1Institute of Physiology and Pathophysiology, Johannes Gutenberg University, D-55099 Mainz; and 2Center of Physiology and Pathophysiology, Georg August University, D-37073 Göttingen, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Kirschstein, Timo,
Wolfgang Greffrath,
Dietrich Büsselberg, and
Rolf-Detlef Treede.
Inhibition of Rapid Heat Responses in Nociceptive Primary Sensory
Neurons of Rats by Vanilloid Receptor Antagonists.
J. Neurophysiol. 82: 2853-2860, 1999.
Recent
studies demonstrated that heat-sensitive nociceptive primary sensory
neurons respond to the vanilloid receptor (VR) agonist capsaicin, and
the first cloned VR is a heat-sensitive ion channel. Therefore we
studied to what extent heat-evoked currents in nociceptive dorsal root
ganglion (DRG) neurons can be attributed to the activation of native
vanilloid receptors. Heat-evoked currents were investigated in 89 neurons acutely dissociated from adult rat DRGs as models for their own
terminals using the whole cell patch-clamp technique. Locally applied
heated extracellular solution (effective temperature ~53°C) rapidly
activated reversible and reproducible inward currents in 80% (62/80)
of small neurons (32.5 µm), but in none of nine large neurons
(P < 0.001,
2 test). Heat and
capsaicin sensitivity were significantly coexpressed in this
subpopulation of small DRG neurons (P < 0.001,
2 test). Heat-evoked currents were accompanied by an
increase of membrane conductance (320 ± 115%; mean ± SE, n = 7), had a reversal potential of
5 ± 2 mV (n = 5), which did not differ from
that of capsaicin-induced currents in the same neurons (4 ± 3 mV), and were carried at least by Na+ and Ca2+
(pCa2+ > pNa+). These observations are
consistent with the opening of temperature-operated nonselective cation
channels. The duration of action potentials was significantly higher in
heat-sensitive (10-90% decay time: 4.45 ± 0.39 ms,
n = 12) compared with heat-insensitive neurons (2.18 ± 0.19 ms, n = 6; P < 0.005, Student's t-test), due to an inflection in
the repolarizing phase. This property as well as capsaicin sensitivity
and small cell size are characteristics of nociceptive DRG neurons.
When coadministered with heat stimuli, the competitive VR antagonist
capsazepine (1 µM to 1 mM) significantly reduced heat-evoked currents
in a dose-dependent manner (IC50 13 µM, Hill slope
0.58, maximum effect 75%). Preincubation for 12-15 s shifted the
IC50 by ~0.5 log10 units to an estimated
IC50 of ~4 µM. The noncompetitive VR antagonist
ruthenium red (5 µM) significantly reduced heat-evoked currents by
33 ± 6%. The effects of both VR antagonists were rapidly
reversible. Our results provide evidence for a specific activation of
native VRs in nociceptive primary sensory neurons by noxious heat. The
major proportion of the rapid heat-evoked currents can be attributed to
the activation of these temperature-operated channels, and noxious heat
may be the signal detected by VRs under physiological conditions.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Polymodal C- and A-fiber nociceptors in the
skin of primates and humans respond within a few milliseconds to fast
temperature increases generated by infrared lasers (Bromm and
Treede 1983
; Tillman et al. 1995
). These short
latencies observed in vivo suggest a rapid direct transduction
mechanism for heat stimuli at the peripheral endings of small dorsal
root ganglion (DRG) neurons (Treede et al. 1995
,
1998
). DRG neurons are primary sensory neurons comparable to olfactory or visual receptor cells, but in contrast to
vision and olfaction, little is known about the transduction mechanisms
of nociception and pain (Belmonte 1996
).
Recent in vitro studies of the underlying membrane currents have
supported the existence of a rapid heat transduction mechanism in
nociceptive primary sensory neurons of the rat (Cesare and McNaughton 1996; Dittert et al. 1998
;
Kirschstein et al. 1997
; Nagy and Rang
1999
). The somata of DRG neurons are used as models for their
own peripheral terminals (Vyklicky and
Knotková-Urbancová 1996
) because the terminals in
the skin are inaccessible for adequate electrophysiological studies
(patch-clamp) due to their small size and tough surrounding tissue.
Brief heat stimuli of <1 s duration were found to elicit inward
currents in DRG neurons of neonatal rats after 4-6 days in primary
culture (Cesare and McNaughton 1996
) and within 4-30 h
after acute dissociation from adult rats (Kirschstein et al.
1997
). The activation was rapid but not instantaneous with a
half-time of maximal activation of 35 ms and with a threshold temperature of ~43°C (Cesare and McNaughton 1996
).
These findings suggest a novel mechanism of rapid cellular signaling by
temperature-operated ion channels.
We have previously demonstrated that heat sensitivity in acutely
dissociated DRG neurons is coexpressed with sensitivity to capsaicin
(Kirschstein et al. 1997), the active ingredient in hot
chili peppers that is a selective activator of nociceptive afferents
(Szallasi and Blumberg 1996
). This observation would be
compatible with two possibilities: 1) capsaicin and heat act on different transduction pathways that are co-localized in the same
nociceptive neurons, or 2) capsaicin and heat act on a
common transduction pathway. The second hypothesis is more likely
because the first cloned vanilloid receptor (VR1) is a nonselective
cation channel that is activated by the vanilloid receptor agonist
capsaicin and by raising the temperature to 40-45°C (Caterina
et al. 1997
; Tominaga et al. 1998
). On the other
hand, additional heat-transduction mechanisms have been described,
which differ from the rapid transduction pathway with respect to
1) threshold temperature and response latency (Treede
et al. 1995
, 1998
), 2) correlation
with VR expression (Nagy and Rang 1999
), and
3) dependence on intracellular calcium (Reichling and
Levine 1997
).
The aim of this study was to test whether and to what extent
heat-evoked inward currents in nociceptive DRG neurons can be attributed to the activation of native vanilloid receptors. For this
purpose, we characterized the electrophysiological properties of
heat-evoked inward currents and of the neurons that express these
currents, tested responses of the neurons to the VR-agonist capsaicin,
and determined the effects of the two known vanilloid receptor
antagonists, ruthenium red (RR) (Amann and Maggi 1991) and capsazepine (CPZ) (Bevan et al. 1992
), on rapid
heat-evoked currents in acutely dissociated DRG neurons of adult rats.
Preliminary accounts of this study have appeared in abstract form
(Kirschstein et al. 1998
).
This paper contains essential parts of the dissertation of T. Kirschstein.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The preparation, neuron dissociation, electrophysiological
recordings, and heat stimulation were done as previously described (Kirschstein et al. 1997). Briefly, adult
Sprague-Dawley rats (120-270 g) of both sexes were deeply anesthetized
with diethylether and rapidly decapitated (see Greffrath et al.
1998
for detail). This method is in accordance with German
national law and the rules of local ethical committees. The spine was
chilled at 4°C in F12-Dulbecco's modified Eagle's medium (Sigma)
saturated with carbogen gas (95% O2-5%
CO2) and additionally containing 30 mM NaHCO3 (Merck, Darmstadt, Germany),
100,000 units l
1 penicillin and 100 mg l
1
streptomycin (Sigma). Thoracic and lumbar DRGs (8-15) were quickly dissected and freed from connective tissue. Neurons were dissociated in
an incubation chamber enriched with carbogen gas at 37°C using collagenase CLS II (5-10 mg ml
1, 40-50 min; Biochrome,
Berlin, Germany) and trypsine (0.2-1 mg ml
1, 10-12 min;
Sigma) dissolved in the F12 medium. After trituration (4-6 times with
a Pasteur pipette) neurons were plated on 35-mm-diam culture dishes,
which also served as recording chambers, and stored at 37°C in a
humidified 5% CO2 atmosphere before being used for electrophysiological recordings 3-12 h (up to 30 h in some cases) after dissociation.
Electrophysiology
Only round or oval-shaped neurons without any processes were
included in this study. The average of the major and the minor diameter
was used to measure the size of oval shaped neurons. Whole cell
patch-clamp experiments were performed in carbogen gas saturated F12
medium (pH 7.4) at room temperature (RT) using an Axopatch 200A
amplifier (Axon Instruments) in voltage-clamp mode at a holding
potential of 80 mV controlled by pCLAMP6 software. Data were also
registered on a chart recorder (L6512, Linseis, Selb, Germany). Patch
pipettes were fabricated from borosilicate glass capillaries
(Hilgenberg, Malsfeld, Germany) using a horizontal micropipette puller
(P-87, Sutter) and filled with a solution containing (in mM) 160 KCl,
8.13 EGTA, and 10 HEPES (pH 7.2; RTip = 5.3 ± 0.2 M
, mean ± SE). Cell diameter,
cross-sectional area, and membrane capacitance were measured, and
excitability was tested by depolarizing voltage steps for each neuron.
Cells lacking a fast inward current with a reversal potential close to
the equilibrium potential of sodium followed by a prolonged outward
current were excluded from further investigation. Experiments in
current-clamp mode were performed to measure the resting membrane
potential (RMP) of each neuron and to investigate single action
potentials (APs) elicited by short (3 ms) depolarizing current pulses
in neurons that were hyperpolarized by constant current injection resulting in membrane potentials between
70 and
80 mV
(n = 18). Inflections in the repolarizing phase were
qualitatively detected as second negative peak in the first derivative
(dV/dt) of each AP, the duration of
repolarization was quantitatively assessed by the 10-90% decay time.
Heat stimulation, drug application, and solutions
Application of ~50 µl of heated extracellular solution
through a puffing system fixed on a micromanipulator was used to elicit heat-evoked currents. Control measurements with a fast temperature sensor (BAT-12, Physitemp; = 5 ms) in place of the neurons
revealed an effective peak temperature of ~53°C, a rise time of
~250 ms, and a decay with a time constant of ~20 s. Effects were
compared with those of application of the same amount of medium at RT. Heat stimuli with or without vanilloid receptor antagonists and control
applications at RT were repeated 2-10 times, and the elicited currents
were averaged. A neuron was considered heat sensitive when the
heat-evoked inward current was significantly greater than any
fluctuations caused by superfusion of solution at RT (unpaired 1-tailed
Student's t-test for each neuron, P < 0.05). Heating the buffered extracellular solution may change its pH, and acid solutions of pH 6.2 are known to activate nociceptive DRG
neurons (Bevan and Yeats 1991
). The pH of a
HEPES-buffered solution decreases while heating (e.g., pH 7.1 at 50°C
according to the Henderson-Hasselbalch equation). In contrast higher
temperatures increase the pH of a
NaHCO3/CO2 buffer, because
the solubility of CO2 is reduced and thus
reverses the HEPES effect. Off-line measurements showed that the pH of
the F12 medium maximally changed in a range of 7.28-7.52 while heating
to 50°C and cooling down to RT. Furthermore, the absence of color
changes of the pH indicator phenol red, which was present in the
extracellular solution, verified on-line that pH throughout all
experiments remained well above 6.8. The membrane conductance was
measured in voltage-clamp mode by hyperpolarizing pulses (5 mV, 10 ms,
50 s
1), and conductance changes were determined
at the maximum amplitude of heat evoked currents.
Reversal potentials of heat- and capsaicin-induced currents were
measured as described by Liu et al. (1997) using fast
depolarizing ramps (
80 to +30 mV in 200 ms every 550 ms). In these
experiments patch pipettes were filled with a potassium-free solution
containing (in mM) 140 CsCl, 10 HEPES, 10 EGTA, and 4 MgCl2 (adjusted to pH 7.2). Tetrodotoxin (TTX,
100 µM; Sigma) and nifedipine (1 µM; Sigma) were added to the
extracellular solution to block voltage-gated Na+
and Ca2+ channels. These neurons were not
included in the general statistics of heat-sensitive cells. To examine
charge carriers of heat-induced currents, Na+ in
the extracellular solution was replaced by
N-methyl-D-glucamine (NMDG; Sigma). In some of
these experiments extracellular Ca2+ was raised
to 10 mM. Reversibility of ion replacement experiments was tested by
final application of heated solution at physiological ion composition.
Capsaicin (Sigma) was initially dissolved in ethanol, diluted to its
final concentration in F12 medium, and applied through the puffing
system as described above. CPZ (dissolved in DMSO; purchased from RBI,
Cologne, Germany) and RR (in extracellular solution; RBI) were prepared
as concentrated stock solutions, diluted to final concentration in F12
medium, and applied either at RT or ~53°C in the same way. Because
CPZ solutions contained final concentrations of 0.01-10% DMSO, the
same amount was added to all solutions applied in CPZ experiments, too.
In the concentration range of 0.01-1%, DMSO alone did not elicit any
visible effects, but with 10% DMSO the proportion of neurons that
could not be kept for the full protocol was increased. Reversibility of
antagonist action was tested by reapplication of heated extracellular
solution without any agents (n = 21). Only neurons
tested for reversibility were included for further statistical analysis.
Data analysis
Off-line measurements and statistical analysis were done using
pCLAMP6 (Axon Instruments) and EXCEL 5.0 (Microsoft). Data are
presented as means ± SE. Treatment effects were statistically analyzed by Student's t-test for paired data and
2 test for analysis of incidences. Dose
dependence of the CPZ-induced inhibition was analyzed by a repeated
measures ANOVA with Student-Newmann-Keuls post hoc test for ordered
means (STATISTICA 4.5, StatSoft). Error probabilities P
0.05 were considered statistically significant. Fitting of the
dose-response function was done using GraphPad Prism 2.01 (GraphPad Software).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electrophysiological properties of heat-sensitive DRG neurons
Whole cell patch-clamp experiments were performed on 89 acutely
dissociated DRG neurons clamped at 80 mV. Brief superfusion of heated
extracellular solution (effective temperature ~53°C) activated a
significantly greater inward current than the same amount of solution
applied at RT (see METHODS) in 62 of the 80 small neurons
(80%; see Fig. 1A for an
example). In contrast, none of nine large neurons (diameter >32.5
µm) significantly responded to heat (P < 0.001,
2 test). In the majority of heat-sensitive
neurons examined (47 of 62) application of solution at RT did not
elicit any change in holding currents. Currents elicited by control
applications at RT were even significantly smaller in heat-sensitive
(10 ± 3 pA, n = 62) than in heat-insensitive
neurons (53 ± 17 pA, n = 18, P < 0.001). Within heat-insensitive neurons, however, the inward currents
elicited by solutions at either ~53°C (62 ± 16 pA) or RT
(53 ± 17 pA) did not differ significantly. Heat-evoked currents
were reproducible at 1 to 15 s interstimulus intervals (1st: 279 ± 43 pA; 2nd: 310 ± 44 pA, n = 62, n.s.) and
were accompanied by an increase in membrane conductance (Fig.
1B), consistent with the opening of temperature-operated
channels. The averaged maximum increase of the membrane conductance
during the peak of heat-evoked currents was 320 ± 115%
(n = 7).
|
Within this subpopulation of small DRG neurons (diameter 32.5 µm),
heat-sensitive and heat-insensitive neurons did not differ in diameter
(27.5 ± 0.3 µm for heat-sensitive, 27.4 ± 0.8 µm for heat-insensitive neurons) nor in whole cell capacitance (23.2 ± 0.9 pF vs. 21.2 ± 1.7 pF). RMP was more depolarized in
heat-sensitive (
46 ± 1 mV) than in heat-insensitive small
neurons (
52 ± 2 mV, P = 0.033), but there was
no correlation between RMP, measured in current-clamp mode, and
heat-evoked current in neurons clamped at
80 mV in voltage-clamp mode
(r =
0.17, n.s., n = 62). Moreover, the three cells with the most negative RMP of about
70 mV exhibited three of the largest heat responses (>600 pA). Therefore heat sensitivity is not a function of a depolarized membrane potential.
A prominent inflection in the repolarizing phase of APs evoked by
constant current injection was observed in 11 of 12 heat-sensitive neurons (Fig. 2), whereas a smaller
inflection was detectable only in 1 of 6 heat-insensitive small neurons
(P < 0.005, 2 test). As a
consequence of this "shoulder," action potentials in heat-sensitive
neurons displayed significantly longer repolarizing phases (10-90%
decay time: 4.45 ± 0.39 ms, n = 12) than
heat-insensitive neurons (2.18 ± 0.19 ms, n = 6;
P < 0.005).
|
To test whether heat and capsaicin sensitivity are coexpressed in DRG
neurons, the effect of capsaicin (1-10 µM) was examined in 29 neurons. None of 5 large neurons (diameter >32.5 µm) but 16 of 24 small neurons (32.5 µm) were excited by capsaicin
(P < 0.01,
2 test). Only 1 of
15 heat-sensitive neurons was not excited by capsaicin, whereas 12 of
14 heat-insensitive neurons were also capsaicin insensitive
(P < 0.001,
2 test). Two
heat-insensitive neurons were excited by capsaicin, but one of those
neurons developed heat sensitivity after the capsaicin-induced
excitation, the remaining neuron was not tested with heat after
capsaicin. Time-to-peak of capsaicin-induced currents varied between
300 ms and 65 s with a mean of 14 ± 5 s
(n = 14). Capsaicin often induced multiple current
peaks, and for the value above the time to the maximum current was
measured (2,950 ± 700 pA). Whereas some capsaicin-induced
currents were almost as fast as heat-evoked currents, others were as
slow as the long-latency capsaicin currents reported by others
(Liu and Simon 1996
; Petersen et al.
1996
).
The reversal potentials of heat-evoked currents and capsaicin-induced currents were determined in five small neurons (diameter 28 ± 0.8 µm) with current-voltage (I-V) curves elicited by fast depolarizing ramps using Cs+ in the intra- and TTX (100 µM) and nifedipine (1 µM) in the extracellular solution. These agents did not abolish all inward currents elicited by the initial depolarizing ramp, which may be due to the presence of, e.g., TTX-resistant sodium channels. However, these small currents disappeared during repeated ramps. Therefore the repeated-ramps protocol was run until those currents had inactivated (within ~100 cycles), before heat or capsaicin were applied to determine reversal potentials. The reversal potential of heat-evoked currents was 5 ± 2 mV, that of the initial phase of capsaicin-induced currents (1 µM) in the same neurons 4 ± 3 mV (n.s.). Within the variability between neurons, the reversal potentials of heat-evoked and capsaicin-induced currents were highly correlated (r = 0.93, P < 0.05, n = 5). The reversal potential near 0 mV can be explained by a nonselective cation channel, that is outwardly permeable to Cs+. In conclusion, heat-evoked currents and capsaicin-induced currents are both carried through similar nonspecific cation channels.
We therefore tested whether the rapid heat-induced inward currents are carried by Na+ and/or Ca2+. When Na+ was replaced by NMDG (n = 15), heat-evoked currents were reversibly reduced to 34 ± 4% of the mean currents before and after ion replacement (P < 0.001). When Ca2+ in the Na+-free extracellular solution was raised to 10 mM (n = 7), heat-evoked currents were enhanced to 478 ± 120% (P < 0.05). Although in these experiments the proportion of putative charge carriers was smaller than in physiological extracellular solution (10 vs. 145 mM) heat-evoked currents did not differ significantly from the normalized controls (129 ± 38% vs. 100%; n.s.), indicating a higher permeability for Ca2+ than for Na+ ions.
Effects of vanilloid receptor antagonists on heat-evoked currents
Heat-sensitive neurons were tested for the effects of vanilloid
receptor antagonists on heat-evoked inward currents to examine pharmacologically, whether and to what extent native vanilloid receptors contribute to rapid heat-transduction mechanisms. When the
competitive antagonist CPZ (10 µM) was added to the heated extracellular solution (Fig. 3,
A-C) heat-evoked inward currents were significantly reduced
by 31 ± 5% (P < 0.01, n = 12).
This inhibition was rapidly and fully reversible, as demonstrated by the response to the application of heated control solution 60 s after
the CPZ application, which did not differ from the initial heat
response (Fig. 3D). Furthermore, CPZ significantly increased the time-to-peak of heat-evoked currents from 124 ± 5 ms to
154 ± 10 ms (P < 0.01; n = 12).
In contrast, in normal extracellular solution, smaller currents were
associated with shorter time-to-peak (r = 0.64, P < 0.05, n = 12).
|
The CPZ-induced inhibition of heat-evoked currents was dose dependent
(1 µM to 1 mM; F4,38 = 6.42, P < 0.001, ANOVA; see Fig. 3E) with an
IC50 of 13 µM (log10
IC50 = 4.88 ± 0.18), a Hill slope of
0.58 ± 0.10, and an extrapolated efficacy of 75 ± 5%, suggesting that most of the rapid heat-evoked current was inhibited by
CPZ, although CPZ binding probably was not at equilibrium with simultaneous application. When neurons were preincubated with 10 µM
CPZ (i.e., at the steepest part of the dose-response function) for
12-15 s, heat-evoked currents were reversibly reduced by 47.2 ± 8% of the normalized controls (P < 0.001;
n = 7). The difference between the inhibiting effect of
CPZ with and without preincubation was marginally significant (47 vs.
31%; P = 0.11), indicating a very fast action of the
competitive antagonist on heat-evoked currents. This difference
corresponded to a shift of the dose-response curve by ~0.5
log10 units resulting in an estimated
IC50 of ~4 µM. Application of CPZ at room
temperature did not elicit any currents (0 ± 0 pA,
n = 2, data not shown).
Application of RR (5 µM) significantly reduced heat responses by
33 ± 6% (Fig. 4; P < 0.005, n = 7), likewise in a fully reversible manner
and without affecting neurons when applied at RT (0 ± 0 pA,
n = 3, data not shown). RR specifically inhibits the
vanilloid receptors at lower concentrations only in a narrow range
(estimated to 0.1-10 µM) and may also reduce intracellular calcium
release (Amann and Maggi 1991; Maggi
1991
). Thus we did not use higher concentrations.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A subpopulation of small neurons acutely dissociated from rat
dorsal root ganglia responded to brief noxious heat stimuli with inward
currents. In contrast to heat-insensitive small and large neurons, this
population was excited by the vanilloid receptor agonist capsaicin and
exhibited a prolonged action potential duration with a prominent
inflection in the repolarization phase. Heat-sensitive DRG neurons
therefore fulfilled several criteria for the somata of primary
nociceptive afferents (Gold et al. 1996; Harper
and Lawson 1985
; Petersen and LaMotte 1991
).
Heat-evoked currents were accompanied by a conduction increase, had a
reversal potential near 0 mV, and were permeable for
Ca2+ > Na+. These findings
suggest that the currents were carried through temperature-operated
nonspecific cation channels. The reversible inhibition of heat-evoked
inward currents by the competitive VR antagonist CPZ and the
noncompetitive VR antagonist RR implies the involvement of vanilloid
receptors in this rapid transduction pathway for noxious heat.
Properties of heat-sensitive DRG neurons
As in our previous study (Kirschstein et al. 1997;
see also Dittert et al. 1998
), we found rapid
heat-evoked currents only in small neurons (
32.5 µm). Nociceptive
neurons are generally small, consistent with their small axons
(Gold et al. 1996
; Harper and Lawson
1985
; Petersen and LaMotte 1991
). In their
pioneering study on heat-evoked currents, Cesare and McNaughton
(1996)
therefore restricted their sample to small DRG neurons.
Two other studies reported heat-evoked currents in some larger DRG
neurons (Nagy and Rang 1999
; Reichling and Levine
1997
), but these responses are likely due to other heat
transduction mechanisms (see Multiple heat transduction
pathways).
Rapid heat-evoked inward currents in DRG neurons were associated with
comparably fast responses to the vanilloid receptor agonist capsaicin,
confirming the results of our previous study (Kirschstein et al.
1997). A similar coexpression has been reported in rat and
monkey nociceptive afferents in vivo (Baumann et al. 1991
; Szolcsányi et al. 1988
). The finding
that vanilloid receptor antagonists substantially reduced all rapid
heat responses in the present study provides another line of evidence
that heat-sensitive neurons express vanilloid receptors.
The repolarization phase of the AP was significantly longer in
heat-sensitive than heat-insensitive small DRG neurons, due to a
shoulder that was exhibited by 11 of 12 heat-sensitive but only 1 of 6 heat-insensitive neurons. This AP shape has been associated with small
soma diameter and slow axonal conduction velocity in the C-fiber range
(Harper and Lawson 1985), the presence of TTX-resistent sodium channels (Waddell and Lawson 1990
), as well as
responsiveness to the vanilloid receptor agonist capsaicin (Del
Mar et al. 1996
). Our data support the view that somal AP shape
is associated with functional characteristics of the cell as a
nociceptive primary sensory neuron rather than simply with its size.
Properties of rapid heat-evoked currents
The suggestion that nociceptive primary sensory neurons possess a
rapid transduction mechanism for noxious heat stimuli that is
independent from tissue damage (Treede et al. 1995) was
supported by the demonstration that noxious heat elicited inward
currents in small DRG neurons (Cesare and McNaughton
1996
). Two possible mechanisms may be responsible for the
inward currents: 1) the heat-induced opening of membrane
channels or 2) the heat-induced inactivation of persistent
outward currents. Because in our data the heat-evoked inward currents
were accompanied by significant increases in whole cell conductance, we
conclude that they were due to channel opening. Likewise, a preliminary
report on cell-attached patch recordings has demonstrated channel
opening by heat stimuli of 40-48°C (Nagy and Rang
1998
).
Noxious heat may either induce a direct opening of temperature-operated
channels or trigger an intracellular signal cascade resulting in
channel opening. A direct transduction mechanism seems more probable,
because the time-to-peak of heat-evoked currents was at least as fast
as the temperature peak of the stimulus used. This finding is
consistent with observations on primary nociceptive afferents in vivo,
that the initial burst of action potentials is already generated during
the rise time of the cutaneous heat stimulus (Treede et al.
1995). To determine the activation kinetics exactly, a heat
stimulus has to be established, which is much faster than the rapid
heat-evoked currents (e.g., a laser-stimulator described by
Baumann and Martenson 1994
).
The reversal potential of heat-evoked currents in our study was similar
to that in cultured DRG neurons of neonatal rats (Cesare and
McNaughton 1996), which resembled that of the rapid component of capsaicin-induced currents (Liu and Simon 1996
;
Oh et al. 1996
). Ion replacement experiments showed that
the temperature-operated channels are permeable to
Na+, Ca2+, and
Cs+ (cf. Cesare and McNaughton
1996
). Finally, rapid heat-evoked currents had a higher
permeability for Ca2+ than for
Na+ ions, which is also typical for
capsaicin-induced currents (Bevan and Szolcsányi
1990
; Jung et al. 1999
). From changes of
reversal potentials of heat-evoked currents after ion replacement, the permeability ratio
pNa+:pCa2+ was calculated
to be 1:1.28 (Cesare and McNaughton 1996
). In addition
to these similarities of heat-evoked and capsain-induced currents
described in different studies, we now have demonstrated that the
reversal potential of both currents is not distinguishable in the same
neuron. Thus both currents may be conducted at least in part by
identical channels.
Vanilloid receptors and heat transduction
A candidate for such a channel is the vanilloid receptor VR1, a
nonselective cation channel that is activated by heating the cell to
40-45°C and that is selectively expressed in DRGs and trigeminal
ganglia (Caterina et al. 1997; Tominaga et al.
1998
). The heterologous expression of VR1 in both human
embryonic kidney cells (HEK 293) and frog oocytes conveyed heat
sensitivity to these cells (Caterina et al. 1997
),
consistent with a direct action of noxious heat on capsaicin-sensitive
membrane channels. Single-channel openings were observed in excised
patches of these VR1-transfected cells to both heat and capsaicin
(Tominaga et al. 1998
). In the present study heat-evoked
currents in DRG neurons were inhibited by 75% by the competitive VR
antagonist CPZ and by 33% by the noncompetitive VR antagonist RR. Thus
we have now provided pharmacological evidence that native vanilloid
receptors are a key element in the responses of nociceptive primary
sensory neurons to noxious heat stimuli.
The reduction in heat-evoked currents by 10 µM CPZ (by 31% with
co-application and 47% with preincubation) was smaller than that
reported for currents elicited by 0.5 µM capsaicin (by 96%) in
neonatal rat DRG neurons (Bevan et al. 1992). Whereas
the reduction of heat-evoked currents by CPZ was fully reversible on
wash out, the capsaicin-evoked currents recovered only by ~50%; this
was attributed to desensitization induced by the first application of
capsaicin (Bevan et al. 1992
). Interestingly, in
Xenopus oocytes expressing the cloned VR1, 10 µM CPZ had
70% efficacy against 44°C (Tominaga et al. 1998
), but
>95% efficacy against 0.6 µM capsaicin (Caterina et al.
1997
). In HEK 293 cells, CPZ had 90% efficacy against moderate
noxious heat and 97% against capsaicin, but the preincubation with CPZ
was four times longer for the heat tests than for capsaicin, recovery
was incomplete, and repetition of the 46°C heat stimulus by itself
reduced the response by 44% (Tominaga et al. 1998
).
Thus a reduced efficacy of CPZ to inhibit VR1 activation by heat
compared with capsaicin is supported by these findings, too. Because
protein structures are highly sensitive to temperature, the affinity of
vanilloid receptors for CPZ is likely to be reduced at the temperature
used in the present study. For enzymes such as lactate dehydrogenase,
the Km for substrate binding is about
three to five times higher at 50°C than at room temperature
(Somero 1995
). The temperature dependence of
ligand-receptor interactions may explain most of the difference between
the IC50 described here at ~53°C (4-13 µM)
and the binding constant of CPZ to vanilloid receptors at room
temperature (Kd of 0.1-0.7 µM),
that was estimated from Schild plots of the competitive antagonism of
CPZ versus capsaicin and resiniferatoxin in rat DRG neurons (Bevan et al. 1992
). Unfortunately, a Schild analysis to
determine the Kd for CPZ-induced
inhibition of heat-evoked currents is impracticable, because the upper
end of useful heat intensities (i.e., the "agonists" concentration)
is limited by activation of another transduction mechanism that is VR
independent (Caterina et al. 1999
; Nagy and Rang
1999
) (see Multiple heat transduction
pathways) and ultimately by cell damage. Therefore we did
not determine, whether CPZ inhibits heat-evoked currents competitively
like it antagonizes capsaicin-induced currents.
Alternatively, the apparent difference in affinity raises the question,
whether the inhibition of heat-evoked currents may have been unrelated
to a specific inhibition of vanilloid receptors. CPZ had no effects on
GABA-receptors, ATP-receptors, and depolarization-induced ion fluxes at
RT (Bevan et al. 1992). Recently, however, nonspecific effects of CPZ have been described on voltage-activated calcium channels with an ED50 = 8 µM (Docherty et al.
1997
) and on nicotinic acetylcholine receptors at 10 µM
(Liu and Simon 1997
). The time to reach half-maximum
effect on voltage-activated calcium channels was ~5 min at 1-10 µM
and 1 min at 100 µM, and this effect was irreversible. Likewise, the
inhibition of nicotinic acetylcholine receptors by 10 µM CPZ was
found with a 2-min preincubation period of the antagonist, and it took
~10 min wash out to reverse this effect. Because in our data the
antagonists were preincubated for a few seconds only and the inhibition
of the "agonist" heat was fully reversible as soon as tested (~1
min or less), the inhibition of heat-evoked inward currents by CPZ is
unlikely to be due to nonspecific effects. A further argument for the
inhibition of heat-evoked currents by a specific interaction with
vanilloid receptors derives from the significant effect of 5 µM RR.
RR interacts with a different site of the vanilloid receptors
(Bevan et al. 1992
; Dray et al. 1990
),
and our dose was within the range that is considered specific for
vanilloid receptors (0.1-10 µM) (Maggi 1991
).
Thus whereas nonspecific effects of CPZ have been reported, the
temporal characteristics of those effects are sufficiently different
from the rapid and fully reversible inhibition of heat-evoked inward
currents by CPZ to warrant the conclusion that vanilloid receptors are
essentially involved in the rapid heat transduction pathway. The
receptor subtype need not necessarily be VR1, because multiple
vanilloid receptors exist (Szallasi 1994), which may be
coexpressed in the same neuron (Liu and Simon 1996
;
Liu et al. 1997
; Petersen et al. 1996
),
and subtype specific antagonists are not available.
Multiple heat transduction pathways
The major proportion of the rapid heat-evoked current of the present study can be attributed to vanilloid receptor activation. However, the extrapolated efficacy of CPZ (75 ± 5%) raises the question of whether this reflects a true incomplete antagonism (e.g., because heat acts at a different site of VR1 than CPZ) or whether the remaining fraction was due to another transduction mechanism that was independent of VRs.
A subpopulation of cultured DRG neurons has recently been described,
that were heat-sensitive but not excited by capsaicin (Nagy and
Rang 1999), and a membrane channel with similar properties has
been cloned and called vanilloid receptor-like channel (VRL-1) (Caterina et al. 1999
). These neurons had a higher heat
threshold, smaller maximum currents, and larger diameters. A
corresponding heat transduction mechanism is present in a subclass of
A
-fiber nociceptors that respond to heat only after prolonged
stimulus duration or subsequent to tissue damage and are called HTM for high-threshold mechanoreceptor or type 1 AMH (the 1st type of A-fiber
mechano-heat nociceptor discovered) (Fitzgerald and Lynn 1977
; Perl 1968
; Treede et al.
1995
). In an in vivo study on the rat saphenous nerve, all
polymodal C-fibers and the majority of polymodal A
-fibers responded
to close arterial injection of capsaicin, whereas the A
-fiber HTMs
were not affected (Szolcsányi et al. 1988
). Thus
in type 1 AMH the slow heat transduction mechanism appears to be
independent of vanilloid receptors and may be mediated by VRL-1.
The remaining current after maximum blockade of VRs in the present
study was significantly delayed, but the time-to-peak was still an
order of magnitude faster than that of currents presented by
Nagy and Rang (1999) and Caterina et al.
(1999)
. Moreover, the size distributions of neurons expressing
VR1 and VRL-1 do not suggest a widespread coexpression but an
expression of either VR1 or VRL-1 (Caterina et al.
1999
). In contrast to Nagy and Rang (1999)
we
found only one capsaicin-insensitive neuron that responded to heat.
This difference between the studies may be due to several reasons.
1) The temperature used by us may not sufficiently activate the high-threshold current. 2) The stimulus used may have
been too short; activation of the corresponding neurons in vivo (i.e., type 1 AMH) depends on a stimulus duration of several seconds (Treede et al. 1998
). 3) Culture conditions
were different (acutely dissociated vs. several days in culture with
nerve growth factor, NGF); in the absence of NGF, type 1 AMHs in vivo
change their phenotype into D-hair receptors (Ritter et al.
1993
).
Still another mechanism seems to underly the heat-induced inward
current (Iheat) reported by
Reichling and Levine (1997). Peak currents (50 vs. 280 pA) and conductance increases (25 vs. 320%) were about one order of
magnitude smaller than in the present and other studies (Cesare
and McNaughton 1996
; Kirschstein et al. 1997
).
Their Iheat was blocked by
extracellular cesium ions, whereas the nonspecific cation channel
activated by brief noxious heat stimuli was even more permeable to
Cs+ than to Na+ ions
(Cesare and McNaughton 1996
). Moreover, their
Iheat exhibited a linear increase from
room temperature to 45°C. This temperature dependence is not easily
compatible with nociceptive neurons.
In summary, rapid heat responses in nociceptive primary sensory neurons appear to depend predominantly on a vanilloid receptor as the heat-sensing element. Conversely, our results support the hypothesis that the physiological function of the native vanilloid receptor VR1 is to detect noxious heat. This mechanism is different from two other heat transduction pathways that have been partly described, and the mechanisms of which await further studies.
![]() |
ACKNOWLEDGMENTS |
---|
The authors thank G. Günther for help with the experiments, J. Rupp and H. Nawrath for the dose-response function analysis, W. Lang for evaluation of pH changes in heated solutions, and G. Böhmer, P. Heusler, and W. Magerl for critical reading of the manuscript.
This research was supported by the Deutsche Forschungsgemeinschaft (Tr236/11-1).
![]() |
FOOTNOTES |
---|
Address for reprint requests: R.-D. Treede, Institute of Physiology and Pathophysiology, Johannes Gutenberg University, Saarstr. 21, D-55099 Mainz, Germany.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 January 1999; accepted in final form 28 July 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|