Department of Anesthesiology and Neurobiology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Liu, L., M. Oortgiesen, L. Li, and S. A. Simon. Capsaicin Inhibits Activation of Voltage-Gated Sodium Currents in Capsaicin-Sensitive Trigeminal Ganglion Neurons. J. Neurophysiol. 85: 745-758, 2001. Capsaicin, the pungent ingredient in hot pepper, activates nociceptors to produce pain and inflammation. However, repeated exposures of capsaicin will cause desensitization to nociceptive stimuli. In cultured trigeminal ganglion (TG) neurons, we investigated mechanisms underlying capsaicin-mediated inhibition of action potentials (APs) and modulation of voltage-gated sodium channels (VGSCs). Capsaicin (1 µM) inhibited APs and VGSCs only in capsaicin-sensitive neurons. Repeated applications of capsaicin produced depolarizing potentials but failed to evoke APs. The capsaicin-induced inhibition of VGSCs was prevented by preexposing the capsaicin receptor antagonist, capsazepine (CPZ). The magnitude of the capsaicin-induced inhibition of VGSCs was dose dependent, having a K1/2 = 0.45 µM. The magnitude of the inhibition of VGSCs was proportional to the capsaicin induced current (for -ICAP < 0.2 nA). Capsaicin inhibited activation of VGSCs without changing the voltage dependence of activation or markedly changing channel inactivation and use-dependent block. To explore the changes leading to this inhibition, it was found that capsaicin increased cAMP with a K1/2 = 0.18 µM. At 1 µM capsaicin, this cAMP generation was inhibited 64% by10 µM CPZ, suggesting that activation of capsaicin receptors increased cAMP. The addition of 100 µM CPT-cAMP increased the capsaicin-activated currents but inhibited the VGSCs in both capsaicin-sensitive and -insensitive neurons. In summary, the inhibitory effects of capsaicin on VGSCs and the generation of APs are mediated by activation of capsaicin receptors. The capsaicin-induced activation of second messengers, such as cAMP, play a part in this modulation. These data distinguish two pathways by which neuronal sensitivity can be diminished by capsaicin: by modulation of the capsaicin receptor sensitivity, since the block of VGSCs is proportional to the magnitude of the capsaicin-evoked currents, and by modulation of VGSCs through second messengers elevated by capsaicin receptor activation. These mechanisms are likely to be important in understanding the analgesic effects of capsaicin.
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INTRODUCTION |
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Nociceptors constitute a
class of peripheral sensory neurons that can be activated by noxious
mechanical, thermal, or chemical stimuli. Exposure of nociceptors to a
noxious stimulus often alters their sensitivity during subsequent
exposure to the same or other stimuli, causing either sensitization or
desensitization (or tachyphylaxis). Activation of nociceptors evokes
action potentials (APs) that transmit information to the CNS to produce
the sensation of pain. Their activation also induces the release of
neuropeptides into the periphery that may result in inflammation and
nociceptor sensitization (Levine et al. 1993).
Nociceptors contain a variety of specialized receptors and
voltage-gated ion channels (McCleskey and Gold 1999
; Pearce and Duchen 1994
; Woolf and Costagin
1999
), including receptors for capsaicin and a several types of
specialized voltage-gated sodium channels (VGSCs), some of which are
relatively insensitive to tetrodotoxin (e.g., TTX-resistant; TTX-R)
(Akopian et al. 1996
, 1999
; Caffrey et al.
1992
; Jeftinija 1994
; Pearce and Duchen
1994
; Porreca et al. 1999
; Roy and
Narahashi 1992
; Sangameswaran et al. 1997
;
Tate et al. 1998
). One mechanism of selectively
inhibiting nociceptors is to inhibit their VGSCs. Indeed, some
compounds like capsaicin can selectively activate and desensitize
nociceptors (Arbuckle and Docherty 1995
; Carstens
et al. 1998
; Caterina et al. 2000
; Dray
et al. 1990
; Foster and Ramage 1981
;
Großkreutz et al. 1996
; Jansco et al.
1980
; Marsh et al. 1987
; Szolcsanyi et
al. 1998
; Torebjork et al. 1992
). That is,
moderate capsaicin concentrations will only desensitize some types of
A
and C fibers without affecting A
mechanoreceptors
(Kohane et al. 2000
; Szolcsanyi et al.
1998
). Based on this latter quality capsaicin is used to treat
a variety of pathologies ranging from arthritis to trigeminal neuralgia
to chronic pain (Szallasi and Blumberg 1999
).
Different types of sensory neurons have been characterized by their
conduction velocity and by their sensitivities to chemical (e.g.,
capsaicin), mechanical, and thermal stimuli. In cultured primary
sensory neurons, different neuronal types can be distinguished on the
basis of the shape of their APs, soma size, the ability to bind lectins
(e.g., IB4), the presence of different types of voltage-dependent sodium, potassium and calcium channels and whether they can be activated by physical (e.g., thermal) and chemical (e.g.,
capsaicin, ATP) stimuli (Cardenas et al. 1995, 1997
;
Caterina and Julius 1999
; Djouhri et al.
1998
; Stucky and Lewin 1999
). Capsaicin-sensitive neurons have been identified in primary cultures of
dorsal root ganglia (DRG), nodose ganglia, and trigeminal ganglia (TG)
(Bevan and Winter 1995
; Caterina et al.
2000
; Liu and Simon 1996
; Marsh et al.
1987
). In this study, we address the relationship between the
types of APs present in TG neurons, their activation by capsaicin, the
modulation of VGSCs and generation of second messengers in different
types of sensory TG neurons. Recently an electrophysiological study
using cultured rat DRGs revealed that activation of capsaicin receptors
leads to the selective inhibition of VGSCs in capsaicin-sensitive
neurons (Su et al. 1999
). We further characterized this
selective inhibitory effect and demonstrated the involvement of cAMP as
an intermediate second messenger between vanilloid receptor activation
and voltage-gated sodium channel modulation.
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METHODS |
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Cell culture
TG neurons were cultured from adult Sprague-Dawley rats as
described previously (Liu and Simon 1996). Trigeminal
ganglia were dissected aseptically and collected in Hank's balanced
salt solution (HBSS). After washing twice in HBSS, the ganglia were
diced into small pieces and incubated for 30-50 min at 37°C in 0.1%
collagenase (Type Xl-S, Sigma) in HBSS. Individual cells were
dissociated by triturating the tissue through a fire-polished glass
pipette, followed by a 10-min incubation at 37°C in 10 µg/ml DNase
I (Type lV) in F-14 medium (Life Technologies, Gaithersburg, MD). After washing three times with F-14 medium, the cells were cultured in DMEM
supplemented with 10% fetal bovine serum and 100 ng/ml NGF/7S. The
cells were plated on poly-D-lysine and laminin-coated glass
coverslips (15-mm diam) and cultured ~24 h at 37°C in a water
saturated atmosphere with 5% CO2. At the
beginning of each experiment, the neurons were placed in a chamber
containing Krebs-Henseleit (KH) buffer on an inverted microscope. The
cell diameter (µm) was measured with a calibrated eyepiece micrometer
under phase contrast illumination and expressed as the average value of
the long and short dimensions of the cell. Only neurons without or with
short processes were used. All experiments were carried out at room
temperature (22-25°C). The composition of the KH buffer was (in mM)
145 NaCl, 5 KCl, 2.0 CaCl2, 1.0 MgCl2, 10 HEPES, and 10 D-glucose;
adjusted to pH 7.4.
Care of animals conformed to standards established by the National Institutes of Health. All animal protocols were approved by the Duke University Institutional Animal Care and Use Committee.
Patch-clamp recording
For whole cell voltage- and current-clamp experiments, glass
pipettes (N-51A borosilicate, Drummond Scientific, Broomall, PA) with
resistance's between 2 and 4 M were used. The pipette solution for
current-clamp experiments contained (in mM) 20 KCl, 130 K-aspartate, 10 EGTA, 10 HEPES, 10 D-glucose, 0.1 Na-GTP, 2 cAMP, 2 Mg-ATP, 5 Na2-creatine
phosphate, and 20 creatine phosphokinase; adjusted to pH 7.4; and for
voltage clamp it was (in mM) 135 CsF, 1.0 CaCl2,
2.0 MgCl2, 10 EGTA, 10 HEPES, and 5 Na2-ATP 5 adjusted to pH 7.3. The signal was
measured using an Axopatch-1D patch-clamp amplifier (Axon Instruments,
Foster City, CA), and the output was digitized with a Digidata 1200 converter (Axon Instruments). In both voltage- and current-clamp
experiments, the liquid-junction potential, capacitance and series
resistance (>80%) was compensated. The leak current, which often
times changed after capsaicin exposure and washout, was subtracted from
the VGSC, as calculated from the resistance before each pulse protocol
for INa activation. Except for the AP recordings, where the sampling
rate was 10 kHz, the sampling rate was 1 kHz.
The whole cell capacitance (pF), resting membrane potential (mV) and
membrane resistance was read at the beginning of the voltage-clamp
experiments and was continuously recorded in the current-clamp
experiments. In current-clamp experiments, the resting potential was
adjusted to 60 mV at the beginning of the experiment. APs were evoked
by step depolarizations of 20 ms with increasing amplitudes ranging
between 0.1 and 2 nA in 50.0-pA steps using pCLAMP 6.04. In
voltage-clamp experiments, the I-V relationship and the Imax
of INa activation was determined by incremental step depolarization's
from
80 or
60 to +60 mV in 5-mV increments as previously described
for DRG or TG (Gold et al. 1998
; Kim et al.
1999
). The peak currents were measured with pCLAMP 6.04 and plotted against the voltage to obtain the I-V of INa
activation. The voltage dependence of inactivation was determined as
the peak of the remaining INa evoked at 0 mV after
80 to 0-mV
prepulse voltage steps of 20 ms. Use-dependent inactivation of VGSC in TG was determined by repetitive INa activation (40-ms pulse of
60 to
0 mV) at various stimulus frequencies (0.2, 0.5, 1, and 2 Hz). TTX-R
INa currents were recorded with the same protocols 3 min after
incubation with 0.1 µM TTX. The maximum sodium current evoked during
a pulse protocol is defined as INap.
Chamber/solution delivery
The chamber containing the neurons had a volume of 500 µl and
was continuously perfused by KH flowing into the chamber at a rate of 6 ml/min. To record VGSCs, the external solution was replaced by (in mM)
30 NaCl, 90.0 Choline-Cl, 20 TEA-Cl, 5 KCl, 5 MgCl2, 2 CaCl2, 20 D-glucose, 10.0 HEPES, 1 CdCl2, and 3 4-aminopyridine (4-AP), pH adjusted to 7.4. With the preceding pipette
solution, the ENa was calculated to be 27.9 mV. The use of this low Na
solution was to reduce the magnitude of the rapidly activating sodium
currents to overcome possible voltage errors that may arise due to poor space clamp during the voltage command. This problem may still be
present in some of the records. However, it does not influence the
results presented in this paper regarding the inhibitory role of
capsaicin on VGSCs. Capsaicin was delivered to the cell using a
multibarreled electrode (Adams and List, Westbury, NY) placed ~50
µm from the cell. The solutions in each barrel were controlled by a
valve (General Valve), and event markers associated with the opening or
closing of the valves signaled the onset and removal times of the
stimuli. In voltage-clamp experiments (HP = 60 mV), neurons were
considered to be capsaicin-sensitive if 1 µM capsaicin evoked inward
currents
25 pA and in the current-clamp experiments if it evoked
depolarizations >5 mV when the holding potential was
60 mV.
Enzyme immunoassay (EIA)
The activation of cAMP by capsaicin was measured by EIA. To
obtain a sufficient yield of cells, newborn (1P) instead of adult TG
cultures were used. TG neurons were cultured as above except the glial
cells were removed using a Percoll gradient and examined for cAMP after
1 day in culture. TG neurons were cultured in 96-well culture plates at
the density of 2 × 105 cells/ml (which was
equivalent to 30-50 µg protein/well) where they were exposed to the
various test conditions. After a 5-min exposure to capsaicin, in the
presence and absence of the vanilloid receptor antagonist, capsazepine
(CPZ) (Maggi et al. 1993), the culture media was
removed, and the TG lysed and assayed for the presence of cAMP using
the EIA kit from Biotrak (Amersham Pharmacia, Piscataway, NJ).
Absorbance was measured and analyzed by comparison with "in plate"
standards with a Dynatech MR5000 plate reader spectrophotometer
(Chantilly, VA) using BioLinx 2.10 software.
Statistics and curve fitting
Data were analyzed and fitted using PClamp (Axon Instruments) or SigmaPlot (SPSS, Chicago, IL) software. Dose-response curves were fitted to the related effect, E, and the capsaicin concentration (cap): E = (Emax * cap)/(cap + K1/2), and characterized by K1/2 (concentration of stimulus producing a half-maximal effect) and Emax (maximum fraction of effect). The Boltzmann relation was fitted to the function: I/INamax or G/Gmax = 1 + exp[(V0.5 - Vm)/k], with V0.5 as the membrane potential at which 50% of activation or inactivation was observed and k as the slope of the function. Data were analyzed for statistical significance using the paired and unpaired (as indicated in the text) Student's t-test, and data are presented as the effects ± SD. The significance was indicated as P < 0.05.
Chemicals
Capsaicin and CPZ were obtained from RBI (Natick, MA). All other chemicals came from Sigma (St. Louis, MO). Cell culture materials were purchased from GIBCO (Life Technologies, Rockville, MD).
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RESULTS |
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Capsaicin inhibits APs only in capsaicin-sensitive neurons
The AP characteristics and capsaicin sensitivity were studied in
TG neurons using current clamp. The resting potential for all the
neurons was 51.8 ± 11.2 mV (n = 87). The
classification of the Cardenas et al. (1995)
neuron
types in DRGs were followed: the potential was set at
60 mV, and APs
were evoked by depolarizing pulses of 20 ms with incremental increases
in the amplitude (of 50 pA) until their shape remained essentially
unchanged. Analogous to characteristics previously found for rat DRGs
(Cardenas et al. 1995
), we distinguished four types of
sensory TG neurons on the basis of their size, their shape, and their
sensitivity to capsaicin (Table 1). Type
I neurons were characterized by a long-duration AP with a prominent
shoulder on the falling limb (Fig. 1). At 50% of the AP amplitude (ADP50%), the duration
was 5.8 ± 1.4 ms (Table 1). Capsaicin (1 µM) induced a
depolarization in 60% of the Type I neurons. The other 40% of Type I
neurons were capsaicin insensitive. Type II neurons had significantly smaller AP durations (ADP50% = 2.6 ± 0.8 ms) than Type I neurons. These Type II neuron also exhibited a small
shoulder on the repolarization phase. About 76% of Type II neurons
were sensitive to 1 µM capsaicin. The other 24% were capsaicin
insensitive. The long AP duration together with shoulders on the
repolarization phases are indicative of the presence of C fibers
(Djouhri et al. 1998
).
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In contrast, neuron Types III and IV had significantly shorter
ADP50%'s and were insensitive to capsaicin
(Table 1). Type III neurons had larger soma diameters than the other
types (P < 0.047), and Type IV neurons were
distinguished from Type III in that repetitive APs could be evoked with
the 20-ms pulse (Cardenas et al. 1995). The APs evoked
in these two neuron types were not changed in the presence of 1 µM
capsaicin. This analysis of APs properties, albeit limited because it
was not the primary goal of this paper, resemble the four DRG Types
previously described, with the major exception that the Types I and II
neurons were not always sensitive to 1 µM capsaicin. In summary, TG
neurons contained at least four clearly distinguishable types of APs, two of which were sensitive to capsaicin.
In agreement with studies from DRGs (Su et al. 1999), we
found that TG neurons that ranged in size from 21 to 47 µm were
capsaicin-sensitive. That is, capsaicin-sensitivity is not an exclusive
property of small diameter neurons.
Of particular relevance to the investigation undertaken in this study
is that 1 µM capsaicin only depolarizes and decreases the probability
of the generation of APs in capsaicin-sensitive Types I and II neurons
(Fig. 1). In capsaicin-insensitive Type I and II neurons, the APs were
unaffected by 1 µM capsaicin as were the APs in capsaicin-insensitive
Types III and IV neurons. This shows that capsaicin, at least at 1 µM, exhibited a marked neuronal type selectivity in its ability to
effect APs (Fig. 1). To eliminate the possibility that an increase in
(VGSC) channel inactivation was solely the cause of the reduced
amplitude of the APs by capsaicin (see following text), the membrane
potential was re-adjusted to 60 mV and the neuron was electrically
depolarized (all the while remaining exposed to capsaicin). Under these
conditions, an AP could be evoked, but its kinetics were delayed and
magnitude decreased. Although there are several possibilities to
rationalize this behavior [e.g., decreased resistance (Marsh et
al. 1987
), inhibition of VGSCs], it is nonetheless clear that
capsaicin increases the threshold for the generation of APs only in
capsaicin-sensitive neurons.
Separation of AP generation from capsaicin receptor desensitization
Under current-clamp conditions, the initial application of 1 µM capsaicin to neuron Types I or II induces a depolarizing potential (Marsh et al. 1987) and a burst of APs (Fig.
2) (Gold et al. 1996a
; Lopshire and Nicole 1997
). In a Type II neuron (Fig.
2A), it was found that the first exposure of 1 µM
capsaicin produced a depolarization (Fig. 2B) that evoked a
spike train when the potential reached
51 mV. When the membrane
potential reached
32 mV, the spike train abruptly ceased. After a
3-min wash, the potential returned to baseline by itself (i.e., it was
not adjusted). The second capsaicin application gave almost as large a
depolarization and with approximately the same rate of depolarization,
but APs failed to be evoked even when the final depolarization
potential far exceeded the threshold for the first application (
51
mV). The fact that the potential returned to baseline and that the
amplitude of the capsaicin response did not change with the second
capsaicin application suggests that the input resistance was similar to that produced by the previous application. Moreover because the resistance decreased during both applications of capsaicin and APs were
generated only in the first and inhibited in the second and subsequent
applications, the failure to evoke APs is not a simple matter of a
decrease in resistance. Also since the rates of depolarization were
about the same for these two applications, the channels should be
inactivated to about the same extent. All subsequent capsaicin
applications (at 3-min intervals) resulted in monotonically decreasing
depolarizations that also failed to evoke APs. In none of the five
neurons tested were APs observed during the second or subsequent
capsaicin exposures.
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To further explore the ability of capsaicin to inhibit the production of APs, we performed experiments similar to those presented in Fig. 2A except that for each 1 µM capsaicin application, APs were evoked at small (100 pA) and large (1000 pA) depolarizing current pulses before (pre-capsaicin) and during the wash phases (post-capsaicin; Fig. 2C). In this experiment, which shows the first and third applications of capsaicin, one could distinguish between APs evoked before capsaicin was applied (pre) and during the wash phase (post) with small (100 pA) and large (1000 pA) depolarizations. The traces in Fig. 2C show a Type II neuron that evoked a burst of APs on the first application of capsaicin. During the wash phase, the 100-pA current injection failed to evoke an AP but the 1,000-pA pulse evoked a long-duration AP with a reduced amplitude. As was seen previously (Fig. 2B), for this capsaicin application, the depolarization produced was significantly larger then the voltage to produce APs in the first application but nonetheless failed to evoke APs. Taken together, it follows that capsaicin increases the threshold for AP generation that is seen during voltage pulses or on more than one application of capsaicin. There are many mechanisms that may underlie the inhibition produced by capsaicin-sensitive neurons. To explore this behavior in further detail, we have undertaken to investigate the effects of capsaicin on the most promising candidate to rationalize the inhibition, namely VGSCs.
Capsaicin inhibits VGSCs
Because only the APs in capsaicin-sensitive neuron Types I and II
were inhibited by capsaicin and because VGSCs are important in the
generation of APs, we explored the effects of capsaicin on VGSCs. VGSCs
were evoked in voltage-clamped TG neurons held at 60 mV (near the
resting potential). The current-voltage (I-V) relationship
of activation of the total peak sodium current
(INap) was measured in the absence and
presence of 1 µM capsaicin. Capsaicin (1 µM) evoked an inward
current of
3.2 ± 1.8 nA (n = 32) in 52% (n = 32) of the neurons, a percentage consistent with
studies in other sensory neurons (Bevan and Winter 1995
;
Marsh et al. 1987
; Su et al. 1999
).
Capsaicin (1 µM) inhibited VGSCs, but only markedly in
capsaicin-sensitive neurons (Figs.
3A, 4, and 5). In
capsaicin-sensitive neurons at the maximum current in the
I-V curve (INamax), 1 and
10 µM capsaicin, inhibited INap by
75 ± 24% (n = 8) and 95 ± 5%
(n = 9), respectively. In the 3-min washout phase, the
current was partially reversible. The capsaicin-induced inhibition of
VGSCs did not significantly change their voltage dependence because the
G0.5 (the half-maximal conductance)
was
17.7 mV in control and
15.8 mV in the presence of 1 µM
capsaicin (Fig. 3C).
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Capsaicin did, however, modulate the time course of the remaining sodium current. As shown in Fig. 3D, 1 µM capsaicin did not affect the rise time but slowed the decay time (compare traces a and c), suggesting a small effect on sodium channel inactivation. In neurons in which 1 µM capsaicin inhibited the INa peak amplitude between 30 and 65%, the time constant of the INa inactivation (at INamax) in the absence and presence of 1 µM capsaicin was 5.4 ± 2.9 ms (n = 5) and 4.4 ± 2.1 ms (n = 5), respectively.
In contrast, in capsaicin-insensitive neurons, INa was only marginally
inhibited by capsaicin (Fig. 4). In this
regard even 10 µM capsaicin only inhibited
INap by 15 ± 9%
(n = 9). The concentration dependence of the
capsaicin-induced inhibition of the
INap (Fig. 5A) shows the marked
difference in sensitivity between capsaicin-sensitive and -insensitive
neurons. In capsaicin-sensitive neurons, the mean apparent
K1/2 fitted to the relative capsaicin
inhibition of the total INamax was
0.45 µM (range, 0.3-1 µM). This variability in sensitivity could
arise from a variety of factors including the different subtypes of
VGSC subtypes expressed (Black et al. 1996), the
different sensitivities of C and A
neurons to capsaicin (Marsh et al. 1987
), and/or on the magnitude of
capsaicin-evoked current. Plotting the amplitude of the
capsaicin-induced inward current (-ICAPS)
against the relative inhibition of the peak VGSC indicates that the
capsaicin-mediated block of INap is
correlated with the amplitude of the capsaicin induced current for
-ICAPS
2 nA (Fig. 5B). At
higher inward currents, the inhibition remains constant at ~100%. We
note that although cell size cannot be ruled out as an additional
factor, the Icaps were measured in capsaicin-sensitive neurons of the
same size range (Table 1). This suggested that the variability of the
INa inhibition by capsaicin is quantitatively related to the capsaicin
sensitivity of TG neurons.
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Effects of capsaicin on VGSC inactivation and use dependence
The effect of capsaicin on the kinetics of the INa (see Fig.
3D) suggested that it delays sodium channel inactivation. In subsequent experiments, the effects of capsaicin were examined on
sodium channel inactivation in capsaicin-sensitive and -insensitive neurons. The voltage dependence of sodium channel inactivation was
determined by measuring the remaining
INap evoked at 0 mV after stepping to
various potentials from 80 to 0 mV in 5-mV steps. For
capsaicin-sensitive neurons in the presence of 1 µM capsaicin, the
inactivation curve was slightly shifted to more negative potentials
(Fig. 6A). In control and
capsaicin-treated neurons, the V0.5
(the voltage where half the current is inactivated) differed slightly,
but not significantly, being
34.9 ± 8.8 mV (n = 5) and
40.4 ± 8.5 mV (n = 5), respectively
(paired t-test of the V0.5
in the same neurons, P > 0.05). These data indicate the presence of more than one type of VGSC in TG neurons (see Fig. 9)
(see also Kim et al. 1999
). In capsaicin-insensitive
neurons, even 10 µM capsaicin did not markedly affect the relative
peak inward sodium current after different depolarizing prepulses (Fig. 6B). In control TGs, the
V0.5 was
43.5 ± 11.6 mV
(n = 6), which was not significantly different from
that in capsaicin-insensitive neurons,
42.6 ± 10.6 mV
(n = 6, unpaired t-test, P = 0.21).
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One well-established mechanism by which anesthetics and other drugs can
inhibit VGSCs is by use-dependent block, a mechanism where the drug
associates preferentially with one of the states of the channels during
periodic depolarizing pulses (Hille 1993). In this
regard, use-dependent block may be a possible reason for the abrupt
inhibition of the spike trains evoked by capsaicin (see Fig. 2). We
found that at a holding potential of
60 mV, neither
capsaicin-sensitive (Fig. 7A)
nor -insensitive (Fig. 7B) neurons exhibit changes in
use-dependent (0.5-5 Hz) inhibition of VGSCs in the presence of 1 µM
capsaicin.
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Taken together, these data demonstrate that in capsaicin-sensitive neurons, capsaicin inhibits VGSCs activation in a dose-dependent manner, with only minor effects on sodium channel inactivation and use-dependent blocking.
Inhibition of VGSCs by capsaicin requires activation of capsaicin receptors
It is established that the vanilloid antagonist, CPZ, blocks
capsaicin-sensitive receptors in a variety of cells (Szallasi and Blumberg 1999). To test whether the inhibitory effect of
capsaicin on VGSCs is mediated by capsaicin receptors in TG neurons as
it is in DRGs (Su et al. 1999
), we tested the inhibitory
effect of capsaicin on VGSCs in the presence of 10 µM CPZ (Fig.
8). The experiment commenced by showing
that capsaicin reversibly inhibited VGSCs. Then we applied 10 µM CPZ
for 2 min and showed that it did not effect the VGSCs. The exposure to
10 µM CPZ together with 1 µM capsaicin caused a slight (10.8 ± 13%; n = 6) inhibition of the VGSCs (Fig. 8). After
wash, rather surprisingly, we found that capsaicin was ineffective in
activating another inward current. Curiously even though capasicin did
not evoke an inward current, its presence induced a smaller but still
marked inhibition of the voltage-gated sodium current, which was
partially reversed on washing.
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Effect of capsaicin on TTX-R and TTX-S VGSCs
Subsets of primary sensory neurons express subtypes of both
TTX-sensitive (TTX-S) and TTX-R VGSCs (Caffrey et al.
1992; Gold et al. 1996b
, 1998
, 1999
; Roy
and Narahashi 1992
). Both types of VGSCs are present in TG
neurons (Fig. 9) (Kim et al.
1999
). We found in TG neurons that the presence of 0.1 µM TTX
inhibited INap max by 45%. Figure 9 shows that capsaicin in
the presence of TTX induced an inward current that after it
desensitized inhibited the TTX-R VGSC. On average, 1 µM capsaicin
inhibited TTX-R currents by 71.0 ± 16.4% (n = 7). In capsaicin-insensitive neurons, capsaicin (10 µM) only
marginally inhibited the maximal TTX-R sodium current (16.7 ± 11.8%, n = 6; data not shown). These inhibitory
effects of capsaicin on TTX-R currents are not significantly different from those found for inhibition of the total VGSCs of 75 ± 24% in capsaicin-sensitive neurons (Fig. 5A).
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Role of cyclic AMP in capsaicin-induced inhibition of VGSCs
Previous studies in DRGs have shown that PKC- and PKA-mediated
pathways can modulate various subtypes of VGSCs (England et al.
1996; Fitzgerald et al. 1999
; Li et al.
1993
). The effects of capsaicin, however, on cAMP content are
unclear. One study showed no effect (Wood et al. 1989
),
while the other showed it increased cAMP (Notham and Jones
1987
). In TG neurons cultured from newborn rats, we found that
capsaicin caused a dose-dependent increase in cAMP (to 1 µM). At
higher capsaicin concentrations (3 µM), the cAMP levels decreased.
The apparent K1/2 for cAMP elevation,
fitted to the maximum effect at 1 µM capsaicin, was 0.18 µM
(n = 5). We also found that 10 µM CPZ inhibited the
capsaicin-induced cAMP elevation (significant inhibition only at
capsaicin concentrations of 0.3-3 µM), suggesting that the increase
in cAMP involved the activation of capsaicin receptors. At 1 µM
capsaicin, where the maximal increase in cAMP was found, 10 µM CPZ
inhibited the increase by 64 ± 9.3% (n = 6).
In voltage-clamped neurons, the addition of the membrane permeable cAMP analogue, CPT-cAMP at 1 mM, but not 0.1 mM, inhibited VGSCs in both capsaicin-sensitive (Fig. 11A), and capsaicin-insensitive neurons (Fig. 11B). CPT-cAMP (1 mM) inhibited the total INap in TG neurons by 33.7 ± 18% (n = 6) but did not affect the kinetics of INa. The time constants fitted to the current decay (at the INamax) were 6.6 ± 2.3 and 6.5 ± 3.2 ms in the control and in the presence of CPT-cAMP, respectively.
Preexposure to CPT-AMP also increased the capsaicin-revoked currents in
TG neurons as was previously found in DRG neurons (Lopshire and
Nicol 1998). In the presence of 100 µM CPT-cAMP, the peak
amplitude of the capsaicin-induced inward current (taken after a 30-s
application) was increased compared with the control value. That is, in
the absence of cAMP, the amplitude of the second capsaicin application
decreases ~28% or to 71.7 ± 26% (n = 8) compared with the first response amplitude (Fig. 12) (Liu and
Simon 1996
). In the presence of 0.1 mM CPT-cAMP, the
capsaicin-induced inward current amplitude was 141.5 ± 101%
(n = 7) of the control value.
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DISCUSSION |
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The pungent compound, capsaicin, activates vanilloid receptors
located primarily on C and A (mechano-heat) sensory nociceptors (Caterina and Julius 1999
; Jansco et al.
1980
; Kumazawa 1996
; Marsh et al.
1987
; Szolcsanyi et al. 1998
). Repeated
applications of capsaicin causes refractoriness to itself and to other
stimuli, while sparing motor function, which is the primary reason that it is used clinically to reduce pain and inflammation arising from the
activation of peripheral nociceptors. The ability of capsaicin to
selectivity desensitize sensory neurons is important to understand for
its clinical efficacy in relieving pain produced by a variety of
ailments (Szallasi and Blumberg 1999
).
Several studies from intact neurons revealed that capsaicin diminishes
the amplitude of action potentials (Gamse et al. 1982; Großkreutz et al. 1996
; Marsh et al.
1987
; Yamanaka et al. 1984
). Here we have
undertaken to understand the factors that underlie capsaicin's ability
to selectivity modulate the APs generated in particular types of
cultured TG neurons that in vivo would presumably be nociceptors
(Baccaglini and Hogan 1983
; Cardenas et al.
1995
; Gold et al. 1996a
;
Pearce and Duchen 1994
; Su et al.
1999
). This report demonstrates that micromolar concentrations of capsaicin selectively inhibits capsaicin-sensitive TG neurons, primarily by inhibiting VGSCs, in a manner that depends on the activation of vanilloid receptors (Figs. 4-8). In this regard, even when the membrane resistance returns to control values after capsaicin application, APs can still be attenuated (Fig. 2
and Marsh et al. 1987
). Although capsaicin may have anesthetic
properties at high concentrations, at low concentrations it selectively
interacts with particular types of neurons (Fig. 1) (see also Kohane et al. 2000
; Su et al. 1999
). That is, at higher capsaicin
concentrations, less specific, effects may be related to the previously
reported inhibition by capsaicin of various types of voltage-gated ion channels [i.e., potassium channels (Kehl 1994
;
Kuenzi and Dale 1996
), and calcium channels
(Docherty et al. 1991
; Kuenzi and Dale
1996
)] in neuronal as well as in nonneuronal cells.
We found that capsaicin selectively diminishes the probability of AP
generation (Figs. 1 and 2) and VGSCs only in TG neurons (Types I and
II) that had CPZ-inhibitable capsaicin-evoked currents (Figs. 4-8)
(Su et al. 1999). The magnitude of inhibition of
INap was found to be proportional to
the amplitude of the capsaicin-evoked currents, at least for currents
less than
2 nA (Fig. 5B). On repeated applications, the
inability of capsaicin to evoke APs can arise from a number of factors,
including decreasing the membrane resistance (Marsh et al.
1987
) although, as noted, the membrane resistance is decreased
on both the first and subsequent applications (Fig. 2). We argue that
long-term inhibition of VGSCs is the likely consequence of the
activation of vanilloid receptors (Figs. 3-8), that activate
second-messenger pathways, including cAMP (Fig. 10). Our studies further show that as
in DRGs, cAMP sensitizes the capsaicin-mediated currents
(Lopshire and Nicol 1998
). Taken together, these data
distinguish two pathways by which sensory sensitivity can be diminished
by capsaicin: by increasing the capsaicin evoked current that increases
the capasicin-induced block of INa (Fig. 5B) and by indirect
modulation of voltage-gated sodium channels through second messengers,
such as Ca2+ (Bleakman et al.
1990
; Cholewinski et al. 1993
), cGMP
(Wood et al. 1989
), and cAMP (Fig. 10), that are
elevated by capsaicin receptor activation. These mechanisms are likely
to be important in understanding the analgesic effects of capsaicin.
|
Types of TG neurons and effects of capsaicin
Primary afferent sensory neurons are classified by their
conduction velocity as C, A, and A
fibers. Functionally, however, different fiber types may respond to the same noxious stimuli, so just
because a fiber responds to a particular stimulus does not, by itself,
specify its classification (Djouhri et al. 1998
; Szolcsanyi et al. 1998
). In an extensive review on the
electrophysiological properties of sensory neurons, it was found that
distinct classes could be distinguished by their AP amplitude,
duration, inflection in the falling phase, and the presence of
voltage-gated sodium, potassium, and calcium channels (Gold et
al. 1996a
; Harper and Lawson 1985
; Pearce
and Duchen 1994
; Rang et al. 1994
). Along these
lines, in cultured rat DRG, Cardenas et al. (1995)
classified neurons into four primary types based on their AP shape and
duration, capacitance, presence of IA and IH
potassium channels, types of calcium channels, TTX-S and TTX-R sodium
channels, and capsaicin sensitivity. Subsequent studies agreed with
this general categorization of DRG subtypes, although some exceptions
were reported (Gold et al. 1996
; Pearce and
Duchen 1994
). Others distinguished distinct small-diameter DRG
based on the binding of isolectin (IB4), presence of peptides, APs and ratios of TTX-R VGSCs (Stucky and Lewin
1999
).
In this study, we followed the classification scheme of Cardenas
et al. (1995) regarding the types of DRG neurons, based on their APs properties and sensitivity to capsaicin. We distinguished the
same four AP shapes and also found, in agreement with others, that
capsaicin-sensitive neurons contain both TTX-S and TTX-R VGSCs (Fig. 9
and Arbuckle and Docherty 1995
; Gold et al.
1996
; Kim et al. 1999
; Stucky and Lewin
1999
; Su et al. 1999
). We also found that
capsaicin inhibits TTX-R VGSCs (Fig. 9).
We have extended the pioneering work of Su et al. (1999)
that showed that capsaicin selectively blocks INa in
capsaicin-sensitive neurons by demonstrating that capsaicin inhibited
the APs (as defined by increasing the current necessary to evoke them)
only in capsaicin-sensitive Type I and II TG neurons (Figs. 1 and 2). Capsaicin (1 µM) also inhibited both the total and TTX-R INa only in
capsaicin-sensitive neurons (Figs. 3 and 9). This specificity, the
blocking action of CPZ, the K1/2 that
approximated that of activation of capsaicin receptors, and the
correlation between the magnitude of the capsaicin response and INa
inhibition, suggest that following capsaicin receptor activation, TTX-R
and TTX-S VGSCs are inhibited by an indirect pathway.
Capsaicin activates cAMP in trigeminal ganglion neurons
In cultured sensory TG neurons, we found that the addition of
capsaicin raised intracellular cAMP concentrations (Fig. 10). The
apparent K1/2 of 0.18 µM for cAMP
activation is lower then that usually required for ion current
activation by vanilloid receptors in DRG and TG neurons (0.5-0.8 µM)
(Jung et al. 1999; Koplas et al. 1997
;
Liu and Simon 1996
, 2000
) and by VR1 receptors expressed
in Xenopus oocytes (0.7 µM) (Caterina et al. 1997
,
2000
). Even lower EC50 values have been reported for
capsaicin-induced changes in intracellular calcium in DRGs (0.07 µM)
(see Cholewinski et al. 1993
). However, comparison of
the concentration dependence of different end points following
capsaicin exposure indicates that such values could still arise from
the activation of VR1 receptors (Szallasi et al. 1999
).
The vanilloid receptor antagonist, CPZ blocks virtually all
capsaicin-activated currents (Caterina et al. 1997
;
Liu and Simon 1996
), but it only partially effective in
blocking the capsaicin-activated increase in cAMP. This suggests that
activation of vanilloid receptors may have released peptides that could
have activated cAMP in a manner that was not inhibited by CPZ.
Presently the ability of capsaicin to increase cAMP are controversial
(Jansco and Wollemann 1977
; Notham and Jones
1987
; Sluka 1997
; Wood et al.
1989
). In rat spinal cord tissue slices, capsaicin induces an
accumulation in cAMP (Notham and Jones 1987
). The
dose-response curve obtained in spinal cord slices shows a similar
biphasic cAMP response as we found in TG (Fig. 10). In contrast, others
found that capsaicin did not produce change in cAMP in cultured DRGs
(Wood et al. 1989
). It is not clear what distinguishes
the latter study from the present and previous findings.
Role of cAMP in the modulation of VGSCs
Activation of cAMP/PKA pathways has physiological implications for
nociceptor sensitivity. Inflammatory mediators like bradykinin (BK) and
PGE2, augmented the sensitivity to noxious
stimuli (Cesare and McNaughton 1997; Gold
1999
). Similarly, cAMP sensitizes the nociceptive response to
heat of unmyelinated afferent nerves (Kress et al. 1996
)
although capsaicin appears to impair thermal nociception (Kohane
et al. 2000
). In the spinal cord, increases in cAMP
concentration, combined with capsaicin exposure, enhanced sensory
sensitivity to mechanical stimulation, resulting in hyperalgesia and
allodynia (Sluka 1997
). Mice mutated for the neuronal
regulatory unit of cAMP-dependent PKA exhibited, on capsaicin exposure,
reduced neurogenic inflammation (Malmberg et al. 1997
).
Thus under physiological conditions in sensory neurons, cAMP pathways
appear to be involved in sensitization rather than desensitization
processes even though we found the total INa is reduced by cAMP (Fig.
11). It follows that capsaicin may
activate other second-messenger pathways (see following text), which
would provide a basis for selective modulation (e.g., sensitization and
desensitization) of voltage gated channels and APs and consequently the
nociceptor sensitivity to endogenous or exogenous compounds.
|
As noted, hyperalgesic agents such as PEG2,
serotonin, and bradykinin, are thought to exert their effect by
increasing cAMP to upregulate PKA that will increase VGSCs
(England et al. 1996; Gold et al. 1996
;
Pitchford and Levine 1991
). However, not every study
found that PEG2 (or BK), affected VGSCs
(Su et al. 1999
). Our data suggest that the
membrane-permeable cAMP analogue, CPT-cAMP (1 mM) depresses the total
VGSC by ~30%, while subsequent exposure of the same
capsaicin-sensitive neuron to 1 µM capsaicin further reduced the
VGSC-evoked currents (Fig. 11). Although we have not explored the dose
dependence of the cAMP effects (see Fitzgerald et al.
1999
; Gold et al. 1998
), the data suggest that
the capsaicin-induced increase in cAMP may only be partly responsible
for the inhibition of VGSCs in capsaicin-sensitive neurons. Besides
cAMP, other second messengers might play a role in the blocking effect
of capsaicin on VGSCs. For instance, both intracellular
Ca2+ (Bleakman et al. 1990
) and
cGMP (Wood et al. 1989
) are elevated during capsaicin
receptor activation by influx through vanilloid receptor-coupled ion
channels or through PKG- or PKC-mediated pathways. Both second
messengers are also known to modulate VGSCs (Fitzgerald et al.
1999
; Marban et al. 1998
).
In inquiring how cAMP may decrease INa, we found that neither 1 mM
CPT-cAMP or 1 µM capsaicin changed the activation or inactivation phases of INa. In contrast, 1 µM capsaicin lengthened the current decay (Fig. 3) and slightly enhanced INa inactivation (Fig. 6). Thus
the effects of cAMP and capsaicin are not equivalent, although they
both block INa to different extents. In cultured neurons, the effects
of cAMP have been shown to be quite variable. For example, brain VGSCs
were inhibited by cAMP without an apparent change in the time course
(Catterall 1999; Gershon et al. 1992
). In
one study using DRGs, the cAMP activator forskolin increased the peak
amplitude as well as delayed the decay of TTX-R sodium currents
(Gold et al. 1998
), whereas in other studies, the
activation of the cAMP/PKA pathway did not alter VGSCs (Cardenas
et al. 1997
; Su et al. 1999
). The differences
may arise from different concentrations tested since the cAMP or
forskolin responses are biphasic (Fig. 10) (Gold et al.
1998
; Notham and Jones 1987
). Altogether various VGSCs can be unaffected, inhibited, or sensitized by activation of PKA
or elevated cAMP (Cantrell et al. 1997
; Gershon
et al. 1992
; Gold et al. 1996
; Narahashi
1999
; Smith and Goldin 1997
). Taking into
account the wide variety of different TTX-S and TTX-R VGSC subtypes
expressed in sensory neurons (Dib-Hajj et al. 1998
; Tate et al. 1998
; Wood and Docherty 1997
;
Woolf and Costagin 1999
), distinct VGSCs may be
differentially modulated by second messengers, which could rationalize
the variability in sodium channel sensitivities.
Capsaicin receptor modulation by cAMP
In mammalian sensory neurons, capsaicin-activated inward currents
are increased by cAMP (Fig. 12 and
Lopshire and Nicol 1998). Preexposure to CPT-cAMP
potentiated the capsaicin response although it did not inhibit
subsequent desensitization or tachyphylaxis (Fig. 12). It appears that
the effect of cAMP or PKA does not arise from either of them activating
directly homomeric vanilloid receptors containing VR1 subunits. This is
because of the results of experiments in which the VR1 receptor was
expressed in Xenopus oocytes and/or Aplysia neurons that
revealed that neither cAMP or PKA directly activated these receptors
and that PKA likely phosphorylated a protein found in sensory neurons
that interacted with the VR1 receptor/channel (Lee et al.
2000
).
|
The initial application of capsaicin to sensory neurons produces an
excitatory response. This is evidenced from current-clamp experiments
in which a burst of APs accompanies the first application (Fig. 2)
(Gold et al. 1996b; Lopshire and Nicol
1997
). We have extended this observation by measuring the
responses of the generated APs to repeated capsaicin applications.
While the amplitude of the response to capsaicin gradually declined
presumably due to receptor desensitization (Figs. 2 and 12), the APs
appeared to be irreversibly blocked after the initial capsaicin
exposure (Fig. 2, A and B). The effect of
capsaicin on AP generation as well as the distinct effects of cAMP
indicate that capsaicin receptor desensitization and capsaicin-mediated
inhibition of APs (and VGSC) are two separate pathways of
capsaicin-sensitive (nociceptor) desensitization.
In summary, these data present one mechanism by which capsaicin may desensitize capsaicin-sensitive trigeminal ganglion nociceptors by inhibiting the generation of APs through the indirect block of voltage-gated sodium channels. This analgesic effect of capsaicin may be useful in defining new compounds that potentially can alleviate pain or sensitization without the initial inflammatory reaction associated with irritants like capsaicin.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute on Deafness and Other Communication Disorders Grant DC-01065 and a grant from the Philip Morris External Research Program.
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FOOTNOTES |
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Address for reprint requests: S. A. Simon, Dept. of Neurobiology, Duke University Medical Center, 427E Bryan Research Bldg., Research Dr., Durham, NC 27710 (E-mail: sas{at}neuro.duke.edu).
Received 24 May 2000; accepted in final form 24 October 2000.
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REFERENCES |
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