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Proc Natl Acad Sci U S A. 2001 June 5; 98(12): 6951–6956. Published online 2001 May 22. doi: 10.1073/pnas.111025298. | PMCID: PMC34459 |
Copyright © 2001, The National Academy of Sciences Physiology Potentiation of capsaicin receptor activity by metabotropic ATP
receptors as a possible mechanism for ATP-evoked pain and
hyperalgesia Makoto Tominaga,*†‡ Makoto Wada,* and Masayuki Masu* *Department of Molecular Neurobiology, Institute of Basic Medical Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan; and †Department of Physiology, Mie University School of Medicine, Edobashi 2-174, Tsu, Mie 514-8507, Japan Received January 16, 2001. The capsaicin (vanilloid) receptor, VR1, is a sensory
neuron-specific ion channel that serves as a polymodal detector of
pain-producing chemical and physical stimuli. It has been proposed that
ATP, released from different cell types, initiates the sensation of
pain by acting predominantly on nociceptive ionotropic purinoceptors
located on sensory nerve terminals. In this study, we examined the
effects of extracellular ATP on VR1. In cells expressing VR1, ATP
increased the currents evoked by capsaicin or protons through
activation of metabotropic P2Y1 receptors in a protein
kinase C-dependent pathway. The involvement of
Gq/11-coupled metabotropic receptors in the potentiation of
VR1 response was confirmed in cells expressing both VR1 and M1
muscarinic acetylcholine receptors. In the presence of ATP, the
temperature threshold for VR1 activation was reduced from 42°C to
35°C, such that normally nonpainful thermal stimuli (i.e., normal
body temperature) were capable of activating VR1. This represents a
novel mechanism through which the large amounts of ATP released from
damaged cells in response to tissue trauma might trigger the sensation
of pain. Pain is initiated when
noxious thermal, mechanical, or chemical stimuli excite the peripheral
terminals of specialized primary afferent neurons called nociceptors
( 1– 4). Many different kinds of ionotropic and metabotropic receptors
are known to be involved in this process ( 5– 7). Tissue damage
associated with infection, inflammation, or ischemia produces an array
of chemical mediators that activate or sensitize nociceptor terminals
to elicit pain at the site of injury. An important component of this
proalgesic response is ATP released from different cell types ( 8– 13).
ATP is released from microvascular endothelial cells during hyperemia,
from nociceptive terminals after noxious stimulation (e.g., with
capsaicin), and from sympathetic nerve terminal varicosities as a
cotransmitter with norepinephrine and neuropeptide Y. ATP also is
released from tumor cells during abrasive activity and from damaged
tissues after trauma or surgery. Extracellular ATP excites the
nociceptive endings of nearby sensory nerves, evoking a sensation of
pain ( 9, 13). In these neurons, the most widely studied targets of
extracellular ATP have been ionotropic ATP (P2X) receptors ( 11– 13).
Indeed, several P2X receptor subtypes have been identified in sensory
neurons, including one (P2X 3) whose expression is
largely confined to these cells ( 14, 15). Our understanding of
purinergic contributions to pain sensation may be incomplete, however,
given that the potential involvement of widely distributed metabotropic
ATP (P2Y) receptors has not yet been well investigated. Vanilloid receptors are nociceptor-specific cation channels that serve
as the molecular target of capsaicin, the pungent ingredient in hot
chili peppers ( 16, 17). We have shown that, when expressed in
heterologous systems, the cloned capsaicin receptor (VR1) also can be
activated by noxious heat (with a thermal threshold of >43°C) or
protons (acidification), both of which cause pain in vivo
( 18, 19). Furthermore, analyses of mice lacking VR1 have shown that VR1
is essential for selective modalities of pain sensation and for tissue
injury-induced thermal hyperalgesia ( 20, 21). These data suggest a
critical role for VR1 in the detection or modulation of pain. To
address whether metabotropic P2Y receptors are involved in VR1-mediated
nociceptive responses, we examined the effects of extracellular ATP on
VR1 expressed in human embryonic kidney-derived HEK293 cells and rat
dorsal root ganglion (DRG) neurons. We report that extracellular ATP
potentiates or sensitizes VR1 responsiveness by any of three different
stimuli through P2Y 1 receptors in a protein
kinase C (PKC)-dependent pathway, resulting in the activation of VR1 at
normal body temperature. Mammalian Cell Culture. HEK293 cells were maintained in DMEM (supplemented with 10% FBS,
penicillin, streptomycin, and l-glutamine) and transfected
with 1 μg of plasmid DNA by using lipofectamine plus reagent (GIBCO).
VR1 cDNA was prepared as described ( 16). M1 muscarinic acetylcholine
receptor cDNA was given generously by Huai-hu Chuang (Univ. of
California, San Francisco). Primary cultures prepared from male adult
Wistar rat DRG ( 22) were incubated overnight (37°C, 5%
CO 2) in medium containing nerve growth factor
(100 ng/ml). All procedures involving the care and use of rats were
carried out in accordance with institutional guidelines. Electrophysiology. Whole-cell patch-clamp recordings were carried out at 1 or 2 days after
transfection of VR1 cDNA to HEK293 cells or dissociation of the DRG
neurons ( 16). Standard bath solution contained 140 mM NaCl, 5 mM KCl, 2
mM MgCl 2, 5 mM EGTA, 10 mM Hepes, and 10 mM
glucose, pH 7.4 (adjusted with NaOH). Bath solution was buffered to
different pH values with either 10 mM Hepes (pH 6.7) or 10 mM Mes (pH
6.2, 5.8, 5.6, 5.3, 4.7, and 4.3). Pipette solution contained 140 mM
CsCl (or KCl), 5 mM EGTA, and 10 mM Hepes, pH 7.4 (adjusted with CsOH
or KOH). All patch-clamp experiments were performed at room temperature
(25°C) unless otherwise noted. When examining the heat-evoked current
responses, bath temperature was increased by using a preheated solution
with the rate of 1.5–2.0°C/sec (about 20 sec). When the
heat-activated currents started to inactivate, the preheated solution
was changed to a 25°C one. Chamber temperature was monitored with a
thermocouple (accuracy ± 0.1°C) placed within 4 mm of the
patch-clamped cell. The solutions containing drugs were applied to the
chamber (180 μl) by a gravity at a flow rate of 5 ml/min. Reverse Transcription–PCR. Total RNA was isolated from male adult rat DRG and HEK293 cells and
reverse-transcribed by using Superscript II (GIBCO). The gene-specific
primers used in this study (5′-GTTCAATTTGGCTCTGGCCG-3′ and
5′-CTGATAGGTGGCATAAACCC-3′ for rat P2Y 1;
5′-GATCTGTATCAGCGTGCTGG-3′ and 5′-CTTGTGCCTTCACAGGCTTG-3′ for human
P2Y 1) were designed from rat and human
P2Y 1 sequences ( 23, 24). PCR was performed with
30 cycles of the following amplification protocol: 94°C, 30 sec;
56.5°C, 30 sec; and 72°C, 90 sec. In voltage-clamp experiments, low doses of capsaicin (10 or 20 nM)
evoked small inward currents in the HEK293 cells expressing VR1. In the
absence of extracellular calcium, no change was observed in the
magnitude of responses evoked by repetitive capsaicin applications. In
contrast, after a 2-min pretreatment with 100 μM extracellular ATP,
the same doses of capsaicin produced much larger current responses
[6.42 ± 1.01-fold (mean ± SEM), n = 52]
(Fig. A). Other
electrophysiological properties of these capsaicin-evoked responses,
including an outwardly rectifying current–voltage relationship,
antagonist (capsazepine) sensitivity, and extracellular
Ca2+-dependent desensitization, were unchanged by
the presence of extracellular ATP (data not shown). A similar
potentiating effect of extracellular ATP was observed on proton-evoked
activation of VR1 (5.68 ± 0.92-fold, n = 32)
(Fig. B). ATP appears to serve as a modulator, rather than
a direct activator of VR1, because no current was observed upon
application of ATP alone (Fig. A and B). In
addition, the ATP-stimulated increase in capsaicin-evoked currents
persisted for several minutes after removal of ATP from the bath (Fig.
A), suggesting the involvement of cytosolic second
messengers in this process.
To examine how ATP changes VR1 responsiveness, we measured VR1 currents
in single cells by serially applying a range of concentrations of
capsaicin or protons in the absence or presence of ATP. The currents
were normalized to the maximal current produced by the application of 1
μM capsaicin or pH 4.3 solution without ATP to each cell. In both
cases, maximal currents in the presence of ATP were almost the same as
those obtained in the absence of ATP. The resultant dose-response
curves clearly demonstrate that ATP enhances capsaicin and proton
action on VR1 by lowering EC 50 values
without altering maximal responses (EC 50 from
114.7 nM to 49.3 nM for capsaicin-activated currents; from pH 5.64 to
pH 5.92 for proton-activated currents) (Figs. C and
D). VR1 potentiation by ATP could be observed at
concentrations as low as 1 μM and saturated at ≈10 μM (Fig.
E), a concentration attainable in the context of
tissue damage ( 25). Potentiating effects of extracellular ATP also were examined on
heat-evoked responses in HEK293 cells expressing VR1. For this
analysis, heat-evoked current responses were compared between different
cells, rather than within the same cell, because repetitive heat-evoked
currents show significant desensitization even in the absence of
extracellular Ca 2+ ( 18) and because the thermal
sensitivity of VR1 increases with repeated heat application ( 26). It is
known that heat-evoked VR1 responses desensitize in an extracellular
Ca 2+-independent manner during relatively long
heat applications ( 18, 27) although brief heat stimuli do not seem to
cause this phenomenon ( 28, 29). This
Ca 2+-independent desensitization also has been
reported in native DRG neurons ( 30). When temperature ramps were
applied to HEK293 cells expressing VR1 in the absence of ATP,
heat-evoked currents developed at about 42°C with an extremely steep
temperature dependence (Fig.
A). ATP treatment lowered the
threshold temperature for VR1 activation significantly (41.7 ±
1.1°C, n = 7, and 35.3 ± 0.7°C,
n = 5, without and with ATP treatment, respectively,
P < 0.001) (Fig. A– C). Thus,
in the presence of ATP, normally nonpainful thermal stimuli (even body
temperature) are capable of activating VR1, a finding consistent with
the observation that exogenous ATP induces pain in human and rats ( 31,
32) at nonelevated body temperatures. These data clearly show that VR1
currents evoked by any of three different stimuli (capsaicin, proton,
or heat) are potentiated or sensitized by extracellular ATP.
To explore the identity of the ATP receptors responsible for VR1
sensitization, we first examined the effects of two P2 receptor
antagonists. In the presence of 100 μM pyridoxal
phosphate-6-azophenyl-2′,4′-disulphonic acid ( 33), the enhancement of
capsaicin-evoked currents by ATP was decreased significantly (2.05
± 0.26-fold increase, n = 13) compared with ATP alone
(5.25 ± 0.83-fold, n = 25) ( P <
0.05) (Fig. E). Another P2
antagonist, reactive blue 2 (RB2, 100 μM) ( 12), almost completely
abolished the effect of ATP (1.36 ± 0.13-fold, n
= 8, P < 0.05 vs. ATP alone) (Fig. A and
E), indicating that P2 receptors mediate the enhancement of
capsaicin-activated currents by ATP. Moreover, we conclude that ATP
effect is mediated by metabotropic ATP (P2Y) receptors, because we
failed to observe ATP-evoked currents indicative of ionotropic ATP
(P2X) receptors in HEK293 cells, consistent with previous reports ( 34).
To distinguish between the subtypes of P2Y receptor that might be
involved in this process, we examined the effect of several ATP-related
reagents (each 100 μM) on the VR1 response (Fig. E).
Among the reagents tested, ATP was the most effective (5.25 ±
0.83-fold, n = 25, P < 0.01 vs.
control), followed by ADP (3.27 ± 1.29-fold, n =
6, P < 0.05). UTP exhibited a small but significant
effect (1.36 ± 0.21-fold, n = 8,
P < 0.05), whereas AMP failed to significantly
potentiate capsaicin-evoked currents (1.13 ± 0.25-fold,
n = 6, P > 0.05). This rank order of
potency (ATP > ADP >> UTP > AMP) is most consistent with
the involvement of P2Y 1 receptors ( 11, 12).
Further evidence for P2Y 1 receptor involvement
comes from our finding that 2-methylthio ATP (2MeSATP), an agonist that
activates P2Y 1 receptors much more effectively
than other P2Y subtypes ( 12), produced a significant potentiation of
capsaicin-evoked currents (2.94 ± 0.85-fold, n =
11, P < 0.05 vs. control) (Fig. B and
E). One major consequence of P2Y 1 receptor
stimulation is activation of phospholipase C through the G protein
G q/11, leading to the production of inositol
1,4,5-trisphosphate (IP 3) and
diacylglycerol (DAG) ( 12, 35). If
G q/11 activation underlies the potentiation of
VR1 by extracellular ATP, stimulation of other
G q/11-coupled receptors might produce similar
effects. When VR1 was coexpressed with the
G q/11-coupled M1 muscarinic acetylcholine
receptor in HEK293 cells ( 36), extracellular application of
acetylcholine significantly increased the magnitude of currents evoked
by either capsaicin or protons (5.30 ± 0.96- and 4.68 ±
1.04-fold for capsaicin- and proton-activated currents, respectively,
P < 0.05 vs. control) (Fig. C), supporting
the involvement of G q/11-coupled signaling in VR1
sensitization. Ca 2+ mobilization by
IP 3 is not a likely mechanism for the
capsaicin-evoked current increase observed in our experiments because
cytosolic-free Ca 2+ is tightly chelated with the
5 mM EGTA included in the pipette solution. Therefore, activation of
PKC by DAG remains a more likely mechanism for ATP-induced potentiation
( 37). To test this possibility, we examined the effect of a highly
potent and selective PKC inhibitor, calphostin C ( 38). When 1 μM
calphostin C was added to the pipette solution, the ATP effect was
almost completely abolished (1.13 ± 0.13-fold, n
= 7, P < 0.05 vs. ATP alone) (Fig. E).
Acetylcholine effects observed in the cells expressing both VR1 and M1
muscarinic acetylcholine receptor were abolished by calphostin C, too
(1.29 ± 0.18-fold for capsaicin-activated currents,
n = 5; 1.22 ± 0.19-fold for proton-activated
currents, n = 4, P < 0.05 vs.
acetylcholine alone) (data not shown). Furthermore, direct activation
of PKC by 100 nM phorbol 12-myristate 13-acetate (PMA) caused a robust
increase in the magnitude of capsaicin-evoked currents (8.09 ±
2.81, n = 10, P < 0.05 vs. control)
(Fig. D and E). The maximal response to
capsaicin was not changed after treatment of PMA, suggesting a further
leftward shift of the capsaicin dose-response curve. When the cells
were exposed to ATP after PMA treatment, no further potentiation of the
capsaicin-activated currents was observed (Fig. D),
indicating that the same pathway is involved in the potentiation by ATP
and PMA. These data clearly indicate the involvement of a PKC-dependent
pathway in VR1 potentiation by ATP and are consistent with the report
that PKC- ![[var epsilon]](/corehtml/pmc/pmcents/epsiv.gif) is specifically involved in sensitization of
heat-activated channels by bradykinin in DRG neurons ( 39). To examine whether the VR1 potentiation by ATP occurs in native
neurons, we performed voltage-clamp experiments on rat DRG neurons
cultured in the presence of nerve growth factor. Because the
sensitivity of these cells to capsaicin appears to be slightly lower
than that of VR1-transfected HEK293 cells (data not shown and ref. 40),
we applied capsaicin to these cells at 50 nM instead of 20 nM.
Nevertheless, in capsaicin-responsive neurons, we found that ATP did,
indeed, potentiate capsaicin-evoked currents (2.04 ± 0.34-fold,
n = 7, P < 0.05 vs. control) (Fig.
A and C). In some
DRG neurons, application of ATP alone caused transient current
responses, most likely mediated by ionotropic P2X receptors. Within
these latter neurons, the magnitude of ATP-induced potentiation of
capsaicin-evoked currents was indistinguishable from that seen in
neurons lacking P2X responses (data not shown). ADP also potentiated
capsaicin-evoked currents in DRG neurons (1.56 ± 0.15-fold,
n = 4, P < 0.05 vs. control), although
less effectively than ATP (Fig. C). Direct activation of
PKC by 100 nM PMA greatly increased capsaicin-evoked currents
(5.38 ± 1.33-fold, n = 5, P <
0.05 vs. control) (Fig. B and C), supporting
the involvement of a PKC-dependent pathway in DRG neurons. Moreover,
expression of the P2Y 1 receptor gene in HEK293
cells and rat DRG neurons was confirmed by reverse transcription–PCR
(Fig. D), consistent with previously reported findings
( 41). Still, a contribution from other metabotropic ATP receptor
subtypes to the potentiation of VR1 responses cannot be excluded.
Inflammatory pain is initiated by tissue
damage/inflammation and is characterized by hypersensitivity both at
the site of damage and in adjacent tissue. Stimuli that normally would
not produce pain do so (allodynia), whereas previously noxious stimuli
evoke even greater pain responses (hyperalgesia). One mechanism
underlying these phenomena is the modulation (sensitization) of ion
channels, such as VR1, that detect noxious stimuli at the nociceptor
terminal ( 1– 4). Sensitization is triggered by extracellular
inflammatory mediators that are released in vivo from
surrounding damaged or inflamed tissue and from nociceptive neurons
themselves (i.e., neurogenic inflammation). Mediators known to cause
sensitization include prostaglandins, adenosine, serotonin, bradykinin,
and ATP ( 1– 5). In the present study, we demonstrated that
extracellular ATP potentiates VR1 responses through metabotropic ATP
(P2Y 1) receptors in a PKC-dependent manner in
both a heterologous expression system and rat DRG neurons. ATP enhances
VR1 responses by lowering the VR1 activation threshold for capsaicin,
proton, and heat stimulation. This effect of ATP might contribute to
ATP-induced hypersensitivity. In addition to potentiating capsaicin- or
proton-evoked currents, ATP also lowers the temperature threshold for
heat activation of VR1, such that normally nonpainful thermal stimuli
(i.e., normal body temperature) are capable of activating VR1, making
ATP act as a direct activator of VR1. This represents a novel mechanism
through which extracellular ATP might cause pain in a pathway distinct
from the activation of P2X receptors. The existence of such a mechanism
is consistent with the recent observation that ATP-evoked nociceptive
behavior in mice is only partially reduced by disruption of the
P2X 3 gene ( 42, 43). In addition, the observation that ATP
can be released from a subset of small, primary afferent nerves in
response to capsaicin ( 9) suggests a possible autocrine mechanism for
the exacerbation of pain. Activation of similar PKC-dependent events
might underlie certain nociceptive effects of other
G q/11-coupled metabotropic receptors such as
bradykinin receptors ( 3– 5, 44) whose occupancy enhances heat-activated
currents in sensory neurons ( 29). Indeed, it has been reported recently
that activation of PKC can induce VR1 channel activity in the absence
of any other agonist ( 45). In our experiments, the involvement of Ca 2+ in
the potentiation of VR1 response could be ruled out because cytosolic
Ca 2+ was tightly chelated by EGTA. Kress and
Guenther ( 28), however, reported a significant potentiation of the heat
response in DRG neurons (likely mediated by VR1) when intracellular
Ca 2+ was raised. Both intracellular
Ca 2+-independent and
Ca 2+-dependent potentiation mechanisms might
occur in native cells. In nociceptors, short-term sensitization by inflammatory mediators is
mediated by two distinct intracellular pathways, activation of protein
kinase A by such agents as prostaglandins ( 46) and activation of PKC by
such agents as bradykinin ( 47). In particular, electrophysiological,
biochemical, and knockout mouse analyses ( 39, 48) have implicated
PKC- in the sensitization of nociceptors whereas a protein kinase
A-dependent pathway has been reported to be involved in the modulation
of capsaicin-gated channel by prostaglandin E 2
( 49). We hypothesize that direct phosphorylation of VR1 or a closely
associated protein changes the agonist sensitivity of this ion channel.
Molecular and biochemical analysis of VR1 should allow this hypothesis
to be tested. It is well accepted that extracellular ATP plays an important role in
nociception because ATP produces a sensation of pain in vivo
( 11– 13, 31, 32) and because some P2 antagonists show analgesic
activity ( 50, 51). Most attention in the pain field has focused on the
role of ionotropic ATP receptors in ATP-evoked nociception. Our
findings suggest that P2Y 1 is also involved in
this process and may represent a fruitful target for the development of
drugs that blunt nociceptive signaling through capsaicin receptors. We thank M. J. Caterina (Johns Hopkins University) and K.
Inoue (National Institute of Health Sciences, Japan) for critical
reading of the manuscript. This work was supported by grants from the
Ministry of Culture, Education, Science and Sports in Japan (to M.T.
and M.M.) and (to M.T.) by Novartis Foundation (Japan) for the
Promotion of Science, The Mitsubishi Foundation, Yamazaki Spice and
Herb Research Foundation, and Takeda Science Foundation. | | VR1 | capsaicin (vanilloid) receptor 1 | | | DRG | dorsal root
ganglion | | | PMA | phorbol 12-myristate 13-acetate | | | PKC | protein kinase C |
1. Fields H L. Pain. New York: McGraw–Hill; 1987. 2. Wood J N, Perl E R. Curr Opin Genet Dev. 1999;9:328–322. [PubMed] 3. Mizumura K, Kumazawa T. Prog Brain Res. 1996;113:115–141. [PubMed] 4. Woolf C J, Salter M W. Science. 2000;288:1765–1768. [PubMed] 5. Cesare P, McNaughton P. Curr Opin Neurobiol. 1997;7:493–499. [PubMed] 6. McCleskey E W, Gold M S. Annu Rev Physiol. 1999;61:835–856. [PubMed] 7. Caterina M J, Julius D. Curr Opin Neurobiol. 1999;9:525–530. [PubMed] 8. Holton P. J Physiol. 1959;145:494–504. [PubMed] 9. Sawynok J, Sweeney M I. Neuroscience. 1989;32:557–569. [PubMed] 10. Li J, Perl E R. J Neurosci. 1995;15:3357–3365. [PubMed] 11. North A N, Barnard E A. Curr Opin Neurobiol. 1997;7:346–357. [PubMed] 12. Ralevic V, Burnstock G. Pharmacol Rev. 1998;50:413–492. [PubMed] 13. Burnstock G. Br J Anaesth. 2000;84:476–488. [PubMed] 14. Lewis C, Neldhart S, Holy C, North R A, Buell G, Surprenant A. Nature (London). 1995;377:432–435. [PubMed] 15. Chen C-C, Akopian A N, Sivilotti L, Colquhoun D, Burnstock G, Wood J N. Nature (London). 1995;377:428–431. [PubMed] 16. Caterina M J, Schumacher M A, Tominaga M, Rosen T A, Levine J D, Julius D. Nature (London). 1997;389:816–824. [PubMed] 17. Szallasi A, Blumberg P M. Pharmacol Rev. 1999;51:159–211. [PubMed] 18. Tominaga M, Caterina M J, Malmberg A B, Rosen T A, Gilbert H, Skinner K, Raumann B E, Basbaum A I, Julius D. Neuron. 1998;21:531–543. [PubMed] 19. Tominaga M, Julius D. Jpn J Pharmacol. 2000;83:20–24. [PubMed] 20. Caterina M J, Leffler A, Malmberg A B, Martin W J, Trafton J, Petersen-Zeitz K R, Koltzenburg M, Basbaum A I, Julius D. Science. 2000;288:306–313. [PubMed] 21. Davis J B, Gray J, Gunthorpe M J, Hatcher J P, Davey P T, Overend P, Harries M H, Latcham J, Clapham C, Atkinson K, et al. Nature (London). 2000;405:183–187. [PubMed] 22. Reichling D B, Levine J D. Proc Natl Acad Sci USA. 1997;94:7006–7011. [PubMed] 23. Tokuyama Y, Hara M, Jones E M C, Fan Z, Bell G I. Biochem Biophys Res Commun. 1995;211:211–218. [PubMed] 24. Janssens R, Communi D, Pirotton S, Samson M, Parmentier M, Boeynaems J-M. Biochem Biophys Res Commun. 1996;221:588–593. [PubMed] 25. Born G V R, Kratzer M A A. J Physiol. 1984;354:419–429. [PubMed] 26. Caterina M J, Rosen T A, Tominaga M, Brake A J, Julius D. Nature (London). 1999;398:436–441. [PubMed] 27. Cesare P, Moriondo A, Vellani V, McNaughton P A. Proc Natl Acad Sci USA. 1999;96:7658–7663. [PubMed] 28. Kress M, Guenther S. J Neurophysiol. 1999;81:2612–2619. [PubMed] 29. Cesare P, McNaughton P. Proc Natl Acad Sci USA. 1996;93:15435–15439. [PubMed] 30. Schwarz S, Greffrath W, Büsselberg D, Treede R-D. J Physiol. 2000;528:539–549. [PubMed] 31. Bleehen T, Keele C A. Pain. 1977;3:367–377. [PubMed] 32. Bland-Ward P A, Humphrey P P A. Br J Pharmacol. 1997;122:365–371. [PubMed] 33. Lambrecht G, Friebe T, Grimm U, Windscheif U, Bungardt E, Hildebrandt C, Baumert H G, Spatz-Kumbel G, Mutschler E. Eur J Pharmacol. 1992;217:217–219. [PubMed] 34. Valera S, Hussy N, Evans R J, Adami N, North R A, Surprenant A, Buell G. Nature (London). 1994;371:516–519. [PubMed] 35. Schachter J B, Li Q, Boyer J L, Nicholas R A, Harden T K. Br J Pharmacol. 1996;118:167–173. [PubMed] 36. Hulme E C, Birdsall N J M, Buckley N J. Annu Rev Pharmacol Toxicol. 1990;30:633–673. [PubMed] 37. Tanaka C, Nishizuka Y. Annu Rev Neurosci. 1994;17:551–567. [PubMed] 38. Kobayashi E, Nakano H, Morimoto M, Tamaoki T. Biochem Biophys Res Commun. 1989;159:548–553. [PubMed] 39. Cesare P, Dekker L V, Sardini A, Parker P J, McNaughton P A. Neuron. 1999;23:617–624. [PubMed] 40. Shin J S, Wang M-H, Hwang S W, Cho H, Cho S Y, Kwon M J, Lee S-Y, Oh U. Neurosci Lett. 2001;299:135–139. [PubMed] 41. Nakamura F, Strittmatter S M. Proc Natl Acad Sci USA. 1996;93:10465–10470. [PubMed] 42. Cockayne D A, Hamilton S G, Zhu Q M, Dunn P M, Zhong Y, Novakovic S, Malmberg A B, Cain G, Berson A, Kassotakis L, et al. Nature (London). 2000;407:1011–1015. [PubMed] 43. Souslova V, Cesare P, Ding Y, Akopian A N, Stanfa L, Suzuki R, Carpenter K, Dickenson A, Boyce S, Hill R, et al. Nature (London). 2000;407:1015–1017. [PubMed] 44. Bhoola K D, Figueroa C D, Worthy K. Pharmacol Rev. 1992;44:1–80. [PubMed] 45. Premkumar L S, Ahern G P. Nature (London). 2000;408:985–990. [PubMed] 46. Aley K O, Levine J D. J Neurosci. 1999;19:2181–2186. [PubMed] 47. Barber L A, Vasko M R. J Neurochem. 1996;67:72–80. [PubMed] 48. Khasar S G, Lin Y H, Martin A, Dadgar J, McMahon T, Wang D, Hundle B, Aley K O, Isenberg W, McCarter G, et al. Neuron. 1999;24:253–260. [PubMed] 49. Lopshire J C, Nicol G D. J Neurosci. 1998;15:6081–6092. [PubMed] 50. Ho B T, Huo Y Y, Lu J G, Newman R A, Levin V A. Anticancer Drugs. 1992;3:91–94. [PubMed] 51. Driessen B, Reimann W, Selve N, Friderchs E, Bültmann R. Brain Res. 1994;666:182–188. [PubMed]
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