Calcitonin gene-related peptide enhances TTX-resistant... : PAIN (original) (raw)
1. Introduction
Calcitonin gene-related peptide (CGRP) is synthesized in about 40% of small- and medium-sized primary afferent neurons many of which are nociceptive (Ju et al., 1987; McCarthy and Lawson, 1990; McNeill et al., 1988). Release of CGRP from sensory endings in the periphery causes vasodilation and produces neurogenic inflammation (Brain et al., 1985; Kilo et al., 1979). Release of CGRP in the spinal cord facilitates spinal nociceptive processing (c.f. Schaible et al., 2004). There are two CGRP receptor subtypes (CGRP1, CGRP2) that are coupled to G proteins and elevate cAMP levels in many cells (Dennis et al., 1989; Poyner, 1992; Quirion et al., 1992; Sexton et al., 1988).
Only few studies have investigated whether CGRP has an effect on primary afferent neurons. Recently, we have shown that CGRP acts on cultured dorsal root ganglion (DRG) neurons from adult rat. About 20% of the DRG neurons bound CGRP-gold complexes suggesting binding sites for CGRP on the cell bodies. In addition, CGRP caused in up to 30% of lumbar DRG neurons an elevation of intracellular calcium. Finally, in patch clamp studies CGRP evoked inward currents in about 30% of the DRG neurons (Segond von Banchet et al., 2002). Some studies in vivo lend support to the presence of CGRP receptors in primary afferent neurons. Ye et al. (1999) suggested that CGRP receptors in the spinal cord are also located in primary afferent neurons. Pokabla et al. (2002) and Ma et al. (2003) identified in many small- to medium-sized DRG neurons the CGRP receptor component protein (CGRP-RCP), which is an intracellular membrane protein that interacts with CRLR (the receptor structure in the membrane) and facilitates CGRP-mediated signaling (Evans et al., 2000; Luebke et al., 1996). Nakamura-Craig and Gill (1991) found sensitization in the paw pressure test after local CGRP injection.
In the present study we began to investigate which currents are affected by CGRP. We focused on TTX-resistant (TTX-R) voltage-dependent sodium currents because TTX-R Na+ channels are expressed preferentially or exclusively in small- to medium-sized nociceptive primary afferent neurons (Akopian et al., 1996, 1999; Dib-Hajj et al., 1998; Sangameswaran et al., 1996) and because CGRP binding has been mainly found in small- to medium-sized DRG neurons (Segond von Banchet et al., 2002). Two types of TTX-R Na+ channels have been identified, namely SNS or PN3 and NaN or SNS2, now Nav1.8 and Nav1.9 (Goldin et al., 2002). Because TTX-R Na+ channels are modulated by inflammatory mediators such as prostaglandin E2 (PGE2), they have been of particular interest in recent pain research (McCleskey and Gold, 1999).
2. Methods
2.1. Primary culture
Adult male Wistar rats (age 60 days) were killed with ether. DRGs were dissected from all spinal segments. The DRGs were incubated at 37°C with 215U/mg collagenase type II (GibcoBRL, D-76344 Eggenstein-Leopoldshafen, Germany) dissolved in Ham's F-12 medium (Sigma, D-82039 Deisenhofen, Germany) for 100min. After washing three times with Ca2+- and Mg2+-free phosphate buffered saline (PBS, pH 7.4, GibcoBRL), the DRGs were incubated for 11min at 37°C in DMEM (Sigma) containing 10,000U/ml trypsin (Sigma). Then the ganglia were dissociated into single cells by gentle agitation and by triturating through a fire-polished Pasteur pipette. The dispersed cells were collected by centrifugation (500×g, 5min), washed three times in DMEM and recentrifuged. In the standard culture the neuron pellets were suspended in Ham's F-12 medium containing 10−3M L-glutamine (Sigma), 10% heat inactivated horse serum (GibcoBRL), 100U/ml penicillin (GibcoBRL), 100μg/ml streptomycin (GibcoBRL), and 100ng/ml nerve growth factor (NGF 7S, Boehringer Mannheim GmbH, D-68298 Mannheim, Germany). In some cultures only 10ng/ml NGF was used. The cells were centrifuged again, resuspended in Ham's F-12 medium (containing L-glutamine, inactivated horse serum, penicillin, streptomycin, NGF, see above) and then plated on 13mm diameter glass cover slips (cells from about 1–1.5 ganglion/cover slip), which had been precoated with poly-L-lysine (50μg/ml, Sigma). The neurons were kept at 37°C in a humidified incubator gassed with 3.5% or 5% CO2 in air. Cultures were fed daily with Ham's F-12 medium.
2.2. Whole-cell patch clamp recordings
Currents were recorded from DRG neurons at room temperature using the whole-cell patch clamp technique (Axopatch 200B amplifier, Axon Instruments, USA). Electrodes (1.0–3.0MΩ) were fabricated from 1.7mm capillary glass (Kimax, Vineland, USA) using a Sutter P-97 puller. The recording pipette solution contained (in mM): 140 CsCl, 10 NaCl, 1 MgCl2, 0.5 CaCl2, 2 Na2-ATP, 5 EGTA, 10 HEPES, pH 7.2. Stimuli were controlled with pCLAMP 7.01 software (Axon Instruments, USA). Voltage errors were minimized using 75–85% series resistance compensation with amplifier circuitry. Current signals were filtered at 1kHz (4-pole Bessel) and sampled at 5kHz with the interface (Digidata 1200, Axon Instruments, USA). For presentation, data were filtered with a Gaussian filter at 500 or 250Hz.
Recordings were performed from DRG neurons that were in culture for 12–48h. Cover slips with cells were transferred to the recording chamber that was mounted on the stage of an upright Nikon microscope (equipped with a 63× water immersion objective). The cells were continuously superfused with a standard HEPES buffer (in mM): 35 NaCl, 72.5 choline-Cl, 5 KCl, 30 TEA-Cl, 2 CaCl2, 2 MgCl2, 0.1 CdCl2, 10 glucose, 10 HEPES, pH 7.4. Cadmium and TEA were included to block calcium and potassium currents, respectively, and 500nM TTX was added to block TTX-sensitive Na+ currents. When compounds (CGRP, the CGRP receptor antagonist CGRP8–37, the protein kinase A (PKA) blocker PKA14–22) were tested, the standard HEPES buffer was switched to a HEPES buffer containing one or two of these compounds. Alpha-CGRP (rat, Bachem, Heidelberg, Germany) was dissolved in ddH2O and then added to HEPES buffer. The final concentration of CGRP in the experiments was 250nM. CGRP8–37 (Bachem, Heidelberg, Germany) was dissolved in the same way. PKA14–22 (500μg, Calbiochem, Bad Soden, Germany) was dissolved in water, and the final concentration in HEPES buffer was 40nM. By contrast, the PKC blocker PKC19–31 (1μM) was added to the pipette solution and thus applied intracellularly during recording.
In further experiments whole-cell patch clamp recordings were used to record action potentials from DRG neurons. After forming the seal under voltage clamp conditions, the amplifier was switched to the current-clamp mode. In these experiments the recording pipette was filled with the following solution (in mM): 140 KCl, 1 MgCl2, 0.5 CaCl2, 2 Na2-ATP, 5 EGTA, 10 HEPES, 10 sucrose, pH 7.2. The cells were superfused with a standard HEPES buffer (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, 10 HEPES, pH 7.4. When the effect of CGRP was to be tested, this solution was switched to a HEPES buffer solution containing 250nM CGRP.
2.3. Experimental protocol
A standard voltage clamp protocol was used to allow comparison of data collected under different experimental conditions. Whole-cell configuration was established after formation of a tight seal (>2GΩ) and compensation of pipette capacitance with amplifier circuitry. Initially the resting membrane potential (_E_res) was measured in the current-clamp mode. After switching to the voltage-clamp mode, Na+ currents were elicited by voltage steps. Starting from a holding potential (_V_H) of −70mV, the neurons were depolarized with 30ms pulses in increments of 5mV up to either +40 or +45mV (interpulse interval=2.0s) (Fig. 1(A,B)). In some experiments a double-pulse protocol was used. The neurons were first depolarized stepwise for 200ms as described before, but after the first pulse they were clamped at −5mV for 40ms. Data from these neurons were used to calculate steady-state inactivation.
Effect of CGRP on TTX-R Na+ currents in DRG neurons. (A) Neuron in which CGRP caused an enhancement of the peak current. Currents were elicited by depolarization from −70 to +45 mV in 5 mV increments (holding potential, V H=−70 mV) before CGRP (top), 5 min after bath application of 250 nM CGRP (middle) and 5 min after washout of CGRP (bottom). (B) Lack of effect of CGRP on TTX-resistant Na+ currents in another DRG neuron.
In order to test the effect of CGRP on TTX-R Na+ currents, neurons were superfused with HEPES buffer. The voltage clamp protocol was applied several times to check whether recording conditions were stable. Then the solution was replaced by a solution containing the HEPES buffer plus 250nM CGRP for 12min. In this time interval the voltage protocol was applied approximately every 2min. Finally the solution was switched to HEPES buffer and recovery was studied. In the recording session a neuron was considered responsive to CGRP when CGRP induced a visible effect on Na+ currents (at least 5–10% change). The final classification of a neuron as being responsive or unresponsive to CGRP was confirmed by off-line analysis (see Section 2.4).
In further experiments we tested whether the CGRP receptor antagonist CGRP8–37 antagonizes the effect of CGRP on TTX-R Na+ currents. Cells were superfused with HEPES buffer for about 5min. When recordings were stable, we switched to HEPES buffer with CGRP8–37 (250nM) for 5–7min. Thereafter HEPES buffer plus CGRP (250nM) and CGRP8–37 (250nM) was administered, and recordings were made at approximately every 2min.
With a similar protocol we tested whether the action of CGRP is dependent on the activation of protein kinase A. After the initial recordings, the standard solution was switched to HEPES buffer with PKA14–22 (40nM), and currents were recorded for 5–7min. Then the solution was changed to HEPES buffer plus PKA14–22 and CGRP (250nM), and currents were measured for about 15min. By contrast, the PKC inhibitor PKC19–31 (1μM) was administered via the recording pipette. After 5–7min of recording, the external standard HEPES buffer was switched to HEPES plus CGRP (250nM).
In a subset of experiments we tested whether CGRP alters the threshold for the generation of an action potential. After the initial procedures the amplifier was switched to the current-clamp mode. At the resting potential, current was applied in 20pA steps (pulse duration 5ms, interpulse interval 1s) until action potentials with the typical overshoot were elicited. Every 2min the stimulation was repeated in 10pA steps several times to determine the firing threshold more precisely. Thereafter the solution for superfusion was changed to a solution containing 250nM CGRP. Within 5–6min after application of CGRP, the current was applied in 10pA steps every 2min to determine the threshold in the presence of CGRP. In control experiments the threshold was repeatedly determined in a period of 15min without changing the solution.
2.4. Data analysis
The data were analysed using the pCLAMP 7.01 software (Axon Instruments, USA) and Origin 6.1 (Microcal Software, Northampton, MA) software programs. Current densities were calculated by dividing the peak current (_I_peak) evoked at each membrane potential (_V_m) by the cell capacitance (_C_m). The peak conductance (G) of Na+ currents at each potential was calculated from the corresponding peak current by using the equation Symbol (_E_Rev: reversal potential of Na+ current; I: peak current amplitude of Na+ current; E: membrane potential). Normalized peak conductance (G/_G_max) and the data describing the fractional decrease in the peak current during the steady-state inactivation (I/_I_max) were fitted with a Boltzmann function I/_I_max or Symbol where _V_½ is the membrane potential generating half maximal current or conductance, respectively, _V_m is the prepulse membrane potential, and k is the slope of the function. Time constants for activation and inactivation were obtained by fitting the activating and inactivating phase of TTX-R Na+ currents with a single exponential function. All data are expressed as means±SEM (_n_=number of tested cells) unless otherwise stated. For the display of I/V curves the average peak currents at each voltage test were used. For statistical comparison of current densities before and after treatment the maximal negative peak currents were taken from each neuron irrespective of shifts of maximum currents with respect to voltage, and the paired _t_-test was used. Significance was accepted at P<0.05. Individual neurons were considered CGRP-responsive when the maximal peak Na+ currents were increased by at least 10% after CGRP application. The size of the neurons was calculated from capacitance that was read out using the specific membrane capacitance (1μF/cm2).
In order to evaluate the effects of CGRP8–37 and of the PKA and PKC inhibitors on CGRP responses, we assessed the proportions of neurons with a response to CGRP under these conditions and compared these proportions to the proportion of responding neurons when only CGRP was applied using Fisher's exact test. The same test was used to compare proportions of neurons that showed a reduction in the current that was necessary for action potential generation in control cells (superfusion without CGRP) and in neurons superfused with CGRP. Significance was accepted at P<0.05.
3. Results
3.1. CGRP enhances Na+ currents in a proportion of DRG neurons
Fig. 1 displays the recording of TTX-R voltage-dependent Na+ currents in two DRG neurons before and during bath application of 250nM CGRP and during washout of CGRP. Bath application of CGRP enhanced the amplitude of Na+ currents in the neuron shown in Fig. 1(A) but not in the neuron shown in Fig. 1(B). Fig. 2 shows the I/V curves (top) of 20 CGRP-responsive (A) and 32 CGRP-unresponsive DRG neurons (B) before and during CGRP application. In the CGRP-responsive neurons the maximal TTX-R Na+ current was −125.2±10.1pA/pF before CGRP and −156.3±13.0pA/pF in the presence of 250nM CGRP (measured 5min after CGRP application), and thus the maximal current density increased by 26±4% (P<0.01, paired _t_-test). The CGRP application caused a small voltage shift of the maximal peak current in the hyperpolarizing direction. In the CGRP-unresponsive neurons we observed a decrease of the maximal current density by about 8.5±1.5% after about 5min. The same effect was observed in control neurons that were not exposed to CGRP (see Figs. 4(A) and 5(A)).
Effect of CGRP on TTX-R Na+ currents in samples of DRG neurons. (A) CGRP-responsive neurons (_n_=20). (B) CGRP-unresponsive neurons (_n_=32). Top graphs: averaged I/V curves before (black squares) and after 250 nM CGRP (circles). Bottom graphs: Membrane conductance (G/G max) before (black squares) and during CGRP (circles).
Time course of TTX-R Na+ currents in CGRP-responsive, CGRP-unresponsive and control neurons and size of neurons. (A) Peak changes in 17 CGRP-responsive neurons, 19 CGRP-unresponsive neurons and 28 neurons not tested with CGRP (control). (B) Cell size of CGRP-responsive, CGRP-unresponsive neurons and neurons not tested with CGRP (control).
(A) Peak current changes in control neurons (not tested with CGRP), in CGRP-responsive neurons, in neurons tested with CGRP in the presence of CGRP8–37, PKA14–22, or PKC19–31. (B) Proportions of neurons that responded to bath application of CGRP in different samples of neurons tested.
CGRP correspondingly influenced the peak conductance (G/_G_max) in CGRP-sensitive cells (Fig. 2(A), bottom, circles). The half-maximal potential (_V_½act) for activation of the TTX-R Na+ current was −8.0±0.3mV in the absence of CGRP and −13.7±0.3mV in the presence of 250nM CGRP (_n_=20 neurons). The slope factor k did not significantly change when CGRP was applied (_k_=4.9±0.2mV before, _k_=5.2±0.3mV during exposure to CGRP). The time constants for activation and inactivation at 0mV were, respectively, 1.02±0.06ms and 4.90±0.20ms in the control period and 0.91±0.05ms and 4.68±0.27ms in the presence of CGRP. In the CGRP-unresponsive neurons (_n_=32 neurons) the peak conductance did not significantly change (Fig. 2(B), bottom), and no significant changes of activation and inactivation were observed.
In a further group of neurons we also studied steady-state inactivation. Fig. 3(A) shows CGRP-responsive neurons (_n_=5) and Fig. 3(B) displays CGRP-unresponsive neurons (_n_=6). In the CGRP-responsive neurons the half-maximal potential for steady-state voltage-dependent inactivation (_V_½inact; I/_I_max) of the TTX-R Na+ current was −25.0±0.2mV before and −30.2±0.5mV during CGRP (P<0.05, paired _t_-test; _k_ was 4.7±0.2mV before and 4.9±0.4mV during CGRP). In the CGRP-unresponsive neurons the half-maximal potential for steady-state voltage-dependent inactivation (_V_½inact; _I_/_I_max) of the TTX-R Na+ current was −24.5±0.4mV before and −25.8±0.5mV during CGRP (_P_>0.05, paired _t_-test; k was 5.7±0.4mV before and 5.9±0.5mV during CGRP).
Steady-state inactivation (I/I max) of TTX-R Na+ currents before (black squares) and during CGRP (circles) in 5 CGRP-responsive neurons (A) and 6 CGRP-unresponsive neurons (B).
Maximal _I_peak changes in CGRP-responsive neurons (dots) were seen 1min after CGRP (Fig. 4(A); CGRP was given at 0min, the last time point of the pre-drug control period). This increase lasted the whole application period of CGRP. In CGRP-unresponsive neurons (triangles), maximal _I_peak showed a slight decrease over time. A similar decrease of maximal _I_peak was seen in control neurons that were not exposed to CGRP (filled squares). Thus, there was no evidence that CGRP reduces currents. When CGRP was washed out substantial or complete recovery of TTX-R currents was usually seen within 5–10min.
Fig. 4(B) plots the changes of maximal _I_peak currents (measured at 5min after CGRP application) versus cell capacitance of the neurons. Cells with and without a CGRP response and neurons not tested with CGRP were in the same size range. In total 20 of 61 neurons (33%) showed an increase (>10%) of the maximal _I_peak after CGRP and were thus considered CGRP-responsive (Fig. 5(B)). Fig. 5(A) shows the maximal _I_peak changes in control neurons and in different groups of neurons tested with CGRP.
Because long-term exposure to NGF in culture has been shown to modulate the expression of mRNA of TTX-R Na+ channels in neurons (Fjell et al., 1999), we studied the effect of CGRP on TTX-R Na+ currents also in DRG neurons that were cultured in the presence of only 10ng/ml NGF instead of 100ng/ml. In this particular sample of 21 neurons tested 5 showed an increase of TTX-R Na+ currents after CGRP application. In the 5 CGRP-responsive neurons maximal current peak densities were −139.3±42.0pA/pF before CGRP and −172.8±39.4pA/pF during CGRP and thus increased on average by 34% (P<0.05, paired _t_-test). Thus the effect of CGRP on TTX-R Na+ currents was also seen after the lower NGF concentration in the culture medium.
3.2. CGRP8–37 blocks the effect of CGRP on TTX-R Na+ currents
In order to test whether CGRP effects can be blocked with CGRP8–37, an antagonist at the CGRP receptor (Dennis et al., 1990; Maggi et al., 1991), 21 neurons were treated with CGRP8–37 (250nM) before CGRP (250nM) was added. An example is shown in Fig. 6(A). It shows Na+ currents evoked by depolarization from −70 to +5mV before CGRP8–37 (control), 7min after application of CGRP8–37 and 7min after additionally adding CGRP. In this sample only one of 21 neurons showed an increase of maximal _I_peak during CGRP (Fig. 5(B)), and this proportion (4.8%) is significantly smaller than the 33% of the neurons that responded to CGRP alone (P<0.05, Fisher's exact test). The I/V curves of the neurons recorded under control conditions, in the presence of CGRP8–37 alone and in the presence of CGRP plus CGRP8–37, are almost overlapping (Fig. 6(B)). Fig. 5(A) shows the maximal _I_peak changes during CGRP in the presence of CGRP8–37.
Effect of the CGRP receptor antagonist CGRP8–37, the PKA inhibitor PKA14–22 and of the PKC inhibitor PKC19–31 on the responses of DRG neurons to CGRP. (A) TTX-R Na+ currents in a DRG neuron elicited by depolarization from −70 to +5 mV before compound application, during CGRP8–37 (250 nM) alone and during CGRP8–37 (250 nM) plus CGRP (250 nM). (B) I/V curves of 19 neurons before compound application, during CGRP8–37 (250 nM), and during CGRP8–37 (250 nM) plus CGRP (250 nM). (C) TTX-R Na+ currents in a neuron before compound application, 7 min after bath application of 40 nM PKA14–22 alone, and 7 min after coapplication of 40 nM PKA14–22 plus 250 CGRP to the bath. (D) I/V curves of 21 neurons before compound application, during PKA14–22 alone, and during PKA14–22 plus CGRP (250 nM). (E) TTX-R Na+ currents in a DRG neuron elicited by depolarization from −70 to +5 mV 2 and 8 min after whole-cell formation, and 5 min after application of 250 nM CGRP to the bath. (F) I/V curves of 21 neurons 2 min after starting of the recording, 8 min after starting of the recording, and during application of PKA19–31 plus CGRP (250 nM).
3.3. Protein kinases A and C are involved in CGRP-induced enhancement of TTX-R Na+ currents
Since CGRP raises the cAMP level, we tested whether the cAMP/PKA pathway mediates the effect of CGRP on TTX-R Na+ currents. Fig. 6(C) shows the testing of a neuron. The application of the membrane-permeant PKA inhibitor PKA14–22 caused a slight decrease of the current, but the subsequent application of PKA14–22 plus CGRP did not increase the current. In the whole sample of neurons tested with this protocol, only one of 21 neurons showed an increase of the peak current after application of CGRP in the presence of PKA14–22 (Fig. 5(B)). This proportion (4.8%) is significantly smaller than the proportion of CGRP-responsive neurons in the control sample (P<0.05; Fisher's exact test). Thus, inhibition of PKA prevented CGRP responses. Fig. 6(D) shows the I/V curves of these neurons under control conditions, in the presence of PKA14–22 alone, and in the presence of PKA14–22 plus CGRP. PKA14–22 itself caused an average reduction of the maximal current density by 9.3±3.0%. A further slight reduction of maximal current density occurred when CGRP was added to PKA14–22 (Fig. 5(A)).
It has been reported that the effect of PKA on TTX-R Na+ currents depends on PKC activity (Gold et al., 1998). We, therefore, tested whether the presence of the PKC inhibitor PKC19–31 in the pipette solution inhibits the response to CGRP. Fig. 6(E) displays the recording from one neuron. During recording with PKC19–31 in the pipette, the currents showed a decrease within 5min before they were stable, and subsequent bath application of CGRP did not increase the currents. In the presence of PKC19–31 an increase of the Na+ currents after CGRP was only seen in 2 of 21 neurons, and this proportion (9.5%) is significantly smaller than that of CGRP-responsive neurons in the absence of other compounds (Fig. 5(B)). The pronounced effect of PKC19–31 can be seen in I/V curves of the neurons 2 and 8min after PKC19–31 in Fig. 6(F). During application of CGRP in the presence of PKC19–31 a further reduction of the peak Na+ current was observed (Fig. 5(A)).
3.4. The effect of CGRP on the generation of action potentials
In order to test whether CGRP would influence the threshold for eliciting an action potential in DRG neurons, recordings were made in the current-clamp mode. In these experiments no TTX and no blockers of K+ and Ca2+ channels were used because it was not the aim to show the contribution of specific currents to threshold changes. Current pulses were applied in 10pA steps. Fig. 7 shows a DRG neuron in which an action potential was elicited at 30pA before CGRP (control), at 20pA during CGRP, and at 30pA after washout of CGRP. In 9 of 28 DRG neurons, 5–6min after the beginning of CGRP application an action potential was elicited with 10pA less current. In the control sample of 14 neurons, in which the threshold was tested repeatedly during superfusion of buffer only, none of the neurons showed a reduction of threshold within 12min. This difference in the proportion of neurons with a reduction in threshold was significant (Fisher's exact test, P<0.05). The average cell diameter of the neurons tested was similar in both groups (29.2±1.0μm in the control group, 30.2±0.7μm in the group with CGRP). Also the initial resting potential was indistinguishable (−53.9±1.6mV in the control group and −56.1±1.2mV in the group with CGRP). The slight hyperpolarization during testing of CGRP (from −48 to −50mV in the neuron shown in Fig. 7) was probably not an effect of CGRP because control neurons showed a similar small hyperpolarization during the recording.
Effect of CGRP on the elicitation of an action potential. (A) Upon application of 10, 20 or 30 pA an action potential was elicited at 30 pA. (B) During CGRP, application of 20 pA elicited an action potential. (C) After CGRP washout, 30 pA were necessary to elicit an action potential.
4. Discussion
This study shows that CGRP significantly increases the amplitude of TTX-R voltage-activated sodium currents in about one-third of isolated small- to medium-sized DRG neurons from adult rats. The effect of CGRP on the neurons was abolished by coadministration of the antagonist CGRP8–37. Furthermore, the effect of CGRP was prevented when PKA or PKC in the neurons was inhibited, suggesting that the effect of CGRP on TTX-R voltage-activated Na+ currents is dependent on second messenger pathways involving PKA and PKC.
4.1. Effect of CGRP on Na+ currents
TTX-R Na+ channels were identified in nociceptive DRG neurons (Akopian et al., 1999; Dib-Hajj et al., 1998), their peripheral axons and spinal processes (Amaya et al., 2000; Jeftinija, 1994; Quasthoff et al., 1995), and in the dorsal horn where C-fibre afferents and other afferents form synapses (Amaya et al., 2000). TTX-R Na+ currents make the largest contribution to the action potential of small- to medium-sized DRG neurons (Blair and Bean, 2002; Fang et al., 2002), and they seem to be sufficient for the generation of action potentials in these DRG neurons (Scholz and Vogel, 2000). In C-type DRG neurons Nav1.8 contributes a substantial fraction of the inward current during the rising phase of the action potential (Renganathan et al., 2001). Furthermore, recordings from sensory endings in the cornea suggest that TTX-R Na+ currents participate in the initial steps of nociceptor activation (Brock et al., 1998). SNS−/− knockout mice exhibit pronounced mechanical hypoalgesia but only small deficits in the response to thermal stimuli (Akopian et al., 1999), suggesting TTX-R Na+ currents are somehow involved in nociceptive encoding of mechanical stimuli.
TTX-R Na+ currents in sensory neurons are a target of mediators that play a role in nociception, e.g. PGE2 (England et al., 1996; Gold et al., 1996, 1998, 2002). After PGE2, the threshold in DRG neurons is lowered, the rate of activation and inactivation is increased and the magnitude of current after depolarization is enhanced (McCleskey and Gold, 1999). On the other hand, TTX-R Na+ currents are inhibited by NO donors (Renganathan et al., 2002), and by the kappa opioid receptor agonist U50,488 through a non-opioidergic mechanism (Su et al., 2002). Here we show that TTX-R Na+ currents are also affected by CGRP. During CGRP application, peak current density increased by 26% on average, and CGRP caused a shift of the conductance towards hyperpolarization, without changing the slope of the peak conductance curve (k). The steady-state inactivation protocol also revealed a significant shift towards hyperpolarization. Thus, CGRP has pronounced effects on DRG neurons that may facilitate neuronal activation. Similar effects have been observed after application of PGE2 to rat sensory neurons although changes of steady-state inactivation have not been observed in earlier studies (Gold et al., 1996, 2002) but only in a recent study on Nav1.9 Na+ currents (Rush and Waxman, 2004).
Positive effects of CGRP on TTX-R Na+ currents have been observed in about one-third of the small- to medium-sized DRG neurons tested. This proportion fits our previous study in which CGRP produced an elevation of intracellular calcium in about 30% of the DRG neurons and in which about 20% of cultured and fixed DRG neurons bound gold-labeled CGRP (Segond von Banchet et al., 2002). Possibly the lower proportion of CGRP-positive neurons in our immunohistochemical study results from our very restrictive definition of a positive labeling. Most of the cultured neurons with CGRP-gold binding were small- to medium-sized and unmyelinated, expressed trkA receptors, and all of them synthesized CGRP (Segond von Banchet et al., 2002).
4.2. Blockade of the CGRP effect by CGRP8–37 and inhibitors of protein kinase A and protein kinase C
Based on the comparison of different samples of DRG neurons we conclude that the effect of CGRP on TTX-R Na+ currents is blocked by the antagonist CGRP8–37. When CGRP was administered following and during the administration of CGRP8–37, only one of 19 neurons showed a response to CGRP whereas CGRP alone increased TTX-R Na+ currents in about one-third of the neurons. In the previous study (Segond von Banchet et al., 2002), binding of CGRP-gold to DRG neurons was prevented by CGRP or CGRP8–37. Collectively these data show that the CGRP effect on DRG neurons and in particular on TTX-R Na+ currents can be blocked by CGRP8–37.
A similar protocol showed that PKA activation is involved in the CGRP effect. We first administered PKA14–22 which is a specific and membrane-permeant blocker of PKA. The application of PKA14–22 by itself caused a small reduction of the TTX-R Na+ currents probably because we reduced basal channel phosphorylation through PKA (Fitzgerald et al., 1999; Gold et al., 1998). When CGRP was administered in the presence of PKA14–22, only one of 21 neurons showed an increase of TTX-R Na+ currents. This is of particular interest because the second messenger pathway cAMP/PKA is critically involved in the action of other mediators that are important for the sensitization of nociceptors during inflammation. For example, PGE2 sensitizes visceral nociceptive primary afferents in vivo to heat stimulation (Mizumura et al., 1993), articular and visceral afferents to mechanical stimulation (Koda and Mizumura, 2002; Schaible and Schmidt, 1988), and chemical stimulation with bradykinin (Schaible and Schmidt, 1988), and cAMP seems to be the relevant second messenger for PGE2 effects (Ferreira and Nakamura, 1979). In DRG neurons cAMP mediates sensitization of TTX-R Na+ currents through activation of PKA (England et al., 1996; Gold et al., 1996, 1998). Similarly, the stimulating effect of cytokines on DRG ganglion neurons is dependent on cAMP and PKA (Zhang et al., 2002).
We also tested the effect of PKC19–31, a PKC inhibitor, on CGRP responses. PKC19–31 had to be included in the electrode, and it caused a decrease of TTX-R Na+ currents similarly as previously reported (Gold et al., 1998). However, CGRP did not elicit the same effect as under conditions where only CGRP was administered, suggesting that PKC inhibition can prevent CGRP effects. The reason for this PKC effect may be an interaction between PKA and PKC. Gold et al. (1998) have observed that forskolin-induced modulation of TTX-R Na+ currents was significantly inhibited by PKC inhibitors whereas PKA inhibitors had little effect on a PDBu-induced increase of TTX-R Na+ currents.
4.3. Effect of CGRP on the action potential
Previously, we observed that the application of CGRP close to neurons in the current clamp-mode elicited a depolarization that was sufficiently high in some neurons to evoke action potentials (Segond von Banchet et al., 2002). Because changes of excitability may occur at lower concentrations of CGRP which do not cause a (substantial) depolarization we tested here whether the much lower concentration of CGRP used in the present study modulates the threshold to elicit an action potential. During bath application of 250nM CGRP we did not observe a depolarization without electrical stimulation. Rather a small hyperpolarization of 2–5mV was seen which was, however, also observed in neurons tested with control solution without CGRP. While repeated current applications under control conditions (solution without CGRP) did not reduce the threshold for an action potential we found that the threshold was lowered in 9 of 28 of the neurons tested with CGRP. At this time, however, this change cannot be attributed to a specific ion channel because we did not use blockers in these experiments. Only occasionally an enhanced firing rate of the neurons was observed when current was injected for 500ms; possibly an increase in the afterhyperpolarization time constant after CGRP may be responsible for this (unpublished observations). Thus, probably various currents are modified by CGRP. In hippocampal pyramidal neurons, CGRP suppresses the slow Ca2+-activated K+ current by activating the cAMP-dependent protein kinase A (Haug and Storm, 2000).
4.4. Functional significance
The spinal cord has been previously identified as a target for CGRP. At this site CGRP is nociceptive (Kawamura et al., 1989) and furthers the development of inflammation-evoked hyperexcitability of spinal cord neurons (Ebersberger et al., 2000; Neugebauer et al., 1996), a large proportion of which express CGRP receptors (Yashpal et al., 1992; Ye et al., 1999). The present study provides evidence that CGRP acts also on DRG neurons and that this action is facilitatory. The data suggest an important role of TTX-R Na+ currents in this process. CGRP is released from primary afferents in vivo by antidromic nerve stimulation (Kress et al., 1999) and by heat (Kessler et al., 1999), and the present data suggest that CGRP does not only act on vessels thus producing neurogenic inflammation but that CGRP also acts back on primary afferent neurons. Future in vivo studies will address whether the application of CGRP to the sensory endings in the tissue will sensitize primary afferent neurons.
5. Conclusions
To our knowledge we have shown for the first time that CGRP enhances TTX-R Na+ currents in a proportion of DRG neurons. This effect is blocked by the CGRP receptor antagonist CGRP8–37 and by inhibition of PKA and PKC. The data further support the presence of CGRP receptors in primary afferent neurons, and they show that TTX-R Na+ currents are a target for CGRP effects.
Acknowledgements
The authors thank Mrs A. Wallner and Mrs F. Diebel for excellent technical assistance, and Prof. K. Benndorf, Department of Physiology, University of Jena, for discussion. The work was supported by the Deutsche Forschungsgemeinschaft (Scha 404/9-2) and the Interdisziplinäres Zentrum für Klinische Forschung.
References
Akopian AN, Sivilotti L, Wood JN. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature. 1996;379:257-262.
Akopian AN, Souslova V, England S, Okuse K, Ogata N, Ure J, Smith A, Kerr BJ, McMahon SB, Boyce S, Hill R, Stanfa LC, Dickenson AH, Wood JN. The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat Neurosci. 1999;2:541-548.
Amaya F, Decosterd I, Samad TA, Plumpton C, Tate S, Mannion RJ, Costigan M, Woolf CJ. Diversity of expression of the sensory neuron-specific TTX-resistant voltage-gated sodium ion channels SNS and SNS2. Mol Cell Neurosci. 2000;15:331-342.
Blair NT, Bean BP. Roles of tetrodotoxin (TTX-) sensitive Na+ current, TTX-resistant Na+ current, and Ca2+ current in the action potentials of nociceptive sensory neurons. J Neurosci. 2002;22:10277-10920.
Brain SD, Williams TJ, Tippens JR, Morris HR, MacIntyre I. Calcitonin gene-related peptide is a potent vasodilator. Nature. 1985;313:54-56.
Brock JA, McLachlan EM, Belmonte C. Tetrodotoxin-resistant impulses in single nociceptor nerve terminals in guinea-pig cornea. J Physiol. 1998;512:211-217.
Dennis T, Fournier A, St Pierre S, Quirion R. Structure-activity profile of calcitonin gene-related peptide in peripheral and brain tissues. Evidence for receptor multiplicity. J Pharmacol Exp Ther. 1989;251:718-725.
Dennis T, Fournier A, Cardieux A, Pomerleau F, Jolicoeur FB, St Pierre S, Quirion R. HCGRP8–37, a calcitonin gene-related peptide antagonist revealing CGRP receptors heterogeneity in brain and periphery. J Pharmacol Exp Ther. 1990;254:123-128.
Dib-Hajj SD, Tyrrell L, Black JA, Waxman SG. NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy. Proc Natl Acad Sci USA. 1998;95:8963-8968.
Ebersberger A, Charbel Issa P, Vanegas H, Schaible H-G. Differential effects of calcitonin gene-related peptide and calcitonin gene-related peptide8–37 upon responses to _N_-methyl-D-aspartate or (R,S)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate in spinal nociceptive neurons with knee joint input in the rat. Neuroscience. 2000;99:171-178.
England S, Bevan S, Dougherty RJ. PGE2 modulates the tetrodotoxin-resistant sodium currents in neonatal dorsal root ganglion neurones via the cyclic AMP-protein kinase A cascade. J Physiol (London). 1996;495:429-440.
Evans BN, Rosenblatt MI, Mnayer LO, Oliver KR, Dickerson IM. CGRP-RCP, a novel protein required for signal transduction at calcitonin gene-related peptide and adrenomedullin receptors. J Biol Chem. 2000;275:31438-31443.
Fang X, Djouhri L, Black JA, Dib-Hajj SD, Waxman SG, Lawson SN. The presence and role of the tetrodotoxin-resistant sodium channel Nav1.9 (NaN) in nociceptive primary afferent neurons. J Neurosci. 2002;22:7425-7433.
Ferreira SH, Nakamura MI. Prostaglandin hyperalgesia, a cAMP/Ca2+ dependent process. Prostaglandins. 1979;18:179-189.
Fitzgerald EM, Okuse K, Wood JN, Dolphin AC, Moss SJ. cAMP-dependent phosphorylation of the tetrodotoxin-resistant voltage-dependent sodium channel SNS. J Physiol (London). 1999;516:433-446.
Fjell J, Cummins TR, Dib-Hajj SD, Fried K, Black JA, Waxman SG. Differential role of GDNF and NGF in the maintenance of two TTX-resistant sodium channels in adult DRG neurons. Mol Brain Res. 1999;67:267-282.
Gold MS, Reichling DB, Shuster MJ, Levine JD. Hyperalgesic agents increase a tetrodotoxin-resistant Na+ current in nociceptors. Proc Natl Acad Sci USA. 1996;93:1108-1112.
Gold MS, Levine JD, Correa M. Modulation of TTX-R _I_Na by PKC and PKA and their role in PGE2-induced sensitization of rat sensory neurons in vitro. J Neurosci. 1998;18:10345-10355.
Gold MS, Zhang L, Wrigley DL, Traub RJ. Prostaglandin E2 modulates TTX-R INa in rat colonic sensory neurons. J Neurophysiol. 2002;88:1512-1522.
Goldin AL, Barchi RL, Caldwell JH, Hofmann F, Howe JR, Hunter JC, Kallen RG, Mandel G, Meisler MH, Netter YB, Noda M, Tamkun MM, Waxman SG, Wood JN, Catterall WA. Nomenclature of voltage-gated sodium channels. Neuron. 2002;28:365-368.
Haug T, Storm JF. Protein kinase A mediates the modulation of the slow Ca2+-dependent K+ current, IsAHP, by the neuropeptides CRF, VIP, and CGRP in hippocampal pyramidal neurons. J Neurophysiol. 2000;83:2071-2079.
Jeftinija S. The role of tetrodotoxin-resistant sodium channels of small primary afferent fibers. Brain Res. 1994;639:125-134.
Ju G, Hökfelt T, Brodin E, Fahrenkrug J, Fischer AJ, Frey P, Elde RP, Brown JC. Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells. Cell Tissue Res. 1987;247:417-431.
Kawamura K, Kuraishi Y, Minami M, Satoh M. Anti-nociceptive effect of intrathecally administered antiserum against calcitonin gene-related peptide on thermal and mechanical noxious stimuli in experimental hyperalgesic rats. Brain Res. 1989;497:199-203.
Kessler F, Habelt C, Averbeck B, Reeh PW, Kress M. Heat-induced release of CGRP from isolated rat skin and effects of bradykinin and the protein kinase C activator PMA. Pain. 1999;83:289-295.
Kilo S, Harding-Rose C, Hargreaves KM, Flores CM. Peripheral CGRP release as a marker for neurogenic inflammation: a model for the study of neuropeptide secretion in rat paw skin. Pain. 1979;73:201-207.
Koda H, Mizumura K. Sensitization to mechanical stimulation by inflammatory mediators and by mild burn in canine visceral nociceptors in vitro. J Neurophysiol. 2002;87:2043-2051.
Kress M, Guthmann C, Averbeck B, Reeh PW. Calcitonin gene-related peptide and prostaglandin E2 but not substance P release induced by antidromic nerve stimulation from rat skin in vitro. Neuroscience. 1999;89:303-310.
Luebke AE, Dahl GP, Roos BA, Dickerson IM. Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocyctes by using a cystic fibrosis transmembrane conductance regulator assay. Proc Natl Acad Sci USA. 1996;93:3455-3460.
Ma W, Chabot J-G, Powell KJ, Jhamandas K, Dickerson IM, Quirion R. Localization and modulation of calcitonin gene-related peptide-receptor component protein-immunoreactive cells in the rat central and peripheral nervous systems. Neuroscience. 2003;120:677-694.
Maggi CA, Chiba T, Giuliani S. Human α-calcitonin gene-related peptide (8–37) as an antagonist of exogenous and endogenous calcitonin gene-related peptide. Eur J Pharmacol. 1991;192:85-88.
McCarthy PW, Lawson SW. Cell type and conduction velocity of rat primary sensory neurons with calcitonin gene-related peptide-like immunoreactivity. Neuroscience. 1990;34:623-632.
McCleskey EW, Gold MS. Ion channels of nociception. Annu Rev Physiol. 1999;61:835-856.
McNeill DLK, Chung K, Carlton SM, Coggeshall RE. Calcitonin gene-related peptide immunostained axons provide evidence for fine primary afferent fibers in the dorsal and dorsolateral funiculi of the rat spinal cord. J Comp Neurol. 1988;272:303-308.
Mizumura K, Minagawa M, Tsujii Y, Kumazawa T. Prostaglandin E2-induced sensitization of the heat response of canine visceral polymodal receptors in vitro. Neurosci Lett. 1993;161:117-119.
Nakamura-Craig M, Gill BK. Effects of neurokinin A, substance P and calcitonin gene-related peptide on peripheral hyperalgesia in the rat paw. Neurosci Lett. 1991;92:325-329.
Neugebauer V, Rümenapp P, Schaible H-G. Calcitonin gene-related peptide is involved in the spinal processing of mechanosensory input from the rat's knee joint and in the generation and maintenance of hyperexcitability of dorsal horn neurons during development of acute inflammation. Neuroscience. 1996;71:1095-1109.
Pokabla MJ, Dickerson IM, Papka RE. Calcitonin gene-related peptide receptor component protein expression in the uterine cervix, lumbosacral spinal cord, and dorsal root ganglia. Peptides. 2002;23:507-514.
Poyner DR. Calcitonin gene-related peptide: multiple actions, multiple receptors. Pharmacol Ther. 1992;56:23-51.
Quasthoff S, Grosskreutz JM, Schröder JM, Schneider U, Grafe P. Calcium potentials and tetrodotoxin-resistant sodium potentials in unmyelinated C fibres of biopsied human sural nerve. Neuroscience. 1995;69:955-965.
Quirion R, Van Rossum D, Dumont Y, St-Pierre S, Fournier A. Characterisation of CGRP1 and CGRP2 receptor subtypes. Ann NY Acad Sci. 1992;657:88-105.
Renganathan M, Cummins TR, Waxman SG. Contribution of Nav1.8 sodium channels to action potential electrogenesis in DRG neurons. J Neurophysiol. 2001;86:629-640.
Renganathan M, Cummins TR, Waxman SG. Nitric oxide blocks fast, slow, and persistent Na+ channels in C-type DRG neurons by S-nitrosylation. J Neurophysiol. 2002;87:761-775.
Rush AM, Waxman SG. PGE2 increases the tetrodotoxin-resistant Nav1.9 sodium current in mouse DRG neurons via G-proteins. Brain Res. 2004;1023:264-271.
Sangameswaran L, Delgado SG, Fish LM, Koch BD, Jakeman LB, Stewart GR, Sze P, Hunter JC, Eglen RM, Herman RC. Structure and function of a novel voltage-gated, tetrodotoxin-resistant sodium channel specific to sensory neurons. J Biol Chem. 1996;271:5953-5956.
Schaible H-G, Schmidt RF. Excitation and sensitization of fine articular afferents from cat's knee joint by prostaglandin E2. J Physiol (London). 1988;403:91-104.
Schaible H-G, Segond von Banchet G, Ebersberger A, Natura G. 2004. Involvement of CGRP in nociceptive processing and hyperalgesia: effects of CGRP on spinal and dorsal root ganglion neurons. In: Brune K, Handwerker HO, editors., Hyperalgesia: molecular mechanisms and clinical implications. Progress in pain research management, vol. 30, IASP Press, Seattle, pp. 201-227.
Scholz A, Vogel W. Tetrodotoxin-resistant action potentials in dorsal root ganglion neurons are blocked by local anesthetics. Pain. 2000;89:47-52.
Segond von Banchet G, Pastor A, Biskup C, Schlegel K, Benndorf K, Schaible H-G. Localization of functional calcitonin gene-related peptide binding sites in a subpopulation of cultured dorsal root ganglion neurons. Neuroscience. 2002;110:131-145.
Sexton PM, McKenzie JS, Mendelsohn FAO. Evidence for a new subclass of calcitonin gene-related peptide binding site in the rat brain. Neurochem Int. 1988;12:323-335.
Su X, Joshi SK, Kardos S, Gebhart GF. Sodium channel blocking actions of the kappa-opioid receptor agonist U50,488 contribute to its visceral antinociceptive effects. J Neurophysiol. 2002;87:1271-1279.
Yashpal K, Kar S, Dennis T, Quirion R. Quantitative autoradiographic distribution of calcitonin gene-related peptide (hCGRPalpha) binding sites in the rat and monkey spinal cord. J Comp Neurol. 1992;322:224-232.
Ye Z, Wimalawansa SJ, Westlund KN. Receptors for calcitonin gene-related peptide: localization in the dorsal and ventral spinal cord. Neuroscience. 1999;92:1389-1397.
Zhang J-M, Li H, Liu B, Brull SJ. Acute topical application of tumor necrosis factor α evokes protein kinase A-dependent responses in rat sensory neurons. J Neurophysiol. 2002;88:1387-1392.
Keywords:
CGRP; CGRP receptor; DRG neurons; TTX-R Na+ current; Protein kinase A; Protein kinase C
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