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Vol. 51, Issue 2, 159-212, June 1999
Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland
I. Foreword: A Brief History of a Really "Hot" Aspect of Pharmacology
II. Introduction
A. Overview
B. Capsaicin: Targets and Actions
C. Early, Indirect Evidence for and against a Vanilloid (Capsaicin) Receptor VR
III. Direct Evidence for a Vanilloid (Capsaicin) Receptor
A. Specific Binding of resiniferatoxin (RTX), a Naturally Occurring Ultrapotent Agonist
B. Development of Capsazepine, a Competitive Vanilloid Antagonist
C. Cloning of the First VR, Termed VR1
IV. Anatomical Localization and Tissue Specificity of VRs
A. VRs in Primary Sensory Neurons; Colocalization with Other Receptors, Neuropeptides, and the Isolectin B4
B. VRs in Vagal (Nodose Ganglion) Neurons
C. VRs in Brain
D. Possible Presence of VRs in Nonneuronal Tissues Such as Mast Cells and Glia
V. Evidence for Multiple VRs
VI. Biochemical Pharmacology of VRs
A. The Cloned VR Is a Nonselective Cation Channel with a Limited Selectivity for Calcium
B. Role of Calcium in Modulating VR Functions
C. Where Is the Vanilloid Binding Site on VRs?
D. VR1 Is Activated by Noxious Heat and Low pH (Protons): the VR as an Integrator of Painful Chemical and Physical Stimuli
E. Ruthenium Red Blocks VRs by an Unknown Mechanism
F. VRs Are Sensitized by Inflammatory Mediators and Proinflammatory Cytokines
G. Aspirin and Related Drugs May in Part Exert Their Analgesic Actions by Blocking VRs
H. Proposed Role of Phosphorylation Sites in Modulating VR1 Activity
I. Regulation of VR Expression
J. VRs Are Thiol Proteins Displaying Positive Cooperativity
VII. Requirements for Ligand Recognition by VRs: Typical and Novel Vanilloids
A. Structure-Activity Relations for Capsaicinoids
B. Structure-Activity Relations for Resiniferanoids for Inducing Calcium Uptake by Sensory Neurons
C. Differences in Structure-Activity Relationships of Vanilloids for Receptor Binding and Calcium Uptake
D. Why Is RTX Ultrapotent as a Vanilloid?
E. Novel Vanilloids Lacking 3-Hydroxy 4-methoxyphenyl (Vanillyl) Functionality
1. Sesquiterpene Unsaturated 1,4-Dialdehydes and Related Bioactive Terpenoids.
2. Triprenyl Phenols as Vanilloids.
3. Implications of the Discovery of Novel Vanilloids Lacking a Vanillyl-Like Functionality.
VIII. Vanilloid Mechanisms
A. Excitation by Vanilloids
1. Stimulation of Vanilloid-Sensitive Neurons and Its Consequences.
2. Hyperalgesia and Allodynia Following Vanilloid Administration.
B. Desensitization to Vanilloids
1. Desensitization.
2. Tachyphylaxis.
3. Is Lasting Tachyphylaxis Possible Without Prior Excitation?
4. Impairment of Neuronal Functions after Vanilloid Treatment.
4. Down-Regulation of VRs as a Mechanism of Long-Term Desensitization to Vanilloids.
6. Messenger Plasticity by Vanilloids as a Novel Mechanism of Analgesia.
C. Neurotoxicity by Vanilloids
IX. Diverse Biological Actions of Vanilloids; VR-Mediated and Independent Mechanism
X. Species-Related Differences in Vanilloid Actions
A. Species-Related Differences in VR Expression
B. Species-Related Differences in Expression of Sensory Neuropeptides and Their Receptors
C. Human VRs
XI. Endogenous Vanilloids: Do They Exist?
XII. Vanilloids in Clinical Practice: Current Uses and Future Perspectives
A. Counterirritation with Capsaicin
B. Desensitization to Capsaicin
C. Adverse Effects of Topical Capsaicin
D. Novel, Innovative Clinical Uses
E. RTX, an Improved Vanilloid Drug Undergoing Clinical Trials
XIII. Vanilloids: Carcinogens, Anticarcinogens, or Neither?
A. Capsaicin
1. Mutagenesis by Capsaicin.
2. Carcinogenesis by Capsaicin: Animal Experiments.
3. May Culinary Hot Pepper Consumption Be a Risk Factor for Stomach Cancer in Humans?
4. Capsaicin: A Potential Antitumor Agent?
B. RTX
XIV. Concluding Remarks, Emerging Principles, and Future Perspectives
Acknowledgments
Appendix
Note Added in Proof.
References
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I. Foreword: A Brief History of a Really "Hot" Aspect of Pharmacology |
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Natural products afford a window of opportunity to study important biology. If the natural product is used or abused by human beings, finding its biological target(s) is all the more significant. Hot pepper is eaten on a daily basis by an estimated one-quarter of the world's population and represents an aspect of pharmacology intimately familiar to most readers of this review. Recall your culinary experiences in, let us say, a Mexican restaurant. Food flavored with jalapeno pepper bites, it induces profuse perspiration, and a subsequent diarrhea is not uncommon (when pepper bites again). However, these symptoms become less intense in regular patrons of the restaurant. In more scientific terms, hot pepper is pungent, affects thermoregulation, activates autonomic reflexes, and is poorly absorbed. However, all these acute effects undergo tachyphylaxis upon repeated applications.
Hot pepper is a native of the Americas (cf. Andrews, 1984
; Naj,
1992
). Aztecs called it chili (this is how hot pepper is mentioned in
the pre-Columbian Aztec manuscripts, known as tlacuilos), a name that
has stuck in Latin America (information from the Internet: Encyclo'Pepr'edia, http://thepeppershop.com/index.html, and Chili Gazette, http://mexicanfood.tqn.com/msubhis.html). But the Old World
adopted a different name, red pepper, instead, erroneously linking chili pepper (Capsicum annuum) to a similarly
hot-tasting plant, black pepper (Piper nigrum) (Garrison,
1929
). Following the discovery of the New World, the habit of the
natives to eat their food hot was first noted by Diego Alvarez Chanca,
a physician to the fleet of Columbus (cf. De Ybarra, 1906
). In Europe,
a depiction of the chili pepper plant appeared for the first time in
the magnificent book of Fuchs (1542). Hot pepper was cultivated in
monastery gardens in Moravia as early as 1566 (cf. Köhler, 1883
).
The Latin name of the plant, Capsicum, was given by the French botanist
de Tournefort for unclear reasons (cf. Naj, 1992
). A popular theory
holds that the name Capsicum was derived from the Greek kapto, meaning
"to bite" (cf. Maga, 1975
), which appropriately describes the main characteristic of the fruit. Others argue that the name Capsicum is
derived from capsa, the Latin word for box, referring to the fact that
the pepper pod is hollow, divided into compartments containing the
seeds (cf. Naj, 1992
).
The active ingredient in hot pepper was first isolated by Thresh
(1846)
more than a century ago. Thresh named this compound capsaicin
and predicted that the structures of capsaicin and vanillin were
closely related. Despite this early discovery, it was not until 1919 that the exact chemical structure of capsaicin (Fig. 1) was determined (Nelson, 1919
). The
complete synthesis of capsaicin took another decade (Spath and
Darling, 1930
). In 1912, Wilbur Scoville (Scoville, 1912
) introduced
his Organoleptic Test to quantitate the pungency of hot peppers (that
is, their capsaicin content) and, although HPLC has long supplanted the
use of the human tongue (cf. Suzuki and Iwai, 1984
), for quantitation
the Scoville Unit remains the measure of hotness. If a pepper has 50,000 Scoville Heat Units, this means that its alcoholic extract needs
to be diluted 1:50,000 for it to cease to be hot-tasting on the human
tongue. The hottest pepper is the Mexican habanero, which boasts
350,000 Scoville Units (cf. Naj, 1992
). The oral test of Scoville,
abandoned by the food industry, survives in laboratories, where it is
still used to detect capsaicin-like compounds in plant extracts.
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With regard to the varied, nonculinary uses of hot pepper, there is
apparently little new under the sun. For example, Incas burned dried
chili peppers to combat the invading Spaniards by temporarily
blinding them (cf. Naj, 1992
). Four centuries later, the first U.S.
patent was issued for the use of capsaicin for martial (tear gas)
purposes (U.S. patent 1,659,158, 1928). Nowadays, capsaicin-containing
sprays, fondly called Sergeant Pepper, are used by law-enforcement
officials in the United States to subdue violent criminals, and in
California are marketed as a "cop-in-a-can" for self-defense
(Hyder, 1996
).
The analgesic use of capsaicin is not novel either (cf. Whittet,
1968
; Lembeck, 1987
; Dasgupta and Fowler, 1997
). Native Americans rubbed their gums with pepper pods to relieve toothache (cf. Naj, 1992
). This practice later also gained popularity in European folk
medicine, as was noted by the Hungarian botanist-turned-clergyman, Hangay (1887)
. As early as 1850, the Dublin Free Press recommended the
use of alcoholic hot pepper extract on sore teeth for instant relief,
recognizing for the first time the therapeutic potential of capsaicin
(Turnbull, 1850
). A fascinating aspect of the rich history of the
analgesic use of capsaicin is that eunuchs serving the Chinese Emperors
were castrated after their scrotums had been repeatedly rubbed with hot
pepper extracts (cf. Anderson, 1990
). As a curiosity, it is also
notable that Native Americans used hot pepper extracts topically
as an aphrodisiac, a practice adopted by early settlers to
the dismay of their priests (Chili Gazette, http://mexicanfood.tqn.com/msubhis.html).
In 1640, Sir John Parkinson noted in his famous Theatricum Botanica
that dogs detest hot peppers (cf. Naj, 1992
). Chickens, by contrast,
can be fed dried pepper powder to turn the yolks of their eggs
orange-red. These observations anticipate the modern use of capsaicin
to make bird food squirrel-free (cf. Rouhi, 1996
). It remains a mystery
why is it that the same hot taste, which repels a variety of mammals
from rats to squirrels to dogs, is found pleasurable by so many humans.
Psychologists speculated that eating hot peppers may be a form of
masochism (Rozin and Schiller, 1980
). This theory, however, is at
variance with the well known geographic pattern of hot pepper
consumption, namely, people living in tropical climates prefer their
food hotter than those residing in temperate climates (Moore, 1970
;
Rozin, 1978
). This pattern gave rise to the theory that eating hot,
spicy food helps combat the warm climate via gustatory sweating
(Haxton, 1948
; Lee, 1954
). This model has gained recent reinforcement
by the cloning of a capsaicin receptor, which seems to be operated by
both heat and capsaicin (Caterina et al., 1997
). Nonetheless, the human
liking of, or aversion toward, the taste of hot pepper is probably far
more complex than this relatively simple physiological model implies.
For instance, Russians like their vodka hot (vodka peperovka), whereas
efforts by the Pabst Brewing Company to introduce a pepper-flavored
beer were unsuccessful (cf. Naj, 1992
).
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II. Introduction |
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A. Overview
If one compares the two previous capsaicin reviews in this Journal
(Buck and Burks, 1986
; Holzer in 1991
), one can notice the steady
evolution of ideas, for instance, the identification of ultrapotent
capsaicin analogs (cf. Szallasi and Blumberg, 1990a
) or the development
of the first competitive capsaicin antagonist, capsazepine (Bevan et
al., 1991
). The past years, however, have witnessed unprecedented
advances that have revolutionized this field. Holzer (1991)
described
the vanilloid receptor
(VR)2 as a
capsaicin-operated conductance, the expression of which is virtually
restricted to a distinct subpopulation of primary sensory neurons. This
is apparently no longer true. In 1997, a VR, termed VR1, was cloned
(Caterina et al., 1997
). The emerging concept is that this VR1
functions as a molecular integrator of painful chemical and physical
stimuli including noxious heat (>48°C) and low pH (Tominaga et al.,
1998
). Apparently, it is not capsaicin but heat that has the capability
of opening the channel pore of VR1, whereas capsaicin and protons only
serve to lower the heat threshold of the receptor. Consequently, even
room temperature is able to gate VR1 in the presence of mildly acidic
conditions and/or capsaicin (Tominaga et al., 1998
). Moreover, the
expression of VRs is not restricted to sensory neurons. In addition to
several brain nuclei (Ács et al., 1996a
; Sasamura et al., 1998
),
nonneuronal tissues, such as mast cells (Bíró et al.,
1998a
) and glial cells (Bíró et al., 1998b
), may express
VRs. There is mounting evidence, both biological and
electrophysiological, to suggest heterogeneity within VRs (cf. Szallasi
and Blumberg, 1996
; Szallasi, 1997
). As yet, it is not known whether or
not all VR subtypes recognize capsaicin. Therefore, we chose to use in
this review the broader term vanilloid-sensitive neuron over the
traditional term capsaicin-sensitive neuron.
It has also been found that ligands with little resemblance to the
vanillyl group in capsaicin can also act as vanilloids (Szallasi et
al., 1996a
, 1998a
, 1999a
), implying that the term VR is somewhat of a
misnomer. Terpenoid unsaturated dialdehydes (Szallasi et al., 1996a
,
1998a
) and triprenyl phenols (Szallasi et al., 1999a
) (Fig. 1) are two
chemical classes of such newly discovered "vanilloids". At present,
resiniferatoxin (RTX; see Fig. 1 for structure), an ultrapotent
capsacin analog (Szallasi and Blumberg, 1989a
, 1990a
), is undergoing
clinical trials, where it proves clearly superior to capsaicin
(Chancellor, 1997
; Cruz et al., 1997a
; Lazzeri et al., 1997
; Cruz,
1998
). At a recent meeting on urinary incontinence (1st International
Consultation on Incontinence; Monaco, 1998), a proposal was
accepted that a Vanilloid Club should be formed. From cloning to
clinic, the vanilloid field is now vibrating with a frenzy of activity,
which makes the life of review writers most complicated. For instance,
rumor has it that additional VR isoforms have been cloned and an
endogenous vanilloid has been isolated.
In this review, we are deliberately focusing on recent developments in
the field. The reader can refer to either the above review by Holzer
(1991)
or the subsequently published book on capsaicin edited by John
N. Wood (1993)
for detailed background information. Excellent, short
reviews are also available from several authors, e.g., Andy Dray
(1992)
, Gábor Jancsó and colleagues (1994)
, Carlo A. Maggi
(1991)
, or János Szolcsányi (1996)
, among others. In any
case, we have tried to provide limited background information to the
degree that it is necessary to provide the context for understanding
recent developments.
The literature on vanilloids is vast. Using the subject word capsaicin, a Medline search yielded 2892 publications that have appeared since 1991, the year when the comprehensive capsaicin review by Holzer was published. On average, one paper dealing with capsaicin actions has thus been published every day over the past 7 years. Approximately one tenth of these papers (245 publications since 1991) deal with some aspect of the therapeutic application of capsaicin in humans. Because a complete overview of this vanilloid literature would be overwhelming, this review tries to provide a selective coverage of the literature, with the focus on breakthrough discoveries, emerging concepts, and provocative new ideas.
B. Capsaicin: Targets and Actions
Capsaicin excites a subset of primary sensory neurons with somata
in dorsal root ganglion (DRG) or trigeminal ganglion (Table 1). As a general rule, these
vanilloid-sensitive neurons are peptidergic, small diameter (>50 µm)
neurons, giving rise to thin, unmyelinated (C) fibers (cf. Holzer,
1991
). Among sensory neuropeptides, the tachykinin Substance P (SP)
shows the best correlation with vanilloid sensitivity (cf. Buck and
Burks, 1986
; Holzer, 1991
). However, on the one hand, not all small
diameter DRG neurons respond to capsaicin, and, on the other hand,
large diameter vanilloid-sensitive neurons, predominantly of the
A
-type, are also known to exist (Table 1). Indeed, in certain
tissues such as the tooth pulp, A
-fibers predominate among the
vanilloid-sensitive nerve population (Ikeda et al., 1997
). Some
small diameter sensory neurons are polymodal nociceptors, whereas
neurons with A
-fibers may function as mechanoheat-sensitive
nociceptors (cf. Meyer et al., 1994
). In other words,
vanilloid-sensitive neurons are heterogeneous morphologically,
neurochemically, and functionally, and they encompass several
subclasses of DRG neurons (Table 1) (cf. Holzer, 1988
, 1991
;
Szolcsányi, 1996
; Holzer and Maggi, 1998
). Because sensitivity to
vanilloids is the only known trait that all of these neurons seem to
share, they are best described as vanilloid-sensitive neurons (cf.
Szallasi and Blumberg, 1990a
,b
; Szallasi, 1996
).
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According to a recent exciting finding by Seybold and colleagues
(Stucky et al., 1998
), vanilloid sensitivity is a plastic property of
DRG neurons. For instance, the algogenic substance bradykinin is able
to recruit intermediate-size neurons (240-320 µm2), normally unresponsive to capsaicin, to
respond to vanilloids (Stucky et al., 1998
). Consequently, the number
of nociceptors that innervate inflamed tissues increases. As we will
see later, this novel mechanism may play an important role in the
development of inflammatory hyperalgesia via spatial summation on
spinal neurons.
Central fibers of vanilloid-sensitive neurons enter the central nervous
system (CNS), where they synapse on second-order neurons of the dorsal
horn of the spinal cord (for DRG neurons) or the spinal nucleus of the
trigeminal tract (for trigeminal ganglion neurons), respectively (cf.
Yaksh and Malmberg, 1994). Generally speaking, vanilloid-sensitive
neurons transmit noxious information (usually perceived as itching or
pain) to the CNS (afferent function), whereas peripherial terminals of
these neurons are sites of release for a variety of proinflammatory
neuropeptides (efferent function; these neuropeptides are summarized in
Table 1). These neuropeptides are believed to play an important role in
initiating the cascade of neurogenic inflammation (Fig.
2) (cf. Foreman, 1987
; Geppetti and
Holzer, 1996
). In most experimental paradigms, capsaicin was found to
activate both afferent and efferent functions, leading to the
adaptation of the "axon reflex" model (cf. Bayliss, 1901
; Bruce,
1910
; Lewis, 1927
; Celander and Folkow, 1953
; Lisney and Bharali, 1989
)
by capsaicin researchers (cf. Holzer, 1988
; Maggi and Meli, 1988
;
Burnstock, 1990
; Lynn and Cotsell, 1991
). However, it has recently been
shown that capsaicin is capable of releasing sensory neuropeptides such
as SP, somatostatin, and calcitonin gene-related peptide (CGRP) from
the peripheral endings of sensitive nerves in the presence of
lignocaine, tetrodotoxin,
-conotoxin, or agatoxin, suggesting a
direct mechanism for peptide release not mediated by axon reflex
(Szolcsányi et al., 1998a
).
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In addition to capsaicin, vanilloid-sensitive neurons are also
activated by a variety of chemical and physical (both heat and
pressure) stimuli (cf. Maggi, 1991
; Lundberg, 1993
). Some of these
compounds, like histamine and bradykinin, have their own receptors.
Others (for example, xylene and mustard oil) are believed to work in a
non-receptor-mediated fashion (N. Jancsó et al., 1968
), possibly
by perturbing membranes. Protons (low pH) are unique in that they have
their own receptors (known as acid-sensitive ion channels or ASICs)
(Waldmann et al., 1997
), but, at the same time, they also act on VRs
(capsaicin) (Bevan and Yeats, 1991
; Petersen and LaMotte, 1993
; Liu and
Simon, 1994
; Martenson et al., 1994
; Kress et al., 1996a
; Caterina et
al., 1997
; Tominaga et al., 1998
). Finally, there are compounds (e.g., sesquiterpene unsaturated dialdehydes; Szallasi et al., 1996a
) and,
more surprisingly, physical stimuli (e.g., noxious heat; Caterina et
al., 1997
) that appear to stimulate sensory neurons in a VR-mediated
fashion. Again, it needs to be emphasized that VR is "a target" but
not "the target" for noxious heat, because the overlap between
heat- and capsaicin-sensitive DRG neurons is only partial (Cesare and
McNaughton, 1996
; Kirschstein et al., 1997
; Reichling and Levine,
1997
).
Among irritant compounds acting on primary sensory neurons, capsaicin
and related vanilloids are unique in that the initial stimulation by
vanilloids is followed by a lasting refractory state, traditionally
termed desensitization (N. Jancsó and A. Jancsó, 1949
; N. Jancsó, 1955
; 1968
; cf. Szolcsányi, 1984
; G. Jancsó,
1994
). As discussed later, this desensitization is a complex process
and has a clear therapeutic potential.
Although capsaicin was regarded as a "remarkably selective tool for
primary sensory neurons" (Holzer, 1991
), it was clear from the
beginning that not all capsaicin actions can be attributed to the
activation of primary sensory neurons. The lack of probes to detect VRs
has led to considerable confusion as to what constitutes vanilloid-sensitive targets. This confusion persists to the present. There was little debate that there are vanilloid-sensitive neurons in
nodose ganglia (Szolcsányi and Barthó, 1978
, 1982
; Marsh et
al., 1987
; Raybould and Taché, 1989
; Waddell and Lawson, 1989
; Sharkey et al., 1991
; Carobi, 1996
). The actual existence of these neurons was recently confirmed by both [3H]RTX
binding (Fig. 3) (Szallasi et al., 1995a
)
and VR1 mRNA in situ hybridization studies (Helliwell et al., 1998a
).
It was also generally accepted that intrinsic sensitive neurons located
in the hypothalamus can mediate the well known effects of vanilloid administration on temperature regulation (Jancsó-Gábor et
al. 1970
; Szolcsányi et al., 1971
). Recent experiments, with both RTX binding (Fig. 4) (Ács et al.,
1996a
) and reverse transcription-polymerase chain reaction using
primers based on the VR1 sequence (Sasamura et al., 1998
), have
confirmed the existence of these VR-expressing hypothalamic neurons.
Other capsaicin actions turned out to be mediated by targets unrelated
to VRs (cf. Holzer, 1991
; Szallasi, 1994
). Nonetheless, the recognition
that nonneuronal tissues may express VRs (Bíró et al.
1998a
,b
) implies that capsaicin effects previously considered to be
nonspecific may, in fact, be mediated by VRs.
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C. Early, Indirect Evidence for and against a Vanilloid (Capsaicin) Receptor VR
From the beginning, three lines of strong evidence pointed to the
existence of a specific capsaicin recognition site. First, capsaicin-like activity required fairly strict structure-activity relations (Szolcsányi and Jancsó-Gábor, 1975
, 1976
;
Szolcsányi, 1982
; Hayes et al., 1984
; Walpole and Wrigglesworth,
1993
). Second, capsaicin sensitivity seemed to be restricted to
well-defined neuronal tissues (cf. Buck and Burks, 1986
; Holzer, 1991
).
Third, susceptibility to capsaicin showed striking species-related
differences. Most authorities agreed that capsaicin sensitivity
occurred only in mammals, and even mammalian species differed
considerably in their responsiveness to capsaicin (cf. Buck and Burks,
1986
; Holzer, 1991
). Furthermore, corroborative evidence for the
existence of a capsaicin receptor was the finding that the inorganic
dye ruthenium red was able to block the capsaicin-induced responses
(cf. Amann and Maggi, 1991
).
If sufficiently high capsaicin doses are used, the above tissue and
species specificities of capsaicin actions are, however, lost. For
example, Ritter and Dinh (1993)
demonstrated capsaicin-evoked silver staining along almost the entire neural axis of the rat, including the retina. In keeping with this, Szikszay and London (1988)
described enhanced glucose use by capsaicin in a wide variety of CNS
structures in the rat. Capsaicin was found to inhibit various enzymes
(Shimomura et al., 1989
; Yagi, 1990
; Teel, 1991
; Wolvetang et al.,
1996
), induce pseudochannel formation in lipid bilayers (Feigin et al.,
1995
), alter membrane fluidity (Aranda et al., 1995
), and block
K+ channels (Dubois, 1982
; Petersen et al., 1987
;
Bleakman et al., 1990
; Castle, 1992
; Baker and Ritchie, 1994
; Kuenzi
and Dale, 1996
), just to name a few characteristic, non-VR-mediated
capsaicin actions.
Early efforts to demonstrate specific binding sites for
[3H]dihydrocapsaicin (Szebeni et al., 1978
)
or photoaffinity-labeled capsaicin-like molecules (James et al.,
1988
) failed due to a combination of the high lipophilicity and
relatively low affinity of these molecules.
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III. Direct Evidence for a Vanilloid (Capsaicin) Receptor |
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A. Specific Binding of resiniferatoxin (RTX), a Naturally Occurring Ultrapotent Agonist
It has been known since the dawn of recorded history that the
latex of the Moroccan cactus-like plant, Euphorbia
resinifera, contains an extremely irritant component (Fig.
5) (cf. Appendino and Szallasi, 1997
).
But it was not until 1975 that the compound responsible for this
irritancy was isolated and named RTX (Hergenhahn et al., 1975
). RTX
(see structure in Fig. 1) combines the structural features of two
classes of natural irritants, phorbol esters and capsaicinoids.
Although RTX did bind to the phorbol ester receptor, protein kinase C
(PKC) (see in Section XIII.B), this low-affinity interaction
could not explain its extreme pungency. In a series of experiments
beginning in 1989 and continuing to date, we have identified RTX as an
ultrapotent capsaicin analog with a unique spectrum of biological
activities (Table 2). Because capsaicin and RTX analogs share a (homo)vanillyl group as a structural motif essential for bioactivity but differ dramatically in the rest of the
molecule (Fig. 1), they are collectively termed vanilloids. Consequently, the primary target for these compounds appears to be best
described as the VR.
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In several assays, RTX is several thousandfold more potent than
capsaicin (Table 2) (cf. Szallasi and Blumberg, 1990a
, 1993b
, 1996
).
This ultrapotency predicted the existence of high-affinity specific RTX
binding sites (VRs). Despite multiple obstacles, in 1990 we finally
managed to demonstrate specific [3H]RTX binding
by rat DRG membranes (Szallasi and Blumberg, 1990b
), providing the
first unequivocal evidence for the existence of VRs (Fig.
6). This initial binding assay was,
however, plagued by a very high nonspecific binding, which prevented
the detection of VRs in the spinal cord or peripheral tissues.
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The methodological means to overcome the problems created by the high
nonspecific RTX binding was furnished by an accidental observation of
ours. In search of endogenous ligands for VRs, we assayed a variety of
body fluids and tissue extracts and found that the acute phase serum
protein,
1-acid glycoprotein (also known as
orosomucoid), binds RTX (Szallasi et al., 1992
).
1-Acid glycoprotein is an important
drug-binding protein (Paxton, 1983
; Kremer et al., 1988
; Maruyama et
al., 1990
). We showed that RTX binds to the well known drug-recognition
domain on
1-acid glycoprotein that it shares
with warfarin (Szallasi et al., 1992
). Plasma binding of RTX to
1-acid glycoprotein may have clear
consequences on pharmacokinetics upon systemic administration that we
will discuss later. RTX binding to
1-acid
glycoprotein, however, differs in two very important aspects from VR
binding. First, it is not markedly influenced by temperature, and
second, it is of an at least 1000-fold lower affinity (Szallasi
et al., 1992
). At 0°C, the dissociation of receptor-bound RTX is
unmeasurably slow (Szallasi and Blumberg, 1993a
). By contrast,
1-acid glycoprotein readily binds unbound RTX
in the aqueous phase (Szallasi et al., 1992
). Because nonspecifically bound RTX in the membrane lipids is in equilibrium with the unbound RTX
in the aqueous phase,
1-acid glycoprotein is
able to extract nonspecifically bound RTX from the membranes without
compromising specific binding (Szallasi et al., 1992
). By centrifuging
the membranes, it is then easy to separate the membrane-, mostly
receptor-bound RTX from the
1-acid
glycoprotein-bound form that remains in the supernatant.
The introduction of
1-acid glycoprotein to the
VR binding assay resulted in a dramatic improvement in the ratio of
specific binding from 50% of the total at concentrations close to the
Kd to 90% of the total binding or
even higher. Using this improved assay, we are now able to detect
specific RTX binding sites in the dorsal horn of the spinal cord
(Szallasi et al., 1993a
,b
; Ács and Blumberg, 1994
; Ács et
al., 1994a
,b
; Szallasi and Goso, 1994
), in various peripheral tissues
[e.g., urinary bladder (Szallasi et al., 1993c
; Ács et al.,
1994a
), urethra (Parlani et al., 1993
), nasal mucosa (Rinder et al.,
1996
), airways (Szallasi et al., 1993b
, 1995b
), colon (Goso et al.,
1993a
)] as well as in several brain nuclei (Fig. 4 and Table
3) (Ács et al., 1996a
).
Furthermore, we developed an autoradiographic approach to visualize VRs
in several species, including human (Figs. 3 and
7) (cf. Szallasi, 1995
).
|
|
B. Development of Capsazepine, a Competitive Vanilloid Antagonist
Capsazepine (Fig. 8), the first and,
as yet, only commercially available competitive VR antagonist comes
from an extensive program at the former Sandoz, now Novartis, Institute
for Medical Research, London, to explore structure-activity
requirements for vanilloid-like activity (cf. Walpole and
Wrigglesworth, 1993
). Capsazepine inhibits vanilloid responses in vitro
with Schild plots consistent with a competitive mechanism (Bevan et
al., 1991
, 1992
). Moreover, capsazepine competes for specific RTX
binding sites in a competitive manner (Szallasi et al., 1993b
). In rat trigeminal ganglion neurons, capsazepine inhibits vanilloid-evoked currents (Liu and Simon, 1994
). In a variety of bioassays, capsazepine is effective against both capsaicin (Dickenson and Dray, 1991
; Urbán and Dray, 1991
; Belvisi et al., 1992
; Perkins and Campbell, 1992
; Maggi et al., 1993
; Santicioli et al., 1993
; Seno and Dray, 1993
;
Ueda et al., 1993
; Lee and Lundberg, 1994
; Lalloo et al., 1995
; Fox et
al., 1995
) and RTX (Ellis and Undem, 1994
; Walpole et al., 1994
;
Ács et al., 1996b
, 1997
; Wardle et al., 1996
, 1997
) but,
surprisingly, not against olvanil (Davey et al., 1994
). The utility of
capsazepine is, however, limited by its moderate potency. At micromolar
concentrations, which are necessary to inhibit capsaicin-evoked responses in most tissues, capsazepine also blocks voltage-gated calcium channels (Docherty et al., 1997
) as well as nicotinic acetylcholine receptors (Liu and Simon, 1997
). Furthermore, recent evidence suggests that capsazepine-insensitive VRs also exist (Liu et
al., 1998
). Therefore one should be very careful when interpreting
results obtained with capsazepine, because positive effects are not
necessarily mediated by VRs, nor do negative data rule out the
involvement of VRs. One more reason for being cautious when working
with capsazepine was furnished by the observation that, at least in the
rabbit, it may act as a weak vanilloid agonist (Wang and Håkanson,
1993
).
|
C. Cloning of the First VR, Termed VR1
Repeated efforts to clone a VR using RTX-like photoaffinity probes
resulted in the identification of several, relatively low-affinity RTX-binding proteins, none of which showed the expected tissue distribution for VRs, nor did they show a VR-like activity in functional assays (Ninkina et al., 1994
; Davies et al., 1997
). David
Julius' group at the University of California in San Francisco chose,
therefore, a different approach. They transfected eukaryotic cells with
pools of a rat cDNA library and used calcium imaging to identify those
cells that responded to capsaicin (Caterina et al., 1997
). Once a
positive pool was found, it was divided into smaller pools (a procedure
known as sib-selection) until they had isolated a single cDNA encoding
the capsaicin-gated channel. They named this receptor VR1.
The rat VR1 cDNA contains an open reading frame of 2514 nucleotides. This cDNA encodes a protein of 838 amino acids with a molecular mass of 95 kDa. At the N terminus, VR1 has three ankyrin repeat domains (Fig. 9A). The carboxy terminus has no recognizable motifs. Predicted membrane topology of VR1 features six transmembrane domains and a possible pore-loop between the fifth and sixth membrane-spanning regions (Fig. 9A). There are three possible protein kinase A phophorylation sites on the VR1 that might play a role in receptor desensitization.
|
VR1 is a distant relative of the transient release potential (TRP)
family of store-operated calcium channels (Montell and Rubin, 1989
;
Hardie and Minke, 1993
; Wes et al., 1995
; Clapham, 1996
; Colbert
et al., 1997
; Roayaie et al., 1998
). There is considerable homology
between VR1 and the drosophila TRP protein in retina (Fig. 9B). This
sequence similarity seems to be restricted to the pore-loop and the
adjacent sixth transmembrane segment in VR1. Interestingly, VR1 also
shows similarity to a Soares human retina cDNA (L. Hillier, N. Clark,
T. Dubuque, K. Elliston, M. Hawkins, M. Holman, M. Hultman, T. Kucaba,
M. Le, G. Lennon, M. Marra, J. Parsons, L. Rifkin, T. Rohlfing, F. Tan,
E. Trevaskis, R. Waterston, A. Williamson, P. Wohldman and R. Wilson,
unpublished observations, Washington University-Merck expressed
sequence tags (EST) Project; Accession: AA047763). Because
capsaicin causes a marked calcium accumulation in rat retina (Ritter
and Dinh, 1993
), it might be speculated that the retina has a site,
related to VR1, that recognizes vanilloids. OSM-9, a novel protein with similarity to rat VR1, plays a role in olfaction, mechanosensation, and
olfactory adaptation in Caenorhabditis elegans (Colbert et al., 1997
). OSM-9, however, does not recognize capsaicin (Cornelia Bargmann, personal communication). These findings imply that 1) in
contrast to previous beliefs, VR isoforms did occur early during evolution, but 2) the capsaicin recognition site is a recent addition to VR1.
It should be noted that a human EST in heart (T12251) displays striking
similarity (68% amino acid identity) to the pore-loop and the adjacent
sixth transmembrane segment in rat VR1 (Fig. 9B). Additional human EST
clones are similar to other regions of the VR1 and could represent
fragments of the human transcript. The presence of a VR1-like EST clone
in heart is surprising but it is entirely in accord with the recent
recognition of nonneuronal VRs (Bíró et al., 1998a
,b
).
Capsaicin has long been known to influence cardiac functions (Fukuda
and Fujiwara, 1969
; Molnár et al., 1969
). Some capsaicin actions
on heart were attributed to an interaction at K+
channels (Castle, 1992
), whereas others were explained by the liberation of neuropeptides, most notably CGRP, from the
vanilloid-sensitive innervation of the heart (Franco-Cereceda et al.
1988
, 1991
; Ono et al., 1989
). It is not impossible that capsaicin can
also act directly on the heart via a cardiac VR.
When expressed in Xenopus oocytes, VR1 is similar in its
electrophysiological properties to native vanilloid-operated channels in sensory ganglia (Caterina et al., 1997
). As observed in cultured DRG
neurons (Baccaglini and Hogan, 1983
; Heyman and Rang, 1985
; Forbes and
Bevan, 1988
; Winter et al., 1990
; Vlachová and Vyklicky, 1993
;
Liu and Simon, 1994
; Petersen et al., 1996
), capsaicin-evoked currents
readily disappear after agonist removal, whereas RTX-induced currents
are much longer in duration and often persist even in the absence of
the agonist. Hill coefficients (approximately 2) derived from the
analysis of capsaicin-induced currents in oocytes injected with mRNAs
encoding VR1 indicate the existence of more than one agonist binding
site (Caterina et al., 1997
). Again, this is in accord with the
properties of native VRs in sensory neurons (Szallasi et al., 1993a
; Oh
et al., 1996
). The implications of this finding are discussed in
Section VI.J.
RTX is, however, only 20-fold more potent than capsaicin to
activate the cloned VR1 (Caterina et al., 1997
), which is at variance with the several thousandfold higher affinity of RTX in the binding assay (cf. Szallasi and Blumberg, 1990a
, 1996
; Szallasi, 1994
). This
apparent contradiction was first explained by postulating the existence
of two distinct classes of VRs, the channel, which represents a
low-affinity site for RTX, and a yet-to-be cloned high-affinity site,
seen in the binding assay. We referred to these receptors as C-type and
R-type VRs, respectively (cf. Ács et al., 1997
;
Bíró et al. 1997
; Szallasi, 1997
). However,
preliminary binding experiments with human embryonic kidney
(HEK)293 cells stably transfected with VR1 cDNA suggests that this is
not the case: the same receptor protein seems to mediate both the
high-affinity RTX binding and the lower affinity calcium uptake
response (A. Szallasi, D. N. Cortright, P. M. Blumberg,
and J. E. Krause, manuscript in preparation).
| |
IV. Anatomical Localization and Tissue Specificity of VRs |
|---|
|
|
|---|
A. VRs in Primary Sensory Neurons; Colocalization with Other Receptors, Neuropeptides, and the Isolectin B4
As first shown by RTX autoradiography (cf. Szallasi, 1995
), VRs
are expressed along the entire length of vanilloid-sensitive sensory
neurons, from the peripheral terminals to the axons to the somata to
the central endings (Fig. 3 and Table 3). In corresponding areas
(compare Figs. 3 and 10) the presence
of VR1-like immunoreactivity has also been demonstrated (Guo et al.,
1999
; Tominaga et al., 1998
). Nerve ligation studies suggest that VRs
are transported from the cell bodies to the periphery in a form capable
of ligand binding (Fig. 3) (Szallasi et al., 1995a
). Peripheral tissues in which VRs were demonstrated by RTX binding to be present are listed
in Section III.A and in Table 3. In the urinary bladder, VRs
seem to be expressed exclusively on nerve endings rather than in
bladder epithelium or the muscle layer, for denervation of the bladder
leads to a complete depletion of specific RTX binding sites (Szallasi
et al., 1993d
). The majority (approximately 90%) of the RTX sites
appear to be present on the pudendal nerve, with a relatively
minor fraction on the hypogastric nerve (Szallasi et al., 1993d
).
|
Northern blot analysis confirmed the presence of VR1 transcripts in
dorsal root and trigeminal ganglia (Caterina et al., 1997
; Helliwell et
al., 1998a
). VR1-like immunoreactivity was detected in more than 50%
of DRG neurons (Fig. 11), with the
expression being most prevalent in small to medium sized neurons (Guo
et al., 1999
). VR1-like immunoreactivity was also observed in both the
central (e.g., dorsal horn of the spinal cord and the caudal nucleus of
the spinal trigeminal complex) and peripheral (e.g., skin and cornea)
processes of primary afferent neurons (Fig. 10) (Guo et al., 1998
). In
DRG neurons, VR1-like immunoreactivity was associated with the Golgi
complex and the plasma membrane (Guo et al., 1999
). In the dorsal horn
of the spinal cord, the VR1 protein was associated with "small clear
vesicles" in preterminal axons and with the plasma membrane of nerve
terminals (Guo et al., 1999
). The latter finding is in line with an
earlier observation of Szolcsányi (Szolcsányi et al.,
1975
), according to which capsaicin depletes "small clear vesicles"
from nerve endings.
|
The distribution of VR1-like immunoreactivity in the spinal
dorsal horn deserves particular attention. The labeling is strongest in
the Lissauer zone (lamina I; Fig. 10C). VR1 protein is also abundant in
the inner, but not in the outer, layer of lamina II (Fig. 10C) (Guo et
al., 1999
). [There is an apparent discrepancy between this finding and
the even distribution of VR1-like immunoreactivity in the substantia
gelatinosa reported by Julius and colleagues (Tominaga et al., 1998
),
the reason for which is not clear.] It should be noted that several
important proteins involved in pain transmission are also enriched in
the inner layer of lamina II (i.e., in close proximity to VR1). Notable
examples include PKC isozyme
(PKC
) (Malmberg et al., 1997a
) as
well as ATP-sensitive P2X3 receptors
(Vulchanova et al., 1998
). P2X3 receptors
colocalize with VR1 both in DRGs, where 75% of the
P2X3 positive neurons also express VR1 (Fig.
11F), and in the inner layer of lamina II (Fig. 11, D and E) (Guo et
al., 1999
). By contrast, PKC
is confined to a population of
interneurons that reside in lamina II of the dorsal horn (Malmberg et
al., 1997a
). The possible role of PKC
and P2X3
receptors in nociception is discussed in Section VIII.
Surprisingly, the colocalization of VR1 with SP in DRG neurons is
limited: for example, only 33% of L5 DRG neurons positive for VR1
contain SP-like immunoreactivity as well (Fig. 11I) (Guo et al., 1999
).
Moreover, much of the VR1 staining in the dorsal horn is concentrated
in the inner layer of lamina II where both SP- (Fig. 11, G and H) and
CGRP-like immunoreactivities are sparse (Fig. 11, J and K) (Guo et al.,
1999
). This limited colocalization between VR1 and SP is at variance
with the profound effects that vanilloids have on SP expression (see in
Section VIII.B.5).
The colocalization of VR1 with the lectin IB4 (Fig. 11, A-C) (Guo et
al., 1999
; Tominaga et al., 1998
) may shed new light on the regulation
of VR expression by trophic factors. Vanilloid-sensitive neurons
require nerve growth factor (NGF) for survival during embryogenesis
(Ruit et al., 1992
), as evidenced by a severe deficit in nociception
and thermoreception in mice with a null mutation in the gene encoding
trkA, the signal transduction receptor for NGF (Smeyne et al., 1994
).
Mature DRG neurons, however, are able to survive in the absence of NGF
(Yip et al., 1984
; Lindsay, 1988
). There is evidence that those neurons
that bind IB4 also express receptors for glial cell-derived
neurotrophic factor, abbreviated GDNF (Bennett et al., 1998
). In 1997, Snider and coworkers (Molliver et al., 1997
) showed in elegant
experiments that IB4-positive DRG neurons switch from dependence on NGF
to dependence upon GDNF during development. Subsequently, McMahon and
colleagues (Bennett et al., 1998
) demonstrated that IB4-positive
neurons may be rescued by exogenous GDNF following nerve injury.
Clearly, it would be interesting to explore the effects of GDNF on VR expression.
B. VRs in Vagal (Nodose Ganglion) Neurons
In addition to DRG (Figs. 3 and 7) and trigeminal ganglion
(Fig. 3) neurons (Szallasi and Blumberg, 1990b
; Szallasi et al., 1993a
,b
, 1994a
; Ács et al., 1994a
), a subset of nodose ganglion neurons also expresses VRs (Fig. 3 and Table 3) (Ács et al., 1994a
; Szallasi et al., 1995a
). A strong autoradiographic signal is
present in the area postrema/nucleus of the solitary tract region (Fig.
3), representing the central termination area for sensory neurons
of the vagus nerve (Szallasi et al., 1995a
). This area is also stained
by an anti-VR1 antibody (Fig. 10A; Guo et al., 1999
).
An unexpected result in the VR1 cloning article by Julius and
colleagues (Caterina et al., 1997
) is the failure of Northern blot
hybridization to detect mRNAs encoding VR1 in nodose ganglia. However,
using a probe corresponding to nucleotides 1513-2482 of the rat VR1
sequence, a strong in situ hybridization signal was detected in nodose
ganglion neurons (Helliwell et al., 1998
). To resolve the apparent
contradiction between these studies, it should be noted that whereas
Julius and coworkers (Caterina et al., 1997
) used the entire VR1
sequence for Northern blot hybridization, Bevan and colleagues
(Helliwell et al., 1998
) generated by polymerase chain reaction a
partial VR1 sequence only. Therefore, it is entirely possible that
nodose ganglion neurons express a VR isoform that differs from VR1 in
nucleotides not included in the probe used for in situ hybridization
histochemistry. The existence of distinct VR isoforms in DRGs and
nodose ganglia would be consistent with the different embryonic origin
of these tissues (cf. Vogel, 1992
). It would also be in accord with
the observations that different neurotrophic factors regulate capsaicin
sensitivity in DRG (NGF and possibly also GDNF) and nodose ganglion
neurons (brain-derived neurotrophic factor), respectively
(Winter et al., 1988
; Winter, 1998
).
C. VRs in Brain
A low to moderate level of specific RTX binding can be detected in
various CNS areas not associated with primary sensory neurons (Ács et al., 1996a
), suggesting the existence of intrinsic brain neurons with VRs (Fig. 4). Among these brain areas, the presence of VRs
in the hypothalamus was expected based on the proposed role of this
structure in the hypothermic action of vanilloids (Jancsó-Gábor et al., 1970
; Szolcsányi et al., 1971
).
The presence of VR1 mRNA in the hypothalamus has recently been
confirmed by reverse transcription-polymerase chain reaction (Sasamura
et al., 1998
). These vanilloid-sensitive terminals in the hypothalamus are probably glutaminergic because capsaicin evokes glutamate release
from slices of hypothalamus (Sasamura et al., 1998
). It is also easy to
visualize how VRs in the reticular formation (Ács et al., 1996a
)
may mediate some vanilloid actions on autonomic regulation
(Jancsó and Such, 1985
; Koulchitsky et al., 1994
; Seller et al.,
1997
). The biological role of specific RTX binding sites in other brain
areas is unclear.
A link between the SP content of sensory neurons and their sensitivity
to capsaicin was postulated as early as the mid-1960s (Ga
parovic
et al., 1964
). In 1976, the high concentration of SP in the substantia
nigra was first reported (Brownstein et al., 1976
) and the search for
capsaicin-sensitive neurons in the basal ganglia began. Five years
later, an enhanced locomotor activity in rats following bilateral
intranigral capsaicin injection and a concomittant decrease in the
cataleptic action of fluphenazine was described (Dawbarn et al., 1981
).
In 1988, capsaicin microinjected into the substantia nigra or caudatus
putamen was reported to induce peripheral vasodilatation and a
subsequent hypothermic response (Hajós et al., 1988
). These
biological actions suggested the existence of intrinsic
vanilloid-sensitive cells in the basal ganglia. The recent detection of
VR1 mRNA in the striatum now firmly establishes the existence of such
neurons (Sasamura et al., 1998
). As yet, it is not known which
subpopulation of nigral neurons express VRs. Neonatal capsaicin
treatment does not deplete SP from the striatum, nor does it alter the
expression of the opioid peptides dynorphin and
met5-enkephalin (Sivam and Krause, 1992
).
Repeated attempts to elucidate the effects of capsaicin treatment on
monoaminergic systems in the brain have yielded controversial results.
For example, increased (Holzer et al., 1981
) or decreased (Dawbarn et
al., 1981
) 5-hydroxytryptamine levels, or no change at all (G. Jancsó et al., 1981
), were reported in the very same year. With
the recent availability of VR1 detecting antibodies as well as probes
for in situ hybridization histochemistry, the identity of the
VR1-expressing subpopulation of basal ganglion neurons will be revealed shortly.
VR1 mRNA seems to be widely present in the brain. Relative levels are
as follows: hypothalamus and cerebellum > cortex, striatum, and
midbrain > olfactory bulb, pons, hippocampus, and thalamus (Sasamura et al., 1998
). Although Northern blot analysis failed to show
VR1 mRNA in the brain (Caterina et al., 1997
), this discrepancy may be
due to the difference in sensitivity between polymerase chain reaction
and Northern blot hybridization. In support of this explanation is the
detection of VR1 mRNA in the rat cortex in solution
hybridization-nuclease protection experiments (D. N. Cortright and
J. E. Krause, personal communication). VR1-positive cells may also
be visualized in the brain by both in situ hybridization and
immunostaining experiments (E. Mezey, A. Guo, D. N. Cortright, R. Elde, J. E. Krause, P. M. Blumberg, and A. Szallasi,
manuscript in preparation).
D. Possible Presence of VRs in Nonneuronal Tissues Such as Mast Cells and Glia
It has been long noted that capsaicin exerts a variety of effects
on nonneuronal tissues (cf. Buck and Burks, 1986
; Holzer, 1991
). These
actions were considered nonspecific because they were at variance with
the prevailing concept of capsaicin being selective for sensory
neurons. In 1998, we showed that vanilloids induce calcium uptake by
mast cells (Bíró et al., 1998a
) and in a glioma cell line
(Bíró et al., 1998b
). The pharmacological characteristics
of this calcium influx response by mast cells and glioma cells were
very similar to those described in DRG neurons (Table
4), with the exception of the magnitude
of the uptake, which was much smaller, implying a VR density lower than
in sensory neurons. In keeping with this, no specific
[3H]RTX binding by mast cells or glioma cells
could be detected (Table 4). We will return to the biological relevance
of such nonneuronal vanilloid interactions later. The novel concept of nonneuronal VRs has received support from the presence of EST clones
homologous to VR1 in the heart and other nonneuronal tissues (Caterina
et al., 1997
).