min in the rat skin, induced vasodilation and plasma
extravasation (Lembeck and Holzer, 1979).
Of great interest is that intradermal injection of SP (30-300 pmol)
induced a dose-dependent edema on wild-type mice, whereas in NK1
receptor knockout mice, the peptide was inactive. The reaction in wild
mice was reduced by the histamine antagonist mepyramine, indicating
that edema induced by the tachykinin, although totally dependent on NK1
receptor-mediated mechanism, contains a mast cell-dependent component
(Cao et al., 1998).
Intravenously injected in the rat, NKA induced a dose-dependent
extravasation of Evans blue in stomach, duodenum, jejunum, caecum, and
colon but had no effect in ileum. NKB equipotently produced
extravasation in the stomach but had no effect in other parts of the
gut. SP was ineffective (Lordal et al., 1996).
NKA (100-400 pmol/kg/min) given by intravenous infusion in an in situ
perfusion model of the anesthetized rat stimulated duodenal motility
and increased duodenal mucosal bicarbonate secretion, fluid output, and
mucosal permeability. Effects were dependent on NK2 receptor activation
(Hallgren et al., 1997). Plasma extravasation was induced by SP and
bradykinin also in mouse gastrointestinal tract and pancreas (Figini et
al., 1997). On the contrary, neither capsaicin nor SP (0.1-1 mg/ml)
nebulized or given intravenously (0.75 nmol/kg) in rabbits produced
significant increases in tracheal or bronchial Evans blue
concentration, indicating that SP does not activate the microvascular
leakage in the major airways of the rabbit (Matheson et al., 1997).
a. In Vitro Experiments.
As many as 16 isolated
gastrointestinal preparations from eight animal species were assayed
with eledoisin and physalaemin (Erspamer and Falconieri Erspamer, 1962;
Bertaccini et al., 1965). Response was always stimulation, but
intensity and reproducibility of response varied conspicuously
dependent on the animal species and the various segments of the
intestinal tract. After synthetic SP and, later on, NKA and NKB became
available, an enormous amount of work appeared on the action of
mammalian tachykinins on isolated preparations of intestinal muscle and
the receptors herein involved (Holzer and Holzer-Petsche, 1997a).
b. In Vivo Experiments.
Dog. The first phenomenon
observed a few minutes after a subcutaneous administration of 25 to 200 nmol/kg eledoisin was vomiting accompanied by profuse salivation.
Vomiting was at first alimentary, and then the emesis episodes of
increasing severity resulted in the ejection of masses of mucus,
sometimes spotted with blood. Shortly after commencement of vomiting,
there was a discharge of formed stools, which was soon followed by
evacuation of watery stools containing mucus and blood accompanied by
violent tenesmus. The tremendous gastrointestinal stimulation and
profound depression of the animal lasted unchanged for 1 h and
then gradually decreased. During the night, there was complete
recovery, and for a few days afterward, the dog showed unusual
voracity. In dogs given a subcutaneous dose of 0.15 mg/kg atropine
sulfate 30 min before the injection of 20 nmol/kg eledoisin, the
alkaloid failed to block stimulation of either salivary glands or
gastrointestinal smooth muscle (Erspamer and Glasser, 1963).
).
The general effects produced by rapid intravenous injection of
physalaemin in dogs were studied by Bertazzoli and Cheli (personal communication). No obvious effects were elicited by 0.1 nmol/kg of
physalaemin; after 0.3 nmol/kg, the only appreciable effect was that
the dog had difficulty in sitting (tenesmus?), and this lasted only for
a few minutes; 0.6 nmol/kg produced the same effect and, in addition,
one or more evacuations of the bowel during the first 5 min; 2.5 nmol/kg caused not only diarrhea but also salivation, lasting 5 to 10 min. Increased salivation and diarrhea were accompanied by vomiting in
dogs treated with 12 nmol/kg of physalaemin. All symptoms disappeared
within 15 to 20 min. Similar doses of physalaemin (2.5 nmol/kg) given
intravenously every day for 10 successive days elicited the same phenomena.
In the anesthetized dog, intravenous infusion of eledoisin at a rate of
40 pmol/kg/min produced neither salivation nor evacuation of the bowel.
Infusion rates of 80 to 250 pmol/kg/min, on the other hand, caused
salivation and evacuation of liquid stools. Stimulation of both
salivary glands and gastrointestinal smooth muscle was
atropine-resistant. High infusion rates produced a more or less evident
cutaneous vasodilation (Erspamer and Glasser, 1963).
In conscious dogs, eledoisin and physalaemin displayed a potent
stimulating effect on the mechanical and electrical activity of the
gut. Low doses of the peptides (2.5-15 ng/kg/min) caused an increase
in frequency and duration of the interdigestive myoelectric complexes,
and an increase in coordinated mechanical activity. High doses provoked
the appearance of a diffuse spike activity, accompanied by intense
local motor activity. Pacesetter potentials were not affected (Caprilli
et al., 1975). After intravenous injection of 1 nmol/kg physalaemin in
anesthetized dogs with a gastric pouch a striking increase in tonus of
the gastric pouch was observed, accompanied by stimulation of rhythmic
contractions, lasting 10 min. In the ileum a slight initial increase in
tonus was followed by intense stimulation of rhythmic activity, lasting
10 to 15 min (Bertaccini, 1982).
In other experiments on the in situ jejuneal loop of the anesthetized
dog, physalaemin was twice as potent as CCK in stimulating motility, 15 times as potent as human gastrin I, 50 to 100 times as potent as either
bradykinin or carbachol, and more than 300 times as potent as
acetylcholine, histamine, and serotonin. Only caerulein overcame
physalaemin (by 3 times) in its stimulating effect (Mantovani et al.,
1969).
In keeping with these results, the tachykinins administered by close
intra-arterial injection increased the motility of gastric antrum and
proximal duodenum. Physalaemin and eledoisin (5 pmol/ml) were the most
potent peptides, followed by SP (50-60%) and neurokinins A and B
(10-12%) (Kuwahara and Yanaihara, 1987). The effect of SP on gastric
motility was found to differ depending on the sites and vagal
innervation of conscious dogs (Shibata et al., 1994).
Cat. Physalaemin, eledoisin, and SP administered via the
splanchnic artery produced both motor and mechanical effects in the stomach of anesthetized cats, characterized by successive phases of
initial distension, sustained contraction, and late distension. At low
doses, distension was the dominant effect. The sustained contraction
and late distension phase were accompanied by phasic contractions.
Atropine abolished the sustained contractions but had no effect on
phasic contractions and distension phases (Lidberg et al., 1983; Barber
et al., 1987).
Guinea pig. Tachykinins influenced peristaltic motor activity
in isolated segments of the guinea pig small intestine. NK2 and
particularly NK3 agonists facilitated intestinal peristalsis, whereas
SP first stimulated and then inhibited peristalsis. The facilitatory
effect of SP was prevented by atropine and seems to involve NK2
receptors, whereas the secondary inhibitory effect is due to NK1
stimulation (Holzer-Petsche, 1995).
Rat. The tachykinins displayed a potent spasmogenic action on
the rat stomach, as measured from the bulk of duodenal effluent. In
terms of threshold doses (0.1-1 nmol/kg intravenous), eledoisin was
the most potent peptide, SP was the least potent, and phyllomedusin was
by far the most effective with regards to the duration of effect
(Bertaccini and Coruzzi, 1977; Bertaccini, 1980). After infusion into
the celiac artery at doses of 0.06 to 20 nmol/min, both SP and NKA
caused contraction of the stomach, NKA being 10 times more potent than
SP (Holzer-Petsche et al., 1987).
The complexity of the effects of the tachykinins on the
gastrointestinal propulsion was evident after the intraperitoneal injection of the peptides. It was shown that 3 min after a test meal,
both SP (> 0.75 nmol/kg) and NKA (> 8.8 nmol/kg) inhibited both
gastric emptying and intestinal transit. The inhibitory effect was
reversed to a stimulant effect by pretreatment with atropine. After 10 min, SP dose dependently enhanced intestinal propulsion, an effect that
was atropine resistant. From the above experiments, it clearly seems
that the gastrointestinal propulsion was dose- and time-dependent, with
variable involvement of the autonomic nervous system (Holzer, 1985).
Sheep. Intravenous eledoisin (50-250 pmol/kg) stimulated
both in anesthetized and in awake and standing animals the motility of
all sections of the stomach producing an increase in tone and in
rhythmic movements. The effect was immediate, atropine resistant, and
lasted from 5 to 30 min, depending on the dose. The omasum was the most
sensitive section. SP was at least 10 times less active than eledoisin
(Ormas et al., 1977).
Toad. On the B. marinus intestine, bufokinin was
the most potent agonist (EC50 = 0.35 µM),
producing a long-lasting contraction similar to that evoked by
physalaemin, SP, and kassinin. Surprisingly, these effects were not
inhibited by highly selective NK1 receptor antagonists (Liu et
al., 1999b).
Fish. The stimulant effect of the tachykinins was also
evident in the fish gut. Both SP and NKA produced contraction of the vascularly preferred cod stomach; SP (pD2 7.05) was almost 6 times more
potent than NKA (Jensen, 1997). The same peptides displayed an
excitatory effect on the circular muscle of the cod intestine, suggesting that tachykininergic neurons are involved in the ascending excitatory reflex of peristalsis (Karila et al., 1998).
2. Secretions.
Salivary secretion. The potent
sialagogic effect of substance P (Haefeli and Hurlimann, 1962) and of
amphibian tachykinins (Bertaccini and De Caro, 1965; Emmelin and
Lenninger, 1967) was recognized several years before the sialagogic
principle in a bovine hypothalamic extract was identified as substance
P by Chang and Leeman (1970).
).
These results were confirmed by Emmelin et al. (1969), who found that
physalaemin stimulated secretion from the dog parotid and submaxillary
glands (threshold doses: 0.5 and 12 nmol/kg, respectively).
Considerably lower doses (threshold: 5-10 pmol/kg) elicited a pressure
increase in both submaxillary and parotid ducts. The contraction of the
myoepithelial cells of the ducts was due to a direct effect of the
peptide, because the usual autonomic blocking agents did not abolish it.
In rats, the threshold sialagogic subcutaneous dose of physalaemin was
0.1 to 0.3 nmol/kg. There was a satisfactory dose-response relationship, and the stimulatory effect could be repeated for 2 to
3 h. By intravenous infusion (threshold: 0.2 nmol/kg), salivary stimulation lasted as long as the infusion was continued (Bertaccini and De Caro, 1965). Increase in salivary flow produced both by intravenous injection (0.1-1 nmol/kg) and infusion (1 nmol/kg/min) was
associated with an increase in amylase and electrolyte secretion. The
flow rate of the submaxillary gland was maintained throughout the
period of infusion, whereas the parotid flow rate tended to decrease,
finally ceasing entirely. Again, autonomic blocking drugs failed to
modify the response to physalaemin (Schneyer and Hall, 1968).
By close intra-arterial injection, the threshold sialagogic dose of
physalaemin was much lower (1.5 pmol) for the submaxillary gland than
for the parotid gland (62 pmol). An increase in flow of saliva occurred
along with a pressure rise in the duct of the submaxillary gland
(Thulin, 1976).
Chronic intravenous administration of physalaemin (10 nmol/kg twice
daily for 15 days) caused a moderate enlargement of the parotid and
submaxillary glands, with a 50% increase in weight. Eledoisin was
inactive at doses up to 500 nmol/kg (Bertaccini et al., 1966;
Cantalamessa et al., 1975). Eledoisin and physalaemin also caused
salivary secretion in the hen at intravenous doses of 0.3 to 0.5 nmol/kg. Physalaemin was 8 to 10 times more potent than eledoisin
(Lembeck and Starke, 1968).
As far as mammalian tachykinins are concerned, results obtained mainly
with SP confirmed and extended results obtained with physalaemin. In
summarizing, it was established that: a) SP potently stimulated
salivation in the dog, ferret, rat, and guinea pig; whereas in humans,
cat, rabbit, mouse, and hamster, it was virtually inactive (Lembeck and
Starke, 1968; Leubeck et al., 1968; Iwabuchi et al., 1992; Tobin and
Ekstrom, 1992). b) The principal effect of SP and NKA was to enhance
the flow of salivary fluid, which is poor in protein, through a
predominant action on the secretory structures of the acini. c) The
secretion of fluid was stimulated in all salivary glands of the rat,
but the submaxillar glands were most sensitive to SP, whereas the
parotid glands and particularly the sublingual glands were less
sensitive (Ekstrom et al., 1983). d) SP also produced changes in the
output of salivary components, as shown by the increase in the
secretion of K+ and Cl
ions, in discharge of proteins, glycoproteins, proteolytic enzymes, amylase, kallikrein, and mucus (Holzer and Holzer-Petsche, 1997b). e)
The rank order of potency in causing salivary secretion in rats after
intravenous injection was: physalaemin = uperolein > eledoisin > SP = kassinin > NKA 
NKB
(Holzer-Petsche et al., 1985).
The greater potency of physalaemin and uperolein in comparison with SP
may be explained by the considerably higher resistance to enzyme attack
offered in the two first peptides by their N-terminal pGlu residue
(Yanaihara et al., 1977). It is generally accepted that the secretory
action of the tachykinins in the rat salivary glands is mediated by the
NK1 receptor (Holzer and Holzer-Petsche, 1997b).
In slices of rat parotid glands, the tachykinins caused a rapid,
concentration-dependent (threshold 10-15 pmol/ml) increase in
K+ efflux and amylase release, independent of any
effect on tissue concentrations of cyclic AMP or cyclic GMP levels.
Simultaneously, there was a stimulation of phosphatidylinositol
turnover (Rudlich and Butcher, 1976; Hanley et al., 1980). Physalaemin
and substance P were emulative, eledoisin was one-half as potent, and
kassinin was 10 times less potent (Brown and Hanley, 1981).
Gastric acid and biliary secretions. Physalaemin did not
influence the gastric acid secretion in fasting dogs at the maximum tolerated intravenous dose (30 nmol/kg). Gastric acid secretion of the
rat was stimulated very little, if at all. In the cod, a teleost fish,
physalaemin and eledoisin potently stimulated gastric pepsin secretion
(ED50 = 1 mg/kg/min); SP was 1000 times less
active (Holstein and Cederberg, 1986). In the isolated porcine nonantral stomach preparation, 0.1 µM SP induced a 2-fold increase in
acid secretion and a 3- to 4-fold increase in pepsinogen secretion. Similar effects were displayed by NKA, whereas capsaicin was inactive (Schmidt et al., 1999). This result may indicate that release of
endogenous SP/NKA is insufficient to affect the above parameters. In
the dog, the peptide caused changes in bile flow that were associated
with contraction of the gall bladder and not with a stimulated
secretory activity. In fact, when the hepatic and cystic ducts were
cannulated separately, physalaemin increased the flow in the cystic
duct but decreased the flow in the hepatic duct. In the rat, the
peptide was completely ineffective (Bertaccini et al., 1967).
In agreement with the above results it was found that SP also
attenuated basal and hormone-stimulated (cholecystokinin and vasoactive
intestinal peptide) overall bile flow and output of bile acids,
Na+, K+,
Cl, and bicarbonate (Starke et al., 1968; Holm
et al., 1978; Konturek et al., 1981).
Intestinal secretion. That physalaemin and eledoisin
stimulate intestinal secretion is shown by the liquid discharges
produced after subcutaneous administration of these peptides. In dogs
with a cannulated 25-cm jejunum segment perfused with saline, the
intravenous injection of 55 pmol/kg/min SP produced an increase in
plasma SP concentration from 6 to 121 fmol/ml, an increase in
intestinal secretion of water (from 102 to 275 µl/min) and
Na+ (from 20 to 23.2 µEq/min), and a decrease
in Cl secretion (from 21.7 to 16.5 µEq/min)
(McFadden et al., 1986).
Close intraarterial infusion of SP (causing a plasma concentration of
1-5 µM) to the in vivo feline small intestine regularly evoked a net
fluid secretion in vivo, accompanied by release into the blood of
vasoactive intestinal polypeptide (Brunsson et al., 1995).
The problem of receptors and mediator systems involved in the in vivo
and in vitro secretory effects of the tachykinins is extremely complex.
NK1 and NK2 receptors seem to play a predominant role. In the canine
intestine, implication of NK2 receptors in addition to NK1 receptors is
demonstrated by the greater potency of eledoisin in comparison with
physalaemin in eliciting watery discharges.
Pancreatic secretion. Physalaemin displayed a moderate,
short-lasting stimulatory action on exocrine pancreatic secretion in
the dog after threshold intravenous doses of 0.05 to 0.5 nmol/kg. Eledoisin was 30 times less active. The content of amylase in pancreatic juice was similar to that present in the secretion elicited
by cholecystokinin (Bertaccini et al., 1967).
Like eledoisin, kassinin was virtually inactive on the dog pancreas. SP
enhanced the basal output of pancreatic juice, amylase, and bicarbonate
in dogs (20 pmol/kg/min, by close intraarterial infusion) and rats
(Thulin and Holm, 1977; Konturek et al., 1981). However, the effect of
the tachykinins was negligible in comparison with that of cholecystokinin.
In dispersed acinar cells prepared from guinea pig pancreas,
physalaemin and eledoisin increased outflow of
Ca2+, accumulated cyclic GMP, and released
amylase without affecting cyclic AMP. The efficacy relative to
caerulein was 29% for eledoisin and 17% for physalaemin (May et al.,
1978; Jensen and Gardner, 1979). In the isolated rat pancreas, 0.1 to 1 nM SP inhibited cholecystochynin-induced amylase release and
secretin-induced flow. Capsaicin displayed the same effects as SP,
probably through release of endogenous SP (Kirkwood et al., 1999).
The functional significance of neuronal tachykinins in the gut has been
the object of an extensive and thorough investigation (Holzer-Petsche,
1995; Holzer and Holzer-Petsche, 1997a, 1997b). All aspects of the
tachykinin function in the gut have been taken in consideration, but it
is extremely difficult to draw some firmly established conclusions from
the enormous amount of data published. Pharmacological "in vitro"
tests have clearly demonstrated that exogenous tachykinins given alone
are among the most active substances on the gastrointestinal motility
of all examined vertebrate species, generally in the sense of clear-cut
stimulation. Things are different in the intact organism. The
tachykinins represent only one of the many active agents of peptide and
nonpeptide nature (gastrin, cholecystokinin, motilin,
enteroglucagon, bombesin, guanylin, neuropeptide tyrosin, opioid
peptides, noradrenaline, histamine, serotonin, acetylcholine,
prostaglandins, ATP, etc.), and it is virtually impossible at present
time to distinguish the part played in the functional control of gut
motility by the tachykinins from that played by the array of the above
active substances. It is highly probable that the tachykinin
co-involvement may be considerably different in the different animal
species and in the different gut sections. The use of tachykinin
antagonists has given only partial and ambiguous results. In
conclusion, whereas, there is no doubt that endogenous SP and NKA may
play an important role by interaction with other enteric transmitters
in the control of gastrointestinal motor activity, the weight of this
role in health and disease remains to be defined.
Exogenous tachykinins seem to cause also secretion of fluid and
electrolytes from the intestinal mucosa, and it has been suggested that
endogenous tachykinins may play a messenger role in intestinal secretory pathway. The increase in the capillary permeability could
contribute to increase in secretions. Moreover, there is evidence that
the tachykinins participate in the hypersecretory, vascular, and
immunological disturbance, associated with infection and inflammatory
bowel disease (Holzer and Holzer-Petsche, 1997a, 1997b). This statement
must be again accepted with caution, keeping in mind that the
tachykinins are also in the case of secretions and, even more so, of
immunological reactions only one of the numerous factors involved in
these processes. To remain in the field of gut secretions, in recent
years a peptide, guanylin, has been extracted and isolated from various
organs, among which is the rat intestine (Currie et al., 1992) in which
it seems to be costored with serotonin in some populations of the
enterochromaffin cells (Cetin et al., 1994). Guanylin exhibits high
sequence and structural homology with Escherichia coli
heat-stable enterotoxin and, like this toxin, guanylin causes secretory
diarrhea in rats, by activation of the guanylate cyclase C receptors.
It is evident that guanylin may play an important role in the
physiological regulation of electrolyte/water secretion in
ion-transporting intestinal epithelium.
Some questions at this point are imperative. Do some enterochromaffin
cell populations costore and cosecrete SP and guanylin? Could guanylin
be costored with SP also in the nonargentaffin, but
argyrophyl/acidophyl cells of the gut? Is it possible that guanylin is
present also in carcinoid tumors, contributing to the diarrhea, which
is one of the most frequent symptoms in the carcinoid syndrome? Should
the answer to these questions be positive, the tachykinin contribution
in the regulation of gut secretion could be attributable not to
neuronal SP but to SP occurring in the highly neglected epithelial
cells of the gut, probably provided with paracrine secretion.
C. Airways System
A detailed description of the effects of the tachykinins on
isolated preparations of tracheal and bronchial musculature in the rat,
guinea pig, ferret, hamster, and man is presented by Frossard and
Advenier (1991), together with a discussion of the receptor types and
subtypes involved in the response to the tachykinins. Under
physiological conditions, tachykinins contribute, to some extent, in
the regulation of the tone of the airways musculature, at least in some
animal species.
In spontaneously breathing guinea pigs, graded doses of eledoisin (2-5 nmol/kg, intravenous) produced a dose-dependent reduction of the tidal volume associated with temporary tachypnoea. With 5 nmol/kg, the tidal volume approached zero, although the movements of the respiratory muscle had increased. Eledoisin was one-half as active as serotonin.
In mechanically respired guinea pigs, eledoisin reduced the inflation
volume in a dose-dependent manner; a 50% reduction was obtained at a
dose of 1 nmol/kg. Serotonin was 3 to 4 times less active (Gjuris and
Westermann, 1965). These results were confirmed by Nilsson et al.
(1977), who observed that intravenous injection of SP produced a
dose-dependent elevation of insufflation pressure. SP (0.4 nmol/kg)
increased the pressure by 100%, an effect that required doses of
histamine 40 times higher. Similarly, Hua et al. (1984), found that
kassinin, eledoisin, and NKA potently increased insufflation pressure.
Physalaemin was less potent, and NKB still less so. Moreover, in guinea
pig airways, physalaemin, eledoisin, and SP provoked an increase in
microvascular permeability to protein by activating receptors localized
in the endothelial cells, as assessed by Evans blue extravasation
(Rogers et al., 1988). It is suggested that both NK1 and NK2 receptors
are involved in the response to tachykinins by the guinea pig
tracheobronchial tree (Ireland et al., 1991; Maggi et al., 1991).
In rats, the broncho-constrictor action elicited by the intravenous
injection of tachykinins was inhibited largely by atropine (suggesting
a release of acetylcholine), and by methysergide (suggesting a release
of 5-HT from pulmonary mast cells). Eledoisin and kassinin were
slightly but significantly more potent than the neurokinins but much
more potent than SP (Joos et al., 1986). In anesthetized rabbits,
peripheral administration of either SP or NKA (0.2-2 nmol/kg) produced
a dose-related increase in rapidly adapting pulmonary stretch receptor
activity without any significant changes in total lung resistance. NKA
was less potent than SP, NKB was practically inactive. This effect is
mediated by activation of both NK1 and NK2 receptors (Matsumoto et al.,
1997).
In the anesthetized dog, challenge with aerosolized NKA (0.1-1%)
produced a dose-dependent increase in lung resistance, a decrease in
dynamic lung compliance, reduced tidal volume, and increased
respiratory rate. Experiments with selective tachykinin antagonists
suggest that in addition to the NK2 receptor the NK1 receptor may also
be involved in the response to NKA by the dog respiratory tract.
Cholinergic reflexes may play a small, but significant role in this
response (Sherwood et al., 1977).
In humans, infusion of SP had little effect on airway function. A small
increase in airway resistance was observed at low dose and converted to
bronchodilation at high doses (3.2 pmol/kg/min) (Evans et al., 1988).
Inhaled SP also failed to increase airway resistance in both normal and
asthmatic subjects. NKA conversely induced a fall in specific airways
conductance in patients with mild asthma, suggesting an activation of
NK2 receptors (Joos et al., 1986). In man, it is rather doubtful from
the available data that the tachykinins play some role in disease, such
as asthma, and that tachykinin receptor antagonists may have a future
in therapeutics of respiratory disease. One of the major symptoms of
the carcinoid syndrome is asthmatic attack. It would be important to
know whether these attacks benefit from administration of tachykinin antagonists.
D. Urogenital Tract
Exogenous tachykinins at extraphysiological concentrations
produced variable degrees of stimulation of smooth muscle preparations of the urogenital tract, especially the urinary bladder and displayed differences in their agonistic potency not only depending on the different animal species but also on the various segments of the urinary tract. The different kinds of responses seem to be mediated through different types of receptors and seem to be brought about both
by a direct effect on the bladder's smooth muscle and by an effect on
intramural sensory nervous pathways ("micturition reflex") (Maggi
and Meli, 1986; Maggi et al., 1986a, 1986b; Maggi 1991). Activation of
rat bladder motility and the micturition reflex may be provoked by
intravenous injection and by topical application of tachykinins on the
serosal surface of the bladder.
Intravenous injection of the peptides elicited a phasic contraction of the bladder (increase of internal pressure) and an activation of a series of rhythmic contractions. Kassinin was the most potent peptide, followed by NKA (30%), NKB (20%), and SP (1%). Thus, endogenous tachykinins together with other active substances may contribute to the tone and motility, including micturition reflex, of the urinary bladder, the ureters, and the urethra, but the part actually displayed by the tachykinins remains to be established.
E. Immune System
The influence of the tachykinins on the immune system has been
carefully reviewed in recent articles (Hartung and Toyka, 1989; McGillis et al., 1990; Eglezos et al., 1991; Maggi, 1997). Although there is increasing evidence that the tachykinins (especially SP and,
subordinately NKA) play a role in neuro-immunomodulation, i.e., in the
control and regulation of the immune response by the central and
peripheral nervous system, the actual relevance and importance of the
tachykinins in immunomodulation is uncertain. Tachykinins are merely
one of the numerous factors that may directly or indirectly influence
the immune system: all or nearly all peptidergic and aminergic
neurotransmitters, the derivatives of arachidonic acid, and all of the
many active substances synthesized and released by the immune cells.
Data supporting the influence of the tachykinins on the immune system are as follows: a) Occurrence of SP immunoreactive fibers in organs of the immune system, such as lymph nodes, thymus, and bone narrow. b) Presence of SP receptors in thymus and spleen and, above all, expression of these receptors in human circulating lymphocytes and monocytes, rabbit polymorphonucleates and leukocytes, guinea pig macrophages. c) Clear-cut action of SP, in vitro and in vivo, on B- and T-cell proliferation, immunoglobulin secretion, cellular chemotaxis, and lymphocyte migration.
The integrity of the immune system is essential for life. The
involvement of tachykinins in the control of the immune system in
health is very difficult to be established and, at any rate, it seems
to be of limited importance: knockout mice with disrupted preprotachykinin A gene were in good health. However, it is necessary to distinguish between physiology and pathology of the immune system.
In pathological conditions, such as inflammatory processes, things are
even more complicated, because of the enormous cascade of biochemical
events that take place during inflammation and immune reaction. It is
certainly possible that certain immune cell types are able to
synthesize and release tachykinins (of extra-neuronal source) and that
NK1 receptors play a role in mediating extravascular migration of
granulocytes into inflamed tissues in response to various stimuli
(Maggi, 1997). In human skin, exogenous tachykinins may cause wheal and
flare, due to release of histamine, and may evoke plasma leakage
through the capillaries in several but not all tissues. SP may also
degranulate mast cells, but this is an effect attributable not to the
intact SP molecule, but to its N-terminal segment acting on receptors
independent from the classical tachykinin receptors. To demonstrate the
complexity of participation of SP in inflammation, it has been shown in
a recent paper (Wallace et al., 1998) that in a model of acute colitis in the rat and guinea pig, NK1 antagonists, although reducing the
infiltration of granulocytes during the first 12 h after induction of colitis, failed after repeated administration during a 3-day period
to affect granulocyte recruitment or severity of tissue injury.
F. Central Nervous System
The overall presence of tachykinins with their receptors in the
CNS of mammals and in that of all examined submammalian species (see
neuronal localization) constitutes the most pregnant and incontrovertible evidence that these peptides play in the CNS a very
important role as neurotransmitter/neuromodulatory agents, as
demonstrated by neurophysiological evidences directly showing this
importance (Otsuka and Yoshioka, 1993).
In the CNS, tachykinins occur in large amounts particularly in areas involved in the central control of several peripheral autonomic functions (blood pressure, respiration, micturition, gastrointestinal motility, etc.), of essential functions (e.g., drinking behavior), of the affective and emotive life (stereotyped behavior, motility, anxiety, aggression, and pain), and of higher cerebral functions (learning and memory).
Blood pressure. Eledoisin (0.1-1 nmol/kg) injected into the
cerebral ventricles of anesthetized rats produced a biphasic
cardiovascular response that consisted of an initial fall of systemic
blood pressure (8-15 mm Hg) followed by a rise (20-22 mm Hg).
Abolition of the fall in blood pressure by phentolamine suggests
central inhibition of sympathetic tone to vessels that afford
peripheral resistance, whereas blockade of the delayed hypertensive
phase by propranolol indicates a central activation of cardiac
adrenoreceptors (Pearson et al., 1969). Different effects were obtained
when 1 µg of eledoisin was intracerebroventricularly administered in
conscious rats. The peptide, in fact, produced a long-lasting rise in
blood pressure (18 mm Hg) that was accompanied by behavioral
excitement. Pretreatment with phentolamine, but not propranolol or
morphine, prevented the pressor response, thus, indicating an
-adrenergic-mediated vasoconstriction (Lambert and Lang, 1970).
Effects produced by intracerebroventricular injection of SP (10 nmol)
were similar: increase in blood pressure, heart rate, and sympathetic
efferent activity with visceral vasoconstriction and hindlimb
vasodilation. The cardiovascular responses were accompanied by a
behavioral defense reaction, including increased locomotion,
scratching, skin biting, and grooming (Unger et al., 1988). Also NKA
(10 nmol) elicited increase in blood pressure and heart rate (via
sympathetic activity) (Takano et al., 1990).
Respiration. Intracerebroventricular injections of SP in rats
(3-30 nmol) induced a dose-dependent stimulation of minute ventilation due to increase in total volume, although respiratory frequency was
slightly reduced (Hedner et al., 1984). Similar respiratory effects
were produced by application of SP on the dorsal surface of the medulla
oblungata in newborn rabbits (Yamamoto and Lagercrantz, 1985).
Gastric acid secretion. The NK3 receptor preferring
tachykinins (kassinin, NKB, and PGKII) given to rats by
intracerebroventricular injection (0.01-10 nmol/rat) elicited a
dose-related inhibition of gastric acid secretion. Kassinin and
eledoisin were the most potent peptides, the other peptides showing the
following order of relative potency: kassinin > NKB = PGKII
NKA > SP and physalaemin (inactive). Subcutaneous doses up to
20 nmol of eledoisin or kassinin were ineffective (Improta and
Broccardo, 1990; Improta et al., 1996).
Gastric emptying. Administration of 0.1 nmol of either
eledoisin or kassinin produced a 35 to 40% inhibition, and 10 nmol caused a 100% inhibition of gastric emptying of a liquid meal. The
relative potency of other examined tachykinins was as follows: NKA,
50%; physalaemin, NKB, and PGKII 0.3%, thus suggesting a predominant
involvement of NK2 receptors (Improta and Broccardo, 1990; Improta et
al., 1996). In general, tachykinins are less potent in their inhibition
of gastric emptying and gastric secretion than either bombesin or
opioid peptides.
Colonic propulsion. Intracerebroventricular injections of
PG-KII (0.1-100 ng/rat; threshold 1 ng/rat), a selective NK3 receptor agonist, produced a dose-related inhibition of colonic propulsion in
the rat. Senktide had a weaker, but evident action, whereas NKB, the
classical mammalian selective NK3 agonist was inactive, up to 10 µg/rat (Broccardo et al., 1999). The interpretation of this
surprising result is obscure. It is tempting to suggest the existence
of different NK3 receptor subtypes.
Food intake. Intracerebroventricular injections of eledoisin
and physalaemin (100-1000 pmol) did not reduce intake of milk or solid
food by rats. Some inhibition of milk intake, observed at 100 ng doses,
was accompanied by increased grooming and locomotion (Massi et al.,
1986).
Thermoregulation. At doses up to 10 nmol,
intracerebroventricular administration of tachykinins (kassinin,
eledoisin, physalaemin, and SP) had no effect on the temperature of
rats kept at room temperature (Broccardo and Improta, 1988).
Sexual behavior. In ovariectomized, estrogen-treated female
rats, bilateral injection of SP (50-1000 pmol) into the midbrain gray
matter produced a rapid, long-lasting (3-h) increase in lordosis score,
similar to that produced by luteinizing hormone-releasing hormone (Dornan et al., 1987). Similarly, injections of SP (10-200 pmol) into the medial-preoptic-anterior-hypothalamic area in rats significantly shortened the interval to initiate copulation and reduced
ejaculation latency (Dornan and Malsbury, 1989).
Drinking behavior. The effects of tachykinins on all aspects of drinking behavior were 1000 times less intense by peripheral than by central administration.
Intracerebroventricular pulse administration of eledoisin (threshold,
10 pmol/rat) potently inhibited water intake evoked by
intracerebroventricular angiotensin II (100 pmol/rat), water deprivation, and cell dehydration. SP and physalaemin were far less
potent; kassinin and NKA caused only a long-lasting inhibition of
drinking due to cell dehydration. Brain areas sensitive to the
antidipsogenic effect of eledoisin versus angiotensin II-induced drinking are the nucleus preopticus medialis, the nucleus anterior hypothalami, and the subfornical organ (Massi et al., 1988, 1990).
Eledoisin, kassinin, and, to a lesser extent, physalaemin caused
release of vasopressin, with ensuing antidiuresis. SP was ineffective.
Vasopressin release (particularly evident upon injection of the peptide
into the hypothalamic paraventricular nucleus) seems to be mediated by
central angiotensin, once NK3 receptors are activated (Polidori et al.,
1989; Massi et al., 1991).
Kassinin, eledoisin, and, to a lesser extent, NKA (100 nmol/rat,
intracerebroventricularly) displayed a potent and long-lasting inhibitory effect on salt intake. SP, physalaemin, and neurokinin B
were far less effective, and thus, did not suggest NK1 receptor involvement, because SP was only poorly effective. The medial region of
the amygdala seems to be main site of action of the tachykinins for
inhibition of salt intake (Massi and Epstein, 1989; Massi et al.,
1990).
The effects of the tachykinins on drinking behavior in other mammalian
species (rabbit and sheep) were considerably less intense and less
constant. However, in cats, intracerebroventricular injections of
eledoisin (100 pmol) caused a remarkable (60%) and long-lasting (over
60 min) inhibition of angiotensin-induced drinking. Eledoisin was at
least four times more potent than SP. Kassinin appeared virtually
inactive (Barocelli et al., 1988).
In sharp contrast to the rat, tachykinins displayed a potent dipsogenic
effect in the pigeon and, less evident, in the duck (De Caro et al.,
1978, 1980). Physalaemin stimulated water intake even at
intracerebroventricular doses as low as 10 pmol/pigeon. At the highest
tested doses (1 nmol), the animal drank more water within a few minutes
than would normally be consumed during a period of 16 to 24 h. The
dipsogenic potency of physalaemin was 10 times less than that of
angiotensin II, similar to that of eledoisin and kassinin, but 10 times
greater than that displayed by NKA, and 100 times greater than that
possessed by NKB. This order of potency does not seem consistent with
the tachykinin receptor subtypes so far proposed. The dipsogenic effect
of the tachykinins cannot be attributed to activation of angiotensin II
receptors, because drinking was not reduced by administration of
angiotensin II antagonists (De Caro et al., 1978, 1988; Massi et al.,
1987).
The selective agonists of NK3 receptors (NKB, senktide, and PG-KII)
potently inhibited ethanol intake in genetically alcohol-preferring rats; at intracerebroventricular doses from 10 pmol/rat PG-KII was 3 times more potent than senktide. At doses of 100 pmol/rat, only alcohol
intake was inhibited in food-deprived rats, not food intake or prandial
drinking, indicating that the effect on alcohol intake was behaviorally
selective. PG-KII, a NK3 receptor tachykinin agonist, inhibited
angiotensin II-induced drinking only at doses of 300 to 1000 pmol,
producing also evident competitive behavior, locomotion, and inhibition
of digestive behavior (Ciccocioppo et al., 1997).
Micturition reflex. Intracerebroventricular injection of SP
(30 nmol) or capsaicin (25 µg) elicited the micturition reflex in the
rat, probably by acting directly on the brain micturitian centers (Dib
et al., 1998).
Stereotyped and motor behavior. At intracerebroventricular
doses of 0.6 nmol, SP(1-7) inhibited not only nociception but also aggressive and grooming behavior, while stimulating, like SP, investigative motor behavior. The C-terminal peptide fragment [pGlu6] SP(7-11) exerted opposite effects
(Hall and Stewart, 1984). In producing a rigorous reciprocal hindlimb
scratching accompanied by extensive grooming behavior, there was an
impressive (approximately 1000 times) difference in responsiveness to
intracerebroventricular injection of SP as a function of the genetic
strain and age of mice, the old animals (4-5 months) being less
sensitive than the young (1-2 months) animals (Hall et al., 1985).
Moreover, whereas the intracerebroventricular injection of all tested
tachykinins (SP, physalaemin, NKA, eledoisin, and kassinin) produced in
mice an enhancement of grooming and scratching behavior and a reduction
of sniffing behavior, only SP increased hindlimb rearing behavior. This
effect, unique to SP, was shared by the N-terminal metabolic fragment
SP(1-7) and, very surprisingly, also by SP(1-6) (Hall et al., 1987).
The intracerebroventricular, but not intravenous, injection in gerbils
of the two SP-like, NK1 receptor agonists,
[Sar9,MetO211]SP
or DAla-[Pro9,Leu10]SP,
and GR 73632, elicited in gerbils a characteristic repetitive hind paw
tapping, which was not associated with an increase in locomotor
activity and seemed to be involuntary. Peak response occurred within 5 to 10 min. GR 73632 was 70 times more potent than
[Sar9,MetO211]SP
(ED50 0.7 nmol/gerbil), but the response induced
by GR 73632 was less intense. Responses were significantly and dose
dependently antagonized only by CNS penetrating NK1 receptor
antagonists (Bristow and Young, 1994; Rupniak and Williams, 1994).
At intracerebroventricular doses of 100 to 400 pmol, the NK1 agonist GR
73632 significantly increased also in the guinea pig the locomotor
activity. The effect was abolished by NK1 receptor antagonists and by
haloperidol (Mason et al., 1992).
Results obtained by subcutaneous or intraperitoneal injection of SP (5 nmol) in mice were at variance. In fact, the peptide decreased
spontaneous locomotor activity and counteracted amphetamine-induced hyperactivity. Spontaneous exploratory behavior was also lowered. It is
possible that brain monoamines are implicated in these effects, with
acceleration of dopamine turnover and retardation of serotonin turnover. Smaller doses of SP showed an antinociceptive morphine-like action in the hot plate test. These results are consistent with SP
having a tranquilizing action in mice (Starr et al., 1978).
Aggressive behavior. The intracerebroventricular injection of
0.6 nmol/kg SP or of the N-terminal fragment SP(1-7) reduced fighting
in mice made aggressive by prolonged isolation. This effect was
enhanced by naloxone. In contrast, the shorter C-terminal analog of SP,
[pGlu6]SP(7-11) increased the
isolation-induced fighting, an effect that was antagonized by naloxone,
demonstrating that the various peptide fragments of the SP molecule can
exert opposite effects on a specific behavior and that the different
effects of naloxone may be modulated by specific mechanisms (Hall and
Stewart, 1984; Hall et al., 1987).
Learning and memory. SP administered subcutaneously
influenced dose-dependently passive and active avoidance conditioning in mice. The retention of a single trial passive avoidance task was
enhanced by 0.75 pmol/g. Higher or lower doses were less active or
ineffective. SP did not alter the rate at which the mice learned an
active avoidance task but increased the extinction of learning (Schlesinger et al., 1983). Similarly, in an appetite motivated learning task, mice injected subcutaneously with 0.75 pmol/g SP retained the task better than control animals, suggesting that SP-treated animals better remembered the original task (Schlesinger et
al., 1986). These results were confirmed by Hasenohrl et al. (1990),
who found that enhancement of inhibitory avoidance learning produced in
rats by SP (40 nmol/kg) was reproduced by the N-terminal fragment
SP(1-7), but not by the C-terminal fragment
[pGlu6]SP(6-11). Higher or lower doses of SP
had no effect, demonstrating that the facilitating effect of the
peptide was reflected by an U-shape dose-response function.
Rats given diazepam 20 min before the training on an inhibitory
avoidance task showed an impaired retention. The amnesic effect of
diazepam was blocked by 50 µg/kg SP and 167 µg/kg SP(1-7) but not
by 134 µg/kg SP(6-11). Thus, the amino acid sequence responsible for
this effect may be encoded by the N-terminal fragment of SP (Costa and
Tomaz, 1998).
Psychological stress: anxiety. The intracerebroventricular infusion of the NK1 agonist GR 73632 (0.1 nmol) in guinea pigs causes not only motor activation but also pronounced and long-lasting audible vocalization, markedly attenuated not only by the antidepressant drugs, but also by the NK1 receptor antagonist L 733.061. Similarly, the CNS-penetrant NK1 receptor antagonists, like the antidepressant and anxiolytic drugs, were able to inhibit vocalization evoked in guinea pig pups by transient maternal separation.
It is concluded that the selective pharmacological blockade of SP
receptors is capable of inhibiting behavioral responses to
psychological stress in a manner resembling the effect of clinically used psychotherapeutic agents (Kramer et al., 1998). However, no
influence on anxiety (upon field assay) was seen in mice with disrupted
gene of NK1 receptor (De Felipe et al., 1998).
Abstinence reaction during opioid withdrawal. It has been
found that SP may modulate the abstinence reaction to opioid
withdrawal. In fact the N-terminal fragment of SP, SP(1-7), may
inhibit the intensity of the withdrawal reaction in morphine-dependent
rats. Moreover, significant increases in concentration of SP(1-7) were observed in different brain areas during morphine withdrawal, indicating the involvement of the SP system during opioid withdrawal (Zhou et al., 1998).
Action on discrete selected brain areas.
Cat
subfornical organ. Neither physalaemin nor eledoisin produced
activation of neurons in the cat subfornical organ upon direct
application onto its surface (Felix, 1967).
).
Rat nigro-striatal system. The bilateral infusion of SP (2.25 nmol) into the substantia nigra produced a strong increase in stereotyped rearing and sniffing, with no concurrent enhancement of
locomotion. After successive infusions, rearing response disappeared. In caudate-lesioned rats, behavioral stimulation by SP was blocked, suggesting that the response to SP is mediated through the
nigro-striatal dopamine system (Kelley and Iversen, 1979).
In accordance with these results Tan and Tsou (1988) found that
intranigral injection of kassinin, eledoisin, and SP (0.5 nmol)
produced a marked dose-dependent increase of 3,4-dihydroxyphenylacetic acid and dopamine concentration in the ipsilateral striatum and a
number of controlateral circlings. The rank order of activity was
kassinin > eledoisin > SP.
SP(1-9) and SP(6-11) at 0.1 to 1 nM concentrations induced an
increase in dopamine outflow from rat striatal slices. The effect of
SP(6-11) was blocked by an NK1 antagonist, whereas the effect of
SP(1-9) was unaffected. The coincubation of the above fragments with
intact SP revealed a negative interaction between fragments and
substance P (Khan et al., 1998).
In fresh striatal slices, (3H)SP bound
specifically to one site from which it was displaced by SP, but not by
SP(1-7) or SP(5-11). In contrast, 10 µM SP(1-7) or SP(5-11)
induced, like SP, a significant internalization of the NK1 receptor. It
is suggested that SP fragments have high affinity with an NK1 receptor
conformance, which is different from that labeled by
(3H)SP (Michael-Titus et al., 1999).
Hippocampus and entorhinal cortical cortex. Intrahippocampal
administration of 10 pmol of SP triggered self-sustained status epilepticus in response to electrical stimulation of the perforant path
for periods too brief (7 min) to have any effect in control rats
(requiring a 30-min stimulation). The seizures were accompanied by
high-amplitude electrographic paroxysmal activity that lasted many
hours. Hippocampal damage resembling that known to occur in human
epileptic seizures was blocked by SP receptor antagonists (RP 67.580).
In addition, in the status epilepticus, a rapid, dramatic increase of
the expression of preprotachykinin A mRNA, of SP in several brain
areas, and of the glutamate release from hippocampal slices has been
shown. It is concluded that enhanced expression of SP during
self-sustained status epilepticus may modulate hippocampal excitability
and play a critical role in the maintenance of status epilepticus
(Liu et al., 1999a). In hippocampal dentate gyrus granule cells,
SP produces a robust enhancement on N-methyl aspartate channel function, prolonging the opening of the channels. Thus, SP
provokes an enhancement of the excitatory amino acid-mediated excitability (Lieberman and Mody, 1998).
In experiments in vitro on slices of rat entorhinal cortical cortex, it
was found that spontaneous epileptiform discharges evoked by the GABA
receptor antagonist bicuculline were reduced in frequency and sometimes
in duration by SP. Excitatory synaptic potentials mediated by aspartate
were not affected by the peptide (Maubach et al., 1998).
Rat ventral tegumental area. SP and related tachykinins
administered either intracerebroventricularly (0.1-20 µg) or
directly into the ventral tegumental area of the rat mesencephalon (0.5 nmol) caused increased locomotor activity, grooming behavior and wet-dog shakes. Kassinin, eledoisin, and NKA elicited the greatest locomotor activity and wet-dog shakes, whereas SP and physalaemin were
not effective in producing full-body grooming (Elliott and Iversen,
1986). Similarly, mice infused with SP (3 nmol) into the ventral
tegumental area exhibited a long-lasting increase in spontaneous
activity with rearing and sniffing (Kelley et al., 1979).
Rat nucleus tractus solitari. Microinjection of SP (0.7 pmol)
or NKA (0.9 pmol) in the nucleus caused prompt, transient hypotension and bradycardia, suggesting that the tachykinins may act as
neurotransmitters of the baroreceptors of the nucleus (Nagashima et
al., 1989). The same results were obtained with microinjection of
either SP or SP(1-7) (60 pmol), and the effects of SP were blocked
when cleavage of SP was inhibited by phosphoramidon, which alone failed to block the depressor and bradycardic effect of SP(1-7), suggesting that only the N-terminal fragment was active and not the intact SP
molecule (Hall et al., 1989).
Rat nucleus accumbens. SP and
[pGlu6]SP(7-11) attenuated passive avoidance
behavior when at picomolar amounts were injected into the n. accumbens.
The N-terminal fragment, SP(1-7) had an opposite effect, facilitating
passive avoidance behavior (Gaffori et al., 1984). However,
intraaccumbens injection of SP at nanomolar doses had no significant
behavioral effects in regard to motility and to conditioned place
preference (Schildein et al., 1998).
Rat nucleus basalis magnocellularis. SP (1 pmol) injected
into the n. basalis magnocellularis region exerted anxiolytic-like effects in the rat as shown by more time spent on the open arms of the
plus-maze test and in social interaction. When administered intraperitoneally, SP had a biphasic dose-dependent effect: anxiolytic action at 40 nmol/kg, and anxiogenic action at 400 nmol/kg (Hasenohrl et al., 1998).
Rat spinal cord. On isolated spinal cord of newborn rats, the
tachykinins at 10 to 100 nM concentrations caused depolarization of
motoneurones. The rank order of potency of the examined tachykinins was
physalaemin > NKB = kassinin = SP > NKA (Matsuto
et al., 1984).
Bullfrog spinal cord. SP, physalaemin, and eledoisin exerted
a strong excitatory action on motoneurones of isolated bullfrog spinal
cord. On a molar basis, SP was about 200 times, physalaemin 1500 times,
and eledoisin 2000 times more active than L-glutamate in
depolarizing the motoneurones. Because the depolarizing action of
substance P and related peptides was blocked by
Ca2+ deficiency by tetrodotoxin, it is likely
that the peptides have a direct action on the motoneurones. Moreover,
Konishi and Otsuka (1974) suggested SP to be an excitatory transmitter
of primary sensory neurons.
Molluscan ganglia. Direct application of physalaemin on
isolated esophageal ganglia of the mollusc Achatina fulica
produced excitation of an identified tonically autoactive, giant neuron (TAN). By bath application of physalaemin (200 µg/ml), the frequency of the TAN's spike discharge increased more than twice. Micro-drop application (3.5 ng) resulted in the biopotential of TAN showing a
slight hyperpolarization followed by a marked depolarization. The
excitatory effect of physalaemin clearly was predominant. No other
peptides examined, including SP and eledoisin, had any effect (Takeuchi
et al., 1976). After trypsin or chymotrypsin treatment, physalaemin
lost its effect, and a clear inhibitory effect emerged on the same TAN.
Among the peptide fragments obtained by chymotryptic digestion, the
tripeptide Lys-Phe-Tyr- appeared to be responsible for the strong
inhibitory effect on TAN. The critical concentration of the tripeptide
by bath application was 6 to 20 µg/ml, whereas by micro-drop, amounts
as low as 0.3-0.5 ng caused a marked inhibition of the TAN
biopotential (Takeuchi and Sakai, 1977; Takeuchi et al., 1977a, 1977b).
From the pharmacological data here presented and from other experiments
on mutant mice, however, it seems clear that the tachykinins are not
essential for life or for most of the above functions and expressions
of the CNS activity. Mutant mice had no gross physical abnormalities,
were similar in size and weight to wild mice, appeared healthy over a
period of at least 6 months, and were fertile with normal litter size
and maternal behavior. They also appeared normal on a rotating rod and
in open field activity (Cao et al., 1998; De Felipe et al., 1998;
Zimmer et al., 1998). Tachykinins are probably a very important link,
but only one link, of the extremely complex chain of events underlying
the chemical conveyance of information within the CNS. The part played
by the tachykinins in the array of transmitters/modulator molecules
occurring in the CNS is not yet completely understood. Thus, as
pharmacological and physiological data on the role of tachykinins in
the CNS are derived essentially from experiments carried out in mice
and rats, it is even harder to conceive that they are transferable, sic et simpliciter, to higher mammals and to man.
Moreover, the SP(1-7) fragment seems to be involved not only in
analgesia but also in aggressive and grooming behavior (Hall and
Stewart, 1984; Hall et al., 1987), aggressive behavior by prolonged
isolation (Hall and Stewart, 1984), hindlimb rearing behavior (Hall et
al., 1987), learning and memory (Hasenohrl et al., 1998), central
elicited hypotension and bradycardia (Hall et al., 1989), diazepam
amnesic effect (Costa and Tomaz, 1998), and the abstinence reaction to
morphine withdrawal in morphine dependent rats (Zhou et al., 1998).
SP(1-7) may be an endogenous modulator of SP actions in the brain
(Herrera-Marschitz et al., 1990). The intranigrally injected fragment
acted as a very potent antagonist against responses induced by
intranigral injection of SP (dopamine release in the striatum with
consequent behavioral effects) and also of physalaemin (Sakurada et
al., 1990b). Thus, it seems demonstrated, beyond any doubt, that
SP(1-7) may act in the CNS as neurotransmitter/neuromodulator.
All of these findings raise the following questions, with the
possibility that the entire problem of the function of tachykinins in
the CNS should be revised:
1) To what extent are the central actions of SP attributable to the
intact SP molecule and to what extent does SP require enzymatic
cleavage with formation of its metabolite SP(1-7) to become active? SP
acts certainly as intact molecule a) in the case that its central
effects are reproducible also by other selective NK1 agonists, such for
example physalaemin; b) in the case that its action is inhibited by
selective NK1 receptor antagonists; and c) in the case that its action
is potentiated by enzyme inhibitors, blocking fragmentation of SP. The
observation that phosphoramidon (2-2000 pmol) (an endopeptidase
inhibitor) and, to a lesser extent, bestatin (an aminopeptidase
inhibitor) remarkably increased and prolonged the behavioral responses
(scratching, biting, and licking) induced in mice by SP suggests the
importance of endopeptidases in terminating the effects of the SP
intact molecule (Sakurada et al., 1990a, 1990b). On the contrary, it is
probable that only SP(1-7) fragment is active a) when its action is
not reproducible by neither SP or other tachykinin agonists; b) when
actions of SP(1-7) are not blocked by NK1 antagonists; and c) when the
effect of SP is blocked by phosphoramidon, which inhibits formation of SP(1-7). For example, hypotension and bradycardia elicited by injection of SP in the rat nucleus tractus solitarius are blocked by
the endopeptidase inhibitors (Hall et al., 1989).
2) Lack of binding of SP(1-7) to any of three classical tachykinin
receptors, presupposes the existence of other selective binding sites.
Data that, however, need confirmation, have demonstrated the existence
of two populations of binding sites in the mouse spinal cord capable of
binding reversibly (3H)SP(1-7) (Igwe et al.,
1990). Specific agonists for NK1, NK2, and NK3 receptors did not
compete at the binding of SP(1-7). These results support the existence
of an N-terminal directed SP-receptor. The fact that DAMGO, a
µ-opioid agonist, was active in displacing the ligand SP(1-7) is surprising.
The existence of different binding sites for the C-terminal fragment
and the N-terminal fragment of SP was indirectly demonstrated also by
Khan et al. (1998) who found that the increased dopamine outflow from
rat striatal slices produced by SP(6-11) was blocked by a NK1
antagonist, whereas the outflow elicited by SP(1-9) was unaffected.
3) At present, it is not clear which of the amino acid residue(s) in
the SP sequence are responsible for the central effects of the
heptapeptide. Generally experiments have been carried out with SP(1-7)
and SP(1-8), suggesting that the Phe7 residue
plays an important role, but in some experiments, SP(1-6) also proved
to be active. SP-like peptides, sharing as many as five to six amino
acid residues with the N-terminal heptapetide of SP, are found in
reptile brain and intestine (bufokinin) and in fish brain (trout SP and
cod SP). It would be of general interest to check whether these
N-terminal fragments (1-7) are active in the CNS of the pertinent
species and of mammals.
4) Transgenic mice with the disrupted the gene encoding the NK1
receptor (De Felipe et al., 1998) could be an excellent material for
investigating if SP (1-7) still displays its central effects and, in
this case, to demonstrate unequivocally, the existence of receptors
activated only by the N-terminal fragment of SP.
G. Pain
Much evidence has accumulated to suggest that SP is synthesized in the periphery by small-diameter sensory "pain fibers" and then, upon intense peripheral stimulation released into the dorsal horn, as a first step, through activation of NK1 receptors of transmission of pain information into the CNS. As a consequence, there is central hyperexcitability and increased sensitivity to pain. However, SP is also largely present, together with its NK1 receptor, in several brain areas. It is beyond question that brain SP contributes to pain perception and elaboration. But how, and to what extent? To answer this fundamental question it seems opportune to discuss separately the problem of SP and pain in the periphery (until the dorsal horns) and in the brain.
Periphery. The first association of SP with pain was made by
Lembeck and Holzer (1979), who suggested that SP, together with other
neuropeptides, may be released from the peripheral sensory nerve fibers
in the skin, muscle, and joints. This release was thought to be
involved in "neurogenic inflammation", a local painful inflammatory
response to certain types of injury or infection, such as that caused
by the classical irritant capsaicin.
By intraperitoneal administration, SP (0.8-3.2 nmol/mouse) displayed
either no analgesic effect (Growcott and Shaw, 1979) or predominantly a
clear antinociceptive action. SP antinociception was found at 10 to 20 pmol in the mouse (Stewart et al., 1976), at 0.8 to 3.2 nmol in the
mouse (Starr et al., 1978), and at 0.2 to 0.8 nmol/kg in the rat
(Mohrland and Gebhart, 1979).
SP injected into the lumbar subarachnoidal space of rats depressed the
tail-flick response in a dose-dependent manner
(ED50 1.2 nmol/rat). Maximum effect was reached
after 20 min and lasted 30 min. The antinociceptice effect of SP was
abolished by naloxone (Doi and Jurna, 1981).
At a dose of 7.5 nmol, SP depressed the motor response evoked by
supramaximal stimulation of the sural nerve and also reduced the
activity of part of the ascending neurons of the spinal cord evoked by
stimulation of C-afferent fibers. The depressive effect in ascending
nociceptive activity was slow in onset, lasted longer than 60 min, and
was abolished by naloxone (Doi and Jurna, 1982). In the superfused
spinal cord of rats and cats, iontophoretic application of SP produced
a long-lasting excitation of the dorsal horn neurons similar to that
elicited by noxious cutaneous stimuli; SP was released from dorsal
horns after stimulation of sensory neurons by capsaicin, and this
release was completely inhibited by morphine (Yaksh et al., 1980).
Intrathecal injection of SP or NKA (10-100 pmol), which in the mouse
caused a dose-dependent reciprocal hindlimb scratching, licking, and
biting response directed to the caudal part of the body, also decreased
latency in the tail-flick assay but did not alter reaction in the hot
plate test. These effects are interpreted as indicative of a
nociceptive behavior (Hylden and Wilcox, 1981; Seybold et al., 1982;
Gamse and Saria, 1986).
SP, physalaemin, and eledoisin intrathecally injected in rat have
been reported to cause hyperalgesia in the tail-flick test. Hyperalgesia produced by the tachykinins was dose-dependent, was maximal 10 to 20 min after injection, and lasted 30 min. The rank order
of potency was: physalaemin > SP > eledoisin. Desensitation to the effects of the peptides was observed after three successive injections of the peptide (Moochhala and Sawynok, 1984).
After microdyalisis of SP or NKA into the dorsal horn of anesthetized
monkeys, it was observed that neither peptide had significant effects
on the background activity or the response to mechanical or thermal
stimulation of the skin. However, each peptide produced significant
increases in the response to simultaneous or subsequent iontophoretic
application of excitatory amino acids (glutamic acid). Thus, it seems
that tachykinins facilitate responses of dorsal horn neurons to
excitatory amino acids or to cutaneous stimuli (Dougherty et al.,
1995).
Similarly, the progressive hypersensitivity of spinal flexor
motoneurons induced by repeated peripheral stimulation of inflamed tissues in decerebrated rats was attenuated by the subcutaneous injection of the NK1 antagonist RP 67580, indicating that SP is involved in mediating progressive hypersensitivity during inflammation (Ma and Woolf, 1997).
Recent, decisive demonstration of the important role of SP in
nociception has been afforded by experiments with nociceptin and by
experiments on mutant mice in which either the preprotachykinin A gene
or the gene encoding the NK1 receptor was disrupted. Inoue et al.
(1998) demonstrated that the nociceptin/orphanin FQ-induced nociceptive
response is brought about in mice by SP release from peripheral nerve
endings of nociceptive primary afferent neurons. After intraplantar
injection into the hindlimb of mice of nociceptin (EC50 = 0.31 fmol), there was a 25 to 70%
increase in the flexor-reflex response, which was abolished by
pretreatment of mice with an NK1 tachykinin receptor antagonist or with
the SP-depleting agent capsaicin, but not by pretreatment with NK2
antagonists. Similarly, nociceptin was completely ineffective in mice
with targeted disruption of the NK1 receptor gene.
It has been demonstrated that the knockout mice, which presented a
disruption of the gene encoding the NK1 receptor (with consequent
blockade of the activity of SP but not of NKA or SP(1-7) (De Felipe et
al., 1998), and the mutant mice, which presented a disruption of the
preprotachykinin A gene (with consequent lack of expression of SP,
SP(1-7), and NKA) (Cao et al., 1998; Zimmer et al., 1998), did not
show any changes to acute pain threshold in mechanical, electrical,
chemical, or thermal nociceptive tests, but their responses were
blunted in tests that involved more intense noxious stimuli. The
importance of SP/NKA seems to apply only to a certain "window" of
pain intensity, and when the intensity of the pain stimulation was
further increased, the response of the knockout mice did not differ
from those of wild mice. The fact that behaviorally acute nociceptive
threshold (tail-flick and hot plate assays) were not affected by gene
disruption would imply lack of any activation of NK1 receptor and of
any involvement of SP in the above assays.
In contrast, when sensory nerves are subjected to an intense period of
noxious stimulation, normal animals show a "wind up phenomenon",
i.e., an amplification and intensity coding of nociceptive reflexes,
which indicate a sensitization of the CNS mechanisms by intense
stimulation. The "wind up" was completely absent in the NK1
receptor lacking mice. Thus, SP seems to play an unexpected role for
full development of stress-induced analgesia and also for the
aggressive response to territorial challenge. Mutant mice did not
present any change in anxiety tests. However, the fact that aggression
but not anxiety (open field assay) was blunted in mutant mice would
again indicate that NK1 receptors and SP are not involved in anxiety
even if, in another anxiety assay (vocalization in guinea pig pups by
transient maternal separation), the NK1 receptor agonists increased
vocalization and the NK1 antagonists remarkably attenuated this effect
(Kramer et al., 1998).
In agreement with the above data, Zimmer et al. (1998) observed
that knockout mice displayed no significant pain responses after
formalin injection, but have an increased pain threshold in the hot
plate test. In addition, the mutant mice reacted normally in the
tail-flick test assay and acetic acid-induced writhing test.
The conclusion is that mutant mice develop hypoalgesia in some assays, but not in others, probably depending on the apparent levels (spinal or supraspinal) in which the involved pain mechanisms are situated. We further suggest that it is possible that enkephalins and SP modulate nociceptive inputs antagonistically and determine whether a nociceptive stimulus is experienced as pain.
Brain. Results obtained by intracerebroventricular injection of SP and other tachykinins, on pain sensation are very complex, conflicting, and open to unexpected speculations.
The intracerebroventricular injection of 2 to 2000 pmol of SP did not
affect the hot plate test in mice (Hayes and Tyres, 1979). Similarly,
intrathecal SP (10-10000 pmol) in rats did not significantly affect
pain threshold in various analgesic tests (paw pressure, tail
immersion, and hot plate test). Moreover, Malthe-Sphirenssen et al.
(1978) found that neither intracerebroventricular injection nor
injection into the periaqueductal gray of high doses of SP (30 nmol)
induced analgesia in rats. Frederickson et al. (1978), in turn, showed
that SP produced analgesia in mice when administered in very small
doses (1.25-5 pmol/mouse) by intraventricular route. The analgesic
effect was blocked by naloxone. At doses greater than 50 pmol, this
effect was lost and hyperalgesia appeared when these doses were
combined with naloxone, analgesia when combined with baclofen. Thus, SP
may have a dual action in brain, releasing endorphin at very low doses
and directly exciting neuronal activity in nociceptive pathways at
higher doses.
All of these results are, however, in conflict with a considerable amount of observations demonstrating that intracerebroventricular SP or SP injected into discrete brain areas is predominantly an analgesic, pain-blunting substance.
Malick and Goldstein (1978) found that, after injection into the
periaqueductal gray, SP (EC50 = 0.7 nmol/rat)
displayed a long-lasting (30-60 min) analgesic effect in the
tail-flick test. SP was approximately 25 times more potent than
morphine, and its effect was significantly antagonized by naloxone.
Similarly, low doses of SP (10 pmol) applied to the subarachnoid space
of the rat potentiated morphine analgesia (0.1 to 0.5 µg) in the rat tail-flick test, either by facilitating release of endogenous opioids
or by modulating opioid receptors.
Stewart et al. (1976, 1982) also demonstrated that centrally
administered SP displayed a clear-cut analgesic action. A first important observation was that SP antinociception appeared after a lag
of approximately 30 min, even after intracerebroventricular injection,
suggesting that the peptide may first require, to become active, an
enzymic cleavage at the
Phe7-Phe8 bond, leading to
release of the N-terminal fragment SP(1-7) (Hall et al., 1989). This
fragment displayed a clear-cut antinociceptive action in the hot plate
test, either by intracerebroventricular injection (5 pmol/mouse) or
intraperitoneal injection (15-20 pmol/mouse). SP(1-6) and SP(1-4)
did not show any significant analgesic effect. Prior treatment with
naloxone abolished the effect of SP(1-7). In comparison with intact
SP, the SP(1-7) fragment displayed its antinociceptive effect only
within a narrow dose range and as expected, had a shorter lag in onset
and a shorter duration of action.
Recently, the effects of two amphibian tachykinins, the NK1 receptor
agonist PG-SPI and the NK3 receptor agonist PG-KII, and the mammalian
tachykinins SP, NKA, and NKB on the reaction time to a painful radiant
heat stimulus (tail-flick test in rats) after intracerebroventricular
injection were investigated and compared (Improta and Broccardo, 2000).
PG-SPI and PG-KII (1, 5 and 10 µg) and SP (10 µg) significantly
increased the reaction time, whereas NKA and NKB did not. Like
analgesia evoked by exogenous SP, PG-SPI-evoked analgesia was blocked
by pretreatment with naloxone. Naloxone left PG-KII antinociception
unchanged, but the NK3 receptor selective antagonist markedly reduced
it. All of these findings suggest NK1 and NK3 tachykinin receptor
system involvement in supraspinal analgesia in rats.
We have discussed in some details this topic on pain because of its
great interest in pharmacology, pathology, and therapeutics even if the
question "SP equals pain substance?" by Iversen (1998) is still
open and has no definite answer, depending on pain intensity (windows!), nature of pain, and methods used to assess response to
painful stimuli.
There is evidence that SP plays a role in transmission of pain sensation and its elaboration in the CNS. Evidence is more convincing in the periphery, from sensory nerve endings to the dorsal horns of the spinal cord (SP = pain substance), less so in the CNS, because of a large number of conflicting results and on the still not clear involvement of SP(1-7).
At any rate, in pain control, there is certainly a close interplay between opioid peptides and SP with the concomitant participation of excitatory and inhibitory amino acids, monoamines and other neuropeptides as well. In human beings, the problem of pain is further complicated by its heavy emotional component. The involvement of SP in defense against stress conditions (anxiety and aggression) is highly probable, but again the importance of this involvement remains to be established. It seems that the monoaminergic system plays here a predominant role and that SP or SP(1-7), like other neuropeptides, displays a modulating effect. Again, results obtained in rats and mice are transferable to human beings with caution.
H. Neurogenic Inflammation
Electrical, mechanical, and chemical stimulation of the C-fibers in sensory neurons causes an axon reflex taking place in the branchings of sensory nerves. The consequence is the neurogenic inflammation: pain, vasodilation (flare), and plasma extravasation.
Antidromic vasodilation is mediated by a neurotransmitter at the
sensory nerve endings in the skin. Similarly, plasma extravasation elicited by antidromic stimulation also seemed to be provoked by a
mediator released from pain sensitive nerve terminals (Jancso et al.,
1967).
Among the many transmitters suggested in this connection were
acetylcholine, noradrenaline, ATP, bradykinin, histamine, 5-HT, and
prostaglandins. At the present time, SP fulfills the criteria for being
accepted as the main mediator for all components of antidromic
stimulation (Lembeck and Holzer, 1979; Pernow, 1985).
| i. | SP is present in the C-fibers of the sensory neurons and is released from these fibers during antidromic stimulation. |
| ii. | Close arterial administration of SP causes vasodilation and plasma extravasation, thus, mimicking the effect of antidromic stimulation. |
| iii. | Capsaicin, which depletes SP in sensory neurons, almost completely blocks vasodilation and neurogenic plasma extravasation. |
The above criteria were completed and remarkably strengthened by
more recent data:
| iv. | The nociceptin/orphanin-induced nociceptive response
is brought about in mice by SP release from peripheral endings of
nociceptive primary afferent neurons (Inoue et al., 1998), supporting
the view that also pain in neurogenic inflammation is due to release of
SP.
|
| v. | In mutant mice with disrupted preprotachykinin A gene,
neurogenic inflammation produced by topical application of capsaicin was almost absent, whereas in non-neurogenic paw edema produced by
complete Freund's adjuvant neurogenic inflammation was the same in
wild-type and mutant mice (Cao et al., 1998). However, there is some
doubt about the fact that SP is the unique direct or indirect (through
release of histamine from the mast cells) agent responsible for the
vasodilation and plasma extravasation seen in neurogenic inflammation.
Two points deserve attention. The first is that SP is costored and
co-released from sensory nerve endings with calcitonin gene-related
peptide, which displays a potent edema producing activity; the second
is that the histamine-releasing activity of SP, which remarkably
contributes to plasma extravasation and edema, has been attributed not
to the intact SP molecule but to its N-terminal fragment (1-7).
Moreover there are data, which need confirmation, showing that
antidromic stimulation may not always release SP but other active agents.
|
In summing up, there is little doubt that neurogenic inflammation represents the most striking and credible example of a decisive, if not unique, involvement of SP in a physiopathological process.
The rat was much less sensitive (intravenous threshold 2.5-5 nmol/kg) and the maximum increase in lachrymal secretion never exceeded 100% (De Caro and Cordella, 1965; Bertaccini et al., 1966).
Lachrymal glands of the rabbit seem to be rather insensitive to
tachykinins. In fact, eledoisin, given by close intra-arterial injection, at doses up to 1 nmol, failed to modify fluid and protein secretion (Dartt et al., 1988). In humans, given by eye drops, 10 to 20 nmol of physalaemin provoked a 95% increase in lachrymal secretion,
with intense conjunctival hyperaemia and sometimes-moderate chemosis
(De Caro and Cordella, 1965). The same amount of eledoisin was
ineffective in normal human subjects but showed striking stimulatory effects in patients suffering from hypofunctional lachrymal glands. Lachrymal secretion increased up to 200% and the effect lasted for
several hours during which it decreased slowly (Impicciatore et al.,
1973).
2. Histamine Release.
After perfusion of the rat isolated
hindlimb with 10 nmol/min of SP, kassinin, eledoisin, and NKA, it was
shown that only SP produced a significant increase in the histamine
concentration in the perfusion liquid, from 263 to 750 ng
histamine/min. The three other tachykinins were inactive (Erjavec et
al., 1981; Holzer-Petsche et al., 1985). Similarly, only SP released
histamine from peritoneal mast cells, whereas eledoisin and kassinin
were ineffective (Erjavec et al., 1981; Pietrowski et al., 1984). In
their ability to release histamine from the rat peritoneal mast cells
neurotensin, kallidin and SP were the most potent agonists.
Surprisingly an undecapeptide SP antagonist behaved as superagonist.
, 1989).
A histamine release by 0.1 to 10 µM SP, with consequent flare, wheal,
and itching, has been also demonstrated in human skin, where the
peptide was 100 times more potent as histamine liberator than in the
rat peritoneal cells. The skin reactions were blocked by antihistaminic
drugs (Hagermark et al., 1978). In more detail, in human skin (volar
surface of the forearm), 6.25 to 25 pmol of SP produced a
dose-dependent flare and wheal response. Only peptides having one or
more basic residues at their N-terminal region were effective in
producing flare: eledoisin and SP(1-7) were 17 and 7 times less active
than SP. Wheal production, on the contrary, was not dependent on basic
residues: physalaemin was the most potent agent, SP was one-half as
potent, and eledoisin was 20 times less potent. Pretreatment with a
histamine antagonist reduced both flare and wheal responses;
pretreatment with capsaicin reduced the flare but not the wheal
response, indicating that in response to the tachykinins, both mast
cells and SP-containing primary neurons are involved (Foreman et al.,
1983). It has been suggested that the histamine-releasing property of
SP derives essentially from cooperation between the basic N-terminal
tetrapeptide and the C-terminal heptapeptide with the two successive
Phe-residues at position 7 and 8 (Mazurek et al., 1981).
To support the validity of this hypothesis, it would be interesting to
check the histamine-releasing activity of carassin(12-21) with three
basic residues in the N-terminal tetrapeptide, but with the sequence
Phe-Val, instead of Phe-Phe and of bufokinin, with 2 basic and 1 acid
residue in the N-terminal pentapeptide and the sequence Phe-Tyr,
instead of Phe-Phe.
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VI. Tachykinins in Human Diseases and Therapeutics |
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Research on this topic is rather scant and at early stage of advancement. Pharmaceutical companies are highly interested in diseases possibly attributable, at least in part, to excess or deficiency in tachykinin production and/or release. However, because no tachykinin agonists are hitherto known to possess an appreciable capacity to cross the blood-brain barrier, the focus of interest lies, at present time, on the tachykinin antagonists, which are generally of nonpeptide nature and are often brain-penetrating molecules.
A. Tachykinin Receptor Agonists
In patients suffering from arteriosclerosis obliterans of the
legs, the effects of eledoisin (15-35 nmol) injected into the femoral
artery have been studied (Szam et al., 1966). The angiorheogram and the
pulse volume increased considerably in the majority of patients.
Fleeting side effects and slight decrease in blood pressure were also
observed. Unfortunately this promising clinical trial was not extended:
inconveniences of intraarterial infusion discouraged continuation of
experiments. Administration, by eye drops, of eledoisin or physalaemin
(10-50 nmol, 1-4 times daily) increased lachrymal secretion and
ameliorated the Sjögren syndrome and other forms of
keratoconjunctivitis sicca due to deficit of lachrymal secretion (De
Caro et al., 1969; Jaeger et al., 1985; Jaeger, 1988). Because of the
relative rarity of the disease and the existence of other therapeutical
approaches, these "orphan" drugs did not further arouse the
interest of the pharmaceutical companies. However, in an organ culture
of rabbit cornea, it was observed that SP alone (not NKA, NKB,
eledoisin, kassinin, or physalaemin) at any concentration (50 ng/ml-50
µg/ml) did not affect epithelial migration but enhanced the stimulant
effect (Nakamura et al., 1997). In the search for the SP fragment
responsible for the above effects, it has been found that both SP and
its C-terminal tetrapeptide, SP(8-11), acted synergistically with
insulin-like growth factor 1 on wound healing of rabbit cornea
(Nakamura et al., 1999). There was both stimulations of epithelial
migration in vitro and of attachment of corneal epithelial cells to a
fibronectin matrix. Moreover, the combination of insulin-like growth
factor 1, SP(8-11), and integrins by topical application facilitated
wound closure in vivo.
As far as it concerns the neurodegenerative and other CNS disorders, it
has been suggested that tachykinins may have both neuroprotective and
neurodegenerative properties (Raffa, 1998). Among the degenerative
diseases of the CNS, in which a deficit of tachykinins is clearly
evident, is Huntington's disease or Chorea. In this autosomal
hereditary disorder, a marked decrease in immunoreactive SP fiber
density in the regions showing the greatest histopathological
destruction, particularly in the substantia nigra, has been reported.
Of course, whereas SP has nothing to do with the etiology of
Huntington's disease, it could be responsible, at least in part, for
the symptoms of this disease: choreiform movements, personality
disturbances, and cognitive decrease to dementia.
The intervention of tachykinins in other CNS disorders is more controversial: amylotrophic lateral sclerosis, Parkinson's disease (decrease of SP in substantia nigra), schizophrenia (no significant change in SP content in brain areas), and depression (predominant data showing elevated levels of SP).
The problem of how tachykinins participate in the cerebral aging is
also a matter of investigation and debate. Here, only a few studies
dealing with the relationship between -amyloid protein and the
tachykinins are reviewed. -Amyloid is a 39- to 43-amino acid
polypeptide that is the primary constituent of senile plaques and
cerebrovascular deposits in Alzheimer's disease and Down syndrome.
Although the protein has been characterized biochemically, neither its
primary biological significance nor its role in the pathogenesis of
Alzheimer's disease is completely known. It has been shown that, in
cultures of rat embryonic hippocampal cells, the -amyloid protein is
neurotrophic in undifferentiated cells and at low concentrations, but
it is neurotoxic in mature neurons and at higher concentrations
(Yankner et al., 1990). Neurotrophic and neurotoxic effects of the
protein were mediated by its fragment 25 to 35, which shows important
homologies to the sequences of SP and other tachykinins. However, the
problem on the possible involvement of tachykinins (namely SP) in the
pathogenesis of Alzheimer's disease cannot be considered solved. In
fact, Zhao et al. (1993) found that amyloid -protein (1-40) was
toxic to NB41A3 neuroblastoma cells in serum-free culture, as judged by decreasing cell number and release of lactic dehydrogenase, and that
this toxicity was inhibited by the concurrent treatment of the cells
with 1 µM physalaemin. In turn, Kimura and Schubert (1993) observed
that amyloid -protein (1-40) weakly activated, for itself, the
tachykinin receptors, but that in the presence of glutamate, the
amyloid -protein produced an activation of both the tachykinin
receptors (especially the NK1 receptors) and the phosphatidylinositol
turnover. There is the possibility that an overproduction of amyloid
-protein disturbs normal neuronal transmission by activating the SP
receptor in synergy with glutamate or by acting as a SP antagonist by
itself. The resulting compromised synapses could lead to the dementia
of the Alzheimer's disease.
B. Tachykinin Receptor Antagonists
Based on the knowledge of distribution of tachykinin receptors and
pharmacological effects of the tachykinins, it may be hypothesized that
receptor antagonists may have several therapeutic applications. With
regard to NK1 receptor antagonists, their therapeutical use has been
hypothesized in the treatment of pain and emesis and, in the periphery,
in the treatment of several inflammatory diseases including arthritis,
inflammatory and motor diseases, and cystitis (Quartara and Maggi,
1998).
At present time, the only documented clinical trial with tachykinin
antagonists, more precisely with a SP antagonist, is that carried out
in the treatment of moderate to severe major depression by a large team
of researchers, starting from the observation that, like clinically
used antidepressant and anxiolytic drugs, also SP antagonists
suppressed isolation-induced vocalization in the guinea pig (Kramer et
al., 1998).
In a placebo-controlled trial, it has been found that in patients
suffering from depression, the SP antagonist MK-869 displayed an
antidepressive effect greater than that displayed by the first choice
drug in depression, paroxetin, and side effects, always in comparison
with paroxetin, were less intense. The mechanism of action of MK-869
is, at present, not completely understood. This TK receptor antagonist
does not interact with monoamine systems (inhibition of re-uptake of
serotonin and/or noradrenaline) like the known antidepressant drugs do;
thus, it cannot be excluded that MK-869 does not act only through NK1
receptor blockade. Moreover, like the known antidepressive drugs,
MK-869 acts only after 2 to 3 weeks, suggesting the possibility that
all antidepressant drugs act via an as yet unclear "common pathway"
mechanism (Wahlestedt, 1998).
The enormous theoretical and therapeutical interest that SP antagonists may represent well tolerated antidepressant drugs is obvious. However, the successful therapeutical use of tachykinin antagonists in humans requires some precise accomplishments: a) knowledge of the tachykinin receptor types and subtypes occurring in the different human organs and tissues and evidence that the antagonists on trial compete exactly with the wanted binding site. This is because of the heterogeneity of all tachykinin receptor types; b) lack of important toxicity (side effects) even by long-term administration. Antagonists are generally synthetic, nonpeptide, and non-natural molecules; c) lack of appreciable agonistic activity; and d) brain-permeability for antagonists destined to act in the CNS.
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VII. General Conclusions |
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We have previously shown that tachykinins constitute one of the largest families of peptides in all of the world whose members are present in all animal species from lower invertebrates to mammals. There is no nervous system, from the most primitive in coelenterates to the most developed and complex human CNS, that is lacking a tachykininergic system.
What is the functional significance of this spectacular display of tachykinin-secreting fibers and their receptors? It is beyond doubt that neuronal tachykinins play an important role in neurotransmission/neuromodulation both in the CNS and in periphery. This is demonstrated by the overall occurrence of tachykinins in the brain and other nervous structures from the lowest invertebrates to mammals. Although important, the tachykinin peptide family represents only one of the numerous peptide and nonpeptide families involved in neurotransmission and neuromodulation. Members of these families are expressed in a variety of tissues, and very frequently a tachykinin is costored and cosecreted by the nerve endings with other peptides or biogenic amines. Moreover, the tachykinins, like all other neuropeptides, may enter in competition, positive or negative, with a number of active extraneuronal compounds originating in blood (bradykinin and angiotensin) or in compact or diffuse endocrine organs.
Tachykinins, with their variable primary structure seem to be adapted to display, in the better way, their function in the different invertebrate and vertebrate phyla. In all examined species, and especially in mammals (the phylum more thoroughly studied), tachykinins elicit a spectrum of biological activity (both in the CNS and in the periphery), which may vary conspicuously in the different species and even in the various strains of single species, again strongly supporting the concept of a general, important functional significance of these peptides.
Transgenic mice with disrupted preprotachykinin A gene or with disrupted NK1 receptor gene are in good health conditions and fertile. This demonstrates that the tachykinins are not essential for life and health, at least in mice but probably in the other mammalian species as well, and points to the well known great adaptability of living organisms and the plasticity of homeostatic mechanisms. At present, we do not know any pathological syndrome attributable entirely or predominantly to excess or defect of tachykinin production and release. No function of the various organs and systems in health and disease seems to depend entirely on the tachykininergic system, and tachykinins seem to be only one arm of the complex mechanism that regulates body functions.
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Footnotes |
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This is the last unfinished review written by professor Vittorio
Erspamer before he died suddenly in October 1999. His collaborators are
proud to present this review on his behalf and to honor his memory as
an enthusiastic and intuitive researcher who enriched the knowledge of
new and unimagined agents and actions all over the world.
Address correspondence to: Cinzia Severini, Consiglio Nazionale delle Ricerche-Institute of Neurobiology and Molecular Medicine, Viale Marx 15/43, I-00137, Rome, Italy. E-mail: c.severini{at}in.rm.cnr.it
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Abbreviations |
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SP, substance P; NKA, neurokinin A; NKB, neurokinin B; RIA, radioimmunoassay; SP-LI, substance P-like immunoreactivity; HPLC, high-performance liquid chromatography; 5-HT, 5-hydroxytryptamine; TK, tachykinin; TAN, tonically autoactive, giant neuron.
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and neurokinin in rat central nervous system.
Neurosci Res
2:
111-120[CrossRef][Medline].-protein activates tachykinin receptors and inositol triphosphate accumulation by synergy with glutamate.
Proc Natl Acad Sci USA
90:
7508-7512
0031-6997/02/5402-285-322$7.00
PHARMACOLOGICAL REVIEWS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
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