Anti-Allodynic Effect of the Flavonoid Myricetin in a Rat Model Of

ARTICLE IN PRESS

European Journal of Pain xxx (2010) xxx–xxx

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European Journal of Pain

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Anti-allodynic effect of the ﬂavonoid myricetin in a rat model of neuropathic pain: Involvement of p38 and protein kinase C mediated modulation of Ca2+ channels

Tim Hagenacker a, Imke Hillebrand a, Andreas Wissmann a, Dietrich Büsselberg b, Maria Schäfers a,* a Department of Neurology, University of Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany b Weill Cornell Medical College at Qatar, Qatar Foundation – Education City, P.O. Box 24144, Doha, Qatar article info abstract

Article history: Flavonoids are increasingly ingested by the population as chemotherapeutic and anti-inflammatory Received 16 December 2009 agents. Myricetin is a naturally occurring flavonoid known for its anti-neoplastic and anti-inflammatory Received in revised form 18 March 2010 effects. Recently, behavioral studies indicate a potential analgesic effect in animal models of pain. Pilot Accepted 14 April 2010 studies suggest a flavonoid-induced modulation of intracellular protein kinases and interactions with Available online xxxx voltage activated calcium channels. The aim of this study was to investigate the analgesic effect of myricetin in a neuropathic pain model Keywords: (spinal nerve ligation, SNL) in rats. To identify potential mechanisms of action, in vitro whole cell patch- Neuropathic pain Myricetin clamp recordings of isolated rat dorsal root ganglia (DRG) neurons were performed to analyze the mod- Voltage activated calcium channel currents ulation of voltage activated calcium channel currents (ICa(V)) and the influence of intracellular kinase Patch-clamp phosphorylation such as p38 mitogen-activated protein kinase (p38) or protein kinase C (PKC). Flavonoids In vivo, a single injection of myricetin (0.1–10 mg/kg i.p.) reduced SNL-induced mechanical allodynia

and thermal hyperalgesia lasting for several hours. In vitro, ICa(V) (depolarization from À80 to 0 mV) were reduced (10–56%) by low (0.1–5 lM) concentrations of myricetin. This decrease was abolished by blockade of PKC (20 lM chelerythrine for 30 min), but not of p38 (10 lM SB203580 for 30 min). In contrast,

higher (10–100 lM) concentrations of myricetin induced an increase of ICa(V) (20–40%), which was blocked by inhibition of p38, but not of PKC. We conclude that myricetin transiently reduces established neuropathic pain behavior. This analgesic

effect may be related to its PKC-induced decrease of ICa(V) in DRG neurons. Ó 2010 Published by Elsevier Ltd. on behalf of European Federation of International Association for the Study of Pain Chapters.

1. Introduction lated to pain development (Cheng and Ji, 2008). Pronociceptive factors activate signaling pathways including p38 mitogen-activated Flavonoids are a c-benzopyrone family that occur naturally and protein kinase (MAPK), a process that has been implicated in the are widely spread in plants. Several biological effects have been as- induction of hyperexcitability on injured dorsal root ganglion cribed to flavonoids (Birt et al., 2001; Havsteen, 2002). Currently (DRG) neurons (Schafers et al., 2003b; Obata and Noguchi, 2004). they are part of experimental cancer therapies (Kumamoto et al., The mechanisms involved in the antinociceptive action of 2009). Recently, behavioral studies indicate a potential analgesic myricetin are unclear. Pilot studies from other non-pain systems effect of myricitrin (glycosylated myricetin) in several animal mod- propose an inhibition of phosphoinositide-3-kinase (Agullo et al., els of pain: myricitrin reduces allodynia caused by intraplantar 1997; Gamet-Payrastre et al., 1999), an inhibition of nitric-oxide injection of complete Freund’s adjuvant or sciatic nerve injury production (Chen et al., 2000; Ferreira et al., 2006) or of several tu- (Meotti et al., 2006b) or intrathecal administration of cytokines mor-progressive signal molecules (Shih et al., 2009; Siegelin et al., (Meotti et al., 2007b), and comprises antinociceptive activity in 2009). In inflammatory pain models, myricitrin inhibited phos- several chemical acute models of nociception (Meotti et al., 2006a). phorylation of p38 in the spinal cord induced by intrathecal cyto- It is known that nerve injury causes the release of pronocicep- kine administration (Meotti et al., 2007b) and prevented protein tive factors that alter ion conductance, leading to sensitization kinase C activation in the hind paw after intraplantar injection of and hyperexcitability of nociceptive neurons, which is closely re- phorbol-myristate acetate (Meotti et al., 2006a,b). In cortical slices of mice with acetic acid-induced viszeral pain, myricitrin blocks Ca2+ influx under a K+-induced depolarization (Meotti et al., * Corresponding author. Tel.: +49 201 7232461; fax: +49 201 7235901. 2007a). Furthermore, in a model of visceral pain screening of E-mail address: [email protected] (M. Schäfers).

1090-3801/$36.00 Ó 2010 Published by Elsevier Ltd. on behalf of European Federation of International Association for the Study of Pain Chapters. doi:10.1016/j.ejpain.2010.04.005

Please cite this article in press as: Hagenacker T et al. Anti-allodynic effect of the ﬂavonoid myricetin in a rat model of neuropathic pain: Involvement of p38 and protein kinase C mediated modulation of Ca2+ channels. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.04.005 ARTICLE IN PRESS

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negative surface charges by CaCl2 pre-treatment prevented the Mechanical sensitivity was first assessed using von Frey hairs and antinociceptive effect of myricitrin (charge-screening effect) (Meo- the up–down method (Chaplan et al., 1994; Lichtman, 1998). The tti et al., 2007a), suggesting a modulation of Ca2+-conductance. 50% probability withdrawal threshold was determined. Thermal The subtypes of voltage activated calcium channels (VACCs) hyperalgesia was then assessed with an algesimeter (Ugo Basile, modulate the intracellular calcium concentration which is a medi- Comerio, Italy) as described previously (George et al., 2000). After ator of pain signals in primary afferent neurons (Damaj et al., 1993; accommodation to the testing apparatus, three consecutive ther- Li et al., 2004; Hagenacker et al., 2008). N-type channels are local- mal tests were applied to the animals’ hindpaws with at least a ized in synaptic nerve terminals of the dorsal horn (Petersen and 5 min interval between tests for an individual paw. Means of three LaMotte, 1991; Hatakeyama et al., 2001). A critical role of N-type tests were calculated. After basal thresholds were determined, rats VACCs in the pain modulation is supported by data from N-type were injected with vehicle or myricetin. For acute application in deficient mice and by the clinical efficacy of N-type channel antag- neuropathic animals, behavioral tests were performed 9 days after onists (Saegusa et al., 2001). T-type VACCs are localized in cell SNL at baseline, 60 min and 120 min after injection. Behavioral bodies and nerve endings where they regulate cellular excitability tests were performed by observers who were unaware of (Schroeder et al., 1990; Jevtovic-Todorovic and Todorovic, 2006; treatment. Todorovic and Jevtovic-Todorovic, 2006). After nerve injury, T-type channel current density is increased (Jagodic et al., 2008). 2.5. Cell culture The goal of this study was to investigate the analgesic effect of myricetin in the model of spinal nerve ligation (SNL) and to analyze DRG were harvested from 21- to 28-day-old Wistar rats from the underlying modulation of VACC currents (ICa(V)) and influence both sides of the spinal column and transferred into ice cooled F- of p38 and PKC activation in DRG neurons. 12 Medium (Biowest, UK). Ganglia were incubated in 0.1 ml colla- genase medium (Sigma Type II, 2612.5 U/ml, Biochrom AG, Ger- 2. Materials and methods many) and trypsin containing saline (2525 U/ml, Sigma Type IX, Sigma, Germany) with intermittent washing. Cells were dissoci- 2.1. Animals ated with a fire polished Pasteur pipette (tip diameter 150 lm) and plated in portions of 50 ll of the resulting suspension on small Experiments with adult male Wistar rats (120–200 g for behav- petri dishes (3 cm; Falcon ‘‘easy Grip’’) and incubated for 4–6 h, ioral procedures and 80–120 g for recording experiments, Charles- allowing cells to attach to the petri dish. Thereafter F-12 (with River, Germany) were approved by the Animal Care and Use Com- 10% horse serum, Sigma, Germany) and nerve growth factor mittees of the University of Duisburg-Essen, Germany. All animals (NGF-7S, Sigma, Germany) were added. Cells were incubated in a were kept on a 14/10 h light/dark cycle with water and food pellets humidified atmosphere (5% CO2)at37°C and used for electrophys- available ad libitum. iological experiments within the next 24 h.

2.6. Electrophysiology 2.2. Surgery Using the voltage-clamp configuration of the patch-clamp All surgical procedures were performed under deep isoflurane technique, whole cell ion currents were recorded from isolated (5% for induction and 2% for maintenance) anesthesia in 50% O . 2 DRG neurons. Only cells of small diameter <30 lm measured by The SNL was performed as described previously (Schafers et al., a stationary magnifier were chosen for the experiments. Routinely 2003a). Briefly, a midline incision above the lumbar spine exposed giga-ohm seals were established. Membrane potential was the left L6 transverse process. This process was then removed and clamped at À80 mV. ICa(V) were elicited by depolarizing command the left L5 spinal nerve was isolated and tightly ligated with 6-0 pulses to 0 mV for 150 ms. To obtain current–voltage relations (IV- silk. curve), depolarizing steps started at À60 mV and were increased stepwise by 10 mV to a maximum depolarization of +30 mV. When 2.3. Chemicals repetitive depolarizations to 0 mV led to a stable inward current,

the application of myricetin was started. ICa(V) were isolated using For in vivo application, myricetin (Sigma–Aldrich, Germany) the following external solution: TEA, 130 mM; glucose, 10 mM; was dissolved in polysorbat 80 and diluted in 0.9% NaCl to final HEPES, 10 mM; MgCl2, 1 mM; BaCl2, 10 mM (all Sigma–Aldrich, concentrations (0.1 mg/kg, 1 mg/kg, 10 mg/kg). Myricetin was gi- Taufkirchen, Germany), tetrodotoxin (TTX; Alomone labs, Jerusa- ven once as a single i.p. injection 9 days after SNL. For in vitro lem, Israel); osmolarity 323 mosM; pH 7.3. recording, myricetin was dissolved in ethanol to stock solutions Patch-clamp pipettes from filament-containing borosilicate with concentration of 10 mM or 1 mM. Final dilutions (ethanol less glass, filled with internal solution (CsCl, 140 mM; HEPES, 10 mM; than 0.001%) were made immediately before use. EGTA, 10 mM; MgCl2, 4 mM; Na-ATP, 2 mM; osmolarity 316 For blocking of p38, SB203580 (Jenabioscience, Germany, mosM, pH 7,2) had a resistance of 4–5 MX. Currents were recorded 10 lM) was dissolved in distilled water and added to the bath vol- using an EPC10 (HEKA, Lambrecht/Pfalz, Germany) and EPC screen ume 30 min before performing patch-clamp experiments. software (Patchmaster, HEKA, Lambrecht/Pfalz, Germany). Data For blocking of PKC, chelerythrine (Alomone labs, Israel, 20 lM) were sampled at 10 kHz, compensated for series resistance. Drugs was dissolved in distilled water and added to the bath volume were applied by a continuous flow bath application system. 30 min before performing patch-clamp experiments. For electrophysiological experiments, the final dilutions of the chemicals 2.7. Data analysis were added in distilled water to the bath solution (for content see Section 2.6). All currents were online corrected for shifts of the resting mem-

brane current and leak corrected using a P/4 protocol. ICa(V) used for 2.4. Behavioral tests calculation of time course, current–voltage relations and concentration–response curves were rundown corrected. Current values Rats were placed in a compartment with a wire mesh bottom were standardized to the time of drug application (=100%). Con- and allowed to acclimate for a minimum of 30 min before testing. centration-response results were obtained by calculating the mean

T. Hagenacker et al. / European Journal of Pain xxx (2010) xxx–xxx 3 percentage of action of the normalized data for each concentration of 1 mg/kg myricetin markedly increased the mechanical with- of myricetin. To compare the mean inhibitory effects (including drawal thresholds (12.8 g ± 0.2 at 60 min), the analgesic effect of standard deviations) of myricetin over the voltage range tested, the higher and lower doses tested was less prominent, indicating currents were normalized to the maximum current of the cur- a bell-shaped dose-dependency for the effect of mechanical allo- rent–voltage relation under control conditions and expressed as dynia in the SNL model. For thermal hyperalgesia, injection of percentage of the maximum current under control conditions. 10 mg/kg myricetin prolonged the withdrawal threshold to 28.5 s ± 3.4 at 60 min after injection, while lower doses (0.1; 2.8. Statistical analysis 1 mg/kg) were less effective. For both mechanical allodynia and thermal hyperalgesia, the analgesic effect of myricetin was signif- Behavioral data are given as mean ± standard error. For differ- icant at 60 min after injection, while at 120 min after injection ences between treatment groups, behavioral data were analyzed there was still a trend of reduced mechanical and thermal by one-way ANOVA with Tukey post-hoc test. For ease of compar- hyperalgesia. ison, data are presented as percentage change from baseline for each rat. Electrophysiological data are given as mean ± standard deviation and were analyzed with double sided Student’s t-test. 3.2. Myricetin modulates ICa(V) in a concentration- and voltage- A difference was accepted as signiﬁcant if p < 0.05. dependent manner

Depolarization of the neurons from holding potential to 0 mV 3. Results resulted in an inward current which inactivates during the voltage step of 150 ms. Under control conditions the mean peak I after 3.1. Single injection of myricetin transiently reduces established pain- Ca(V) depolarization was À1.36 ± 0.61 nA (Fig. 2A and D). Vehicle (etha- associated behavior in SNL rats nol) alone did not affect ICa(V) in the concentration used (data not shown). Application of 5 lM myricetin (Fig. 2A–C) reduced peak Prior to injection, mean withdrawal thresholds and latencies I by À56.3 ± 8%. The reduction of the peak calcium current 9 days after SNL were similar for all test groups (5.7 g ± 2.4 for Ca(V) started 35 s after application and it took up to 480 s until a steady mechanical withdrawal thresholds and 17.4 s ± 2.2 for thermal state was reached. Threshold concentrations were below 0.1 lM withdrawal latencies). Injection of vehicle did not change (data not shown). Over the entire voltage range, the largest peak mechanical withdrawal thresholds or thermal withdrawal I was elicited by depolarization to À10 mV. Five micromolar latencies. In contrast, injection of myricetin dose-dependently Ca(V) myricetin reduced peak I from À60 mV to +30 mV, except enhanced mechanical withdrawal thresholds and thermal with- Ca(V) depolarizations to À30 mV, where I was unaffected. The reduc- drawal latencies at 60 min after injection (Fig. 1). While injection Ca(V) tion was most prominent at depolarizations >À10 mV. In contrast,

application of 10 lM myricetin (Fig. 2D–F) increased ICa(V) by 40.1 ± 6.1%. The increase began 20 s after application and reached

steady state after 150 s. The current–voltage relation of ICa(V) after the application of 10 lM concentrations of myricetin displayed a voltage-dependent effect. While in the lower voltage range

(À60 mV to À30 mV) ICa(V) was reduced, depolarizations from À20 mV to +30 mV led to an increase of ICa(V). This effect was more pronounced in the higher voltage range depolarizations. Reversal potentials were not signiﬁcantly changed with either high or low concentrations of myricetin. Comparing different concentrations of myricetin (Fig. 3), appli-

cation of 0.1–5 lM myricetin reduced peak ICa(V) (from À10% to À56.3%) concentration-dependently. In contrast, concentrations

of myricetin higher than 10 lM led to opposite effects: peak ICa(V) was increased concentration-dependently (from +20.5% to +40%).

3.3. p38 MAPK mediates increase of ICa(V) by high concentrations of myricetin

To test whether intracellular signal cascades are involved in the

effect of myricetin on ICa(V), p38 phosphorylation was inhibited by preincubation with SB203580 30 min before application of myrice-

tin. Mean ICa(V) after incubation was 1.23 ± 0.66 nA and not significantly different from control-ICa(V). With preincubation of SB203580, the reduction of ICa(V) by myricetin at low concentrations (1 lM) is not signiﬁcantly affected (from À20 ± 7% to À32 ± 8%) (Fig. 4). In contrast, at high concentrations of myricetin (10 lM) inhibition of p38 completely abolished the increase of

ICa(V) seen before and changed to a decrease of the current (from +40 ± 6% to À37 ± 7%). The current–voltage relation after inhibition Fig. 1. Transient reversal of established neuropathic pain behavior in rats with SNL. of p38 and application of 10 lM myricetin modulated the voltage- A single intraperitoneal injection of myricetin dose-dependently and transiently dependent effect observed before. Then I was reduced starting attenuates established mechanical allodynia and thermal hyperalgesia in rats with Ca(V) SNL 60 min after injection (1 mg/kg myricetin for mechanical allodynia, 10 mg/kg at a depolarization to À30 mV to +30 mV. Reversal potential was myricetin for thermal hyperalgesia) (n = 6 per group). not affected.

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Fig. 2. Biphasic modulation of ICa(V) by different concentrations of myricetin. Modulation of ICa(V) by 5 lM (Fig. A–C) or 10 lM (Fig. D–F) myricetin: raw trace of ICa(V) during depolarization from the holding potential (À80 mV) to 0 mV. Black line marks control current, grey line marks the current after application of myricetin (A; 5 lM) (D; 10 lM).

Current–voltage relation of ICa(V). Bold line marks the current during stepwise depolarization from the holding potential to +30 mV under control conditions. Dashed line marks the current after application of myricetin. Over the entire voltage range, the current is mainly reduced (B; 5 lM) /induced (E; 10 lM) in the higher voltage range. Time course of ICa(V) during repetitive depolarization to 0 mV. For 5 lM myricetin (C), the peak current is reduced by À56.3 ± 8.3% (n = 10). For 10 lM myricetin (F), the peak current is increased by +40.1 ± 6.1% (arrows marks point of application) (n = 14).

Fig. 3. Concentration-dependency for myricetin induced modulation of ICa(V). Concentrations of myricetin inbetween 0.1 lM and 5 lM reduce I by À10.6% Ca(V) Fig. 4. Preincubation with the p38 inhibitor SB203580 abolishes the myricetin- to À56.3% (n = 19). In marked contrast, concentrations of myricetin of 10 lM and ÃÃ ÃÃÃ induced increase of ICa(V) peak current. With 1 lM myricetin inhibition of p38 does higher increase ICa(V) by 20.5–40.1% (n = 46) ( p < 0.01; p < 0.001). not affect the reduction of ICa(V). In contrast, with 10 lM myricetin, inhibition of p38

completely reverses the ICa(V) from induction to reduction (À37.6 ± 7.6%) (n = 32) 3.4. Protein kinase C mediates decrease of ICa(V) by low concentrations (Ãp < 0.05; ÃÃp < 0.01; ÃÃÃp < 0.0001). of myricetin incubation with chelerythrine 30 min before application of

To analyse a potential involvement of protein kinase C in mod- myricetin. Application of the inhibitor alone did not affect ICa(V) ulation of ICa(V) by myricetin, protein kinase C was inhibited by pre- (data not shown). Mean ICa(V) after incubation was 1.41 ± 0.65 nA

T. Hagenacker et al. / European Journal of Pain xxx (2010) xxx–xxx 5

Busselberg, 2007). In our study, low concentrations of myricetin re-

duced ICa(V) in DRG neurons, whereas higher concentrations of myricetin induced an increase of ICa(V), suggesting a biphasic concentration–response relation. A similar dose–response was found for the antinociceptive effect of myricetin on mechanical allodynia

in vivo, supporting the hypothesis that modulation of ICa(V) may contribute to the analgesic effect of myricetin: at low doses/concentrations, it may reﬂect myricetin induced inhibitory effects on VACCs. At high doses/concentrations, it may reﬂect myricetin-in-

duced excitatory effects on VACCs. As a reduction of ICa(V) lowers 2+ 2+ intracellular Ca , an increase of ICa(V) elevates intracellular Ca , which may increase the cell membrane excitability, trigger more neurotransmitter to be released and thus possibly decrease pain thresholds, supporting reduced analgesic effects of high-dose myricetin in vivo. Fig. 5. Preincubation with the PKC inhibitor chelerythrine (20 lM) abolishes the Interestingly, the stimulatory effect of concentrations >10 M reduction of ICa(V) peak current (myricetin 1 lM). The effect of 1 lM myricetin on l ICa(V) is abolished by PKC inhibition. The inhibition of PKC does not influence ICa(V) myricetin on peak ICa(V) was abolished by preincubation with the after application of 10 lM myricetin (Ãp < 0.05; ÃÃp < 0.01;ÃÃÃp < 0.0001). p38 inhibitor SB203580, suggesting a p38-mediated modulation of VACCs by myricetin. A previous study has shown that myricitrin is capable of abolishing p38 phosphorylation in the spinal cord in and not significantly different from control-I . With inhibition Ca(V) response to intrathecal cytokine application (Meotti et al., of protein kinase C, 1 lM myricetin abolished the reduction of I Ca(V) 2007a). p38 belongs to the MAPK that play a critical role in cell sig- completely (from À20 ± 7% to À1.8 ± 2.4%) (Fig. 5). In contrast, naling and gene expression (Sorkin et al., 2009). There is increasing 10 lM myricetin did not significantly affect the increase of I Ca(V) evidence that indicates that p38 activation plays an important role with inhibition of protein kinase C. in the development of neuropathic pain: phospho-p38 is increased in DRG neurons following peripheral inflammation and nerve inju- 4. Discussion ries (Ji et al., 2002; Jin et al., 2003; Obata and Noguchi, 2004), and intrathecal inhibition of p38 inhibits nerve injury induced pain Myricetin is a flavonoid that is increasingly discovered for its behavior (Ji et al., 2002; Schafers et al., 2003b; Obata and Noguchi, potential analgesic effects in mostly inflammatory and acute pain 2004; Suter et al., 2007). Although p38 signaling is typically asso- states. We show here that myricetin attenuates pain-associated ciated with long-term changes in cell function because of its role in behavior in a neuropathic pain model in rats. Our in vitro data regulating gene expression, recent data also support the hypothe- demonstrate for the first time direct evidence that in DRG neurons sis of p38-induced modulation of ion channels via posttransla- myricetin modulates ICa(V), and thus provides a rapid mechanism tional regulation: for VACCs, only data from non nociceptive that may influence nociceptive neuron excitability and therefore systems are available so far. In hippocampal neurons the activation neuropathic pain behavior after nerve injury. of N-type channels is required as a co-factor for the activation of The present study demonstrates that myricetin given intraper- p38 (Katoh-Semba et al., 2009), and in cardiomyocytes p38 is nec- itoneally significantly alleviates already established mechanical essary for the expression of T-type channels (Morishima et al., allodynia and thermal hyperalgesia after SNL in rats. This is in 2009). p38 is also involved indirectly in signal transmission pro- agreement with a recent pilot study which demonstrated transient cesses. It has been shown that N-type channel modulating effects analgesic effects of a single injection of myricitrin on established of bradykinin, which is relevant for molecular surrogates of ther- mechanical allodynia in mice after partial sciatic nerve injury mal hyperalgesia, depend on p38 (Wilk-Blaszczak et al., 1998). (Meotti et al., 2006b). The short duration of the analgesic effect ob- Whereas p38 activation appears to transmit the stimulatory served in our study is also comparable to previous reports (Meotti effect of concentrations >10 lM myricetin on peak ICa(V), PKC acti- et al., 2006b) which describe a maximum effect 1 h after injection vation seems to transduce the inhibitory effect of lower concentra- in our SNL model, that was abolished 6 h after injection in the tions of myricetin on peak ICa(V). Several studies have shown mouse sciatic nerve injury model (Meotti et al., 2006b). The tran- experimental evidence for a myricetin-induced inhibition of PKC sience of the behavioral effect of myricetin may argue for direct activation before: flavonoids inhibit phosphoinositide 3-kinases inhibition of excitability of sensory neurons rather than involve- and consequently PKC isoenzyme activation (Gamet-Payrastre ment of intracellular signal transduction pathways. Apart from et al., 1999; Aslam et al., 2007). Flavonoids can also inhibit PKC this, analgesic effects of myricitrin have been described in mostly directly (Agullo et al., 1997), as shown in many non-neuronal sys- inflammatory and acute pain models (Meotti et al., 2006b, tems such as the intestinal barrier, mast cells, colorectal carcinoma 2007a,b). cells, glioblastoma cells, fibroblasts, and vascular smooth muscle At present, it is unknown whether the essential inhibitory ac- cells (Herrera et al., 1996; Jimenez et al., 2002). tion of intraperitoneally applied myricetin is in the spinal cord, But PKC has been shown to play a crucial role in pain transmis- DRG or both. Systemic effects of myricetin could occur through ac- sion, especially regarding modulation of ion channel activity: for tions on DRGs because the blood–nerve barrier is incomplete at VACC, it has been shown that inhibition of T-type currents, which this site (Devor, 1999). In this study, we give in vitro evidence are mediated by neurokinin-1 activation, depends on PKC, as well for myricetin induced modulation of ion channel currents in DRG as regulation of L-type subforms (Galizzi et al., 1987; Doerner et al., neurons. 1988; Schroeder et al., 1990; Rangel et al., 2009; Yang et al., 2009). Previous studies suggested Ca2+ channels as promising targets Barium currents, which are experimentally used for the analysis of of myricetin (Meotti et al., 2007a). It is well-established that intra- calcium channel currents, are reduced by a PKC-mediated bradyki- cellular Ca2+ represents a key role for neurotransmitter release, cell nin effect (Boland et al., 1991). Especially L-type channel currents membrane excitability, activation of intracellular proteins and pain are affected by bradykinin (Robson and Burgoyne, 1989; Baumgar- thresholds (Silinsky et al., 1977; Clement, 1981; Welch et al., 1992; ten et al., 1992; Toselli and Taglietti, 2005). In central neurons, T- Prado, 2001; Cervero and Laird, 2003; Ward, 2004; Hagenacker and and L-type channels are involved in the gating of pain signals. This

6 T. Hagenacker et al. / European Journal of Pain xxx (2010) xxx–xxx gating is modulated by PKC activity (Cheong et al., 2008). In other Clement JG. BaCl2-induced contractions in the guinea pig ileum longitudinal 2+ pain associated brain areas like the periaqueductal gray, HVA-cur- muscle: role of presynaptic release of neurotransmitters and Ca translocation in the postsynaptic membrane. Can J Physiol Pharmacol 1981;59(6):541–7. rents were reduced by PKC mediated effects of opioid-receptor- Damaj MI, Welch SP, Martin BR. Involvement of calcium and L-type channels in mu-agonists (Cho et al., 2001; Ingram and Traynor, 2009). In addi- nicotine-induced antinociception. J Pharmacol Exp Ther 1993;266(3):1330–8. tion PKC is involved in opioid-receptor controlled internalization of Devor M. Unexplained peculiarities of the dorsal root ganglion. Pain 1999(Suppl. ):S27–35. N-type channels in DRG neurons and blockade of L-type channels Doerner D, Pitler TA, Alger BE. 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J Biol Chem 1987;262(15):6947–50. this effect is diminished by an opposing mechanism, the p38-med- Gamet-Payrastre L, Manenti S, Gratacap MP, Tulliez J, Chap H, Payrastre B. iated increase of VACC. This may not contribute to a direct analge- Flavonoids and the inhibition of PKC and PI 3-kinase. Gen Pharmacol sic effect by inhibition of excitation, but may have impact on 1999;32(3):279–86. George A, Marziniak M, Schafers M, Toyka KV, Sommer C. Thalidomide treatment in intracellular calcium-dependent processes. Our results thus give chronic constrictive neuropathy decreases endoneurial tumor necrosis factor- new evidence for a potential role of myricetin as a therapeutic alpha, increases interleukin-10 and has long-term effects on spinal cord dorsal agent in neuropathic pain. horn met-enkephalin. Pain 2000;88(3):267–75. Hagenacker T, Busselberg D. Modulation of intracellular calcium influences While our results may help to explain parts of analgesic effects capsaicin-induced currents of TRPV-1 and voltage-activated channel currents it is clear that the overall role of myricetin and related flavonoids in in nociceptive neurones. J Peripher Nerv Syst 2007;12(4):277–84. modulating pain is much more complex. Further studies are Hagenacker T, Ledwig D, Busselberg D. Feedback mechanisms in the regulation of intracellular calcium ([Ca2+]i) in the peripheral nociceptive system: role of needed to investigate which specific VACC subtypes are affected TRPV-1 and pain related receptors. Cell Calcium 2008;43(3):215–27. by myricetin. Additionally, recent studies also suggest myricitrin- Hatakeyama S, Wakamori M, Ino M, Miyamoto N, Takahashi E, Yoshinaga T, et al. induced interactions with other ion channels, such as K+-channels Differential nociceptive responses in mice lacking the alpha(1B) subunit of N- type Ca(2+) channels. Neuroreport 2001;12(11):2423–7. (Meotti et al., 2007a). Finally, a considerable number of studies re- Havsteen BH. The biochemistry and medical significance of the flavonoids. ported other effects of flavonoids, and many of them could also Pharmacol Ther 2002;96(2–3):67–202. contribute to their antinociceptive action (Agullo et al., 1997; Ga- Herrera MD, Zarzuelo A, Jimenez J, Marhuenda E, Duarte J. Effects of flavonoids on rat aortic smooth muscle contractility: structure-activity relationships. Gen met-Payrastre et al., 1999; Chen et al., 2000; Meotti et al., Pharmacol 1996;27(2):273–7. 2006a). Therefore, further studies are needed to define the antino- Ingram SL, Traynor JR. Role of protein kinase C in functional selectivity for ciceptive mechanism of myricetin as this may offer a novel pain- desensitization at the mu-opioid receptor: from pharmacological curiosity to alleviating target without major side effects. therapeutic potential. Br J Pharmacol 2009;158(1):154–6. 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