|
|
Proc Natl Acad Sci U S A. 1996 December 24; 93(26): 15435–15439. | PMCID: PMC26422 |
Copyright © 1996, The National Academy of Sciences of the USA Neurobiology A novel heat-activated current in nociceptive neurons and its
sensitization by  bradykinin Paolo Cesare and Peter McNaughton* Department of Physiology, Division of Biomedical Sciences, King’s College London, Strand, London WC2R 2LS, United Kingdom Received July 1, 1996; Accepted September 16, 1996. Pain differs from other sensations in many respects. Primary
pain-sensitive neurons respond to a wide variety of noxious stimuli, in
contrast to the relatively specific responses characteristic of other
sensory systems, and the response is often observed to sensitize on
repeated presentation of a painful stimulus, while adaptation is
typically observed in other sensory systems. In most cases the cellular
mechanisms of transduction and sensitization in response to painful
stimuli are not understood. We report here that application of pulses
of noxious heat to a subpopulation of isolated primary sensory neurons
rapidly activates an inward current. The ion channel activated by heat
discriminates poorly among alkali cations. Calcium ions both carry
current and partially suppress the current carried by other ions. The
current is markedly increased by bradykinin, a potent algogenic
nonapeptide that is known to be released in vivo by
tissue damage. Phosphatase inhibitors prolong the sensitization caused
by bradykinin, and a similar sensitization is caused by activators of
protein kinase C. We conclude that bradykinin sensitizes the response
to heat by activating protein kinase C. Keywords: pain, sensory transduction, protein kinase
C, sensory neuron The dorsal root ganglion (DRG) contains a mixed population of
primary sensory neurons that send myelinated and unmyelinated axons to
the skin surface and elsewhere within the body, and convey information
about a wide variety of sensory stimuli, including touch, temperature,
and noxious stimuli. Noxious stimuli are detected both by terminals of
unmyelinated C fibers, which typically respond to noxious chemical,
mechanical, and heat stimuli, and by terminals of small myelinated A
fibers, which often exhibit more specific responses ( 1– 5). From this
mixed population of sensory neurons, a population consisting mainly of
nociceptors can be selected simply by choosing small neurons, because a
large fraction of the smallest neurons (<25 μm in diameter) in the
DRG possess unmyelinated axons and respond to noxious stimuli ( 6,
7). The cellular basis of the response to painful stimuli has, in contrast
with other sensations, been little investigated. This is partly because
of the difficulty of isolating the fine pain-sensitive nerve terminals,
and partly because of the difficulty of defining what is a painful
stimulus. Rather than attempting to study nociceptive nerve terminals
themselves, we have used cultured neurons from the DRG as a model
because many of the properties of nociceptive neurons in
vivo are known to be replicated in small cultured neurons from the
DRG ( 8– 12). We have investigated the responses of these neurons to
heat stimuli of an intensity that would produce a sensation of pain and
avoidance responses in a human or animal subject. One objective was to
investigate at a cellular level how noxious heat stimuli excite
nociceptive neurons. To our knowledge, no studies to date have
investigated the transduction pathways underlying the excitation of
nociceptors by heat. A second objective was to investigate the cellular
basis of the process of sensitization, which in vivo
produces an increasing sensation of pain in response to repeated
application of a noxious heat stimulus. Preparation and Recording. Neonatal (<5 days old) Wistar rats
were decapitated, and about 40 DRG were removed. Neurons were isolated
and cultured in the presence of nerve growth factor (100 ng/ml,
Promega) in 35-mm-diameter plastic culture dishes for 4–6 days ( 12).
Small neurons (<25 μm diameter) were selected and whole-cell
voltage-clamped at −70 mV using patch-clamp electrodes of tip diameter
≈0.7 μm (bubble number ≈5.5) and a List EPC7 amplifier. Pipette
solution contained 135 mM KCl, 0.2 mM CaCl 2, 1.6 mM
MgCl 2, 2 mM EGTA, 2.5 mM MgATP, 0.2 mM LiGTP, and 10 mM
Hepes (pH 7.3). Hanks’ solution used for experiments contained 130 mM
NaCl, 4 mM KCl, 5 mM CaCl 2, 1 mM MgCl 2, 4 mM
d-glucose, and 10 mM Hepes (pH 7.4). Solutions were changed
within 20 msec (see Fig. B) using an automated multipipe
solution changer (BioLogic RSC-100). One solution was heated using a
Peltier device regulated to within ±0.1°C by a proportional gain
feedback controller. Temperature was monitored by a miniature
thermocouple immediately before the solution entered the bath. Linear
temperature ramps changing from 28°C to 49°C in 10 sec were
generated by positioning the neuron in front of the thermally
controlled pipe and switching on the Peltier device; data such as that
in Figs. A and C are plots of current
versus temperature measured by the miniature thermocouple and corrected
for the 200 msec transit time from the thermocouple to the cell. Data
were acquired at (typically) 10 kHz using pclamp software
(Axon Instruments, Foster City, CA).
For experiments on ionic selectivity, the internal solution was
modified by substituting Cs+ for K+ to suppress
voltage-activated K+ currents and by reducing divalent ion
concentrations to avoid possible block of the heat-sensitive current
from the inner membrane surface. Pipette solution composition was 135
mM CsCl, 2 mM EGTA, 2.5 mM MgATP, 0.2 mM LiGTP, and 10 mM Hepes (pH
7.3). External solutions contained 100 μM Cd2+ to
suppress calcium currents and 10 mM Hepes and 4 mM
d-glucose, in addition to the stated concentrations of NaCl
and CaCl2. Nominal zero-Ca solutions do not contain EGTA,
which would chelate Cd2+, and [Ca2+] was
found to be 10 μM by atomic absorption spectrophotometry.
N-Methyl-d-glucamine was used a Na+
substitute. Basic Characteristics of the Response to Heat. Recordings of
membrane current obtained from small DRG neurons using the whole-cell
patch-clamp technique are shown in Fig. . In
all neurons, a rapid increase in inward current was observed in
response to a noxious thermal stimulus, but two different types of
response were clearly distinguishable. In neurons that we classified as
heat-insensitive, the only current change was a small increase in
inward current, which was activated as rapidly as the solution could be
changed. The instantaneous current change was also observed in neurons
classified as heat-sensitive, but in these latter neurons a much larger
increase in inward current was activated with a delay following the
instantaneous current change (see Fig. B). Neurons were
considered to be heat-sensitive if the delayed membrane current
exceeded 50 pA in response to a temperature step to 49°C.
Approximately 56% of small neurons (<25 μm in diameter) were
heat-sensitive. The form of the dependence of the delayed heat-sensitive current on
temperature is shown in Fig.
A (trace 1). The activation of current in
heat-sensitive neurons was strikingly nonlinear, with the major change
in inward current occurring above 42°C. A similar dependence on
temperature of both the subjective sensation of pain and of the
frequency of action potentials in nociceptive nerve fibers is observed
in vivo ( 1– 4). The responses of receptors sensitive to
non-noxious warmth, in contrast, increases over the range 30–40°C
and declines above ≈44°C ( 13). No responses to temperature with
properties similar to those of warm-sensitive neurons were observed in
the present study. The current versus temperature relation of
temperature-insensitive neurons was much more linear than in
heat-sensitive neurons (trace 2 in Fig. A), as
expected if the current in these neurons resulted from a physical
process such as a nonspecific change in membrane resistance or in the
pipette seal resistance. Fig. B (solid circles) shows that a monotonic
relationship between the magnitude of the current increase and the
half-time of activation was observed in different neurons, with the
largest current associated with the shortest activation time. The mean
time to half-activation was 34.8 ± 2.6 msec (mean ± SEM,
n = 36), comparable to the observed activation time in
heat-sensitive C-fiber afferents in vivo of <100 msec ( 32). Ionic Selectivity of the Heat-Sensitive Channel. The ionic
selectivity of the current activated by heat is examined in Fig.
. The reversal potential of the current with
Na+ as the principal extracellular cation and
Cs+ as the principal internal cation was close to 0 mV,
showing that the ion channel activated by heat discriminates poorly
between Na+ and Cs+. When the larger cation
N-methyl-d-glucamine replaced Na+ in
the external solution, however, the reversal potential was more
negative (see Fig. legend), showing that NMG is much less permeable
than Na+ or Cs+. Calcium passes through the
channel, as shown by its effect on the reversal potential in the
absence of Na+. From the values of reversal potential given
in the legend to Fig. and using the constant-field equation (see ref.
14, equations 1-13 and 13-11), we calculate
PNa:PCs:PNMG:PCa
= 1:1.24:0.08:1.28, respectively.
Calcium ions also have a blocking effect on the current carried by
other cations, as can be seen from the observation that addition of 2
mM Ca 2+ reduces the heat-sensitive current when compared
with that observed in nominal zero-Ca 2+ solution (Fig. ).
Calcium, therefore, competes with Na + for a binding site in
or near the channel. In both the ionic selectivity and the block by
calcium, the ion current activated by heat resembles most closely the
current gated by cyclic nucleotides in a wide variety of sensory and
other neurons ( 15, 16), although it also has some similarity to the
voltage-activated calcium channel, where calcium both carries current
and blocks current carried by alkali cations. Sensitization of the Heat Response. No sensitization of
the response of the isolated neurons used in the present study was
observed on repeated application of heat, in contrast to the behavior
of heat-sensitive neurons in vivo in which strong
sensitization is observed ( 1– 4). However, application of bradykinin
(BK), a potent pain-inducing nonapeptide generated by the release of
proteolytic enzymes from damaged cells in injured or inflamed tissues
( 17– 21), caused a striking sensitization of the response to
noxious temperatures (Fig. A). As described by other authors ( 22, 23), BK was observed to activate an
inward current in some, but not all, small DRG neurons (e.g., Fig.
). About half (27 of 65) of heat-sensitive
neurons responded to BK with a current exceeding 10 pA in magnitude.
The heat-sensitive current elicited by a 49°C temperature step in
these BK-responsive neurons increased to 209 ± 19% of control
(mean ± SEM; n = 12) at the peak of the response
to BK. In the same cells, BK speeded the response to a heat pulse, with
the time to half-activation of current decreasing to 65 ± 2.9%
of its value before BK application (see inverted triangles in Fig.
B). To rule out the possibility that the
sensitization of the heat response was directly related to the
activation of membrane current by BK, we also measured sensitization 2
min after BK application, when the BK-induced current had declined to
zero (see Fig. A). Sensitization at this time was still
significant, at 155 ± 15% of control ( n = 15).
The sensitization caused by BK was limited to those neurons in which BK
caused a significant membrane current response, because no enhancement
of the heat response was observed in 38 of 38 heat-sensitive neurons in
which BK application did not elicit a significant inward current.
Sensitization Caused by Activation of Protein Kinase C
(PKC). A possible mediator of the sensitizing effect of BK is
PKC, which is known to be activated by BK via a G protein-coupled
pathway ( 22, 24– 26). Like BK, the specific PKC activator phorbol
12-myristate 13-acetate (PMA) both increased the membrane current
activated by noxious heat and speeded the rate of activation (Fig.
B). The mean increase in current in nine neurons was to a
value 532 ± 123% of control, and the half-time of activation was
reduced to 39 ± 7% of control (see also Fig.
B, open circles). Both effects are similar to those
of BK, although the magnitude is larger, presumably because a more
complete activation of PKC is obtained with PMA than with BK. Fig.
C demonstrates that the threshold for activation of
membrane current by temperature is shifted to lower temperatures by PKC
activation, an effect that is similar to that observed in
vivo after sensitization of the skin by noxious heat or injection
of BK ( 1– 4, 27). The time course of sensitization of the response to heat by BK is shown
in Fig. A. The time course of onset of the sensitization
was rapid, but the effect considerably outlasted the transient inward
current elicited by BK. The sensitization therefore is not simply
connected to the opening of ionic channels by BK, as might occur if the
current activated by BK was heat-sensitive. In Fig. B,
application of the phosphatase inhibitor calyculin A greatly slowed the
rate of recovery of sensitization after BK application, as expected if
the sensitization caused by BK is due to phosphorylation of the
heat-sensitive channel or of a component in the pathway modulating the
channel. Note also that calyculin A did not affect the time course of
current elicited by BK, showing that the deactivation of the membrane
current activated by BK does not depend on a process involving
dephosphorylation. Further evidence in support of a role of phosphorylation by PKC in
sensitization of the heat response is shown in Fig.
A. Application of PMA caused a
long-lasting sensitization, and further applications of PMA were
without effect, as expected from the known long-term activation of PKC
by PMA ( 28). Staurosporine, which inhibits PKC by competing for the ATP
binding site ( 29), rapidly reverses the sensitization caused by PMA
(Fig. B).
The results reported here show that primary nociceptive neurons
respond to noxious heat with a rapid, but not instantaneous, activation
of an inward membrane current. The ion channel activated by heat
distinguishes poorly between monovalent cations. Calcium ions can also
carry current through the channel, but calcium has in addition a
blocking action, because the current carried by monovalent ions is seen
to be partially suppressed by the presence of external calcium ions.
These observations suggest that the heat-activated channel belongs to
the same family as the channel activated in visual and olfactory
receptors by cyclic nucleotides, both of which exhibit qualitatively
similar properties. It remains an open question whether the
heat-sensitive channel is gated directly by heat or whether an
intracellular messenger is involved, as in the case of the cyclic
nucleotide-gated channels. Unlike nociceptive neurons in vivo, isolated neurons do not
exhibit sensitization in response to repeated application of heat,
suggesting that the process of sensitization is not intrinsic to the
neuron but is instead caused by factors released by damage to adjacent
cells. One such factor known to be released by tissue damage is BK, a
potent algogenic nonapeptide that acts at B 2 receptors to
release intracellular calcium and activate PKC. We find that BK
sensitizes the heat response and that the mechanism involves activation
of PKC. PKC activation produces a shift in the threshold of the
heat-sensitive current to lower temperatures, in agreement with
in vivo observations in which damaged tissue becomes
sensitive to normally non-noxious temperatures ( 1– 4). The experiments reported here show that BK has two distinct actions of
excitation and sensitization on nociceptive neurons. ( i) BK
excites neurons by eliciting an inward current, which depolarizes the
neuron and generates action potentials ( 9, 10, 22, 23). ( ii)
Sensitization of the heat response is caused by PKC activation, and
recovery from sensitization depends on dephosphorylation because it is
prevented by phosphatase inhibitors (Fig. B). Excitation
and sensitization follow different time courses (Fig. ). Although the
internal transmitter mediating the activation of inward current by BK
remains to be established, it is clear from the results reported here
that sensitization of the heat response is mediated by PKC. The sensitizing effect of BK on the heat response is distinct from the
sensitization caused by prostaglandins, in which the excitability of
the nociceptive neuron is increased by lowering the threshold of
activation of a tetrodotoxin-insensitive Na + current ( 30).
The current modified by prostaglandins is strongly voltage-sensitive,
is not activated at the resting potential and is inactivated on
maintained depolarization. The heat-sensitive current modified by BK,
on the other hand, is neither activated nor inactivated by changes in
membrane potential. It is perhaps not surprising, in view of the range
of algogenic and sensitizing stimuli to which nociceptive neurons must
respond, that different sensitizing agents act by distinct pathways. The importance for inflammation of the heat response of nociceptors is
underlined by the recent observation that neurogenic inflammation (the
flare response) depends on activation of a specific class of
heat-sensitive nociceptor and is not elicited by stimulation of
polymodal nociceptors ( 5). A further link between the heat response and
neurogenic inflammation is suggested by the finding that activation of
PKC in isolated neurons, which from the results in the present paper
will sensitize the heat response, enhances the release of the
neuropeptides thought to be responsible for eliciting neurogenic
inflammation ( 31). The results reported here raise the possibility of biophysical study of
the processes underlying activation and sensitization of pain-sensitive
neurons by heat. They also suggest possible therapeutic approaches to
the relief of pain by the use of PKC inhibitors, perhaps by targeting
particular isoforms of PKC involved in the sensitization process. We thank Prof. Carlos Belmonte for helpful discussions and Roger
Stoughton for participation in early experiments. This work was
supported by the European Commission. 1. Besson J M, Chaouch A. Physiol Rev. 1987;67:67–155. [PubMed] 2. Treede R D, Meyer R A, Raja S N, Campbell J N. Prog Neurobiol. 1992;38:397–421. [PubMed] 3. Levine J D, Fields H L, Basbaum A I. J Neurosci. 1993;13:2273–2286. [PubMed] 4. Meyer R A, Campbell J N, Srinivasa N R. In: Textbook of Pain. 3rd Ed. Melzack R, Wall P, editors. Edinburgh: Churchill Livingstone; 1994. pp. 13–44. 5. Lynn B, Schutterle S, Pierau F K. J Physiol (London). 1996;494:587–593. [PubMed] 6. Lawson S N, Perry M J, Prabhakar E, McCarthy P W. Brain Res Bull. 1993;30:239–243. [PubMed] 7. Hoheisel U, Mense S, Scherotzke R. Anat Embryol. 1994;189:41–49. [PubMed] 8. Baccaglini P I, Hogan P G. Proc Natl Acad Sci USA. 1983;80:594–598. [PubMed] 9. Rang H P, Bevan S, Dray A. Br Med Bull. 1991;47:534–548. [PubMed] 10. Rang H P, Bean S, Dray A. In: Textbook of Pain. 3rd Ed. Melzack R, Wall P, editors. Edinburgh: Churchill Livingstone; 1994. pp. 57–78. 11. Gold M S, Dastmalchi S, Levine J D. Neuroscience. 1996;71:265–275. [PubMed] 12. Gilabert, R. & McNaughton, P.A. (1996) J.
Neurosci. Methods, in press. 13. Darian-Smith I. Handbook of Physiology. Vol. 3. Bethesda, MD: Am. Physiol. Soc.; 1984. pp. 879–913. 14. Hille B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer; 1992. 15. Hodgkin A L, McNaughton P A, Nunn B J. J Physiol (London). 1985;358:447–468. [PubMed] 16. Finn J T, Grunwald M E, Yau K W. Annu Rev Physiol. 1996;58:395–426. [PubMed] 17. Steranka L R, Manning D C, DeHaas C J, Ferkany J W, Borosky S A, Connor J R, Vavrek R J, Stewart J M, Snyder S H. Proc Natl Acad Sci USA. 1988;85:3245–3249. [PubMed] 18. Dray A, Perkins M. Trends Neurosci. 1993;16:99–104. [PubMed] 19. Walker K, Perkins M, Dray A. Neurochem Int. 1995;26:1–16. [PubMed] 20. Hall J M, Geppetti P. Neurochem Int. 1995;26:17–26. 21. Dray A. Br J Anaesth. 1995;75:125–131. [PubMed] 22. Burgess G M, Mullaney I, McNeill M, Dunn P M, Rang H P. J Neurosci. 1989;9:3314–3325. [PubMed] 23. Nicol G D, Cui M. J Physiol (London). 1994;480:485–492. [PubMed] 24. Thayer S A, Perney T M, Miller R J. J Neurosci. 1988;8:4089–4097. [PubMed] 25. McGuirk S M, Dolphin A C. Neuroscience. 1992;49:117–128. [PubMed] 26. Jones S, Brown D A, Milligan G, Wilier E, Buckley N J, Caulfield M P. Neuron. 1995;14:399–405. [PubMed] 27. Kumazawa T, Mizumura K, Minagawa M, Tsujii Y. J Neurophysiol. 1991;66:1819–1824. [PubMed] 28. Nishizuka Y. Nature (London). 1988;334:661–665. [PubMed] 29. Wilkinson S E, Hallam T J. Trends Pharmacol Sci. 1994;15:53–57. [PubMed] 30. Gold M S, Reichling D B, Shuster M J, Levine J D. Proc Natl Acad Sci USA. 1996;93:1108–1112. [PubMed] 31. Barber L A, Vasko M R. J Neurochem. 1996;67:72–80. [PubMed] 32. Treede R D, Meyer R A, Raja S N, Campbell T N. J Physiol (London). 1995;483:747–758. [PubMed]
|
This article has been 
