Capsaicin Differentially Modulates Voltage-Activated Calcium Channel Currents in Dorsal Root Ganglion Neurones of Rats

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Brain Research 1062 (2005) 74 – 85 www.elsevier.com/locate/brainres Research Report Capsaicin differentially modulates voltage-activated calcium channel currents in dorsal root ganglion neurones of rats

Tim Hagenacker a, Frank Splettstoesser a, Wolfgang Greffrath b, Rolf-Detlef Treede b, Dietrich Bu¨sselberg a,*

aInstitut fu¨r Physiologie, Universita¨tsklinikum Essen, Universita¨t Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany bInstitut fu¨r Physiologie und Pathophysiologie, Universita¨t Mainz, Saarstrasse 21, 55099 Mainz, Germany

Accepted 25 September 2005 Available online 2 November 2005

Abstract

It is discussed whether capsaicin, an agonist of the pain mediating TRPV1 receptor, decreases or increases voltage-activated calcium channel (VACC) currents (ICa(V)). ICa(V) were isolated in cultured dorsal root ganglion (DRG) neurones of rats using the whole cell patch 2+ clamp method and Ba as charge carrier. In large diameter neurones (>35Am), a concentration of 50AM was needed to reduce ICa(V) (activated by depolarizations to 0 mV) by 80%, while in small diameter neurones (30Am), the IC50 was 0.36 AM. This effect was concentration dependent with a threshold below 0.025 AM and maximal blockade (>80%) at 5AM. The current–voltage relation was shifted to the hyperpolarized direction with an increase of the current between À40 and À10mV and a decrease between 0 and +50 mV. Isolation of L-, N-, and T-type calcium channels resulted in differential effects when 0.1 AM capsaicin was applied. While T-type channel currents were equally reduced over the voltage range, L-type channel currents were additionally shifted to the hyperpolarized direction by 10 to 20 mV. N- type channel currents expressed either a shift (3 cells) or a reduction of the current (4 cells) or both (3 cells). Thus, capsaicin increases ICa(V) at negative and decreases ICa(V) at positive voltages by differentially affecting L-, N-, and T-type calcium channels. These effects of capsaicin on different VACCs in small DRG neurones, which most likely express the TRPV1 receptor, may represent another mechanism of action of the pungent substance capsaicin in addition to opening of TRPV1. D 2005 Elsevier B.V. All rights reserved.

Theme: Sensory systems Topic: Pain modulation: pharmacology

Keywords: Voltage-activated calcium channel current; Capsaicin; Pain; TRPV1; Dorsal root ganglion neurones; L-type calcium channel; N-type calcium channel; T-type calcium channel; Small diameter neurones; Electrophysiology; Patch clamp technique

2+ Abbreviations: [Ca ]i, intracellular calcium concentration; Calciseptine, specific L-type calcium channel blocker; Caps, Capsaicin (8-methyl-N-vanillyl-6- nonenamide); DRG, dorsal root ganglion; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,NV,NV-tetraacetic acid; FS-12, specific L-type calcium channel blocker; IC50, inhibitory concentration reducing 50%; ICa(V), voltage-activated calcium channel currents; ICa(L), long lasting voltage-activated calcium channel current (‘‘L-type’’); ICa(N), neither–nor voltage-activated calcium channel current (‘‘N-type’’); ICa(T), transient voltage-activated calcium channel current (‘‘T- type’’); ICa(P + Q), purkinje voltage-activated calcium channel current (‘‘P/Q-type’’); IV–Curve, current–voltage relation curve; Pimozide, specific T-type calcium channel blocker; TEA, tetraethylammonium; TTX, tetrodotoxin; TRPV1, transient receptor potential channel of the vanilloid receptor subtype, type 1 (formerly VR1); VACCs, voltage-activated calcium channels; N-Conotoxin GVIA, specific N-type calcium channel blocker; N-Conotoxin MVIAA, specific N- type calcium channel blocker * Corresponding author. E-mail address: [email protected] (D. Bu¨sselberg).

1. Introduction calcium channels to the hyperpolarizing range [30], indicating a facilitated activation of these channels with smaller Capsaicin (8-methyl-N-vanillyl-6-nonenamide), the ‘‘hot’’ depolarizations. This results in an intracellular rise of calcium substance in chilli peppers (Capsicum frutescens), is naturally within this voltage range. Furthermore, Cheng and colleagues present in the food consumed by humans, being commonly [10] demonstrated that myocytes might react differently, used as a spice. Capsaicin binds to a specific receptor, TRPV1 since they found an increase of the voltage-activated calcium (formerly VR1-) receptor, a member of the vanilloid receptor channel current after the application of nanomolar concen- subfamily of the transient receptor potential (TRP) channel trations of capsaicin. superfamily [21]. Capsaicin-sensitive neurones that express To shed light on the possible different effects of capsaicin TRPV1 have a large range of physiological functions on ICa(V), we describe a reduction in small DRG neurones (reviewed in [35]). For example, capsaicin acts in human (IC50 = 0.36 AM), while large neurones were six times less bronchi, where it inhibits the fenoterol induced relaxation of sensitive. To analyze the effect of capsaicin in small DRG smooth muscles [14]. Capsaicin-sensitive neurones play a neurones in more detail, we isolated currents through the L-, major role in the urinary bladder for the maintenance of N- and T-type calcium channels and found differential normal function [3,4] and are involved in the regulation of effects on these calcium channel subtypes. These results blood pressure [36]. But most important are the effects in the might give hints to understand pain modulating processes nociceptive system (reviewed by [35]). following the initial transduction process. Functional TRPV1 receptors are associated with a calcium A preliminary report has been published in abstract form permeable channel pore and are located in the plasma [20]. membrane of neurones as well as in the membrane of the endoplasmatic reticulum of these cells. These TRPV1 receptors are mainly expressed in a functionally distinguish- 2. Methods able subpopulation of dorsal root ganglia (DRG) neurones. These neurones are small and presumably have also 2.1. Preparation of dorsal root ganglion neurones nociceptive receptors [1,6–8,18]. TRPV1 is activated by a variety of chemicals and by thermal stimuli and was therefore Dorsal root ganglion neurones were isolated from 21- to suggested to represent a transducer molecule for noxious heat 28-day-old ‘‘Wistar’’ or ‘‘Lewis’’ rats. Animals were deeply [6,21]. Accordingly, small capsaicin-sensitive DRG neurones anaesthetized with Isofluran (Curamed), until pinching the rat were shown to specifically respond to noxious heat pulses tail and feet revealed complete analgesia. Thereafter, the rats [19,22,23,27,32], whereas large DRG neurones, that do not were decapitated, and the vertebral column was opened by a express TRPV1, are not heat-sensitive [22,23,27]. Thus, dorsal approach, starting at the cranial end. Spinal cord was activation of TRPV1 seems to play an essential role in removed, and dorsal root ganglia (DRGs) were collected by nociception, e.g., in the transduction process for noxious heat fine forceps from both sides of the spinal column and stimuli and in capsaicin-induced hyperalgesia [9,24,35]. transferred into ice cooled F-12 Medium (Biowest and As a main second messenger intracellular calcium Sigma, Taufkirchen, Germany). Spinal nerves were cut off 2+ ([Ca ]i) is tightly regulated and plays a major role, in the under optical control using fine ophthalmological scissors. mediation of nociception. Nonetheless, in a defined range, Thereafter, the ganglia were transferred into a mixture of 0.9 2+ changes of [Ca ]i are allowed and were shown to have ml F12 medium and 0.1 ml collagenase medium (2612.5 U/ functional relevance for the transduction of painful stimuli ml, Sigma Type II) and digested in a humidified atmosphere [19]. The activation of the TRPV1 receptor results in a rise (5% CO2)at37-C for 40–55 min. In the next step, the of the intracellular calcium [6,17,29]. This calcium could collagenase was removed by washing the ganglia with 1.5 ml either have been released from internal stores or could enter of F12 medium for three times. Then the ganglia were through calcium permeable pores from the extracellular transferred into trypsin containing saline (2525 U trypsin per space [13,17]. While the amount of calcium entering ml in F12 medium, Sigma Type IX) and incubated for 2–3 through the TRPV1 associated pore was relatively low in min under the same condition. Trypsinated ganglia were voltage-clamp experiments at À80 mV [40], larger amounts washed two times. After adding F12 medium (final volume of extracellular calcium may enter the cell when voltage- 0.7 ml), the DRG neurones were triturated with a fire polished activated calcium channels (VACCs) are activated, e.g., by Pasteur pipette (tip diameter 150 Am) until the ganglion action potential discharges [17]. capsules were opened and the neurones were released from It has been shown that capsaicin directly blocks calcium the ganglia. A portion of 50 Al of the resulting suspension was entry through voltage-activated calcium channels in a variety placed in the middle of small petri dishes (3 cm; Falcon ‘‘easy of different cell types [25,33] with effective concentrations Grip’’) and incubated for 4–6 h, allowing cells to attach to the between 5.8 AM and 20 AM (IC50 values [25]), while it seems petri dish. Thereafter, 1 ml F12 (with 10% horse serum, to be highly effective in sensory neurones at a concentration Sigma) and 100 Al nerve growth factor were added to each of 1 AM [38]. It has also been shown that capsaicin (30 AM) petri dish. Cultures were used for electrophysiological shifts the current–voltage relation of voltage-activated experiments within the next 24 h. 76 T. Hagenacker et al. / Brain Research 1062 (2005) 74–85

2.2. Recording techniques and isolation of the different through the bath (bath volume 1 ml) with a continuous flow calcium channel currents of 5 ml/min. To investigate the blocking effects of capsaicin on the With the whole cell patch clamp technique, membrane different types of calcium channel currents, we recorded currents of dorsal root ganglion neurones were recorded time dependence with standardized protocols. Starting from using a HEKA EPC9 (Germany) amplifier and EPC screen the holding potential at À80 mV voltage was increased to 0 software (HEKA, PULSE). mV for 150 ms. After ten control depolarizations, capsaicin Microelectrodes were pulled from borosilicate glass with was applied. Current–voltage relations were frequently inside filaments (o.d.: 1.5 mm and i.d.: 0.86 mm; Hilgen- recorded to investigate the shift of the current–voltage berg, Malsfeld, Germany) with a Sutter electrode puller relation of calcium channel subtypes. (Model P-87). Electrodes were fire polished with a micro To illuminate the function of the intracellular calcium, forge (MF-830, Narashige, Japan) to a final resistance of 3– which might enter through the TRPV1 receptor channel 7MV and filled with a internal solution of the following complex or through voltage-gated calcium channels as well composition: CsCl, 140 mM; HEPES, 10 mM; EGTA, 10 as by a release from the intracellular calcium stores, the mM; MgCl2, 4 mM; Na-ATP 2 mM; osmolarity 316 mosM. calcium chelator (EGTA), generally used in the recording The pH was adjusted to 7.2. electrode, was replaced by adding 10 mM cesium chloride. Before starting the experiment, the F12 medium of the In addition, the extracellular barium was replaced by the culture was changed, against a external medium, containing same concentration of calcium for these experiments. tetraethylammonium (TEA), 130 mM; glucose, 10 mM; HEPES, 10 mM; MgCl2, 1mM; BaCl2, 10 mM (all Sigma- 2.3. Data analysis Aldrich, Taufkirchen, Germany), tetrodotoxin (TTX; Alo- mone labs, Jerusalem, Israel), 1 AM; osmolarity 323 mosM; All currents were online corrected for shifts of the resting pH 7.3. TTX, TEA and cesium were used to block TTX- membrane current and leak corrected using a P/3 or P/4 sensitive sodium channels and potassium channels, respec- protocol. All calcium channel currents (ICa(V)) used for tively. Barium was used as a charge carrier. For the recording calculation of time course, current–voltage relations and of T-type channel calcium currents, calcium (99.9%) was dose–response curves were rundown corrected, assuming a used as charge carrier. For isolating defined calcium channel linear rundown. The rundown was calculated from 10 to 15 currents, pharmacological blockers were applied. For block- currents elicited before application. All current values were ing the T-type channel, pimozide (100 nM; Alomone labs, standardized to the time of drug application (=100%). Jerusalem, Israel; [31]) was used. For blocking L-type The capsaicin-induced current through the TRPV1 pore channels, calciseptine (2 AM; Alomone labs, Jerusalem, was calculated by measuring the leak currents, with EPC9 Israel; [11]) or nimodipin (2 AM; Alomone labs, Jerusalem, software before and after the application of capsaicin. While Israel) and – in some cases – FS-2 (2 AM, Alomone labs, the capsaicin-induced current is reflected in the increase of Jerusalem, Israel; [39]) were added. For blocking N-type the leak currents, the subtraction of the leak currents before calcium channels, N-Conotoxin GVIA (1 AM; Alomone and after application of capsaicin were taken as the current labs, Jerusalem, Israel) or MVIIA (2 AM; Alomone labs, which was directly induced by capsaicin. An experimental Jerusalem, Israel) was used. All blockers were pre-incubated approach by blocking all voltage-activated calcium channels for 5 min, before starting the experiment. by cadmium was not taken into account, due to the After a giga-ohm seal was obtained and a stable access to numerous unspecific effects of cadmium, and its additional the neurones had been established, membrane potential was influence on the intracellular Ca2+concentration [26] which routinely clamped at À80 mV. Voltage-activated calcium would result in a change of the current inactivation. channel currents were routinely elicited by depolarizing The current–voltage relation of the capsaicin-induced command pulses to 0 mV (to À20 mV for T-type channel current was measured by calculating the difference of the currents) for 150 ms. Data were sampled at 10 kHz, leak currents before and after application of capsaicin, over compensated for series resistance and stored on a hard disc. the voltage range between À80 to +20 mV. To obtain current–voltage relations (IV curve) depolarizing Dose–response curves were obtained by calculating the steps started at À50 mV and were increased step wise by 10 mean percentage of action of the normalized data as well as the mV to a maximum depolarization of +40 mV. standard deviation for each concentration of capsaicin. Data Capsaicin (Sigma Aldrich, Taufkirchen, Germany) was were fitted using the Langmuir equation: y = sch/(kh + ch) dissolved in ethanol 100% at a concentration of 10 mM. For where c is the concentration, s the saturation (here = 100), k recordings, this mixture was diluted in the external solution. the concentration at half-saturation and h the Hill coefficient. Final dilutions (0.025 AM–50 AM, therefore, ethanol was To compare the mean inhibitory effects (including 0.001% at the usually used concentration of 0.1AM standard deviations) of capsaicin over the voltage range capsaicin) were made immediately before use. Drugs were tested, currents were normalized to the maximum current of applied by a bath application system. To achieve total the current–voltage relation under control conditions and exchange of the bath saline, a volume of 10 ml was flushed expressed as a percentage of the current. T. Hagenacker et al. / Brain Research 1062 (2005) 74–85 77

The shift of the current–voltage relation after the depolarizing the neuron to 0 mV) which was reversible application of capsaicin for the different calcium channel within 200 s after withdrawal of the drug (Fig. 1c). The types was calculated by determining the minima of the amplitude of this capsaicin-induced current was concen- current–voltage relation with one data point before and after tration dependent: an inward current of 156 pA T 12 pA was the minimum using a polynomium of 2nd degree. The voltage induced at À80 mV with a concentration of 0.1 AM and a differences of the calculated minima under control conditions current of 309 pA T 37 pA at a concentration of 0.5 AM. and after the application of capsaicin – as a value of the However, even during application of 0.5 AM capsaicin, no shift – were calculated to quantify the degree of the shift. such capsaicin-induced current was observed in large To calculate the activation of calcium channel subtypes, neurones (changes of À2.4% to +2.6% compared to the the depolarization where the largest current (under control peak calcium channel current; n =6;Fig. 1d). This finding conditions or after the application of capsaicin) was elicited is compatible with the suggestion that the capsaicin receptor was set to 1. All other currents were calculated in relation to TRPV1 is mainly expressed in small nociceptive DRG this current, the sigmoid fit (3 parameters) was made with neurones (e.g., [7,18]). Sigma Plot 8.0. All other calculations were made using In large neurones application of capsaicin (0.5 AM; Microsoft Excel software. Data are given as means T SD. n = 6) led to a minor reduction of the calcium channel ‘‘P values’’ were calculated with the Student’s t test current (Fig. 1b, grey trace), while a concentration of 50 AM (double sided, unpaired, type 3) compared to control data capsaicin resulted in an almost complete reduction of the ( P < 0.001 (***); P < 0.005 (**); P < 0.01 (*)). current (not shown). This effect of capsaicin on calcium channel currents was clearly more pronounced in small neurones (10 Amto30 3. Results Am). Here, ICa(V) was already reduced by 24 T 3.6% at a concentration of 0.1 AM capsaicin (Figs. 1a, e, 2). Voltage-activated calcium channel currents were success- The time dependence of the reduction of the standardized fully recorded from 82 dorsal root ganglion neurones. In all peak calcium channel current after application of capsaicin in experiments, we clearly distinguished between large (diam- large (Fig. 1f; n = 6) and small cells (Fig. 1e; n = 5) illustrate eter >35 Am, n = 12) and small neurones (30 Am, n = 70). the different efficiency. After 10 voltage jumps to 0 mV under ICa(V) was recorded from 32 small neurones, in the other 38 control conditions, the application of capsaicin was started. recordings from small neurones calcium channel subtypes The reduction of the channel current was rapid, while it took were isolated (L-type, n = 9; N-type, n = 17; T-type, n =7; several minutes until a steady state was reached. In large cells, none of these types, n = 5; compare Table 1). an application of 0.5 AM capsaicin reduced the current by Depolarizing the neurones from the holding potential to 0 11.6 T 3% (Fig. 1f). The effect was more pronounced in small mV resulted in an inward current which slightly inactivated neurones, where a capsaicin concentration of 0.1 AM led to a during the voltage step of 150 ms (black traces in Figs. 1a maximum reduction of the peak current after 620 s (Fig. 1e; and b). n = 5). In these small cells, this effect was partially reversible Application of capsaicin (0.1 AM for >600 s) led to (¨52%) upon wash out of capsaicin (0.1 AM) but not at changes of the holding currents in small cells, exclusively. higher concentrations. We did not test the reversibility with Calculation of this current in 5 small neurones revealed that high concentrations in large neurones. capsaicin (0.1 AM) induced an inward current in the range of To obtain the concentration dependency of the reduction 9.5–10.5% of the peak calcium channel current (when of calcium channel currents (activated by a depolarization to 0 mV) in small cells, the reduction of the peak current by Table 1 different concentrations of capsaicin was fitted to the Number of experiments with the application of capsaicin conducted on Langmuir equation (Fig. 2). The sigmoid dose–response small and large neurones with and without the isolation of the current curve showed a threshold concentration below 0.025 AM through different calcium channel subtypes and an IC50 value of 0.36 AM. A nearly complete block IV TD I NIC A Caps (>80% reduction) was reached at concentrations above 5 Small cells ICa(V) 4195 6 34 AM. The Hill coefficient was calculated to be 1, indicating I 45 9 Ca(L) that capsaicin most likely binds to one binding site. ICa(N) 10 7 17 ICa(T) 34 7 In large neurones, higher concentrations were needed to ICa(nn) 5 block the calcium channel current. A concentration of 0.5 Large cells 6 12 AM had only a small effect on the current (6.72 T 6.12%; Further specifications are given in the text. P = 0.0002 vs. small neurones), and a full block (>80%) IV—Current voltage relation. was reached with 50 AM (diamonds in Fig. 2). This is a five TD—time dependence. to ten times lower sensitivity of the ICa(V) compared to small ICaps—capsaicin-induced membrane current. NIC—no internal EGTA as chelator was used, and barium in the external DRG neurones. solution was replaced by calcium. These recordings were also used to Since the high sensitivity of small neurones to capsaicin calculate the concentration dependence of the capsaicin effect. might be a key function for processing pain sensation, all 78 T. Hagenacker et al. / Brain Research 1062 (2005) 74–85

Fig. 1. Raw traces of voltage-activated calcium channel currents in small (a) and large (b) neurones, elicited by a depolarization from the holding potential of À80 mV to 0 mV. Currents under control conditions (black; lower traces) and after application of capsaicin (grey; upper trace) are superimposed. Note that currents were strongly reduced by 0.1 AM in small cells, whereas even 0.5 AM had little effect in large cells. Capsaicin induced an inward membrane current in small neurones (c; n = 5, 0.1 AM), but not in large neurones (d; n = 6, 0.5 AM), consistent with differential expression of TRPV1 according to neuron size. The current is expressed as a percentage of the total peak channel current. Averaged time dependences of voltage-activated calcium channel peak currents in small (e; n = 5, diameter < 30 Am) and large neurones (diameter >35 Am) (f; n = 6), Currents were elicited by a depolarization from the holding potential of À80 mV to 0 mV. The first arrow in both panels points on the time of the capsaicin application with 0.1 AM on small and 0.5 AM on large cells. The second arrow in panel (e), points on the washout of capsaicin. The interruptions of the time dependence result from measurements of current–voltage relations at these times. Means T SD. further experiments were carried out on these capsaicin- effect of capsaicin was observed at +40 mV with a reduction sensitive neurones. of the current of 83%. The current was nearly unaffected at Comparing the current–voltage relation of voltage- À10 mV. In the voltage range of À30 mV to À10 mV activated calcium channel currents before and after the capsaicin increased the channel current. At À20 mV, the application of 0.1 AM capsaicin (n = 4), the effects were current was increased by the factor 3–4, but we did not clearly different in the lower voltage range (À30 mV to À10 obtain a significant voltage dependency of the I–V shift mV) compared to the effect in the more depolarized range since this shift differed from cell to cell. This was taken as a (À10 mV to +50 mV; Fig. 3b). While under control possible indication that subtypes of VACCs (T-, N-, or L- conditions, the largest channel current was elicited by a type) might be affected differentially and therefore generate depolarization step to +10 mV, the maximum of the a more or a less pronounced shift, depending on the current–voltage relation curve shifted to the left and thus composition of the different channel subtypes in the cell to more hyperpolarized voltages (À10 mV). The maximum recorded. In regard to the diversity of DRG neurones, a T. Hagenacker et al. / Brain Research 1062 (2005) 74–85 79

maximum of the current–voltage relation shifted by À6.69 mV T 8.42 mV to the hyperpolarized direction (black bars). This large standard deviation reflects the big difference of the occurrence of calcium channel subtypes in the different cells recorded. The situation is different when comparing the shifts for the isolated channel subtypes. The maximum of the current–voltage relation of L-type channel currents shifted by À7.11 mV T 1.66 mV (dark grey bar; P = 0.0012 compared to non-shifter N-type) and for a subset of N-type channel currents (ICa(N-shift)) were shifted for À9.49 mV T Fig. 2. Dose–response relation for the reduction of the peak of voltage- 2.23 mV ( P = 0.002 compared to the non-shifter), while in ? activated calcium channel currents by capsaicin in small cells ( ) for a three out of seven recordings, no shift of the current– depolarizing voltage jump from holding potential of À80 mV to 0 mV for voltage relation of N-type channel currents was observed 150 ms. IC50 has been calculated with 0.36 AM (0.025 AM, n = 3; 0.1 AM, n = 5; 0.5 AM, n =4;1AM, n =3;5AM, n = 4). Threshold (medium grey bars). There was also no significant shift in concentration was below, the lowest concentration of 0.025 AM. Maximal the current–voltage relation after the application of cap- block (more than 80% reduction) was reached above concentrations of 5 saicin for T-type channel currents (light grey bar). A > M capsaicin. Diamonds ( ) are showing the reduction of ICa(V) in large The steady-state activation for the presented calcium cells, for concentrations of 0.5 AM and 50 AM capsaicin. Difference of channel subtypes under control conditions (bold lines), and reduction of ICa(v) is highly significant in small vs. large neurones ( P = 0.0002). Means T SD. after application 0.1 AM capsaicin (dashed lines) is illustrated in the right part of Fig. 5. For the L- and the N1-subtype, the activation clearly shifts to less depolarized varying composition of VACC subtypes from cell to cell is voltages (Fig. 5b, d), while we did not observe a shift for the granted. To illuminate whether capsaicin differentially N2- and the T-type channel currents after the application of affected these subtypes, we isolated L-, N-, and T-type capsaicin (Fig. 5f, h). channel currents and tested them separately for their L-type calcium channel currents expressed the highest sensitivity to capsaicin. reduction of the current when capsaicin (0.1 AM) was The raw traces of the L (Fig. 4a)-, N (Fig. 4c)-, and T- applied, and the channel was activated by a depolarization type (Fig. 4e) calcium channel current before and after the to 0 mV (35.5 T 11.3%; Fig. 6, grey bars). Three neurones application of capsaicin (0.1 AM) illustrated that all three where ICa(N) was isolated were less sensitive to the same subtypes could be reduced by capsaicin. The time depend- concentration of capsaicin (19.3 T 7.1%; dark grey bars) ence of the peak channel currents illustrates that a steady while in four the current was not reduced at all (À6.8 T state of the effect was reached within several minutes for all 5.1%; light grey bars). The isolated T-type channel current three subtypes (Figs. 4b, d, and f, for number of experi- was less sensitive than the L-type and the effected N-type ments, see Table 1). However, in three (out of seven) channel currents (12.4 T 10.4%, black bars). neurones in which the time dependence for N-type channel In total, in all of the 10 experiments where current– currents were recorded, no reduction of the calcium channel voltage relations for N-type channels were analyzed before currents was observed after the application of capsaicin and after the application of capsaicin, there was a clear (Fig. 4d, grey line). effect. These effects could be divided in three subgroups: (1) To highlight the effects of capsaicin on the current– shift and reduction by capsaicin (n = 3), (2) shift but no voltage relation of the different channel subtypes, subsets of reduction (n = 3; the apparent reduction of the current at a experiments were performed, where only current–voltage relations were recorded. Therefore, these data could not be corrected for a linear rundown and could not be taken into account to calculate the reduction of the channel current. These data (before and after the application of capsaicin) were standardized to the largest peak channel current of the IV under control conditions. A shift of the current–voltage relation in the hyperpolarized direction was observed when capsaicin was applied when L-type (Fig. 5a) and a subgroup of N-type channel currents (Fig. 5c) was activated. Another subgroup of N-type channel currents (Fig. 5e) and ICa(T) (Fig. 5g) did not express any shift after capsaicin was applied. Fig. 3. Normalized current–voltage relation of the effect of capsaicin on voltage-activated calcium channels in small DRG neurones. Control IV is The averaged shift of the largest current in the current– represented by solid line, IV under 0.1 AM capsaicin by dashed line (n = 4). voltage relation over ICa(V) and of the different calcium Shown is the effect of a capsaicin concentration of 0.1 AM on 4 small channel subtypes is illustrated in Fig. 6a. For ICa(V), the neurones. Means T SD. 80 T. Hagenacker et al. / Brain Research 1062 (2005) 74–85

Fig. 4. Raw traces of isolated L-type (a), N-type (c) (both elicited by depolarizing to 0 mV), T-type (e) (elicited by a depolarization to À20 mV) calcium channel currents. The black lines represent the current elicited under control conditions and the grey line after the application of capsaicin. Time dependence of voltage-activated calcium channel current of the different types are shown for, L-type (b), two subtypes on N-type (d), and T-type (f). Used capsaicin concentration was 0.1 AM. Arrows point to the time of application. Means T SD. depolarization to 0 mV is not taken into account here), and +20 and +30 mV (n = 5). While we have not analyzed this (3) current reduction of the channel current without a shift of current in detail, there was no shift in the current–voltage the current–voltage relation (n = 4). relation, and this current was only slightly reduced when The shift of the current–voltage relations, as well as the capsaicin was applied (0.1 AM; data not shown). reduction of the calcium current through the isolated It is known that activation of TRPV1 receptor elevates calcium channel subtypes, was significantly different intracellular calcium by a transmembranous flow of calcium between the subtypes. This is illustrated by the vertical bars from the extracellular space as well as by activation of (Figs. 6a, b). The reduction of the total calcium current is capsaicin-sensitive intracellular stores (e.g., [13,17]). There- not the sum of the L-, N-, and T-type currents, since these fore, the reduction of the calcium channel current by channel subtypes are not equally distributed in the neurones. capsaicin could be mediated by an increase of the intra- Furthermore, there might be also other calcium channel cellular calcium concentration. A rise of the intracellular subtypes which were not further analyzed (see below). calcium concentration could be mediated by calcium entry After blocking L-, N-, and T-type calcium channels a as well as by a calcium release from the stores. To meet voltage-activated calcium channel current remained which these concerns, calcium in the external solution was used as had a maximum in the current–voltage relation between a charge carrier, and the intracellular calcium chelator T. Hagenacker et al. / Brain Research 1062 (2005) 74–85 81

Fig. 5. Illustration of the effect of 0.1 AM capsaicin in the current–voltage relation in the subtypes of voltage-gated calcium channels (a: L-type; c, e: N-type; g: T-type), in representative neurones. To highlight only the shift (but not the reduction of the current, since these data were not corrected for a linear run-down) the maximum current of the control IV was set to 100%. While the IVof L-type channel currents was significantly shifted, there was no shift of the IV when T- type currents were isolated. N-type currents expressed a shift in 60% of the neurones (c), while there was no shift (but a reduction of the current) in 40% of the experiments (e). Note that for this set of experiments only current–voltage relations were recorded during the application of capsaicin, therefore, the IVafter the application of capsaicin could not be corrected for a linear rundown. Calculation of the steady-state activation of the different channel subtypes (b: L-type; d, f: N-type; h: T-type) shows that capsaicin modulates only in some subtypes the activation. While the L-type and the N1-type express a modulation of their activation by capsaicin, this was not observed for the N2- or the T-type.

EGTA was replaced by adding 10 mM cesium chloride. reduction of calcium channel currents is at least partially Overlaying the time dependence of these experiments with mediated by the rise of internal calcium (Fig. 7). the time dependence of the other experiments during the application of 0.5 AM capsaicin displays a highly significant difference ( P < 0.001, t-test) between both experimental 4. Discussion conditions (18.8% T 4.3% without chelator but with calcium; 53.5% T 6.4% with EGTA as chelator and barium We found that voltage-activated calcium channel currents as the charge carrier), indicating that the capsaicin-induced of small as well as large DRG neurones are affected by 82 T. Hagenacker et al. / Brain Research 1062 (2005) 74–85

3. Compared to the peak ICa(V), the current induced by capsaicin is relatively small (¨8% at À80 mV, comp Fig. 1c). 4. The current–voltage relation of the capsaicin-induced current [28] indicates that this current decreases in direction to 0 mV (where the reversal potential is; and therefore is negligible at this potential) before it increases at more depolarized potentials.

Unfortunately a direct reduction of the TRPV1 induced current the TRPV1 antagonists capsazepine or ruthenium red was not advisable, since both antagonists are relatively unspecific and in addition have the ability to block calcium channels by themselves. Capsaicin may bind directly to VACCs, since its analog capsazepine (a competitive TRPV1 receptor antagonist) is known to bind and block VACCs [12]. Nevertheless, our results suggest that the modulation of ICa(V) depends on the presence of the TRPV1 receptor, since the effect is clearly more pronounced in small DRG neurones – which express this receptor – compared to large DRG neurones (which do not express this receptor). The reduction of ICa(V) at relatively high concentrations, as it occurs in large neurones, is in agreement with the findings of Kopantisa and co-workers [25] and most likely reflects a direct action at the channel, while it is intriguing to speculate that the high sensitivity of ICa(V) in small neurones is modulated by the activation of the TRPV1 Fig. 6. (a) Shift of the maximum value of the current–voltage relation for receptor.

ICa(V) and the isolated calcium channel subtypes after the application of Our findings on small DRG neurones are in agreement capsaicin (0.1 AM). (b) Reduction of voltage-activated calcium channel with the experiments of Wu an co-workers [38]. But Wu and currents (and the isolated subtypes) when the currents were activated by a co-workers found a similar reduction of the calcium current depolarization to 0 mV (for ICa(V), ICa(L), and ICa(N))ortoÀ20 mV (for through all channel subtypes, while our results demonstrate ICa(T)). Means T SD. that the action of capsaicin on ICa(V) in small DRG neurones elicits differential effects in different calcium channel capsaicin, with the small neurones being more sensitive by a factor of 5 to 10. Previously, we and others have demonstrated that small cells are far more sensitive to heat stimuli compared to large DRG neurones [15,22,23,27], and we demonstrated in addition that such small DRG neurones express the TRPV1 receptor [7,18]. While capsaicin induces a voltage-dependent inward current through TRPV1 in small DRG neurones, the question arises whether this current itself might have a major impact on ICa(V). While certainly the capsaicin-induced current results in a reduction of the membrane resistance, the following observations favor the conclusion that this current does not significantly interfere with our finding, that capsaicin differentially modifies currents through VACCs: Fig. 7. The reduction of ICa(V) is significantly different when a possible rise of intracellular calcium due to calcium entry or a release from intracellular 1. The time dependence of the reduction of the ICa(V) is stores is inhibited by intracellular calcium chelator EGTA (as routinely slower than the one we observed for the capsaicin- used) in comparison to conditions where calcium is present in the induced current (comp. Fig. 1c). extracellular solution and no calcium chelator is used in the intracellular solution. Shown are averaged time dependences of the peak calcium 2. The Hill coefficient for the capsaicin-induced reduction channel currents after the application of 0.5 AM capsaicin (black), recorded of IVCa was about 1, whereas it was about 2 for activating under standard conditions (black) and using calcium extracellularly and no TRPV1 [6]. chelator in the recording electrode (grey). Means T SD. T. Hagenacker et al. / Brain Research 1062 (2005) 74–85 83 subtypes. T-type currents and the remaining current after calcium from the internal stores [13]; similarly, we have blockade of L-, N-, and T-type channels (possibly a P-, Q-, suggested previously that noxious heat activates these and/or R-type, which was found only in 5 out of 38 intracellular compartments [17]. That the calcium entry neurones and therefore was not further analyzed) were not and/or the calcium release from the stores clearly reduces effected in the low concentration range of capsaicin. This is the effects on voltage-activated currents could be clearly in agreement with the conclusions drawn by Borgland and demonstrated. On the one side, the intracellular calcium co-workers [5] who also found a smaller sensitivity of T- rise will inactivate the voltage-activated calcium channels. type channels to capsaicin. Furthermore, calcium dependent intracellular signalling L-type channels were most sensitive to capsaicin (0.1 pathways could be modulated by the rise of calcium. This AM). The current through these channels was reduced by is in clear contradiction to the findings of Wu and co- nearly a third with a stepwise depolarization to 0 mV, workers [38], who could abolish the capsaicin-induced while this current was increased by nearly 80% by a effect when BAPTA was used in their intracellular depolarization to À20 mV. In general, the current was solution. Unfortunately, this difference in the result is reduced in the voltage range between 0 and +40 mV but not easy to explain. Our major concern with their results increased at depolarizations between À60 and À10 mV. A is that they have used routinely the calcium chelator possible explanation is that capsaicin facilitates the EGTA (10 mM) in their recording electrode. Therefore, activation as well as the inactivation of the channel. the addition of another calcium chelator BAPTA (10 mM) Therefore, the current–voltage relation shifts to the in their electrode should not have given any change of hyperpolarized direction with the result that the channel their result. current is larger at smaller depolarizations but appears to The influence of capsaicin on pain-modulation has been be smaller when the cell is more depolarized. Interestingly, discussed for a long time (reviewed in [35]): it can shift activation of L-type calcium channels seems to play a the heat sensitivity curve of TRPV1, it can transiently or 2+ major contribution to the transient rise in [Ca ]i when irreversible damage TRPV1 expressing neurones. Here, we unclamped DRG neurones were activated by noxious heat propose an additional mechanism of action, by modulation pulses [17]. of VACCs. The action of capsaicin on voltage-activated N-type It is open for discussion whether the increasing calcium channel currents is the most intriguing result of this reduction of calcium channel currents with higher concen- study. This channel is of special interest since it has been trations of capsaicin possibly suppresses either intensive discussed to be a modulator of pain sensation [2,16]. While pain signals themselves and/or calcium-dependent sensiti- in half of the experiments, there was also a shift of the zation of TRPV1 [34,37]. The shift of the current–voltage current–voltage relation as described for the L-type relation as seen in L- and partially in N-type channel currents, both shifts being most likely triggered by the currents magnifies the calcium response for slight depola- same mechanism. The other half of the neurones with rizations, while the calcium entry is depressed with larger isolated N-type ion current did not express such a shift. depolarizations. This is noticeable since depolarizations in Here, the current was equally reduced over the entire the lower voltage range between À50 and À10 mV occur voltage range. These two different effects occurred inde- frequently during receptor and/or postsynaptic potentials. pendently of each other, but we do not know whether these Therefore, a facilitated calcium entry at this voltage range two effects are the result of two different mechanisms will modulate such responses and possibly increase the initiated by the activation of the TRPV1 receptor. Since the sensitivity of neurones involved. N-type currents in the capsaicin-insensitive subpopulation In general, the capsaicin-induced shift of the current– displayed a more hyperpolarized peak, which was not voltage relation of L- and N-type channel currents, the further affected by capsaicin, one may speculate whether reduction of specific channel currents (N-type), and the there may be an endogenous pathway for the reduction of distribution of these channel subtypes in sensory DRG the N-type channel currents in isolated neurones. neurones could result in a modulation of the afferent pain While Wu and co-workers [38] did not find a signal at the spinal cord. modulation on the activation for calcium channel subtypes, our study clearly points out that the steady-state activation of the L- and the N-subtype is affected by Acknowledgments capsaicin. A possible explanation could be that Wu and co-workers used calcium as charge carrier in most of their We like to thank Prof. Dr. S. Cleveland of the Institute experiments, while we used barium. The extracellular of Physiology at the University of Du¨sseldorf for fitting calcium possibly influences the charge screening of the the dose–response data to the Langmuir equation. Mr. D. membrane. Ledwig was involved in the final stage of this study. We The application of capsaicin results in an increase of also thank Mrs. K. Go¨pelt for her excellent technical cytosolic calcium not only by a calcium entry through assistance in preparing the cultures of dorsal root ganglion associated membrane channels but also by releasing neurones from rats. 84 T. Hagenacker et al. / Brain Research 1062 (2005) 74–85

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