Capsaicin Inhibits Activation of Voltage-Gated Sodium Currents in Capsaicin-Sensitive Trigeminal Ganglion Neurons

L. Liu, M. Oortgiesen, L. Li, and S. A. Simon

Department of Anesthesiology and Neurobiology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Adelta and C fibers without affecting Abeta 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 MOmega 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[(V0.5Vm)/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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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1. Properties of TG neurons



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Fig. 1. Four types of action potentials in trigeminal ganglion (TG) neurons. TG neurons were current-clamped at -60 mV, and action potentials (APs) were evoked with 20-ms depolarizing current pulses with increasing amplitude (0.1-2 nA in 50.0-pA steps). The APs shown were those that corresponded to the lowest value of injected current in which the AP shape did not markedly change. Different APs types were distinguished according to the classification scheme of Cardenas et al. (1995) (see Table 1). The cells were exposed to 1 µM capsaicin for 2 min. If the neuron was capsaicin sensitive, the voltage depolarized and eventually returned to baseline when the channels desensitized. During these periods, inward currents of the same magnitude used to generate the control APs were applied and the ensuing changes in voltage were recorded. After a several-minute wash (last traces in Types II-IV), the reversibility of the capsaicin-induced effect was tested. For the Type II neuron in the presence of capsaicin, the first evoked potential was elicited while the cell was depolarized, and the second, after the potential was reset to -60 mV. After wash, an AP similar to the control could once again be evoked. Under the similar conditions, APs in neuron Types III and IV were unaffected by 1 µM capsaicin.

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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Fig. 2. Capsaicin desensitization of APs and receptors. A: a Type II neuron. B: during the 1st capsaicin exposure, the capsaicin-induced depolarization was accompanied by a burst of APs. During the 2nd and subsequent applications of capsaicin, APs failed to be evoked even though the potential was the same (-60 mV). Each capsaicin application was separated by a 3-min wash. Bars indicate duration of capsaicin applications. C: a similar experiment to one in B, but one in which APs were evoked by 20-ms pulses of currents of 100 and 1,000 pA before and after the application of 1 µM capsaicin. The 1st application of capsaicin activated a burst of APs. Before the 2nd application (not shown), the APs were slightly reduced in amplitude as was the capsaicin-induced depolarization, which did not evoke APs (data not shown). The 2 modified APs shown in the 100- and 1,000-pA pulses before the third capsaicin application are greatly attenuated demonstrating that the threshold for generating them was increased both by current pulses and by application of capsaicin. As shown, no APs were generated on the capsaicin application. Bar indicates duration of capsaicin application.

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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Fig. 3. Effects of capsaicin on voltage-gated sodium channels in capsaicin-sensitive TG neurons. A: voltage-gated INa were evoked by 40-ms step depolarizations from -60 to +60 mV with 5-mV increments. In this neuron, 1 µM capsaicin induced an inward current with a peak amplitude of -1.3 nA (not shown). After 2 min, the holding current had returned to the control level, and the same pulse sequence was applied. The INa was reduced in the presence of capsaicin. After wash subsequent exposure to 10 µM capsaicin further reduced the INa. After a 3-min wash the inhibition was partly reversed. B: the peak sodium currents (INap) from A were plotted vs. the step depolarization from -60 mV. Shown are the I-V curves in control, in the presence of 1 and 10 µM capsaicin, and after wash. C: averaged G-V curves in control and in the presence of 1 µM capsaicin. ---, fits to the Boltzmann equation with V0.5 = -17.7 and -15.8 mV and k =8.5, and 8.8 mV for controls and in the presence of 1 µM capsaicin. Capsaicin did not produce a change in the voltage dependence. D: the 3 superimposed currents showing the changes in the time course between the INa in control solution (a) and during capsaicin exposure but after the current returned to baseline (b). The INa in the presence of capsaicin was normalized (c) to the peak of the control peak INa. These data show no change in rise time, but a delayed inactivation.

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 Adelta 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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Fig. 4. Effects of capsaicin on voltage-gated sodium channels (VGSCs) in capsaicin-insensitive TG neurons. Voltage-gated INa were evoked by 40-ms step depolarizations from -60 to +60 mV with 5-mV increments. In this TG neuron, capsaicin (10 µM) was ineffective in inducing an inward current (data not shown). After 2 min capsaicin exposure, the same protocol was applied, showing a small reduction of the INa. The inhibition was not reversible after a 3-min wash. The I-V curves were depicted with the peak currents (INap) against the step depolarization from -60 mV showing the small inhibition by capsaicin at all potentials <0 mV. See text for further details.



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Fig. 5. Dose-dependent block of VGSCs by capsaicin. A: plots of the inhibitory effects of capsaicin in capsaicin-sensitive and -insensitive TG neurons. The relative inhibition of the INap at the maximum depolarization (INamax) in the presence of capsaicin compared with the control INap. The K1/2 was fitted to the data on capsaicin-sensitive neurons was 0.45 µM. The numbers above each data point represent individual experiments done at these concentrations. B: graph of the relationship between the peak inward current of the capsaicin-induced response (ICAPSp) and the relative inhibition of the voltage-gated INap at the INamax. Each data point represents the results from an individual capsaicin-sensitive TG neuron. The inhibition of the INap increased with increasing capsaicin-induced response amplitudes and reached a maximum of ~90% for capsaicin currents greater than -2 nA.

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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Fig. 6. Effects of capsaicin on sodium channel inactivation. A: the voltage dependence of INa inactivation was determined by applying 40-ms prepulses from the -60-mV holding potential first to -80 mV and then to 0 mV. Subsequent pulses were reduced from -80 mV with 5-mV incremental steps (every other step shown). After those variable prepulses, the test pulse of 40 ms to 0 mV was applied. The relative peak current (INap) of the test pulse compared with that after the -60-mV prepulse was depicted for the different potentials. The INap-V curves depict the relative peak current in controls () and in the presence of 1 µM (A) or 10 µM (B) capsaicin (). In capsaicin-sensitive TG neurons (A), the amplitude of the INa was reduced. In addition, the V0.5 was shifted from -34.9 ± 8.8 mV (n = 5) and -40.4 ± 8.5 mV (n = 5). The shapes of the response indicate the presence of 1 type of VGSC. In capsaicin-insensitive TG neurons, even at 10 µM capsaicin, the amplitude of the INa was not or only slightly affected. The fits to these data (---) to Boltzmanns for controls and capsaicin-insensitive neurons had V0.5 = -45.0 and -43.3 mV and k factors of 8.1 and 8.5, respectively.

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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Fig. 7. Effects of capsaicin on use-dependent inactivation in TG neurons. Voltage-gated INa were evoked from TG neurons held at -60 mV by 40-ms pulses to -10 mV applied at 1-s intervals. The INap at different times is depicted relative to the 1st INap, indicating the reduction in VGSC activation in control solution () and in the presence of 1 µM capsaicin (open circle ). A: use dependence of INa in a capsaicin-sensitive neuron. Capsaicin inhibited the INa and slightly enhanced use-dependent block. After a 3-min wash, the use-dependent blockage returned to control values (not shown). The averaged responses of 5 experiments are shown in the right panel. B: use-dependent blockage of INa in a capsaicin-insensitive neuron. Capsaicin (1 µM) neither affected the INap nor use-dependent blockage. The averaged responses of 5 experiments are shown in the right panel.

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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Fig. 8. Capsazepine (CPZ) inhibits the capsaicin-induced block of VGSCs. After obtaining VGSC currents the addition of 1 µM, capsaicin induced a transient inward current that after it desensitized markedly blocked VGSCs. The inhibitory effect of capsaicin was reversible after 5-min wash. The presence of 10 µM CPZ did not affect the INa. The subsequent application of 1 µM capsaicin plus 10 µM CPZ evoked a small, but transient, inward current (not shown). In the presence of CPZ and capsaicin, VGSCs were not inhibited. After a 3-min wash, the subsequent application of capsaicin did not induce a detectable inward current. However, capsaicin still reversibly inhibited the INa. Between exposures and during stimulation with capsaicin, the neuron was voltage-clamped at -60 mV. The current amplitude and time marks are indicated for the sodium currents, the brackets at the capsaicin-induced inward current define -1.5 nA and 10 s.

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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Fig. 9. Effects of capsaicin on TTX-R VGSCs in capsaicin-sensitive TG neurons. VGSCs were evoked by 40-ms step depolarizations from -60 to +60 mV with 5-mV increments. Exposure to 0.1 µM TTX inhibited the INa by 70%, leaving the TTX-R component. Application of capsaicin (1 µM) induced a transient inward current. When the holding current returned to the control level, the same protocol was applied in the presence of TTX and capsaicin. The TTX-R current was reduced 61% in the presence of capsaicin. It was partly reversible on wash.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The pungent compound, capsaicin, activates vanilloid receptors located primarily on C and Adelta (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.



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Fig. 10. Capsaicin increases cAMP in cultured TG neurons. Exposure of cultured TG neurons to capsaicin elevates cAMP as measured by enzyme immunoassay (EIA). The increase was concentration dependent, having an apparent K1/2 of 0.18 µM that was fit to the concentration-response curve (assuming the values at 1 µM capsaicin are maximal). CPZ (10 µM) partially inhibited the capsaicin-induced elevation of cAMP. At 1 µM capsaicin, CPZ significantly inhibited the increase in cAMP (paired t-test, P < 0.05).

Types of TG neurons and effects of capsaicin

Primary afferent sensory neurons are classified by their conduction velocity as C, Adelta , and Abeta 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.



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Fig. 11. Modulation by cAMP on VGSCs in TG neurons. Voltage-gated INa were evoked by 40-ms step depolarizations from -60 to +60 mV with 5-mV increments. A: preexposure to the membrane permeable CPT-cAMP (1 mM) for 2 min, reduced the INa. After wash, the application of capsaicin (1 µM) induced an inward current and further reduced the sodium current. B: in a capsaicin-insensitive neuron, the INa was also inhibited by 1 mM CPT-cAMP. The inhibition was partially reversible. Capsaicin (10 µM) was ineffective in inducing an inward current and only marginally reduced the sodium current.

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).



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Fig. 12. Effect of cAMP on capsaicin response in TG neurons. Top: applications of 1 µM capsaicin evoke inward currents. In all experiments, 1 µM capsaicin was applied 30 s every 3 min. Preexposure to CPT-cAMP (100 µM, 2 min) itself did not evoke a current, but the maximum amplitude of the capsaicin-induced inward current was over this time period was increased. Shown in this figure are the currents evoked by the 2nd capsaicin application in the presence of CPT-cAMP (at 6 min) and the 4th application (at 12 min). Bottom: averaged data for experiments performed in the absence of CTP-cAMP () and in the presence of 0.1 mM CPT-cAMP (). Receptor desensitization (or rundown) was not prevented by the presence of CPT-cAMP. The ordinate shows that the relative (to the initial application) of the peaks of the capsaicin evoked currents (ICAPSp). HP = -60 mV. Data for controls from Liu and Simon (1966).

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society